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Endothelium and smooth muscle function in rat mesentric vasculature He, Yi 2001

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ENDOTHELIUM AND SMOOTH MUSCLE FUNCTION IN RAT MESENTERIC VASCULATURE by  YI HE M.D., Bethune Medical University, 1983 M.Sc, The University of British Columbia, 1994  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Pharmacology & Therapeutics, Faculty o f Medicine) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A April 2001 ©Yi H e , 2001  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  of  T h e U n i v e r s i t y o f British Vancouver, Canada  DE-6  (2/88)  Columbia  I  further  purposes  gain  be  It  is  shall  that  agree  may  representatives.  financial  permission.  Department  study.  requirements  not  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  head  make  it  extensive of  my  copying  or  my  written  11  ABSTRACT  The mesenteric arterial bed ( M A B ) comprises medium and small arteries as well as arterioles. They are important in generating and controlling peripheral resistance, thereby regulating blood flow and maintaining blood pressure. This hemodynamic function is mainly determined by the smooth muscle tone and contractility o f mesenteric arteries and arterioles. Endothelial cells lining blood vessels help smooth muscle in this function by releasing various vasoactive substances.  Abnormal vascular reactivity and impaired endothelium  function has been found in several forms o f hypertension. The purpose o f the research in this dissertation was to study some cellular mechanisms involved in regulating smooth muscle reactivity and endothelium vasodilator function in rat mesenteric vasculature, and their abnormalities in hypertensive states. CI" currents represent a depolarizing mechanism in vascular smooth muscle cells, thus in the first part o f the study the contribution o f CI" channels to cti-adrenoceptor-mediated vasoconstriction was studied in mesenteric arteries in vitro and in vivo  from  sham  normotensive and two-kidney, one-clip (2K1C) hypertensive rats. Blockade o f CI" channels with niflumic acid ( N F A ) significantly inhibited cirazoline-induced vasoconstriction in isolated M A B from both groups o f rats. Cirazoline-evoked vasoconstriction was also significantly inhibited following removal o f CI" from the perfusion buffer. Removal o f CI" resulted in a significantly greater inhibition o f cirazoline-mediated vasoconstriction in M A B from sham rats as compared with 2 K 1 C rats. In vivo, intravenous infusion o f cirazoline caused a dose-dependent decrease in superior mesenteric vascular conductance. Pretreatment with N F A significantly attenuated the cirazoline-mediated decrease in vascular conductance.  Ul  To further investigate how the CI" channel blockade impaired ai-adrenoceptor-mediated vasoconstriction, the inhibitory effect o f N F A on cirazoline-induced vasoconstriction in isolated M A B was compared with that produced by the voltage-operated calcium channel ( V O C ) blocker nifedipine ( N F D P ) . The extent to which the contractions to cirazoline were reduced by nifedipine compared to N F A plus N F D P was similar. Thus, effects o f N F A and N F D P were not additive.  In addition, in the absence o f extracellular C a , the transient 2 +  phasic contraction to cirazoline was not affected by N F A , or by N F D P . N F A also had no effect on contraction induced by the depolarizing agent KC1. These observations suggest that CI" channels  play an important role in ai-adrenoceptor-induced  vasoconstriction in  mesenteric blood vessels. They may act by producing membrane depolarization, thereby indirectly inhibiting  activation o f V O C s .  The contribution o f CI" channels  in cti-  adrenoceptor-mediated vasoconstriction in mesenteric blood vessels from 2 K 1 C hypertensive rats appears to be reduced. This effect may reflect an adaptive change due to increased vascular resistance in hypertension. In the second part o f the dissertation, the role o f CI" channels in endotheliumdependent relaxation to acetylcholine (ACh) in superior mesenteric artery and the factors that mediate the endothelium-dependent relaxation were investigated. The aorta was also studied as a comparison. A C h concentration-dependently relaxed phenylepinephrine (PE)-induced tone in rat endothelium-intact mesenteric arteries and aorta. Inhibition o f CI" channels with N F A had no effect on the dilator responses to A C h in either mesenteric arteries or aorta. The BK  C a  antagonist, T E A , decreased the potency (pD ) to A C h without affecting the maximum 2  response (Rm ) in mesenteric arteries, whereas it had no effect in aorta. In the presence o f ax  N F A plus T E A , there was no further inhibition seen in mesenteric arteries as compared to  iv  T E A alone. In contrast, in the aorta, the pD2 to A C h was significantly inhibited by N F A plus T E A although without changing the Rmax- In addition, neither N F A nor T E A alone, nor T E A plus N F A had any effect on relaxation to the C a  2 +  ionophore A23187 in aorta. These data  suggest that besides B K c , CI" channels play a functional role in ACh-induced endotheliuma  dependent  relaxation  in aorta,  inactivation o f receptor-operated C a  possibly by 2 +  preventing  the  depolarization-mediated  channels ( R O C ) , thereby resulting in a sustained C a  influx and N O synthesis. B y contrast, in mesenteric arteries, K  2 +  channels, but not CI"  +  channels, mediated the relaxation. In addition, we found that indomethacin has no effect on, while L - N M M A only slightly impaired, the relaxation to A C h , suggesting that the effect o f PGI2 is negligible, while the contribution o f N O is small in mesenteric arteries. Furthermore, the L - N M M A / indomethacin-insensitive component o f the relaxation response o f mesenteric artery to A C h was greatly inhibited in the presence o f S K c and B K c a  a  antagonists. H i g h K  +  (30 m M ) further decreased the maximum relaxation to A C h , but did not abolish it. Thus, the observations  suggest  that E D H F  contributes  to  a large  part  o f the  ACh-induced  vasorelaxation in rat superior mesenteric arteries. Another relaxing factor or (possibly more than one) that is distinct from E D H F , such as N O and PGI2, may also play a role. In the third part o f the dissertation, the contribution o f endogenous E D R F ( N O ) and endothelium-derived contraction factors (prostaglandins) to reactivity to N E in M A B  from  hypertensive Zucker obese rats with hyperinsulinemia and insulin resistance was studied. The influence o f insulin on the N E response was examined. There was no major difference in pressor responses to N E in M A B between hypertensive Zucker obese and hormotensive Zucker lean rats, except for a small decrease in responsiveness to the highest concentration o f NE  (90  nmol) tested.  Inhibition o f N O  synthesis  with  L-NMMA  enhanced  the  vasoconstriction to N E , while blockade o f prostanoid production by indomethacin decreased the N E response. A pathophysiological concentration o f insulin (200 mU/1) potentiated responses to the two lowest concentrations o f N E (0.3 and 0.9 nmol) used in M A B from Zucker obese rats, but not lean rats. The potentiating effect o f insulin was further enhanced after blockade o f N O synthesis, while it was prevented by inhibition o f prostanoid production. These data suggested that NE-induced vasoconstrictor responses are normally modulated by concurrent release o f N O and vasoconstrictor cyclooxygenase product(s) in M A B from both obese and lean Zucker rats. Insulin increases the release o f contracting cyclooxygenase product(s) and enhances reactivity to N E in M A B from obese rats. This altered action o f insulin may play a role in hypertension in this hyperinsulinemic/insulin resistant model.  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E OF C O N T E N T S  vi  LIST OF T A B L E S  xii  LIST OF FIGURES  xiii  LIST OF A B B R E V I A T I O N S  xv  ACKNOWLEDGEMENTS  xviii  DEDICATION  xix  INTRODUCTION I.  1  OVERVIEW  II.  1  CHARACTERISTICS OF RAT MESENTERIC VASCULATURE  3  1.  Structure and Constituents  3  2.  Localization of Peripheral Resistance  5  3.  The Control of Mesenteric Circulation  6  4.  Sympathetic Vasoconstriction in Mesenteric Arterial B e d  6  III.  ALPHAI-ADRENOCEPTORS AND  VASOCONSTRICTION  7  1.  The cti-Adrenoceptors  7  2.  Alphai-Adrenoceptor Subtypes  8  3.  Alphai-Adrenoceptor Signaling and C a M o b i l i z a t i o n  4.  Calcium, and oci-Adrenoceptor-Induced Contraction  5.  Calcium Influx Channels, Voltage Dependence and Activation  2+  by ai-Adrenoceptors 6  Possible Role o f CI" Channels in C a  11  15 2 +  influx and Smooth Muscle  Contraction IV.  9  18  ENDOTHELIUM-MEDIATED REGULATION O F MESENTERIC ARTERIAL TONE 26 1.  Endothelium-Derived Vasorelaxing Factors 1.1  Nitric Oxide (NO)  26 26  vii  1.2  Prostacyclin (PGI2)  37  1.3  EDHF  39  2.  V.  Endothelium-Derived Contracting Factors  45  2.1  Endothelin-1  45  2.2  Prostanoids: PGH2, TxA  48  2.3  Superoxide Anion (0 )  49  2  2  ABNORMALITIES IN HYPERTENSION  52  1.  Goldblatt 2 K 1 C Renovascular Hypertension  52  2.  Hypertension and Hyperinsulinemia/Insulin Resistance  58  VI.  SUMMARY  PARTI.  66  T H E CONTRIBUTION OF CHLORIDE CHANNELS T O a i - A D R E N O C E P T O R M E D I A T E D V A S O C O N S T R I C T I O N IN RAT MESENTERIC ARTERY  68  I.  RATIONALE  68  II.  WORKING HYPOTHESES AND SPECIFIC RESEARCH OBJECTIVES  73  III.  METHODS AND MATERIALS  76  1.  Surgical Preparation o f Hypertensive Rats  76  2.  Measurement o f Plasma Renin Activity  77  3.  Perfused Isolated Mesenteric Artery Preparation  77  4.  Experimental Protocols in Perfused M A B  78  5.  In vivo Measurement o f B l o o d F l o w and Vascular Conductance  81  6.  Experimental Protocols for in vivo Experiments  81  7.  Isolation o f Small Mesenteric Arteries  82  8.  Experimental Protocols for Measurement o f  1 2 5  I Efflux in  small mesenteric arteries  82  9.  Chemicals  83  10.  Data and Statistical Analysis  83  IV.  RESULTS 1.  Characteristics o f 2 K 1 C Hypertensive Rats  2.  Effect o f N F A on Cirazoline-Induced Vasoconstriction in  85 85  via  Isolated Mesenteric Arteries Perfused with Normal Krebs 3.  85  Effect o f N F A on Cirazoline-Induced Vasoconstriction in Isolated Mesenteric Arteries Perfused with Cl"-Free Buffer  4.  91  Influence o f N F A on Cirazoline-Induced Change in Mesenteric Vascular Conductance in Anaesthetized 2 K 1 C Hypertensive and Sham Normotensive Rats  5.  100  Effect o f Nifedipine and Nifedipine Plus N F A on Cirazoline-and KCl-Induced Vasoconstriction in Isolated M A B Perfused with Normal Krebs  6.  104  Effects o f N F A on Cirazoline- and KCl-Induced Vasoconstriction in Isolated M A B Perfused with L o w C a  7.  2 +  Solution  107  Effect o f N F A on Cirazoline-Induced Vasoconstriction i n Isolated M A B Perfused with C a - F r e e - E G T A Solution  107  2+  8.  1 2 5  I Efflux from Small Mesenteric Arteries  116  D I S C U S S I O N  V.  120  The Role of Ct Channels in ccj-Adrenoceptor-Induced Vasoconstriction 120 The Selectivity of NFA  125  Altered Function of Ct Channels in Mediating ai-Adrenoceptor-Induced Vasoconstriction in MAB from 2K1C Hypertensive Rat  126  VI  S U M M A R Y  131  VII  C O N C L U S I O N S  133  P A R T 2.  T H E MECHANISMS OF ACETYLCHOLINE-INDUCED RELAXATION IN R A T M E S E N T E R I C A R T E R Y : A COMPARISON WITH AORTA  I.  R A T I O N A L E  II.  W O R K I N G  H Y P O T H E S E S  in.  M E T H O D S  A N D  134 134  A N D  S P E C I F I C  R E S E A R C H  O B J E C T I V E S  M A T E R I A L S  13  8  140  1.  Isolated Artery R i n g Preparation for Isometric Tension Measurement  140  2.  Experimental Protocols  140  3.  Chemicals  141  ix  4. IV.  Statistical Analysis  142  RESULTS 1.  143  ACh-induced Relaxation 1.1.  The Effect of NFA and TEA on ACh-induced Relaxation in Rat A orta and Mesenteric Arteries  143  1.2.  Effect of L-NMMA on A Ch-Induced Relaxation of PE-Evoked Tension 147  1.3  Effect of Indomethacin on ACh-induced Relaxation of PE-Evoked Tension  2.  A23187-Induced Relaxation 2.1  2.2  151  151  Effect of L-NMMA and iC on A23187-Induced Relaxation of PE-Evoked Tension  3.  151  The Effect of NFA and TEA on A23187-Induced Relaxation in Rat Aorta and Mesenteric Arteries  152  Effect o f KC1 and K(c > Channel Blockade on ACh-induced a  NO-Independent Relaxation V.  143  DISCUSSION Aorta  152 161 161  Effect of NFA and TEA NO-Mediated and NO Independent Relaxation Mesenteric Artery Effect of NFA and TEA NO-Mediated and NO Independent Relaxation Aorta and Mesenteric Artery  161 164 167 167 168 171  Effect ofPGh in ACh- Induced Relaxation in Aorta and Mesenteric Arteries  171  EndotheHum-dependent relaxation to A23187 in aorta and mesenteric arteries  172  Vi.  SUMMARY  177  vn  CONCLUSIONS  179  VIII  PHYSIOLOGICAL SIGNIFICANCE  180  X  PART. 3  N O R E P I N E P H R I N E - I N D U C E D V A S O C O N S T R I C T I O N IN ISOLATED PERFUSED M A B F R O M OBESE Z U C K E R RATS: T H E E F F E C T O F INSULIN  181  I  RATIONALE  181  II.  WORKING HYPOTHESES AND SPECIFIC RESEARCH OBJECTIVES  184  III.  METHODS AND MATERIALS 1.  .  General Methodology  8  6 186  Animals  186  B l o o d pressure measurement  186  Biochemical analysis o f blood samples  186  Perfused isolated M A B preparation  187  2.  Experimental Protocols  187  3.  Chemicals  188  4.  Statistical Analysis  189  IV.  RESULTS 1.  General Characteristics o f Zucker Rats  2.  NE-Induced Vasoconstriction in Isolated Perfused M A B from  193 193  Obese and Lean Zucker Rats  193  3.  Effect o f N O S and/or C O X Inhibition on NE-Induced Responses  193  4.  Effect o f Insulin on NE-Induced Vasoconstriction in Isolated Perfused M A B  197  Influence o f N O S , C O X , PGH2/TXA2 Receptor and E T Receptor  5.  Inhibition on Insulin-Potentiation o f N E Responses V.  1  DISCUSSION  202 205  Characteristics of Zucker Obese Rats  205  NE-Induced Vasoconstriction in MAB of Zucker Rats  206  1.  Reactivity to NE and KCl in isolated perfused MAB  2.  Blockade of the NO synthesis enhanced vasoconstrictor responses to NE  3.  206  207  Blockade of COX Pathway Suppresses Pressor Responses to NE  210  xi  4.  Effect of COX Inhibition on Pressor Responses to NE After Blocking of NO Synthesis  5.  Lack of Influence of Endothelin on Responses to NE  213 213  Insulin Effect on Vasoconstrictor Responses to NE in MAB of Zucker Rats 1.  Hyperinsulinemia elevated pressor responses to NE in MAB from obese rats  2.  214  214  Blockade of NO synthesis enhanced vascular effect of insulin in obese rats  3.  Inhibition of COX blocked insulin effect in MAB  4.  ET-1 contributing to potentiating effect of insulin on responses to NE in obese rats  215 216  217  VI.  SUMMARY  220  VII.  CONCLUSIONS  222  CONCLUDING REMARKS  223  BIBLIOGRAPHY  225  c  Xll  LIST OF TABLES Table  Page  1.1  Physiological characteristics o f 2 K 1 C and sham rats  86  1.2  Effects of N F A (3 mg/kg) or vehicle on mean blood pressure, superior mesenteric artery blood flow and conductance in anesthetized 2 K 1 C or sham rats  101  Effects of N F A (3 mg/kg) on cirazoline-induced changes in M A P in anesthetized 2 K 1 C and sham rats  102  Effects o f N F A (3 mg/kg) on cirazoline-induced decreases in vascular conductance (% o f control) in superior mesenteric artery in anesthetized 2 K 1 C and sham rats  103  Effects o f prazosin and N F A on cirazoline-induced isolated small mesenteric arteries  119  1.3  1.4  1.5  2.1  2.2  2.3  3.1  1 2 5  I efflux in  Sensitivity and maximum relaxation to A C h or A23187 in the absence and in the presence of N F A , T E A or N F A plus T E A in isolated aortic and mesenteric artery rings with intact endothelium  146  Effect of L - N M M A ( A ) and L - N M M A plus indomethacin (B) on sensitivity and maximum relaxation to A C h in the absence and in the presence o f T E A or N F A plus T E A in intact rat aortic and mesenteric artery rings  150  Effects of K(ca) channel blockers or KC1 (30 m M ) on sensitivity and maximum relaxation to A C h in intact mesenteric artery rings  160  Physiological characteristics o f lean and obese Zucker rats  194  xiii  LIST OF FIGURES Figure  Page  0.1  Alphai-adrenoceptor signaling pathway in vascular smooth muscle cells  12  0.2  ACh-induced release o f E D R F s in endothelial cells  28  1.1  Effect o f vehicle on vasoconstrictor responses to cirazoline in isolated M A B from 2 K 1 C or sham rats perfused with normal Krebs  87  Effect o f N F A on pressor responses to cirazoline in M A B from 2 K 1 C or sham rats perfused with normal Krebs  89  Effect o f N F A on vasoconstriction to KC1 in M A B from 2 K 1 C or sham rats perfused with normal Krebs  92  Effects of Cl"-free buffer and vehicle on pressor responses to cirazoline in M A B from 2 K 1 C or sham rats  94  Effects o f N F A (3 u M ) on pressor responses to cirazoline in M A B from 2 K 1 C or sham rats perfused with CI" -free buffer  96  Effects o f N F A (10 u M ) on pressor responses to cirazoline in M A B from 2 K 1 C or sham rats perfused with Cl"-free buffer  98  Effect of nifedipine ( N F D P ) and N F D P plus N F A (10 u M ) on contraction to cirazoline (A) or KC1 (B) in M A B from S D rats perfused with normal Krebs  105  Effect o f low C a buffer and vehicle on pressor responses to cirazoline (A) and KC1 (B) in M A B from S D rats  108  Effect o f N F A (3 u M in A , 10 u M in B ) on pressor responses to cirazoline in M A B from S D rats perfused with low C a buffer  110  Effect o f N F A (3 u M in A , 10 u M in B ) on contraction to KC1 in M A B from S D rats perfused with low C a buffer  112  Effect of N F A (10 u M ) on pressor response to cirazoline (0.3 nmol) in M A B from S D rats perfused with C a free-EGTA (1 m M ) solution  114  Effects o f cirazoline on arteries o f S D rats  117  1.2  1.3  1.4  1.5  1.6  1.7  1.8  1.9  2 +  +  1.10  2 +  1.11  2 +  1.12  125  I efflux in isolated small mesenteric  XIV  2.1 A 2.1 B  2.2 A 2.2 B  2.3 A 2.3 B  2.4  2.5  3.1  3.2  3.3  3.4  3.5  Effect of N F A , T E A or N F A plus T E A on relaxation responses to A C h in intact rat aortic rings Effect of N F A , T E A or N F A plus T E A on A C h - induced relaxation in intact rat mesenteric artery rings Effect of L - N M M A on relaxation responses to A C h in intact rat aorta, in the absence and presence o f T E A or N F A plus T E A Effect of L - N M M A on relaxation responses to A C h in intact mesenteric artery rings, in the absence and presence o f T E A or N F A plus T E A  144 144  148 148  Effects of L - N M M A and KC1 on A23187-induced relaxation in intact rat aorta Effect o f T E A , L - N M M A and KC1 on A23187-induced relaxation in intact rat mesenteric arteries  153  Representative traces showing the relaxation responses to A23187 in intact rings from aorta (A) and mesenteric artery (B)  155  Effect of KC1 and K ^ ) channel blockers on L-NMMA/indOmethacinresistant responses to A C h in intact rat mesenteric artery rings  158  Control experiments for responses to N E in isolated M A B obtained from lean or obese Zucker rats  191  Initial concentration-response curve to N E (A) and responses to KC1 (B) in isolated M A B obtained from lean or obese Zucker rats  195  Contraction to N E in the absence and presence of L - N M M A , or indomethacin or L - N M M A plus indomethacin in M A B from lean or obese Zucker rats  198  Concentration-response curves to N E in the absence and presence o f insulin (200 mU/1) in isolated M A B from lean or obese Zucker rats  200  153  Influence of various inhibitors on the potentiating effect o f insulin on contraction to N E in isolated M A B from obese Zucker rats  203  XV  LIST OF ABBREVIATIONS 125j  125  2K1C  Two-kidney, one-clip  ACh  Acetylcholine  Angll  Angiotensin II  ANOVA  Analysis o f variance  BKca  large conductance calcium-activated potassium channels  BP  B l o o d pressure  Ca  2 +  iodine  Calcium ion  cr  Chloride ion  cox  Cyclooxygenase  CTX  Charybdotoxin  Eci  Chloride equilibrium potential  ECs  Endothelial cells  ED  The molar concentration o f agonist which produces 50% o f the maximum effect  5 0  EDHF  Endothelium-derived hyperpolarizing factor  ER  Endoplasmic reticulum  Er  Reverse membrane potential  ET-1  Endothelin-1  ETOH  Ethanol  IbTX  Iberiotoxin  Id(Ca)  Calcium-activated chloride current/channel  iK(Ca)  Calcium-activated potassium current  Indo  Indomethacin  n>3  Inositol 1,4,5-triphosphate  k  Ion efflux rate (elimination constant per  K( a)  Calcium-activated potassium channels  K  Potassium ion  C  +  KC1  Potassium chloride  L-NMMA  N^-monomethyl-L-arginine  MAB  Mesenteric arterial bed  MAP  M e a n blood pressure  Na  Sodium ion  +  NE  Norepinephrine  NFA  Niflumic acid  NFDP  Nifedipine  NO  Nitric Oxide  NOS  Nitric oxide synthase  0 "  Superoxide anion  2  pD  -log E D  2  PGH PGI  2  2  5 0  Prostaglandin endoperoxides H  2  Prostacyclin  Popen  Open probability  Rmax  The maximum response o f an agonist  ROCs  Receptor-operated calcium channels  SKca  Small conductance calcium-activated potassium channels  SMAC  Superior mesenteric arterial conductance  SMAF  Superior mesenteric arterial blood flow  SOD  Superoxide dismutase  SR  Sarcoplasmic reticulum  TEA  Tetraethylammonium  TxA  2  Thromboxane A2  v  Membrane potential  VOCs  voltage -operated calcium channels  VSMC  Vascular smooth muscle cell  m  XV111  ACKNOWLEDGEMENTS I would like to give my profound gratitude to my supervisor Dr. Kathleen M . M a c L e o d for her accepting me as her student to complete my doctoral degree program, for her advice, understanding, consideration and constant support throughout the last course o f my research. I am also greatly indebted to Dr. Casey van Breemen, my co-supervisor, for giving me the opportunity to continue my study under his supervision as a P h . D . student in the Department o f Pharmacology. H i s brief but crucial advice always greatly inspired me. Without his consideration and support it is hard to imagine that I could have completed my program successfully. I would like to thank the members o f my research committee: D r . John H . M c N e i l l and D r . Ismail Laher for their support and advice. M y special thanks to my former supervisor D r . Reza Tabrizchi. W i t h his instruction I started my doctoral program and completed most o f my initial research. I would like to thank D r . David V . Godin, the head o f the Department o f Pharmacology, for his financial support and for his constant concern with my progress. I am also thankful to all the professors in the department who gave me courses in the past years for their valuable knowledge and helpful advice. I would also like to thank the Faculty o f Pharmaceutical Sciences, especially my home division: the Division o f Pharmacology and Toxicology. Everyone there has been very friendly so that I feel it as though I was at my home department. Thanks to B i l l y C h o w and Violet Y u e n for their help with glucose and insulin assay; to D r . Linfu Y a o for his technical advice, and to B i l l y Chow, Swamy Subramanian, L i l i Zhang and Andrea Bardell for making our laboratory a happy and warm place to work.  xix  DEDICATION  In the memory of my Mom & To my Dad and my brothers Ningyi, Xian and Yong  & To Helai for the love in the long journey of my study towards both Master's and Ph. D. degree in Canada  1  INTRODUCTION  I.  O V E R V I E W  In normal circumstances, the cardiovascular system delivers blood to the tissues in amounts corresponding to the metabolic demand, and at a pressure that allows appropriate diffusion across the capillaries. A n important part o f this process is mediated by the resistance vessels, which measure 20 to 500 pm in lumen diameter and consist o f small arteries (with a lumen larger than 100 pm) and arterioles (with a lumen smaller than 100 pm) (Davis et al. 1986; Mulvany and Aalkjaer 1990; Schiffrin 1992). Adjustment o f the resistance of these vessels through changes in their lumen diameter permits regulation o f tissue blood flow and aids in control o f blood pressure, thus allowing appropriate distribution o f cardiac output. This hemodynamic characteristic o f the resistance vessels is mainly determined by the smooth muscle tone o f the resistance arteries, which is governed by local, neuronal and humoral factors. Endothelial cells lining blood vessels, in particular, help smooth muscle in this function by producing and releasing various vasoactive substances. Knowledge o f mechanisms involved in the regulation o f smooth muscle tone in resistance arteries is thus o f major importance for our understanding o f the regulation o f peripheral resistance under normal conditions and the pathogenesis o f diseases such as hypertension where the peripheral resistance is altered. The splanchnic circulation receives about 25% o f total cardiac output in resting man (Folkow and N e i l 1971). It may possibly receive up to 30% o f cardiac output in rats under resting conditions (Folkow and N e i l 1971; Nilsson 1985). This makes it an important region  2  for maintaining cardiovascular homeostasis (Lundgren 1983). The mesenteric vascular bed, one o f the major vasculature beds within the splanchnic circulation, possesses great potential for demand related up- or down-regulation o f the blood flow to the intestines (Mitchell and Blomqvist 1971). Resistance to blood flow in the mesenteric vascular bed is therefore o f great hemodynamic importance. A s representatives o f resistance vasculature and highly reactive muscular vessels, rat mesenteric arteries have been extensively used in research in past decades. Besides their use in in vivo studies o f relationships between blood pressure, blood flow and vascular resistance, various preparations o f isolated mesenteric arteries have been used in in vitro work. For instance, the isolated perfused mesenteric arterial bed (McGregor 1965) is frequently used to study the regulation o f the integrated contractile activity o f the mesenteric arterial vasculature as a whole. The isolated small arteries (usually the 2  n d  and 3  r d  order branches o f the superior mesentery) are widely used to investigate the structure and function o f the individual resistance arteries (Bevan and Osher 1972; Duling et al. 1981; Halpern et al. 1984; Mulvany and Halpern 1976). The superior mesenteric artery, while in all likelihood a conduit artery, is also frenquently used to compare the properties o f muscular arteries with aorta and other elastic arteries, as well as with those muscular arteries in different tissues. The overall purpose o f the studies presented in this dissertation is to investigate some of the cellular mechanisms that regulate smooth muscle contraction and relaxation in mesenteric arteries, and to study how neurotransmitters, circulating hormones such as insulin, and vasoactive substances released from endothelium interact to modulate mesenteric vascular tone in normal and hypertensive states.  3  Specifically, the study consisted o f 3 parts. The purpose o f the first part was to investigate the contribution o f CI" channels to ai-adrenoceptor-mediated vasoconstriction in mesenteric arteries from normal and hypertensive rats. The second part o f the study was designed to examine the role o f CI" as well as K  +  channels in endothelium-dependent  relaxation in superior mesenteric artery, and the factors that mediate the endotheliumdependent relaxation. A n d finally, the purpose o f the third part o f the study was to evaluate the regulation o f NE-induced vasoconstriction in the perfused mesenteric arterial bed from obese rats with hypertension. The effect o f insulin was examined in the latter investigation. The following review focuses mainly on oti-adrenoceptor-mediated excitationcontraction coupling mechanisms and on mechanisms by which the endothelium modulates vascular tone, and how these are altered by hypertension. The general characteristics o f mesenteric vasculature that may be distinct from other systemic vascular beds and that may help to understand the mechanisms that regulate vascular tone in the mesenteric vasculature are also addressed.  n.  C H A R A C T E R I S T I C SO F R A T  M E S E N T E R I C  1.  Structure and Constituents  V A S C U L A T U R E  Generally, the arterial network in the rat mesenteric vasculature comprises the superior mesenteric artery, a medium-sized artery with a diameter range greater than 460pm, and 16-20 freely dividing small arteries, each o f which further subdivides into a few branches before joining the mesenteric arterial arcade (with a lumen diameter about 200pm) that runs parallel to the intestinal wall. F r o m the arcade arise many smaller arteries, which further  4  branch out, as arterioles, over the intestine (Hebel and Stromberg 1976; Lee et al. 1983b; Mulvany et al. 1978). Similar to other arteries, the vascular wall o f mesenteric arteries consists o f an outer tunica adventitia, a central tunica media, and an inner tunica intima. The main cellular constituents o f the vessels are the smooth muscle in the media and endothelium in the intima. The superior mesenteric artery is more muscular in structure than aorta. The smooth muscle in the superior mesenteric artery consists o f 60% o f the total volume o f tunica media. There are approximately 6-8 layers o f smooth muscle cells ( S M C ) arranged between 4-6 layers o f elastic laminae around the vessel wall. (Lee et al. 1983a; Lee et al. 1983b). The walls o f small mesenteric arteries become thinner as the arteries narrow, and there are only fragmented or no elastic laminae within the media (Lee et al. 1983a; Lee et al. 1983b). Smooth muscle cells in the small arteries and arterioles are arranged circumferentially. The number o f S M C layers decreases with the decrease in diameter. The small arterioles have only a single layer o f smooth muscle cells (Lee et al. 1983a; M i l l e r et al. 1987); however the volume fraction o f smooth muscle cells within the media increases with the decrease i n the diameter o f the vessels, being 70% in small arteries and 85% in larger arterioles (Lee et al. 1983a). The endothelial cells are separated from the media by an internal elastic lamina and form a continuous cover. Parts o f endothelial cells project into the vascular smooth muscle layer forming myoendothelial junctions at various points along the arteries and arterioles (Lee et al. 1983a). Perivascular nerves are localized near the media within the adventitia. (Furness 1973; Lee et al. 1983a).  5  2.  Localization of Peripheral Resistance Localization o f peripheral resistance  is important in understanding how  the  mesenteric arterial bed regulates peripheral resistance. Therefore, measurements o f pressure in individual vessels have been made. The percentage drop in pressure is indicative o f the portion o f resistance formed in the specific vessels. Based on the results from direct measurements o f the microcirculatory pressure in intestinal vasculature o f anaesthetized rats, early work indicated that small arteries and the largest arterioles in the mesentery contribute approximately 2 3 % to 57% and the small arterioles in the intestinal wall 18% to 47%, o f the total vascular resistance (Bohlen 1983; Gore and Bohlen 1977; Meininger et al. 1986). Recently, in conscious freely moving rats, Fenger-Grone et al (Fenger-Gron et al. 1995) measured the pressure along the mesenteric vascular bed and showed that about 3 1 % o f the systemic blood pressure drop occurs in the arcade small arteries, about a 5 1 % drop occurs in the intestinal microcirculation including arterioles, capillaries, venules and small veins, while 5% o f systemic pressure dissipates in superior mesentery, 6% in arcade veins and 7% in remaining veins plus the hepatic circulation. The results indicate that both small mesenteric arteries and microcirculatory vessels contribute significantly to peripheral resistance. In addition, when pressure was measured simultaneously with superior mesenteric blood flow and then vascular resistance was calculated (Fenger-Gron et al. 1997), both small mesenteric arteries and microvessels were shear to contribute to increased resistance during norepinephrine (NE)-induced smooth muscle contraction in conscious freely moving rats, indicating that the mesenteric arterial bed not only contributes to generating peripheral vascular resistance but also contributes to its control.  6  3.  The Control of Mesenteric Circulation Smooth muscle tone and the resistance of the mesenteric arterial bed, as in other  resistance vessels, are controlled by various intrinsic and extrinsic factors including physical forces, such as blood pressure and blood flow (Bevan and Laher 1991; Busse and Fleming 1998; Schubert and Mulvany 1999; Sun et al. 1992), neural stimuli (Bevan et al. 1980; Kawasaki et al. 1988; Nilsson et al. 1986), circulating hormones, and locally synthesized vasoactive  substances,  especially  endothelium-derived substances  (Sowers  1996 and  references therein). The influence of these factors varies in different vascular beds. For instance, the strength of intrinsic myogenic responses that are evoked by transmural pressure is much less in rat mesenteric vessels as compared with the same size skeletal muscle and cerebral vessels, where myogenic responses are predominant (Coombes et al. 1999; Lagaud et al. 1999; Osol et al. 1991; Watanabe et al. 1993; Wesselman et al. 1996). On the other hand, the fact that the neural regulation of blood flow is predominant in the splanchnic region has long been recognized. (Bohlen 1984; Furness and Marshall 1974)  4.  Sympathetic Vasoconstriction in the Mesenteric Arterial Bed Rat mesenteric blood vessels are densely innervated with sympathetic nerves that  mediate vasoconstriction (Furness and Marshall 1974; McGregor 1965; Nilsson et al. 1986). Normally occurring sympathetic vasoconstriction tonically regulates mesenteric vascular tone (Altura 1967). It has also been suggested from in vitro studies that the sympathetic neurotransmitter N E augments the myogenic response regulating mesenteric arterial tone (Chlopicki et al. 1996; Wesselman et al. 1996). In anaesthetized rats, direct observation under a microscope showed that sympathetic nerve stimulation constricts all the mesenteric arteries except precapillary arterioles that have  7  no adrenergic nerves in close association with them; the magnitude o f the contraction is greater in arteries than in arterioles (Furness and Marshall 1974). In vitro studies on isolated arteries have shown similar results, in which the maximal neurogenic response paralleled the sympathetic innervation density, being greater in the small mesenteric arteries, less in superior mesenteric artery and least in aorta (Nilsson et al. 1986). The mechanisms by which sympathetic nerve stimulation causes vasoconstriction involve in both a - and non-ctadrenergic mechanisms (Hirst and Edwards 1989 and references therein). The latter produce excitatory junction potentials (e.j.ps.); while the former release N E that activates postjunctional a-adrenoceptors (Bevan et al. 1980; B o w m a n and Rand 1980). The importance o f these two mechanisms varies both from artery to artery and from species to species and depends on the parameters o f stimulation used. It has been demonstrated that in rat mesenteric arteries, sympathetic vasoconstrictor responses are predominantly mediated by N E released from adrenergic nerve terminals (Kawasaki et al. 1988; Nilsson 1984; Nilsson et al. 1986). The a-adrenergic receptors activated by N E in rat mesenteric artery appear to be predominantly oti (Chen et al. 1996; Colucci et al. 1980; Colucci et al. 1981). Thus, the activation o f cti-adrenoceptors by N E is vital to control mesenteric vascular resistance, thereby regulating blood flow and blood pressure.  HI.  ALPHAI-ADRENOCEPTORS AND VASOCONSTRICTION  1.  The cti-Adrenoceptors In blood vessels, cti-adrenoceptors are present throughout the vasculature but are  more prominent on the arterial side. Besides the neurotransmitter N E , oti-adrenergic receptors are also activated by circulating catecholamines, N E and epinephrine. M a n y  g  observations have demonstrated that N E released from sympathetic neurons activates the receptor in much the same way it would i f applied exogenously (Raat et al. 1998). In addition, a wide variety o f antagonists such as phentolamine and prazosin, and agonists such as phenylepinephrine (PE), methoxamine and cirazoline can selectively block or stimulate oti-adrenergic receptors and distinguish them from other adrenergic receptors (Bylund et al. 1998; Ruffolo et al. 1991). In vascular smooth muscle, cxi-adrenoceptors serve a primary role in the control o f smooth muscle constriction (Vargas and Gorman 1995).  2.  Alphai-Adrenoceptor Subtypes Three oti-adrenoceptor subtypes have been identified by cloning. These were  originally named ai , an,, c  oiia/d,  and were subsequently renamed oti , an,, aw, respectively. a  They now have been classified on the basis o f pharmacological evidence as aiA ( a i ) , a i e c  (aib) and  aw  (aia/d)  adrenoceptors (Bylund et al. 1998 and references therein). A fourth a i -  adrenoceptor subtype has been postulated and is designated as a i x based on its l o w affinity for prazosin (Oshita et al. 1991). Recently it has been suggested that the aiL subtype may represent a particular conformational state o f the aiA-adrenoceptor (Ford et al. 1997). The three adrenoceptor subtypes a ^ , aie and a m differ in their amino acid sequence and their affinity for a variety o f synthetic agonists and antagonists (Bylund et al. 1994; Graham et al. 1996; H w a et al. 1995). They were reported to be heterogeneously distributed along the rat arterial tree (Piascik et al. 1994). According to pharmacological studies, contractions induced by exogenous N E and/or peripheral nerve stimulation are believed to be predominantly mediated by the activation o f aiA-adrenoceptors in the perfused mesenteric arterial bed o f rats (Chen et al. 1996; K o n g et al. 1994; Williams and Clarke 1995; Z h u et al. 1999). A small  9  number o f a m -adrenoceptors may also be activated by N E release in this vascular bed (Kong  et  al. 1994). The ctiA-adrenoceptor has  been  found to  be  responsible for  vasoconstriction evoked by application o f N E or peripheral nerve stimulation in other resistance vascular beds (Blue et al. 1992; Eltze et al. 1991; Z h u et al. 1997). In superior mesenteric artery, as well as in other conduit vessels, the am-adrenoceptor subtype has been suggested to mediate N E - and PE-induced contraction, at least in part (Buckner et al. 1996; Hussain and Marshall antibodies as tools,  1997; Y o u s i f et al. 1998).  O I I A - ,  Recently, using subtype-selective  a m - , and am-adrenoceptors were all detected in aorta, caudal,  femoral, iliac, renal, superior mesenteric, and mesenteric resistance arteries. However, despite the expression o f all adrenoceptors, only a single adrenoceptor seems to mediate the contractile response in the renal  (GCIA)  and femoral  (OCID)  arteries (Hrometz et al. 1999). Based  on our current knowledge, the role o f the other a i - adrenoceptor subtypes, i f they exist in the mesenteric resistance arteries, is not clear.  3.  AIphai-Adrenoceptor Signaling and C a  2 +  Mobilization  ai-adrenoceptors are intrinsic membrane glycoproteins and members o f the  G  protein-coupled receptor ( G P C R ) super family. Stimulation o f ai-adrenoceptors results in activation o f various effector enzymes including P L C , P L A  2  and P L D v i a different G  proteins, (Insel et al. 1998; L l a h i and Fain 1992; Nishio et al. 1996; Perez et al. 1993; Ruan et al. 1998; Schwinn et al. 1991; W u et al. 1992). It has been generally accepted that the major effector that transduces cti-adrenoceptor signals is the enzyme P L C (Cotecchia et al. 1990), which is likely to be P L C - P in vascular smooth muscle (Lee and Severson 1994). a i adrenoceptors couple to P L C predominantly via pertussis toxin-insensitive G proteins o f the  10  G / i i family (Boyer et al. 1992; Smrcka et al. 1991; Taylor et al. 1991; W u et al. 1992). q  Activated P L C catalyzes the hydrolysis o f the membrane lipid, phosphatidylinositol-4,5bisphosphate (PIP2), to yield the second messengers, diacylglycerol ( D A G ) and inositol1,4,5-triphosphate (JP ) (Abdel-Latif 1986). D A G activates protein kinase C ( P K C ) , which 3  may play a central role in phosphorylation o f variety o f cellular proteins that involved in the transduction o f oci-adrenoceptor activation into the final biological response (Berridge 1981; Horowitz et al. 1996; Mironneau et al. 1991; Nishizuka 1995; Walsh et al. 1994). A t the same time, IP3 binds to specific receptors (IP3 receptor) on sarcoplasmic reticulum (SR) and causes C a r e l e a s e from the intracellular stores (Berridge 1993; lino 1990; Lepretre et al. 2+  1994). The IP3- released C a  2 +  can in turn activate a calcium-induced calcium release channel  (CICR), which causes calcium release from a second S R pool (Baro and Eisner 1995; Karaki et al. 1997), and can activate several other classes o f Ca -sensitive ion channels on the cell 2+  membrane, such as calcium-activated K ( K (c )) and CI" (Ici(Ca)) channels which modulate the +  a  cell membrane properties (Amedee et al. 1990a; Amedee et al. 1990b; Byrne and Large 1988b; Pacaud et al. 1989a). Activation o f ai-adrenoceptors also leads to influx o f C a  2 +  from  the extracellular space (Ruffolo et al. 1991 and references therein). Depending on the species and tissue, a i - adrenoceptors are directly or indirectly coupled to several different channels including voltage-operated C a  2 +  Ca  2 +  channels ( V O C s ) (Bolton 1979; Bulbring and  Tomita 1987; Nelson et al. 1988; V a n Breemen et al. 1978), receptor-operated Ca channels 2+  (ROCs) (Bolton 1979; Bulbring and Tomita 1987; Ruegg et al. 1989; V a n Breemen et al. 1978), and non-selective cation channels (Amedee et al. 1990a; Byrne and Large 1988b; Loirand et al. 1991). However, the mechanism o f signaling from the activation to C a  2 +  influx is still not very clear. Another C a  2 +  ai-adrenoceptor  influx pathway is the C a  2 +  11  release-activated C a  channels ( C R A C ) , which are activated by S R depletion after receptor  stimulation, and have a variable sensitivity to dihydropyridine C a  2 +  channel blockers ( L o w et  al. 1991; Putney 1990). The channels that are insensitive to dihydropyridines have been suggested to be non-specific cation channels (Wayman et al. 1996). This pathway is believed to be important for the refilling o f the depleted S R and makes a variable contribution to contractile response depending on the smooth muscle type (Gibson et al. 1998; Karaki et al. 1997; Putney 1987 and references therein). C a  2 +  influx from the extracellular space can also  activate C I C R in intact guinea pig aorta, rat portal vein and rat mesenteric artery (Gregoire et al. 1993; Ito et al. 1991). A n increase in free cytosolic C a activation plays a predominant  2 +  level ([Ca ]i) by cti-adrenoceptor 2+  role in cti- adrenoceptor-mediated  biological  events,  especially in regulation o f smooth muscle contraction. (Fig 0.1)  4.  Calcium and ai-Adrenoceptor -Induced Contraction The contraction o f vascular smooth muscle by activation o f ai-adrenoceptors mainly  depends upon the increased [Ca ]i that results from both C a 2+  2 +  release from intracellular  organelles (i.e. SR) and influx from extracellular space (Ruffolo et al. 1991; Somlyo and Somlyo 1994). This is thought to occur by the Ca -calmodulin dependent activation o f 2+  myosin  light chain kinase, which then  phosphorylates  myosin light chain  (MLC).  Phosphorylated myosin can interact with actin and so induce contraction. In addition, ctiadrenoceptor agonists can increase the C a  2 +  sensitivity o f M L C phosphorylation by inhibition  of M L C phosphotase activity, and therefore increasing contraction at a constant level o f [Ca ]i (Somlyo and Somlyo 1994). The enhancement o f contractile filament Ca sensitivity, 2+  2+  and the fact that this enhancement may be mediated through P K C during oti- adrenoceptor  12  Ca  2 +  Figure 0.1 Major a [-adrenoceptor signaling pathways that mediate vascular smooth muscle contraction. (1) I P receptor, (2) voltage-gated C a channels, (3) receptoroperated C a channels (4) I i (Please see text for details). 2 +  3  2 +  C  ( C a ) i  13  activation, have been demonstrated in rat mesenteric arteries (Buus et al. 1998; Drenth et al. 1989; Jensen et al. 1992; Raat et al. 1998). Ca  2 +  release from the S R by IP3 produced on receptor activation is mainly responsible  for the initial peak o f the agonist-induced contraction. This initial response is referred to as the "phasic" response. The sustained contraction, which is referred to as the "tonic" response, is caused mainly by C a  2 +  influx (Karaki et al. 1997; Minneman 1988). This conclusion is  based on the observations that in vascular smooth muscle N E and other agonists induce only a transient contraction in the absence o f external C a , and that agonist-induced IP3 2 +  production is also transient (Abdel-Latif 1986 and references therein). In addition, depletion of SR C a  2 +  stores by ryanodine, a drug with selectivity for the S R (Julou-Schaeffer and  Freslon 1988; Sutko et al. 1985), inhibited only the initial portion, but not the sustained portion o f agonist -induced contraction (lino et al. 1988; Julou-Schaeffer and Freslon 1988; Kanmura et al. 1988). O n the other hand, it has been found that the tonic responses are largely inhibited in Ca -free solution or in the presence o f C a 2+  2 +  channel blockers, without  affecting the initial phasic response (Somlyo 1985; van Breemen and Saida 1989). C a  2 +  influx may also contribute to the initial portion o f contraction, whereas intracellular release could also contribute to the tonic response depending on the concentration o f the agonist and the arteries (Weber et al. 1995). Several studies indicate that the contribution o f C a  2 +  from different sources to  contraction in resistance vessels may differ from that in large conduit arteries. (Ashida et al. 1988; Cauvin et al. 1984; Jensen et al. 1992; L o w et al. 1996; Sato et al. 1988). It has been reported that ryanodine inhibited the NE-induced contraction by 52% in rat aorta and 14% in bovine tail artery without changing high K -induced contractions. A C a +  2 +  channel blocker  c  14  almost completely abolished high K -induced +  contractions and reduced  NE-induced  contractions by 4 5 % in the aorta and 82% in the tail artery (Ashida et al. 1988). In rabbit mesenteric arteries (Cauvin et al. 1984), NE-induced contractions and NE-stimulated  4 5  Ca  efflux decreased in Ca -free 2 m M E G T A solution, while the sensitivity o f NE-induced 2+  contractions to inhibition by the C a  2 +  channel blocker diltiazem increased, in a graded  fashion from proximal to distal arteries. It was also shown that C a influx induced by N E i n 4 5  the resistance vessels was approximately 10,000 fold more sensitive to the action o f diltiazem than that in rabbit aorta (Cauvin et al. 1984). These data indicate a decreasing release o f intracellular C a  2 +  and an increasing dependence on extracellular C a  2 +  for NE-induced  contractions as one proceeds from proximal to distal arteries. In addition, a functional study on the relative contribution o f extracellular C a  2 +  and C a  2 +  stores to N E - induced smooth  muscle contraction in arteries and arterioles from different species also suggested that although an agonist-releasable C a  2 +  pool is present at all levels o f the vasculature, the role o f  the S R diminishes as the arteries become smaller, while C a  2 +  fluxes across the plasma  membrane predominate during oti- adrenoceptor activation ( L o w et al. 1996). In the perfused rat hindlimb, Z h u et al (Zhu et al. 1998) demonstrated that the NE-induced maximum response was decreased by 92% following perfusion with Ca -free medium. Nifedipine 2+  concentration-dependently inhibited NE-induced contractions with a maximum inhibition o f 65% and the residual nifedipine-insensitive response was further  inhibited by  Cd , 2 +  suggesting the N E response in this preparation is mediated largely v i a an influx o f extracellular C a , mainly v i a nifedipine-sensitive C a 2 +  channels.  2 +  Furthermore, in rat  mesenteric small arteries, the main effect o f N E on [Ca ]i was suggested to be mediated 2+  through voltage-dependent, dihydropyridine-sensitive C a  2 +  channels, because when force,  15  membrane potential and [ C a ^ j i were measured simultaneously during stimulation with N E or potassium, a similar relationship between [ C a ] , and membrane potential was seen for both 2+  forms o f activation (Nilsson et al. 1994). The inhibition o f NE-induced contraction by nifedipine was also reported in these blood vessels (Chen et al. 1996). These results support the view that the contractile responses o f resistance vessels are heavily dependent on the presence o f extracellular C a  2 +  and that C a  2 +  entry occurs mainly though the V O C s during cti-  adrenoceptor activation.  5.  Calcium  Influx  Channels,  Voltage  Dependence  and  Activation  by  oti-  Adrenoceptors. W i t h patch-clamp and molecular biology techniques, six subtypes o f V O C s including L - , N - , P - , Q - , R-, and T-type have been demonstrated  in vascular smooth muscle  (Ganitkevich and Isenberg 1990; Hofmann and Klugbauer 1996; Nelson et al. 1990b), whereas no direct evidence for existence o f R O C s has been observed (Droogmans et al. 1987; Karaki et al. 1997; Nelson et al. 1990b; Nilsson et al. 1994). The predominant V O C s in arterial smooth muscle have been found to be L-type C a  2 +  channels, which are selectively  sensitive to inhibition by the dihydropyridines (Tsien et al. 1988). A t present, the major C a influx pathway is considered to be voltage-dependent L-type C a called C a  2 +  2 +  2 +  channels (hereafter simply  channels), since both the maintained arterial tone and the increase in [ C a ] i upon 2+  cti-adrenoceptor activation can be strongly inhibited by dihydropyridines (Kuriyama et al. 1995; Minneman 1988; Nilsson et al. 1994) (and references mentioned above). It is well known that membrane depolarization opens C a  2 +  channels (Godffaind 1986;  Kuriyama et al. 1995). The steady-state fractions o f time that a C a increase  exponentially with  membrane  depolarization  from  2 +  channel is open  (P  ope  n)  relatively hyperpolarized  16  potentials. That the voltage dependence o f P  o p e  n is over a wide voltage range has been  demonstrated (Nelson et al. 1990b; Nelson et al. 1988). This increase in P  o p e  n with membrane  potential is limited by the promotion o f a long-lived closed state called inactivation. The C a  2 +  channel is rapidly desensitized during prolonged depolarization, but is not completely inactivated (Fleischmann et al. 1994; Imaizumi et al. 1991; Nakayama et al. 1996). It has been shown that the relationship between C a  2 +  influx through C a  2 +  channels and membrane  potential ( V ) can be very steep, with 3 m V depolarization or hyperpolarization increasing or m  decreasing C a  2 +  influx as much as two-fold (Nelson et al. 1990b; Nelson et al. 1988). In  addition, the relationship between smooth muscle V between the V  m  and C a  2 +  m  and arterial tone as the relationship  (see above) is also very steep, so that even V  m  changes o f a few  millivolts cause significant changes in blood vessel diameters (Nelson et al. 1990b and references therein). Mesenteric artery tone is very sensitive to V  m  in the range between -46  and -20 m V (Cheung et al. 1999). The threshold for contraction induced by K C 1 is approximately  -46mV.  Maximum  contraction was  attained  when  the  arteries  were  depolarized to -20 m V . Thus, l m V depolarization resulted in an approximately 4% increase in tone. (This relationship was not altered in spontaneously hypertensive rats). Smooth muscle cells in arteries and arterioles, in vitro, have stable membrane potentials between -60 and -75 m V (Hirst and Edwards 1989). V  m  values measured in vivo are in the range o f - 4 0 to  -55 m V (Nelson and Quayle 1995 and references therein). The membrane potential o f arterial smooth muscle cells in vivo falls in the same range in which the current through C a  2 +  channels is strongly voltage dependent (Nelson et al. 1990b). It has long been known that in vascular smooth muscle,  oti-adrenoceptor-mediated  contraction is usually accompanied by a depolarization (Bolton et al. 1984; Byrne and Large  17  1988a; Mulvany et al. 1982; Nanjo 1984; Suzuki and K o u 1983; Takata 1980). For example, N E depolarized mesenteric arteries that were not subjected to transmural pressures, with the degree o f depolarization depending on the concentration [2-4 m V with 0.5 u M N E (Nelson et al. 1990a); <5 and 25 m V with 10 u M N E in guinea pig (Bolton et al. 1984) and rat (Mulvany et al. 1982) mesenteric arteries, respectively]. A steep relation between membrane depolarization and tension development in rat mesenteric arteries was also found on exposure to N E (Cheung et al. 1999; Mulvany et al. 1982). Thus it is not surprising that otiadrenoceptor stimulation activates C a  2 +  channels by causing depolarization (Pacaud et al.  1991). It has been suggested that agonists open L-type channels by depolarizing the cell membrane through activation o f nonselective cation channels (Amedee et al. 1990a), inhibition o f K  +  channels and/or activation o f CI" channels (Pacaud et al. 1992; Suzuki 1981).  However, it has been shown that N E , which was applied to the solution bathing the extrapatch membrane, also increased P  o p e  n o f single C a  channels in patches on single cells  2 +  isolated from rabbit mesenteric artery without any change in membrane potential (Nelson et al. 1988). It has been suggested that agonists may open C a  2 +  channels directly or indirectly  through an intracellular second messenger and GTP-binding protein in the absence o f membrane depolarization (Karaki et al. 1997 and references therein; Nelson et al. 1988). The reason for the discrepancy is not clear. It may be due to functional differences between species or tissues, or differences in the methods used, or the existence o f the two mechanisms, parallel and synergistic. Nevertheless, in rat mesenteric small arteries, challenge with N E caused membrane depolarization, elevated [Ca ];, and induced contraction. There 2+  was a strong correlation between membrane potential and [ C a ] ; when membrane potential, 2+  [Ca ]i and force were simultaneously measured, suggesting that cti-adrenoceptor activation 2+  18  elevated [Ca ]i by depolarization-induced calcium influx through voltage-sensitive channels in these vessels (Nilsson et al. 1994).  6.  Possible Role of CI" Channels in C a  6.1.  Increase in CI Conductance Resulting in Membrane Depolarization in VSMC  2 +  Influx and Smooth Muscle Contraction  A possible role o f CI" ions in agonist-induced C a  2 +  entry is suggested by the fact that  the manner in which V S M cells handle CI" sets up an ideal system for producing and maintaining membrane depolarization (see below). It is known that the intracellular CI" level ([Cl"]i) is many times higher than that predicted by passive distribution in smooth muscle ( A i c k i n and Brading 1982; Casteels 1981; K o n c z and Daugirdas 1994). Vascular smooth muscle cells accumulate CI" intracellularly through several processes (Chipperfield et al. 1993; Davis 1992; Davis et al. 1993), including N a - K - 2 C 1 " cotransport, +  +  C17HC03"  exchange, and a third component, possibly an ATP-dependent transporter (Davis 1996). Estimates o f the CI" equilibrium potential { E i = -60 log (extracellular CI" concentration C  /intracellular CI" concentration)}, measured using either radiolabeled CI" flux (Kreye et al. 1977; Wahlstrom 1973a) or ion selective microelectrodes (Davis 1996), range between -11 and -50 m V . In any given vascular tissue, E c i has always been measured to be roughly 15-30 m V more positive than resting V  m  (-45 to -65 m V approximately). This is consistent with  values measured directly with ion selective microelectrodes in other types o f smooth muscle cells  (Eci between  -30 to  -20  m V ) (Aickin  and  Brading  1990).  Therefore,  any  neurotransmitter or local mediator which increases CI" conductance w i l l produce efflux o f C f , drive the membrane potential toward E i and hence evoke depolarization in V S M . I f the C  CI" conductance-mediated depolarization is sufficient to increase significantly the open  19  probability o f V O C s , it w i l l result in an increased C a  2 +  entry and subsequent smooth muscle  contraction.  6.2.  Activation of ai-Adrenoceptors Increases CT Conductance and Induces Membrane  Depolarization in VSMC. Adrenergic stimulation has  frequently  been shown to increase total  membrane  conductance while inducing depolarization and vasoconstriction (Bolton et al. 1984; Byrne and Large 1987; Casteels et al. 1977; Mekata and N i u 1972; Takata 1980). Radiolabeled ion flux studies have shown that stimulation o f the a-adrenoceptor increases the  membrane  permeability to CI" ions in veins and arteries (Casteels et al. 1977; Smith and Jones 1985; Videbaek et al. 1990; Wahlstrom 1973b; Wahlstrom and Svennerholm 1974). Besides CI" conductance, an increase in a nonspecific cation conductance may also contribute to N E induced depolarization in rabbit portal vein (Amedee and Large 1989). In rat small mesenteric arteries N E increased CI" efflux when producing depolarization, without altering the rate o f K  +  efflux or N a  +  influx, indicating that N E increased the membrane CI"  permeability (Videbaek et al. 1990). A similar result has been shown in rat portal vein (Wahlstrom 1973b). That the ai-adrenoceptor-induced  depolarization resulted from an  increase in conductance to CI" ions has also been confirmed by microelectrode recording and patch pipette studies in guinea pig intact mesenteric veins, and isolated cells from rat and rabbit portal vein (Amedee and Large 1989; Byrne, Large 1988b; Pacaud et al. 1989b; V a n Helden 1988). In short segments o f guinea pig mesenteric vein, it was found that N E evoked a depolarization, and stimulated an inward current with a reversal potential (Er) about -22 m V (close to the expected Eci), which was shifted to more positive values when CI" i n the external solution was replaced with an impermeant anion ( V a n Helden 1988). The alteration  20  o f E r is in the same direction as the change in E c i on substitution with a low-Cl" solution ( A i c k i n and Brading 1982), indicating N E activates  a CI" conductance  to  produce  depolarization. In addition, microelectrode recording in whole tissues has also shown that the depolarization produced by N E was greatly attenuated on prolonged exposure to low CI" solution. (Van Helden 1988). This observation is consistent with that in other types o f smooth muscles (Large 1984). In low external CI", it is expected that the [Cl"]i w i l l also fall (Aickin and Brading 1982; Davis et al. 1991; M c M a h o n and Jones 1988), and therefore the overall membrane CI" conductance is low when external CI" is reduced. Consequently, the reduction in the depolarization to N E in low-Cl" conditions implies that a CI" conductance increase is responsible for NE-induced depolarization. Moreover, alteration o f the CI" equilibrium potential produced similar changes in the reversal potential o f the NE-induced response recorded with microelectrode or patch pipette techniques in V S M cells (Amedee and Large 1989; Byrne and Large 1988b; Pacaud et al. 1989b). The increased CI" conductance leading to membrane depolarization thus may be an important mechanism that indirectly opens V O C s and induces Ca -dependent vasoconstriction in response to a i 2+  adrenoceptor activation (Mironneau and Macrez-Lepretre 1995).  6.3.  Agonist-Activated CT Channel in VSMC. Agonist-induced CI" currents have now been identified in isolated vascular myocytes  from several types o f blood vessels, and can be activated by a number o f agonists that depolarize and contract arteries. NE-activated CI" currents, as mentioned above, were also found in cells o f rabbit ear artery (Amedee e t a l . 1990b) and pulmonary artery (Wang and Large 1993). The Ici(Ca) was blocked by the selective ai-adrenoceptor antagonist prazosin  21  (Amedee et al. 1990b; Pacaud et al. 1989b), suggesting N E evokes Ici(Ca) v i a a i adrenoceptors. Endothelin elicited a similar CI" current in pig coronary, human mesenteric artery and rat renal resistance artery and aortic smooth muscle cells (Gordienko et al. 1994; Klockner and Isenberg 1991; V a n Renterghem and Lazdunski 1993), as did vasopressin and A T P in cultured aortic cells (Droogmans et al. 1991; V a n Renterghem and Lazdunski 1993), as well as histamine in freshly isolated cells from rabbit pulmonary artery (Wang and Large 1993). These agonist-activated CI" currents are C a  2 +  dependent (Ici(Ca)) (Droogmans et al.  1991; Lamb et al. 1994; Pacaud et al. 1992; Pacaud et al. 1989a; Hirakawa et al. 1999). W i t h simultaneous  patch-clamp  recording  and  intracellular  demonstrated in rat portal vein cells that N E did not open C a  Ca 2 +  2 +  measurements,  it  was  channels, but increased [ C a ] i 2+  and evoked a Ca -activated CI" current at a holding potential o f -50 rriV, which is about the 2+  resting potential in physiological conditions. These effects were blocked when heparin, an IP3 receptor inhibitor, was included in the pipette solution (Pacaud et al. 1991). In addition, when intracellular C a  2 +  stores were depleted by caffeine, subsequent application o f the  agonists in the presence o f caffeine failed to evoke a significant rise in [Ca ]i and did not 2+  induce Ici(Ca) (Pacaud et al. 1992). Furthermore, in Ca -free external solution, N E induced a 2+  transient rise in [Ca ]i and was still able to activate a CI" current (Pacaud et al. 1992; Pacaud 2+  et al. 1989b). NE-induced Ici(Ca) was blocked by caffeine, but could be recorded in a Ca -free 2+  ( + E G T A ) bath solution in rabbit ear artery and pulmonary artery cells (Amedee et al. 1990b; Wang and Large 1993). Taken together, these data suggest that the CI" current evoked by otiadrenoceptor activation results from an increase in the intracellular concentration o f calcium released from internal stores. I i(Ca) activated by the release o f calcium from intracellular C  stores stimulated by A T P and histamine was also reported in V S M C from pig aorta and  22  rabbit pulmonary artery (Droogmans et al. 1991; W a n g and Large 1993). Although it has been demonstrated that prolonged exposure to Ca -free solution gradually reduced and 2+  eventually abolished Ici(Ca) (Amedee et al. 1990b; Droogmans et al. 1991; W a n g and Large 1993; Hirakawa et al. 1999) and that depolarizing pulses which produced C a  2 +  entry through  V O C s also activated I i ca) (Lamb et al. 1994; Pacaud et al. 1989a), the extent to which C a C  2 +  (  entry from extracellular sources can sustain activation o f these channels is unknown. It has been suggested that i n smooth muscle, pharmacological receptors are linked to Ici(ca) by a G protein-nVintracellular C a  2 +  store pathway (Loirand et al. 1990; Pacaud et al. 1993). Thus,  agonists which stimulate nVdependent mobilization o f C a  2 +  from intracellular stores could  activate Ici(Ca)- O n the other hand, Pacaud (Pacaud et al. 1991) suggested that the activation o f CI" channels in rat portal vein is a prerequisite for enhanced opening o f voltage-dependent Ca  2 +  6.4.  channels in response to N E .  Physiological Role of I i(Ca) C  6.4.1. Ici(Ca) blockers The evaluation o f the physiological role o f Ici(Ca) has been slow, since available Ici(Ca) blockers are relatively non-selective. The most commonly used CI" channel blockers, including anthracene-9-carboxylic acid (A-9-C), the stilbene derivatives, 4-acetamido 4isothocyanostilbene-2,2'-disulfonic  acid  (SITS)  and  4,4'-diisothiocyanostilbene-2,  2'disulfonic acid (DJDS) and the fenamate, niflumic acid ( N F A ) , have been characterized as Ici(ca) inhibitors i n V S M (Large and Wang 1996 and references therein); (Kirkup et al. 1996a). In addition, 5-nitro-2-(3-phenylpropylamino)-bezoic acid ( N P P B ) (Kirkup et al. 1996b),  ethacrynic  acid,  indanyloxyacetic  acid  ( I A A ) (Greenwood  et  al.  1995),  diphenylamine-2, 2'-dicarboxylic acid ( D P C ) (Baron et al. 1991) and another two fenamates,  23  flufenamic acid and mefenamic acid (Greenwood and Large 1995) were also found to inhibit Ici(Ca) in V S M . None o f these compounds are specific for Ici(Ca)- For example, fenamates are also found to activate large conductance, Ca -sensitive K 2+  +  channels (BK(Ca)) in porcine  coronary artery membranes (Ottolia and Toro 1994) and in whole-cell recordings from canine coronary and rabbit portal vein (Greenwood and Large 1995; X u et al. 1994). A - 9 - C , I A A and ethacrynic acid were reported to do the same in rabbit portal vein (Toma et al. 1996). However, there are marked differences in the concentrations required to inhibit Ici(Ca) and evoke lK(Ca) for N F A . In rabbit portal vein, N F A inhibited spontaneous transient inward currents (STICs, calcium-activated CI" currents) with an IC50 of approximately 2 x 10" M but 6  evoked potassium current only at concentrations greater than 1 x 10" M (Greenwood and 4  Large 1995), indicating a high selectivity for Ici(Ca)- With A - 9 - C , I A A and ethacrynic acid, there is either no concentration difference or a slight concentration difference (around 2 fold, for ethacrynic acid) between inhibition o f Ici(Ca) and activation o f BK(c ), indicating these a  antagonists exhibit a lower degree o f selectivity for Ici(ca)- (Greenwood et al. 1995; H o g g et al.  1994b; Toma et al. 1996). In addition, ethacrynic acid and I A A also evoked a  glibenclamide-sensitive current (Toma et al. 1996), and mefenamic acids have been reported to potentiate the ai-adrenoceptor-activated nonselective cation channels in rabbit portal vein (Yamada et al. 1996), and in the concentration range that flufenamic acid decreases S T I C (Ici(Ca)) amplitude, these compounds also reduced both the amplitude and frequency o f spontaneous  transient  outward  currents  (STOCs,  Ca -activated K 2+  +  currents,  IK(C )) 3  (Greenwood et al. 1995). The potency o f other channel blockers, such as D I D S and SITS, against Ici(Ca) (IC50 greater than TO" M ) is less than that o f their well-established effects on 4  CI-HCO3" exchange in smooth muscle e.g. see (Aickin and Brading 1983). In addition, A T P -  24  induced cation currents in rabbit ear artery are potently inhibited by stilbene derivatives, demonstrating that these agents are unsatisfactory as selective Ici(Ca) blockers, at least i n some V S M (Amedee et al. 1990b). N P P B , another potent compound against Ici(Ca), at 10 u M was selective for the Ici(ca), but at 30 u M also inhibited the calcium current by around 70% in rat portal vein (Kirkup et al. 1996b). In contrast, in rat cerebral arteries, a significant block o f calcium channels was observed even at 10 u M N P P B (Doughty et al. 1998). These results suggest there may be different effects or varied degree o f activity for some o f these compounds in different tissues. A s mentioned above, it seems that N F A is the most potent and selective inhibitor o f Ici(Ca). It has also been demonstrated that in rabbit and rat portal vein N F A potently inhibited NE-evoked Ici(Ca) with IC50 values o f 6 x 10" M and 1-100 u M 6  respectively. A t concentrations up to 5 x 10' M it did not inhibit the influx o f divalent 5  cations (measured using B a  as a carrier) induced by membrane depolarization, and at  concentrations between 10 to 100 u M , it did not inhibit V O C s at all, suggesting N F A did not inhibit voltage-gated calcium channels (Hogg et al. 1994a; Kirkup et al. 1996a). These observations offer the possibility that N F A may be a useful tool to evaluate the role o f Ici(ca) in agonist-induced vasoconstriction. However, N F A (2x 10" and 5 xlO" M ) has been shown 6  5  to enhance NE-stimulated iK(Ca), and it has been suggested that N F A may increase the amount o f C a  released from the intracellular store i n response to stimulation with N E i n  rabbit portal veins (Hogg et al. 1994a). However, this characteristic o f N F A may be tissuespecific, since in rat portal vein at concentrations less than 3 xlO" M , N F A had no effect on 5  the magnitude o f the caffeine- or NE-stimulated B K ( a ) (Kirkup et al. 1996a). C  6.4.2. Ici(Ca) blockers inhibit agonist-induced vasoconstriction.  25  Given the general lack o f selectivity o f CI" channel blockers, it is important to study the effects o f these blockers with a carefully controlled experimental design to assess the role o f Ici(Ca) in contractile mechanisms under physiological condition. There have been several experiments with CI" channel antagonists in whole tissue preparations,  suggesting the  involvement o f a CI" conductance in agonist-induced contraction o f smooth muscle. F o r instance, it was demonstrated that I A A - 9 4 reduced endothelin-evoked contraction in rat aorta and renal arteries (Iijima et al. 1991; Takenaka et al. 1992) and the contraction induced by angiotensin II in rat renal afferent arterioles (Carmines 1995). It was also shown that N F A reduced NE-induced contraction in rat aorta and mesenteric arteries, as well as E T - 1 - and A n g II-induced contractions in rat pulmonary arteries. (Criddle et al. 1996; Criddle et al. 1997; Guibert et al. 1997; H e and Tabrizchi 1997; H y v e l i n et al. 1998; Lamb and Barna 1998).  N F A and  DDDS  were  also  reporte  to  inhibit  oti-adrenoceptor-mediated  vasoconstriction i n mesenteric vascular bed i n anesthetized rats (He and Tabrizchi 1997; Lamb et al. 2000). Recently, it has been reported that N P P B (3 u M ) inhibited the contractile response and increase in C a 4 5  2 +  influx produced by P E , a selective cti-adrenoceptor agonist,  in rat caudal arteries ( M i n et al. 1999). Removal o f chloride ions also impaired PE-induced contractions and  4 5  Ca  2 +  influx, while N P P B had no effect on PE-induced contraction in Cl"-  free buffer. These results thus provide some evidence o f the role o f CI" channels in ctiadrenoceptor-mediated C a  influx and contraction. It is o f interest to further assess the  contribution o f CI" conductance to contractile responses to cti-adrenoceptor in mesenteric arteries since, as mentioned previously, i n mesenteric arteries the membrane potential has an important modulating influence on the tension response to N E (Cheung et al. 1999; Mulvany  26  et al. 1982), and the main effect o f N E on [Ca ]i has been found to be mediated though 2+  voltage-dependent, dihydropyridine-sensitive C a  IV.  2 +  channels (Nelson et al. 1988).  E N D O T H E L I U M - M E D I A T E D R E G U L A T I O N O FM E S E N T E R I C A R T E R I A L  TONE  The single layer o f endothelial cells (ECs) that lines the luminal side o f mesenteric arteries and all other blood vessels plays an important role i n regulating blood vessel function. The E C layer serves in part as a protective covering and permeability barrier to the movement o f substances through the blood vessel wall. In addition, E C s also have an active role i n regulating vascular tone by releasing various vasoactive substances: relaxing and contracting factors. Vessel tone is dependent on the balance between these factors, as well as on the ability o f the smooth muscle cells to respond to them.  1.  Endothelium-Derived Vasorelaxing Factors A large body o f evidence shows that E C s synthesize and release nitric oxide ( N O ) ,  prostacyclin (PGI2) and an unidentified endothelial-derived hyperpolarizing factor(s) ( E D H F ) that cause blood vessels to dilate (Furchgott and Vanhoutte 1989; Furchgott and Zawadzki 1980; Garland et al. 1995; Moncada and Vane 1978b; Palmer et al. 1987 and references therein).  1.1.  Nitric Oxide (NO):  1.1.1. N O synthesis N O is formed from the guanidine-nitrogen terminal o f L-arginine plus molecular oxygen by a heme-containing enzyme called N O synthase ( N O S ) (Ignarro 1990a; Palmer et al. 1988). There are three isoforms o f N O S : the constitutive endothelial N O S (eNOS, N O S  27  III), and neuronal N O S (nNOS, N O S I), which are mainly present in endothelial and neuronal cells, and inducible N O S (iNOS, N O S II) that is only found in cytokine-activated cells and does not seem subject to any cellular control mechanisms (Forstermann et al. 1994; Marsden et al. 1992; Nishida et al. 1992; Palmer et al. 1988). Activation o f the constitutive N O S is Ca -calmodulin-dependent and requires reduced nicotinamide adenine dinucleotide 2+  phosphate ( N A D P H ) ,  5,6,7,8-terahydrobiopterin  ( B H 4 ) and flavin mononucleotide  for  optimal activity (Busse et al. 1993; Knowles and Moncada 1994; Mayer and Werner 1995; Moncada et al. 1991). The production o f N O can be inhibited by interfering with any o f the above factors (Moncada et al. 1991; X i e et al. 1992). Analogues o f L-arginine, such as N ° monomethyl-L-arginine ( L - N M M A ) , N^nitro-L-arginine ( L - N N A ) , N ° - nitro-L-arginine methyl ester ( L - N A M E ) , are potent competitive inhibitors o f N O S activity and selectively inhibit N O formation. These have been very useful in providing insight into the role of N O in the vasculature (Knowles and Moncada 1994; Mayer e t a l . 1989; Moncada et al. 1991; Rees et al. 1989; Rees et al. 1990).  1.1.2. Mechanism of N O release in endothelial cells a).  Agonist-stimulated N O release In endothelium, N O generation by eNOS is stimulated by various neurohumoral  substances including A C h , bradykinin, histamine, A D P , A T P , etc., which strictly depend on an increase in [ C a ] (Busse et al. 1989; Busse et al. 1993; Freay et al. 1989; Furchgott and 2+  ;  Vanhoutte 1989; Griffith et al. 1986; L o n g and Stone 1985; Lopez-Jaramillo et al. 1990; Luckhoff and Busse 1986; Luscher 1990; Singer and Peach 1982) (Fig. 0.2). The agonistinduced increase in [ C a ] i involves both a transient nVmediated release o f C a f r o m 2+  2+  28  EC  VSMC  Figure 0.2 Schematic diagram illustrating pathways o f Ach-induced release o f E D R F s in endothelial cells and relaxation o f smooth muscle by the E D R F s . Modified from Vanhoutte (1997) . A C h binding to muscarinic receptor increase i n I P which contrabutes to the increase in cytoplasmic C a by releasing it from endoplasmic reticulum (ER). Muscarinic receptor activation induces an influx o f C a into the cytoplasma. The resulting increase in [Ca ]j activates N O S to produce N O and leads to the release o f E D H F . The increased [ C a ] i also accellerates the formation o f P G I from arrachidonic acid ( A A ) by C O X . Stimulation with A C h also produces hyperpolarization o f membrane potential (Vm) due to activation o f K and CI" channels by the rise in C a - The resuling hyperpolarization accentuates C a influx due to the increased electrochemical gradient for C a and thereby by a positive feed back loop potentiaes the release o f E D R F s . N O causes relaxation by activating the formation o f c G M P from G T P . P G I causes relaxation by activating the formation of c A M P from A T P . E D H F cause hyperpolarization and relaxation by opening K + channels. 3  2 +  2 +  2+  2+  2  ( C a )  2+  2 +  2 +  2  29  intracellular stores (Freay et al. 1989; Jaffe et al. 1987; Pirotton et al. 1987; Wang et al. 1995b) and a small but more sustained transmembrane influx o f C a  2 +  from the extracellular  space (Wang et al. 1995a). Several lines o f evidence have shown that release o f N O absolutely requires C a  2 +  influx from the extracellular space (Luckhoff and Busse 1990;  Luckhoff et al. 1988; White and Martin 1989). However, the regulation and pathway(s) for agonist-induced C a  2 +  entry remain to be elucidated (Nilius et al. 1997b). It has been  suggested that agonists binding to their receptors in the plasma membrane either directly gate the C a  2 +  channels (receptor-operated  channels), or indirectly couple to the C a  channels  2 +  through a G protein (Chen and Rembold 1995) or second messenger such as IP3 (Vaca and Kunze 1995) or IP4 (Luckhoff and Clap ham 1992) to cause C a is that agonist-induced C a Ca  2 +  2 +  (via store -operated C a  2 +  entry. Another suggestion  entry is a consequence o f depletion o f an endoplasmic reticulum 2 +  channels) (Putney 1991), which can be achieved in the absence  o f agonist (Hallam et al. 1989). Recently it has been further demonstrated that in freshly isolated aortic endothelial cells, A C h and store depletion activated the same C a  2 +  entry  pathway but through parallel mechanisms (Wang and van Breemen 1997). Although in vascular endothelial cells there exists a variety o f Ca -permeable 2+  responsible for receptor-mediated C a Ca  2 +  2 +  channels that  are  entry, it is generally accepted that agonist-induced  influx is controlled by membrane potential (Demirel et al. 1994; N i l i u s et al. 1997b and  references therein). A membrane hyperpolarization caused by agonist opening K provides an electrochemical gradient for maintained C a  2 +  +  channels  entry during agonist stimulation. A  similar mechanism for modulation o f the driving force has also been proposed for CI" channels (Hosoki and Iijima 1994; Hosoki and Iijima 1995; Wang and van Breemen 1999;  30  Yumoto et al. 1995) (Fig. 0.2). It has been reported that depolarizing endothelial cells by increasing the extracellular K arteries with K induced C a  2 +  +  +  concentration or preincubation o f endothelial cells or intact  channel blockers decreased the duration and the magnitude o f agonist-  influx. This in turn reduced the production o f N O (Luckhoff and Busse 1990)  and inhibited vasorelaxation (Demirel et al. 1994). However, little information is available on whether CI" channels modulate N O synthesis and NO-mediated vasorelaxation.  b).  Basal and constitutive release o f N O The continuous basal release o f N O represents a sizable portion o f the total N O -  releasing capacity o f native endothelial cells (Busse et al. 1993). However, the rate o f N O formation under basal conditions seems to be substantially smaller in cultured endothelial cells, implying that native endothelial cells may be continuously exposed to a stimulus, such as shear stress, which affects N O synthase expression. Evidence has accumulated that mechanical force generated at the endothelium by fluid shear stress and pulsatile stretch are important in ensuring the continuous release o f vasoactive endothelial autacoids (Busse and Fleming 1998). It has been suggested that e N O S may be differentially activated by receptordependent agonists and mechanical stimuli (Fleming et al. 1997). It has been observed that a rapid N O release in response to an onset o f flow or an increase in flow above preexisting levels, like in response to receptor-dependent agonists, was  Ca /camodulin-dependent 2+  (Busse et al. 1993; Kuchan and Frangos 1994). O n the other hand, the constitutive sustained release o f N O from the endothelium by physical stimuli such as shear stress exerted by the flowing blood, as well as mechanical stress induced by isometric contraction, may involve [Ca ]i redistribution within the cytoskeleton/caveolae and the activation o f one or more 2+  31  regulatory eNOS-associated proteins without any apparent rise o f [Ca ]i (Busse and Fleming 2+  1998; Fleming et al. 1999; Hutcheson and Griffith 1996; Kuchan and Frangos 1994).  1.1.3. Mechanism of NO-mediated relaxation N O released from vascular endothelial cells diffuses rapidly to and acts in a paracrine fashion on adjacent vascular smooth muscle cells. The smooth muscle relaxation caused by N O was first described to be mediated mainly by the activation o f soluble guanylate cyclase in the smooth muscle cells o f vascular wall, leading to increase in guanosine 3',5'-cyclic monophosphate ( c G M P ) and the subsequent activation o f cGMP-dependent protein kinases, such as protein kinase G ( P K G ) , which may modulate C a  2 +  metabolism resulting in smooth  muscle relaxation (Cornwell et al. 1991; Ignarro 1990b; L i n c o l n and Cornwell 1991; Salomone et al. 1996; Tewari and Simard 1997). The reliance o f endothelium-dependent vasodilation on this mechanism is based on the parallel drawn between the increase in c G M P content o f arterial tissue caused by endothelium-dependent  vasodilators and those o f  nitrovasodilators, whose action is based on releasing N O (Gruetter et al. 1981; Ignarro 1989; Martin et al. 1985; Rapoport et al. 1985). In addition, endothelium-dependent relaxation to N O may be reduced by hemoglobin or methylene blue, which antagonize the rise in c G M P either by inhibiting guanylate cyclase, or by scavenging N O and preventing its stimulation o f the enzyme (Edwards et al. 1986; Gruetter et al. 1981; Ignarro et al. 1987; Ignarro et al. 1986; Kruszyna et al. 1987; Martin et al. 1986; W o l i n et al. 1990) Recently, N O has been shown to produce hyperpolarization in resting tissue or to repolarize smooth muscle cells previously depolarized by an agonist (Cohen et al. 1997; Garland and McPherson 1992; Krippeit-Drews et al. 1992; Murphy and Brayden 1995a;  32  Parsons et al. 1994; Plane et al. 1995; Plane et al. 1998; Tare et al. 1990). K  +  channel  activation by N O (either directly or via c G M P ) has been observed in a number o f isolated arteries (Archer et al. 1994; Bolotina et al. 1994; B y c h k o v et al. 1998; George and Shibata 1995; Mistry and Garland 1998; Peng et al. 1996; Plane et al. 1998; Quignard et al. 1999; Robertson et al. 1993). In rat mesenteric arteries, N O and/or N O donor hyperpolarization o f the resting membrane potential have been observed, and the hyperpolarization was sensitive to glibenclamide, implicating  K  A  channels (Garland and Plane 1996; Garland and  T P  McPherson 1992). Electrophysiological experiments have revealed that N O and N O donors produced a cGMP-independent activation o f large conductance Ca -activated K 2+  +  channels  (BKca) in isolated smooth muscle cells from rat small mesenteric arteries (Mistry and Garland 1998). In microvessels o f rat mesentery, N O donors activated B K (c ), but this effect a  was mimicked by c G M P and inhibited by blocking the activity o f P K G (Carrier et al. 1997). In addition, A C h hyperpolarized smooth muscle in intact rat small mesenteric  arteries  tonically, by activating both A T P - and Ca -dependent K current (Weidelt et al. 1997). The 2+  +  hyperpolarization was completely blocked by an inhibitor o f N O S but not by methylene blue, a guanylate cyclase inhibitor, suggesting the non-involvement o f the soluble guanylate cyclase. Functional studies showed that in rat endothelium-intact isolated  mesenteric  resistance arteries, full relaxation to N O donors can be accounted for by a charybdotoxin (CTX)-sensitive, cyclic GMP-independent mechanism (Plane et al. 1996). C T X is an inhibitor o f large and intermediate Ca -activated K+channels. 2+  Evidence from the literature suggests that N O operates at multiple sites in vascular smooth muscle cells. The extent to which different mechanisms contribute to relaxation may depend on the contractile agonist, the specific endothelium-dependent relaxant used, the  33  tissue and the species (Ghisdal et al. 2000; Plane et al. 1998; W o l i n et al. 1998) (and references above).  1.1.4. N O regulates mesenteric vascular tone a).  Effect o f basal N O release: Several lines o f in vivo evidence suggest that constitutive levels o f expression o f N O S  in endothelium are sufficient to influence tone in mesenteric blood vessels under basal conditions. Inhibition o f N O S has been shown to produce constriction o f mesenteric blood vessels and decrease mesenteric blood flow under basal conditions (Gardiner et al. 1990) and following ganglion blockade (Fozard and Part 1991). In isolated superior mesenteric arteries, the basal tone was enhanced in the presence o f the N O S inhibitor, N^-nitro-L-arginine ( L N N A ) or the guanylate cyclase inhibitor, methylene blue ( W u et al. 1997). In addition, when the N O concentration was measured with an NO-specific microelectrode, the N O scavenger oxyhaemoglobin reduced the N O signal below baseline in the absence o f vasoconstrictor (Simonsen et al. 1999). These results suggest the presence o f continuous basal release o f N O in these preparations. However, in isolated perfused  mesenteric arterial beds and in  pressurized and perfused mesenteric resistance arteries, N O S inhibitors did not have any effect on basal tone, but enhanced responses to vasoconstrictors, suggesting there is no basal N O release, whereas the liberation o f N O requires active tone (Adeagbo et al. 1994; Amerini et al. 1995; Baisch et al. 1994; D o h i et al. 1990; Ebeigbe et al. 1990; L e Marquer-Domagala and Finet 1997; Tatchum-Talom and Atkinson 1997). In isolated mesenteric arteries, shear stress induces relaxation and this effect is totally endothelium-dependent in both large (400500 pm) and small (150-250 pm) arteries (Takamura et al. 1999). The contribution o f N O ,  34  which was evaluated by the use o f N O S inhibitors, was found to be more prominent in large arteries than in small arteries,  whereas the NO-independent component  was equally  distributed in both sizes o f arteries and was inhibited by K channel blockers (Takamura et +  al. 1999).  b).  Effects o f agonist-induced N O release In the isolated perfused mesenteric arterial bed, as in other arteries, A C h , histamine,  AVP  and the C a  2 +  ionophore A23187 all induced endothelium-dependent  relaxation  (Adeagbo and M a l i k 1990; Bhardwaj and M o o r e 1988; Furchgott et al. 1987; Randall et al. 1988). However, it has been found since the pioneering work by Furchgott and colleagues that hemoglobin and methylene blue inhibit ACh-induced relaxation to lesser extent i n the mesenteric arterial bed as compared to large blood vessels such as aorta (Furchgott et al. 1987; K h a n et al. 1992). Later, the possibility that both N O and E D H F are involved in the responses o f the rat mesenteric arterial bed to A C h and histamine was suggested by Adeagbo & Triggle (Adeagbo and Triggle 1993). They showed that in physiologic salt solution (PSS), A C h - and histamine-induced vasodilation o f cirazoline-preconstricted mesenteric arterial beds were only partially attenuated by the N O S inhibitor N ° - nitro-L-arginine methyl ester ( L - N A M E ) . Changing the membrane potential by varying extracellular K [K ] +  0  +  concentration  decreased L-NAME-resistant vasodilation, indicating a role o f the putative E D H F .  These observations were supported by other investigators (Kamata et al. 1996a; M c C u l l o c h et al. 1997; Parsons et al. 1994; Randall et al. 1997). Since A C h and carbachol were less potent as vasorelaxants in the presence o f K or K channel blockers than i n the presence o f +  +  L - N A M E , a greater contribution o f E D H F than N O to relaxation induced by muscarinic  35  receptor stimulation in rat mesenteric arterial bed has been suggested (Adeagbo, Triggle 1993; Kamata et al. 1996a; Kamata et al. 1996b; M c C u l l o c h et al. 1997; Parsons et al. 1994; Randall et al. 1997). The relative contribution of N O to endothelium-dependent relaxation on stimulation by muscarinic agonists was small, with only a minor decrease in potency o f the relaxant, or with a small reduction in maximum relaxation in the presence o f N O S inhibitors among these studies. In addition, Kamata et al showed that the effects o f the endotheliumdependent relaxation induced by platelet-activating factor (PAF) (Kamata et al. 1996b), or by the C a - A T P a s e inhibitor cyclopiazonic acid (Kamata et al. 1996a) were different as 2+  compared to A C h , and suggested that a novel-relaxing factor (Kamata et al. 1996a) may exist. Furthermore, Parsons et al (Parsons et al. 1994) also compared ACh-induced relaxation in perfused mesenteric arterial beds with that in isolated second, third and fourth order sequential branches and concluded that the relative contribution o f N O and NO-independent components was similar in sequential branches. However, different results have been presented by other researchers (see below). In isolated small mesenteric arteries, Shimokawa et al (Shimokawa et al. 1996) demonstrated that the contribution of N O decreases as the vessel size becomes smaller under both basal  conditions and  on  stimulation with  A C h . They also  showed that  the  immunoreactivity o f eNOS was strongest in aorta and decreased as the vessel size became smaller in the mesenteric vasculature. Using L - N A M E  to inhibit N O S , Garland and  McPherson (Garland and McPherson 1992) concluded that release of N O was not involved in ACh-induced vasorelaxation in isolated small mesenteric arteries from Wistar K y o t o rats. Gustafsson et al (Gustafsson et al. 1993) also failed to find any effect with another N O S inhibitor L - N N A in number o f their experiments with small mesenteric arteries from Wistar  36  rats, as did Zygmunt et al (Zygmunt et al. 1995), using female Sprague-Dawley (SD) rats. O n the other hand, it has been reported that L - N N A inhibited ACh-induced relaxation in small mesenteric arteries from Sprague-Dawley and Wistar rats, although N O contributed only a small part o f the A C h - induced relaxation ( H w a et al. 1994; Plane and Garland 1996; Waldron and Garland 1994; W u et al. 1993). In isolated superior mesenteric artery, the relative contribution o f N O also varied among different studies. It has been reported that N O solely contributed to ACh-induce relaxation in S D rats ( H w a et al. 1994), whereas other studies showed that both N O dependent and independent relaxation contribute to ACh-induced responses in Wistar rats (Chen and Cheung 1997; Fukao et al. 1995; Nagao et al. 1992). The  results  for  the  Ca  2 +  ionophore  A23187-induced  endothelium-dependent  relaxation were also conflicting in mesenteric vasculature. N o A23187-stimulated relaxation effect in small mesenteric arteries (Zygmunt et al. 1995), or only a NO-independent relaxation (Parsons et al. 1994), or both NO-dependent and independent relaxation evoked by A23187 (Nagao et al. 1992; White and H i l e y 1997) were reported in rat mesenteric arteries. In addition, Kamata (Kamata et al. 1996b) reported that A23187-induced relaxation was not affected by either depolarization with high K  +  or by exposure to e N O S - c G M P pathway  inhibitors, whereas the vasodilation was slightly but significantly inhibited by treatment with a combination o f methylene blue and L - N N A in isotonic high K  +  solution, suggesting that  A23187 may also produce a novel E D R F or more than one. The reasons for the diversity o f the contribution of N O to receptor-dependent agonistinduced relaxation in mesenteric vasculature among different studies is not entirely clear, but the contributing factors as mentioned above may be the differences in the strain and gender  •  •  ' 7 37  o f rats, in the way o f handling the tissues, such as the initial stretch o f vessels (Parkington et al. 1993; Zygmunt et al. 1994a), the nature o f the relaxing agonists, and that o f the contractile agonist that was used to precontract the blood vessels. Indeed, it has been observed that N E induced contractions were reversed by A C h v i a both N O and N O  synthase-independent  smooth muscle repolarization, whereas the reversal o f contraction to the thromboxanemimetic U46619 by A C h was entirely mediated by the action o f N O , independently o f a change in membrane potential (Plane and Garland 1996).  c).  Vasoconstrictor-induced N O release Recently it has been demonstrated that in rat perfused mesenteric arterial bed,  stimulation o f perivascular sympathetic nerves releases N E and induces vasoconstriction which triggers a secondary release o f endothelial N O coupled to c G M P production. In addition, exogenous NE-induced vasoconstriction is also coupled to increases in N O and c G M P release. The electrically evoked vasoconstriction and N O release were abolished by blocking either sympathetic exocytosis with guanethidine or ai-adrenoceptors with prazosin, suggesting the N O release is stimulated by N E binding to ai-adrenoceptors (Boric et al. 1999).  1.2.  Prostacyclin (PGh)  1.2.1. Synthesis and release P G I was discovered in 1976 (Moncada et al. 1976a). P G I production is initiated by 2  2  the enzyme P L A , which liberates arachidonic acid from membrane phospholipids. The 2  enzyme prostaglandin G / H synthase, which possesses cyclooxygenase ( C O X ) activity,  38  converts arachidonic acid into prostaglandin endoperoxides. Subsequently, PGI2 synthase forms PGI2 from the endoperoxide prostaglandin H2, which is the precursor o f all prostanoids. PGI2 is the major vasodilator prostaglandin (PG) produced by E C in most blood vessels including mesenteric arteries (Carter and Pearson 1992; Moncada and Vane 1978a; Peredo et al. 1997; P i p i l i et al. 1988). Inhibition o f C O X activity with C O X inhibitors, such as indomethacin, w i l l effectively block synthesis o f PGI2 and other prostanoids, thereby preventing their actions. L i k e N O , PGI2 synthesis/release  is also stimulated by variety o f  endogenous  mediators and drugs, as well as physiological stimuli (Bhagyalakshmi and Frangos 1989; Piper and Vane 1971). The release o f PGI2 is also believed to be triggered by an increase in [Ca ]i (Hallam et al. 1988; L o n g and Stone 1985). However, in bovine cultured aortic 2+  endothelial cells, N O release correlates most closely with transmembrane C a than C a  2 +  2 +  influx rather  release from intracellular stores, while PGI2 release is entirely dependent on C a  2 +  release from the stores (Luckhoff et al. 1988). Parsaee (Parsaee et al. 1992) have shown that higher levels o f [Ca ]i are required for P G I 2+  2  release than for N O release. Furthermore,  inhibition o f intracellular Ca mobilization by T M B - 8 attenuated bradykinin-induced PGI2 2+  release (Whorton et al. 1984), while exerting a minimal effect on N O release (Peach et al. 1987). Therefore, it seems that there is a difference in the C a  2 +  source required for the release  of N O and P G I (Fig. 0.2). 2  1.2.2. Actions Physiologically, PGI2 is a local autacoid (Blair et al. 1982). In the lumen o f blood vessels PGI2 prevents platelet aggregation, and thus the release o f vasoconstrictor and  39  growth-promoting agents. It acts in concert with nitric oxide, which also inhibits platelet aggregation (Radomski et al. 1987).  PGI2  also acts on smooth muscle cells to exert a  vasorelaxant effect (Moncada and Vane 1978b).  PGI2  contributes to endothelium-dependent  relaxation o f several isolated blood vessels and to vasodilation o f perfused  organs  (Forstermann et al. 1986; Holtz et al. 1984; Lamontagne et al. 1992; Vegesna and Diamond 1986). The mechanisms by which PGI2 mediates smooth muscle relaxation involve the stimulation o f specific receptors and activation o f adenylate cyclase leading to an elevation of intracellular cyclic adenosine monophosphate ( c A M P ) (Halushka et al. 1989; Luscher and Vanhoutte 1990) (Fig. 0.2). PGI2 was reported to activate glibenclamide-sensitive K  +  channels via the c A M P pathway, leading to smooth muscle hyperpolarization and relaxation (see review for Vanhoutte et al. 1996). However, the relative importance o f PGI2 in relation to endothelium-derived nitric oxide and other endothelium-derived vasorelaxants such as E D H F , at the level o f resistance arteries, is currently unclear, both physiologically and in hypertension (Schiffrin 1996).  1.3.  EDHF  1.3.1. Identity In many blood vessels, inhibition o f the synthesis o f N O and P G I does not result in 2  complete loss o f endothelium-dependent relaxation in response to variety o f agonists, such as ACh,  histamine, bradykinin, or substance P . A putative n o n - N O / P G I  2  mediator which  hyperpolarizes vascular smooth muscle cells has been termed the endothelium-derived hyperpolarizing factor (Feletou and Vanhoutte 1999; Mombouli and Vanhoutte 1997; Quilley et al. 1997; Waldron et al. 1996). The chemical identity of E D H F is not yet established. It has been proposed that E D H F could be K  +  in small resistance arteries o f rats (Edwards et al.  40  1998) , or may be a metabolite o f arachidonic acid produced by cytochrome P-450-dependent monooxygenase in coronary, mesenteric and carotid arteries o f several species (Adeagbo and Henzel 1998; Chen and Cheung 1996; Hecker et al. 1994; Popp et al: 1996; Triggle et al. 1999) , Alternatively, it has been suggested to be anandamide, an arachidonic acid derivative and endogenous cannabinoid, in isolated perfused mesenteric and coronary arterial beds o f rats (Randall et al. 1996; Randall and Kendall 1997; Randall and Kendall 1998). However, evidence has also been presented that neither K (Quignard et al. 1999; Vanheel and V a n de +  Voorde 1999), nor a cytochrome P450 metabolite (Chataigneau et al. 1998a; Corriu et al. 1996; Fukao et al. 1997b; V a n de Voorde and Vanheel 1997), nor a cannabinoid (Chataigneau et al. 1998b; Plane et al. 1997; White, Hiley 1997) meets the pharmacological criteria o f an E D H F . Taken together, the collective data suggest that E D H F is not one substance, and there may be a considerable number o f different mechanisms that mediate endothelium-dependent  hyperpolarization in different vascular beds. Indeed, it has been  suggested that endothelium-dependent hyperpolarization could involve electrical coupling through the myo-endothelial junctions, not only in small resistance arteries (Edwards et al. 1999; Yamamoto et al. 1999) but also in conduit arteries (Chaytor et al. 1998; Edwards et al. 2000) . In a very recent report, Edwards et al (Edwards et al. 1999) compared responses putatively mediated by E D H F in guinea pig internal carotid and rat hepatic and mesenteric arteries, and evaluated the effect o f gap junction inhibitors. They concluded that gap junctions play some role in the E D H F response in rat arteries, but the primary mechanism would appear to be mediated by K . In contrast, in the guinea-pig internal carotid artery, gap +  junctions may be the sole mechanism underlying the response attributed to E D H F , indicating that the nature o f E D H F shows considerable tissue and species variability.  41  1.3.2. [Ca ]i dependency of E D H F release In common with the release o f N O and PGI2, elevation o f [Ca ]i in endothelial cells 2+  has also been proposed to be essential for the release o f E D H F (Chen and Suzuki 1990). This hypothesis has been supported by the finding that the C a endothelium-dependent  membrane hyperpolarization  2 +  ionophore A23187 induces  (Chen and Suzuki 1990; Nagao et al.  1992; Nakashima and Vanhoutte 1993). Recently, Fukao etal (Fukao et al. 1997c) measured ACh-induced  endothelium-dependent  NO/PGFrindependent  hyperpolarization  i n rat  mesenteric artery as a marker for E D H F release. They reported that the ACh-induced release of E D H F is possibly initiated by C a  2 +  release from an HVsensitive C a  2 +  pool as a  consequence o f stimulation o f phospholipid hydrolysis due to phospholipase C activation, and is maintained by C a that the C a Ca  2 +  2 +  2 +  influx via a N i - and Mn -sensitive pathway. They also indicated 2 +  2+  influx mechanism seems to be activated following H V i n d u c e d depletion o f the  pool. Thus, the E D H F release from mesenteric arteries seems to rely on both C a  release from intracellular stores and C a  2 +  2 +  entry from the extracellular space (Fig. 0.2).  1.3.3. E D H F - mediated vasodilation in mesenteric arteries. a).  Mechanisms o f EDHF-mediated relaxation The action o f E D H F is believed to occur via the activation o f K channels, leading to +  hyperpolarization o f the vascular smooth muscle membrane and vasorelaxation. This is based on evidence that variations in the extracellular K concentration ( [ K ] ) control the amplitude +  +  0  o f the endothelium-dependent hyperpolarization, and that the hyperpolarization is associated with enhanced K conductance across the membrane and is blocked by some K +  +  channel  42  blockers (Adeagbo and Triggle 1993; Chen and Suzuki 1989; Chen et al. 1991; Fukao et al. 1997a; Nagao and Vanhoutte 1992; Taylor and Weston 1988). In addition, EDHF-mediated relaxation correlates  well with EDHF-mediated hyperpolarizationj  implying a causal  relationship (Chen and Cheung 1997) and is also blocked by elevated [ K ] or K +  +  D  channel  antagonists (Adeagbo and Triggle 1993; Fukao et al. 1995; Garland and McPherson 1992; Hansen and Olesen 1997; M c C u l l o c h et al. 1997; Randall et al. 1997; Waldron and Garland 1994; Zygmunt et al. 1994b). However, the K channels mediating the response to E D H F in +  vascular smooth muscle have not been characterized (Fig. 0.2). In  superior  mesenteric  artery,  when  membrane  potential  and  tension  were  simultaneously measured, tetraethylammonium T E A (5mM), a relatively selective inhibitor of BKca, and apamin, a small-conductance C a  2 +  activated K  +  channel ( S K c ) antagonist, a  significantly inhibited ACh-induced smooth muscle hyperpolarization and relaxation that were resistant to the action o f N O S inhibitors. C T X , a large- and intermediate- Ca -activated 2+  K  +  channel antagonist, marginally inhibited both responses. However, the combination o f  apamin and C T X abolished both the hyperpolarization and the relaxation (Chen and Cheung 1997). In the perfused  mesenteric arterial bed, information regarding the specific K  +  channels that mediate the response to E D H F is limited. Only Adeagbo (Adeagbo and Triggle 1993) reported that apamin completely blocked the EDHF-induced relaxation to A C h . M o s t o f the studies that characterize the EDHF-mediated relaxation are carried out in small resistance mesenteric arteries. Vasorelaxation to A C h was reported to be attenuated by iberiotoxin (IbTX), a selective large-conductance Ca -activated K channel ( B K c ) blocker, 2+  +  a  T E A (5mM), 4-aminopyridine (4-AP), a blocker o f delayed rectifier, voltage-dependent, K  +  43  channels (Kv), and BaCi2 (100 u M ) , a selective blocker for inward rectifier K  +  channels  (Nelson and Quayle 1995 and references therein). Combined pretreatment with I b T X plus L N N A completely blocked the vasorelaxation (Hansen and Olesen 1997). In addition, apamin, T E A ( I m M ) and 4 - A P each significantly reduced N O - and P G M n d e p e n d e n t relaxations to carbachol, but had no significant effect on the response to A23187 (White and Hiley 1997). Although B a C b and C T X alone did not show any effect, exposure o f arterial segments to the combination o f apamin and C T X abolished EDHF-mediated hyperpolarization (Chataigneau et al. 1998b) and N O - and PGI -independent relaxation (Plane et al. 1997; White and Hiley 2  1997) evoked by A C h or carbachol. In contrast, I b T X had no significant effect on the relaxation to carbachol either alone or in combination with apamin (White and H i l e y 1997). Furthermore, in pressurized small mesenteric arteries (diameter: 70-120 u M ) , Lagaud et al (Lagaud et al. 1999) showed that apamin alone completely blocked, while I b T X had no effect on,  ACh-induced,  NO/PGl2-independent  relaxation. In contrast,  the EDHF-mediated  response to C P A was abolished only in the presence o f apamin plus I b T X , although either apamin or I b T X alone significantly inhibited it. In addition, neither T E A , C T X , 4 - P A nor B a C b , had any effect on the response to C P A . In another study, using pressurized small mesenteric arteries of the same size, Doughty (Doughty et al. 1999) reported that apamin plus C T X abolished ACh-induced dilation o f either PE-stimulated or myogenic tone in the presence o f L - N A M E and indomethacin when the drugs were applied intraluminally. Since supervision with both C T X and apamin was without effect on the EDHF-mediated relaxation, the authors concluded that apamin and C T X block EDHF-mediated relaxation by an action on the endothelium, and not an action in the smooth muscle.  44  Shear stress also induces E D H F release in mesenteric arteries. The released E D H F was noted in both large (400- 500 urn) and small vessels (150-250 urn). The EDHF-mediated component o f the shear stress-induced relaxation was almost abolished by T E A and was significantly inhibited by the combination o f C T X and apamin (Takamura et al. 1999). M o s t studies in the mesenteric vascular bed demonstrate that the ATP-sensitive K  +  channel blocker glibenclamide has no effect on EDHF-mediated hyperpolarization and relaxation (Adeagbo and Triggle 1993; Garland and McPherson 1992; Hansen and Olesen 1997; Kamata et al. 1996a; Lagaud et al. 1999; M c C u l l o c h et al. 1997). Collectively, the data suggest that in the mesenteric vascular bed, several K are involved in EDHF-mediated relaxation, particularly Ca -activated K 2+  +  +  channels  channels. The  heterogeneity o f K channels, especially with different agonists, suggests that E D H F activity +  may be due to more than one chemical entity.  b).  Functional contribution o f E D H F to endothelium-mediated relaxation It is evident that EDHF-induced relaxation assumes a greater functional importance  than N O as artery size decreases (Hwa et al. 1994; Nagao et al. 1992; Shimokawa et al. 1996). However, the importance o f E D H F is not just related to the vessel size, but may also be vascular region-dependent (Clark and Fuchs 1997; Zygmunt et al. 1995). Experiments in coronary and pulmonary arteries indicate that E D H F may represent a reserve mechanism in some large arteries under certain conditions (Drummond and Cocks 1996; Kemp et al. 1995; Kilpatrick and Cocks 1994). In the mesenteric vascular bed, E D H F seems to play an important role in mediating endothelium-dependent vasodilation (see above), in the main mesenteric artery (Chen and Cheung 1997; Fukao et al. 1995; Nagao et al. 1992) and its  45  small branches (Adeagbo and Triggle 1993; H w a et al. 1994; Kamata et al. 1996a; Kamata et al. 1996b; M c C u l l o c h et al. 1997; Parsons et al. 1994; Plane and Garland 1996; Randall et al. 1997; Waldro and Garland 1994; W u et al. 1993) although some conflicting results have been reported ( H w a et al. 1994).  2.  Endothelium-Derived Contracting Factors Vasoconstrictors derived from E C have been identified and characterized to some  degree, including endothelin-1 (ET-1) and endothelium-derived contracting factor(s) ( E D C F ) (Luscher and Vanhoutte 1990; Vanhoutte 1989). The nature o f E D C F varies with the species and anatomical site o f its production. Prostaglandin endoperoxides H2 ( P G H ) , thromboxane 2  A  2  (TxA2) and superoxide anion (O2") have been suggested as possible candidates (Luscher  et al. 1992). In most situations, contractions initiated by vasoconstrictors derived from endothelial cells are found in pathological conditions, particularly in hypertension, while under normal conditions relaxing factors are predominantly released from endothelial cells (Luscher et al. 1993b; Mistry and Nasjletti 1988; Purkerson et al. 1986; Vanhoutte 1996; W i l c o x etal. 1996).  2.1.  Endothelin-1  2.1.1. Synthesis and release The endothelins are a family o f contractile peptides made up o f 21-amino acids (Yanagisawa et al. 1988). They are synthesized from larger precursors and expressed in different tissues. E T - 1 is synthesized in endothelial cells, and its expression is induced by several factors including hypoxia, N E , angiotensin n,  vasopressin, thrombin, insulin,  cytokines and growth factors (see review for Masaki 1995). Physical stimuli, such as shear  46  stress, and other factors such as N O and PGI2 decrease E T - 1 production and release. E T - 1 release from perfused rat mesenteric arterial bed and its enhancement by hypoxia has been observed (Rakugi et al. 1990). The circulating levels o f E T - 1 are low under physiological conditions, since most peptides are secreted toward the abluminal side, i.e. toward the smooth muscle cells (Wagner et al. 1992). Therefore, E T - 1 mainly acts in a paracrine and autocrine manner through two subtypes o f receptors: E T A and E T B , which have been cloned (Arai et al. 1990; Sakurai et al. 1990).  2.1.2. E T receptors and their function in rat mesenteric vascular bed ET  A  receptors,  which are present in vascular smooth muscle cells,  mediate  vasoconstriction and cellular proliferation (Luscher et al. 1993a), while E T B receptors, which were thought to occur mainly on endothelial cells, mediate vasodilation by generation o f N O and P G I (Luscher et al. 1993a; Matsuda et al. 1993; Warner et al. 1989; Wright and Fozard 2  1988). It has now been demonstrated that E T B receptors are also found on vascular smooth muscle and mediate vasoconstriction in some blood vessels (Batra et al. 1993; Moreland et al. 1992; Sumner et al. 1992). A t lower concentrations (comparable to physiological plasma levels), E T - 1 causes vasodilation by activation o f endothelial E T B receptors, while at higher concentrations it provokes sustained contractions by activation o f E T A (and in some blood vessels, also E T B ) receptors on the smooth muscle cells (Luscher et al. 1996; Masaki 1995; Mehta et al. 1992). In addition, E T - 1 may interact with other vasoactive substance to affect smooth muscle function. It was shown that threshold doses o f E T - 1 potentiated responses to N E or sympathetic nerve stimulation in several blood vessels o f different species including  47  rat mesenteric arteries (Henrion and Laher 1993; Tabuchi et al. 1989b; Wong-Dusting et al. 1991; Y a n g et al. 1990). Recently K i t a etal (Kita et al. 1998) confirmed that E T - 1 at subpressor doses enhances contractile responses to N E , and further demonstrated that this effect was mediated by E T B  receptors, in perfused rat mesenteric arterial bed. In addition, in  hypertension, endothelin may also stimulate release o f C O X pathway-derived contracting factors to mediate endothelium-dependent stimulated T x A  2  vasoconstriction. It has been shown that E T  release and evoked an endothelium-dependent contraction in aorta from  spontaneously hypertensive rats ( S H R ) but not Wistar-Kyoto ( W K Y ) rats (Taddei and Vanhoutte 1993). E T - 1 stimulated release o f P G E  2  from the perfused rat mesenteric artery  was reported (Tabuchi et al. 1989a). E T B receptors that are responsible for endothelial-dependent relaxation have been characterized in isolated perfused mesenteric vascular bed (D'Orleans-Juste et al. 1993; Warner et al. 1993). E T  A  and E T B receptors that induce vasoconstriction have also been  found in perfused mesenteric vascular bed (D'Orleans-Juste et al. 1993; Warner et al. 1993), as well as in endothelium-denuded intact small mesenteric arteries and primary cultures o f smooth muscle cells isolated from the mesenteric resistance arteries (Touyz et al. 1995). In small mesenteric arteries, E T receptors were thought to predominate and seemed to be the A  critical ones involved in vasoconstriction (Deng et al. 1995). However, a clear role for E T B receptors in mediating constrictor responses to E T - 1 in small mesenteric arteries without endothelium, which was only revealed when both E T A and E T B receptors were blocked, was demonstrated (Mickley et al. 1997). A similar phenomenon has also been reported in rabbit pulmonary artery (Fukuroda et al. 1994) and other non-vascular tissues (Clozel and Gray 1995; Fukuroda et al. 1996). It was suggested that a receptor crosstalk occurs and therefore  48  blockade o f both E T and E T B receptors may be required for effective inhibition o f E T - 1 A  induced vasoconstriction. (Fukuroda et al. 1996; M i c k l e y et al. 1997).  2.2.  Prostanoids:  PGH2, TxA2  2.2.1. Synthesis /release and receptor blockade The prostaglandin endoperoxide P G H , as mentioned above in section III. 1.2, is an 2  intermediate in the C O X pathway o f arachidonic acid metabolism (Moncada and Vane 1978a). Like PGI2, thromboxane A ( T x A ) is transformed enzymatically from P G H , but v i a 2  an alternative metabolic pathway. T x A  2  2  2  synthase, which catalyzes the transformation o f P G  endoperoxides into T x A , has been found mainly in platelets, as well as in blood vessels 2  (Moncada and Vane 1978a). In blood vessels, prostaglandins are mainly synthesized i n and released from endothelial cells (Smith 1986). T x A  2  and P G H  2  stimulate contraction o f  vascular smooth muscle v i a interaction with a common receptor (Mais et al. 1985), which can be blocked by specific receptor antagonists, such as SQ 29,548 (Auch-Schwelk et al. 1990).  2.2.2. Effect of  PGH2/TXA2  in normal and hypertensive mesenteric vascular bed of rats  Endothelium-dependent contractions with a variety o f stimuli including arachidonic acid, A C h , the C a  2 +  ionophore A23187, 5-HT and sudden stretch, that are sensitive to  inhibitors o f C O X , occur in veins (De M e y and Vanhoutte 1982; M i l l e r and Vanhoutte 1985), cerebral (Katusic et al. 1988; Katusic and Vanhoutte 1989; Shirahase et al. 1987; Toda et al. 1988), and pulmonary arteries (Altiere et al. 1986), and diabetic  aorta  (Tesfamariam et al. 1990; Tesfamariam et al. 1989), as well as in aorta (Kato et al. 1990; K u n g and Luscher 1995; L i n et al. 1994; Luscher and Vanhoutte 1986) o f aging and  49  hypertensive rats. Both  P G H 2  and T x A  2  have been implicated as the endothelium-derived  contracting factor. In mesenteric resistance arteries, P G H in endothelium-dependent  2  and T x A have been reported to be involved 2  vasoconstriction (Lang et al. 1995; N o l l et al. 1997), or in  impaired endothelium-dependent relaxation (Carvalho et al. 1997; Diederich et al. 1990; Jameson et al. 1993; L i and Bukoski 1993; Luscher et al. 1990; Sunano et al. 1999; Takase et al. 1994; Watt and Thurston 1989) to A C h in genetic or experimental hypertensive rats. In addition, these contracting factors may also be responsible for the change in the vascular responsiveness to some contractile agonists such as A n g II and N E in hypertensive mesenteric arteries (Carvalho et al. 1997; N o l l et al. 1997). The contribution o f P G H TxA  2  2  and  may be different in different models o f hypertension. F o r example, in mesenteric  resistance arteries o f adult SFfR and D O C A - s a l t hypertensive rats, P G H the endothelium-dependent  2  seems to mediate  contraction elicited by A C h , which opposes relaxation by  endothelium-derived nitric oxide (Diederich et al. 1990; Luscher et al. 1990). In 2 K 1 C renovascular hypertensive rats, the blockade o f T x A / P G H 2  inhibition o f T x A  2  2  receptors with ridogrel and  synthase with dazoxiben normalized the impaired relaxation response to  A C h in the perfused mesenteric arterial bed, while the smooth muscle response to nitric oxide, tested with sodium nitroprusside, was unaltered. This suggests that the decreased responsiveness o f smooth muscle to A C h resulted from an increase in T x A formation rather 2  than a decrease in sensitivity to N O in the mesenteric resistance vessels o f this model o f hypertensive rats (Carvalho et al. 1997).  2.3. 2.3.1  Superoxide Anion (Oi) Formation  50  0 " is generated though one-electron reduction o f 0 2  2  by N A D ( P ) H oxidases, and other  enzymes, such as xanthine oxidase (Land and Swallow 1971). It is known that 0 " is 2  produced via the side-chain reaction o f P G H synthase in the presence o f N A D H or N A D P H (Kukreja et al. 1986). The rate o f 0 " generation was markedly inhibited by C O X inhibitors 2  when arachidonate was used as substrate (Kukreja et al. 1986). It has been demonstrated that cultured endothelial cells could produce O2" under basal conditions and during such stimulation as reperfusion or treatment with bradykinin, A23187, interferon-y, interleukin-1 or angiotensin II ( A n g II) (Katusic 1996 and references therein; Zhang et al. 1999). Recently accumulated evidence has shown that A n g II stimulates production o f O2" in blood vessels throughout the vascular wall, especially in the endothelium and adventitia ( D i Wang et al. 1999; Nakane et al. 2000). A n g II also increased O2" production in cultured vascular smooth muscle cells (Griendling et al. 1994; Touyz and Schiffrin 1999). C O X , xanthine oxidase and N A D H oxidoreductase have all been identified as sources o f 0 " in the vascular endothelium (Cosentino et al. 1994; Holland et al. 1990; 2  Kontos 1985; Mohazzab et al. 1994; Munzel et al. 1999; Rajagopalan et al. 1996; White et al. 1996).  2.3.2. Mechanism of action O2" can be inactivated by superoxide dismutase ( S O D ) or may react with other free radicals, such as N O (Pryor 1994). Interaction o f 0 " with N O is very rapid and leads to 2  production o f an oxidant, peroxynitrite (Beckman et al. 1990). Thus, O2" may decrease the concentration o f N O , favoring an increase in arterial tone, and increase formation o f a potentially toxic free radical that may cause oxidative injury. In addition, increased  51  production o f O2" in the blood vessel wall inhibits synthesis o f PGI2, but not that o f TxA2 (Katusic and Vanhoutte 1989; Moncada et al. 1976b). This effect may also contribute to impairment o f endothelium-dependent relaxation and favor an increase in arterial tone.  2.3.3. Effects of 0 ~ on endothelial function in mesenteric vasculature 2  C V has been proposed as a possible endothelium-derived contracting factor (Katusic and Vanhoutte 1989; Vanhoutte and Katusic 1988), but evidence for a direct vasoconstrictor effect o f 0 " in vascular smooth muscle cells is missing (Katusic 1996). However, it has been 2  repeatedly reported that vasoconstriction in response to agonists, such as A23187, U46619, N E and A n g II, could be inhibited by S O D or potentiated by S O D inhibitors in canine basilar artery, rabbit renal afferent arterioles and rat aorta, respectively (Katusic et al. 1993; Katusic, Vanhoutte 1989; Kawazoe et al. 2000; Laight et al. 1998; Schnackenberg et al. 2000). The effects  were  endothelium-dependent,  and  N O S inhibitors could  either  restore  the  vasoconstriction in the presence o f S O D or abolish the potentiation produced by inhibitors o f S O D (Laight et al. 1998; Schnackenberg et al. 2000). The results suggested that the major mechanism responsible for participation o f O2" in endothelium-dependent inactivation o f N O . endogenous  contractions is  O2" was also reported to suppress the modulatory influence o f  N O on A n g U-induced afferent  arteriolar constriction in diabetic  rats  (Schoonmaker et al. 2000) and a NE-induced pressor response in aorta from S H R rats ( W u et al. 1998). O2" effects on endothelium function by increasing the breakdown o f N O were also found in rat mesenteric vasculature. In rat small mesenteric arteries, it was shown that S O D caused  an  endothelium-dependent  relaxation  o f NE-induced  tone  and  potentiated  52  endothelium-dependent relaxation to A C h (Sunman et al. 1993). This effect o f S O D has been attributed to its ability to scavenge O2" that inactivates basal and ACh-induced N O release. In the perfused mesenteric microcirculation o f rats, application o f A n g II induced an immediate production o f O2" and vasoconstriction that was inhibited by S O D (Kawazoe et al. 1999). In addition, when N O was directly measured in isolated mesenteric small artery rings, an increased N O decomposition by O2" was observed i n adult (15 week-old) stroke-prone spontaneously hypertensive rats ( S H R S P ) as compared with age-matched Wistar-Kyoto rats, although N O release remained unaffected  normotensive  (Tschudi et al. 1996).  Furthermore, the endothelium-dependent vasoconstriction to high concentrations o f A C h seen in small mesenteric arteries from prehypertensive S H R rats (4 week-old), but not in normotensive Wistar-Kyoto rats, seems to be mediated by O2', which interferes with the effects o f N O (Jameson et al. 1993).  V .  A B N O R M A L I T I E S I N  H Y P E R T E N S I O N  The hemodynamic characteristic o f established hypertension is an increase i n total peripheral resistance. Factors thought to contribute to the increased resistance include: 1) augmented  humoral responses  and increased sympathetic nerve activity, 2) impaired  endothelium function, and 3) structural changes, particularly in peripheral resistance vessels (Conway 1984; F o l k o w 1982; Mulvany 1994). This section w i l l mainly describe the changes in smooth muscle and endothelial function in renovascular hypertension and insulin resistance with hypertension.  1.  Goldblatt 2K1C Renovascular Hypertension  1.1.  2K1C Rat Model of hypertension  53  The 2 K 1 C hypertensive rat is one o f the models o f Goldblatt hypertension that also includes the one-kidney, one-clip ( 1 K 1 C ) model o f experimental hypertension. Goldblatt and his colleagues (Goldblatt et al. 1934), in 1934, produced a reliable model o f renal hypertension by constricting the renal arteries o f a dog with adjustable silver clamps. Very quickly, this technique was adapted for use in other small mammals. In 1939 W i l s o n and B y r o m (Wilson and B y r o m 1939) adapted the silver clip technique to rat, and successfully produced persistent hypertension by partially occluding the left renal artery and leaving the other kidney untouched (Goldblatt two-kidney one-clip hypertension). It is well established that unilateral renal ischaemia causes hypertension in humans. The 2 K 1 C renal hypertensive rat is the experimental counterpart o f human renovascular hypertension, and has been widely used for exploring the mechanisms  o f the genesis  and sustenance o f  hypertension in past decades (Martinez-Maldonado 1991).  1.2.  Peripheral Resistance in 2K1C Hypertensive Rats The majority o f hemodynamic studies in the 2 K 1 C model indicate that the elevated  blood pressure in both early and chronic phases depends on an increased peripheral vascular resistance (Averill et al. 1976; Hallback-Nordlander et al. 1979; Russell et al. 1983). It has been shown that mesenteric vasculature is a key area for the increased peripheral resistance in renovascular hypertension (Faber and Brody 1983; Meininger et al. 1984; Meininger et al. 1985; Teranishi and Iriuchijima 1985). The increased peripheral resistance o f early phase 2 K 1 C hypertensive rats could due to a neurohumoral mechanism causing a functional increase in vascular smooth muscle tone, but in the established phase there may be  54  reinforcement by structural changes within the blood vessels. This is discussed in the next section.  •1.3.  Structural Alterations Structural changes develop rapidly in 2 K 1 C hypertensive rats. Left ventricular  hypertrophy could be demonstrated within 7 days o f renal artery constriction and vascular changes after approximately 3 weeks (Lundgren and Weiss 1979). Later, these indirect observations using the isolated perfused hindlimb preparation were confirmed by in vitro morphological  measurements  o f isolated  mesenteric  resistance  arteries from 2 K 1 C  hypertensive rats, 4 weeks after renal artery constriction (Mulvany and Korsgaard 1983). In addition, another study (Deng and Schiffrin  1991), which investigated and quantified  alterations in structure as well as in reactivity to different agents, also reported a significant reduction in external and lumen diameters, increased media width and increased media-tolumen ratio i n small mesenteric arteries from 2 K 1 C rats. The alteration occurred within 6 weeks o f development o f hypertension and was accompanied by significant increases in active wall pressure produced i n response to N E and arginine vasopressin ( A V P ) . Thus, it was suggested that rapid and early structural changes, which enhance vascular reactivity to vasoconstrictors, might contribute to the maintenance o f the elevated blood pressure. However, structural changes i n the resistance vessels cannot be the sole determinant o f the raised peripheral resistance, because removal o f the constricting clip i n 2 K 1 C hypertensive rats resulted i n a rapid fall i n blood pressure to normal levels within 24h (Ferrario 1974; Russell et al. 1983; Thurston et al. 1980), whereas structural vascular changes take several  55  weeks to resolve after reversal o f hypertension (Lundgren and Weiss 1979; Watt and Thuston 1990)  1.4.  Changes in Contractile Response in Mesenteric Vasculature of 2K1C Hypertensive Rats It  is  well  accepted  that  the  renin-angiotensin  system  participates  in  the  pathophysiology o f the 2 K 1 C hypertension (Carretero and Gulati 1978; Martinez-Maldonado 1991). It is known that dramatically parallel increases in B P , plasma renin activity and circulating angiotensin II ( A n g U ) concentrations occur in the period immediately following constriction o f the renal artery, whereas over the course o f 6 weeks, plasma A n g II levels tend to fall back to near normal but B P remains elevated or continues to increase (Morton and Wallace 1983). It has been shown that blockade o f the renin-angiotensin system with antibody to A n g II, a specific competitive antagonist o f A n g II or an angiotensin-converting enzyme inhibitors, lowers the blood pressure o f 2 K 1 C hypertensive rats in the early phase (Bennett and Thurston 1996; B i n g et al. 1981; Brunner et al. 1971; Pals et al. 1971; Tokioka et al. 2000). In addition, an increase in vascular responsiveness to A n g II has been demonstrated in the rat isolated perfused mesenteric arterial bed (McGregor and Smirk 1968) and perfused untouched kidney (Collis and Vanhoutte 1978), and in isolated rabbit aorta, renal, and iliac strips in both early and/or later phases o f 2 K 1 C hypertension (Yoshida et al. 1987). Moreover, studies in whole animals showed an increase in the pressor response to low levels o f A n g II in the chronic phase (Melaragno and Fink 1995) or after the constricting clip had been removed from the renal artery, which leads B P fall (Skulan et al. 1974). B y contrast, vascular reactivity to A n g II was reported to be decreased in rat mesenteric arteries in the early phase o f 2 K 1 C hypertension (Benedetti and Linas 1987). Another study also  56  showed that the pressor response to A n g JJ was decreased in both early and chronic 2 K 1 C hypertensive rats (Marks et al. 1979). In addition, inhibition o f the renin-angiotensin system with A n g II antagonists  or by angiotensin-converting enzyme ( A C E ) inhibition only  produced a partial fall in blood pressure in early-phase hypertension (Benetos et al. 1986), and only the converting enzyme inhibition reduced the blood pressure in rats with chronic 2 K 1 C hypertension (Bing et al. 1981; Dickinson and Y u 1967). The latter phenomenon has been attributed to the fact that during A C E inhibition, tissue bradykinin levels increase, causing vasodilation and the subsequent lowering o f B P (Benetos et al. 1986; Lindsey et al. 1983). Thus, other factors may also contribute to sustained B P in both early and chronic renovascular hypertension.  A n increased vascular reactivity to N E , either in vivo (Carvalho et al. 1997; Fortes et al. 1992; Fortes et al. 1990) or in vitro (Carvalho et al. 1997; Fortes et al. 1992; Haeusler and Haefely 1970; McGregor and Smirk 1968; Tsuda et al. 1989), in the rat mesenteric arterial bed from the early phase (3-6 weeks after surgery) in 2 K 1 C hypertensive rats has been reported. The increased responsiveness to N E was also observed in perfused rat hindquarters (Baum and Shropshire 1967; M c Q u e e n 1961; Mistry et al. 1983) and isolated rabbit renal and iliac arterial strips (Yoshida et al. 1987) from 2 K 1 C models. In addition, Collis (Collis and Alps 1975) found a significant positive correlation between B P and vascular reactivity to N E in the mesenteric arterial bed from renal hypertensive rats, as did M c Q u e e n (McQueen 1961) in hindquarters. Furthermore, it was found that intravenous injection o f nanonmolar concentrations o f neuropeptide Y or N E caused a greater dose-dependent pressor response in anesthetized 2 K 1 C hypertensive rats as compared to normotensive controls (Mezzano et al. 1998). The mechanism that produced exaggerated responsiveness to N E in mesenteric  57  vasculature from 2 K 1 C rats has not been thoroughly studied. Apart from the structural alterations (see above), whether or not there is altered peripheral adrenergic function is not clear. One study showed that the enhanced superior mesenteric resistance to flow in 2 K 1 C hypertensive rats was largely ascribable to a sympathetic neural mechanism (Shimamoto and Iriuchijima 1989). Schiffrin (Schiffrin 1984;) reported that the density o f ai-adrenergic receptors was significantly decreased, while the affinity was significantly increased in the mesenteric vascular bed o f renal hypertensive rats (both 2 K 1 C and 1K1C), and suggested this might be secondary to increased sympathetic nervous activity. In contrast, Tsuda et al (Tsuda et al. 1989) showed that N E release and pressor responses during periarterial nerve stimulation were unchanged in isolated mesenteric vasculature during the acute phase (3 weeks after surgery) o f hypertension, and were rather reduced during the chronic phase (7-8 weeks after surgery) in 2 K 1 C compared to sham normotensive control rats, while responses to exogenous N E were significantly increased. In contrast, Cauvin and Pegram (Cauvin and Pegram 1983) found that in isolated mesenteric small resistance arteries, responses to exogenous N E were comparable in 2 K 1 C hypertensive and sham-operated normotensive rats after a 2-week period o f clipping. However, few studies have been carried out to clarify the post-adrenergic receptor alterations, including calcium handling, in mesenteric arteries from 2 K 1 C hypertensive rats. Impaired endothelial function could also contribute to the abnormal vasoconstriction. It has been reported that endothelium-dependent relaxation to A C h was attenuated in aorta (Heitzer et al. 1999; V a n de Voorde and Leusen 1986) or mesenteric arteries from 2 K 1 C hypertensive rats (Bennett et al. 1993; Carvalho et al. 1997; Cauvin, Pegram 1983; Fortes et al. 1992). In mesenteric arteries, most o f the studies found that incubation with the C O X  58  inhibitor  indomethacin  restored  the  A C h relaxation,  suggesting  that the  abnormal  endothelial-dependent relaxation is due to an increased release o f an E D C F (Bennett et al. 1993; Fortes et al. 1992), probably TxA2, as mentioned above (Carvalho et al. 1997). These in vitro results are consistent with studies in whole animals with N O S inhibitors, which have suggested that there was no deficiency in N O but rather that N O serves as an important buffer mechanism by counterbalancing COX-sensitive vasoconstriction, thereby lessening renal artery clipping-induced blood pressure elevation (Huang et al. 2000; Sigmon and Beierwaltes 1995; Sigmon and Beierwaltes 1998). However, the contribution o f C O X pathway products to NE-induced responses is uncertain. It has been reported that incubation with indomethacin had no effect on the increased reactivity to N E either in vivo or in vitro in the mesenteric arterial bed (Fortes et al. 1992), while T x A receptor antagonists inhibited the 2  potentiated response to N E in vitro, but not in vivo in mesenteric arterial bed (Carvalho et al. 1997).  2.  Hypertension and Hyperinsulinemia /Insulin Resistance  2.1.  Insulin Hypothesis of Hypertension An  independent  association o f hypertension  with hyperinsulinemia in insulin  resistance states is well established from epidemiological studies (He et al. 1999; Lissner et al. 1992; Manicardi et al. 1986; Masuo et al. 1997; M o d a n et al. 1985; Salonen et al. 1998; Skarfors et al. 1991; Tsuruta et al. 1996). Primary insulin resistance is defined as a reduced ability o f insulin to stimulate glucose uptake, principally in skeletal muscle. Insulin resistance is an abnormal state and a common feature o f type 2 diabetes mellitus and obesity, that also share an association with hypertension (Manicardi et al. 1986; M o d a n et al. 1985; Reaven 1988). Compensatory hyperinsulinemia is a signal o f the presence o f insulin resistance, and  59  is also found in essential hypertension regardless o f obesity and glucose intolerance (Ferrannini et al. 1987). It has been reported that high B P is positively correlated with fasting plasma insulin levels independently o f the effect o f age, obesity, fasting glycaemia and antihypertensive medications (Denker and Pollock 1992; M o d a n et al. 1985; Salonen et al. 1998; Tsuruta et al. 1996). In addition, hyperinsulinemia occurs in the young normotensive offspring o f patients with essential hypertension (Ferrari et al. 1991; Grunfeld et al. 1994), and is associated with an increased incidence o f hypertension in men (Salonen et al. 1998; Skarfors et al. 1991) and women (Lissner et al. 1992), in both African Americans and whites (He et al. 1999), as well as in non-obese and non-diabetic Japanese (Tsuruta et al. 1996). Furthermore, several genetically and experimentally hypertensive animal models, such as SHR  and  fructose  fed  hypertensive  rats,  also  demonstrate  insulin  resistance  and  hyperinsulinemia (Hwang et al. 1987; M o n d o n and Reaven 1988). Interestingly, M c N e i l l ' s group has repeatedly reported that chemically diverse drugs that have the common property of attenuating hyperinsulinemia also lower B P in both S H R and fructose hypertensive rats, and the antihypertensive effects o f these drugs could be reversed by simply restoring the plasma insulin levels in the drug-treated rats to those that existed before drug treatment (Verma and M c N e i l l , 1999 and references therein). These findings have led to the hypothesis that insulin may be o f primary pathophysiological importance in the development o f hypertension.  2.2.  Possible Mechanisms of Association Between Hyperinsulinemia/Insulin Resistance and Hypertension: Vascular Action of Insulin A number o f mechanisms may contribute to the development o f hypertension  associated with hyperinsulinemia/ insulin resistance. Hyperinsulinemia and insulin resistance  60  may independently alter vascular reactivity o f arterial blood vessels, and thereby B P , although it is not known yet i f this relationship is causal. To  ascribe a causal role to hyperinsulinemia in the pathogenesis  o f essential  hypertension, sensitivity to the possible blood pressure elevating actions o f insulin should be preserved despite resistance to the glucose-lowering action o f the hormone. It has been demonstrated that insulin can promote renal tubular sodium reabsorption (DeFronzo 1981; DeFronzo et al. 1975), stimulate sympathetic nerve activity and increase catecholamine levels both in humans and in animals (Dornfeld et al. 1987; Liang et al. 1982; R o w e et al. 1981; Sowers et al. 1982). Thus, hyperinsulinemia could increase vascular resistance and arterial pressure (Landsberg 1986; Reaven 1988). However, evidence against this notion also exists (Anderson et al. 1992; Anderson et al. 1991). Recently, it has been suggested that direct interaction o f insulin with blood vessels may be a potentially important link to hyperinsulinemia/insulin resistance to hypertension (Brands et al. 1998; Yki-Jarvinen and Utriainen 1998). In the isolated rat mesenteric arterial bed, insulin at physiological concentrations was shown to significantly increase pressor responses to exogenous N E (Townsend et al. 1992; Verma and M c N e i l l , 1999), and to potentiate arginine vasopressin (AVP)-induced vasoconstriction ( W u et al. 1994). The insulin-induced potentiation o f the response o f M A B to N E has been reported to be further augmented i n arteries from fructose hypertensive rats, suggesting chronic hyperinsulinemia may serve to increase peripheral vascular resistance (Verma and M c N e i l l 1999). Insulin has also been shown to enhance proliferation o f vascular smooth muscle cells (Ridray 1995; Stout et al. 1975) and to increase ET-1 gene expression (Oliver et al. 1991) and E T - 1 release (Hattori et al. 1991; H u et al. 1993). In rat femoral arteries, an insulin-mediated increase in  61  contraction to KC1 was significantly reduced in the presence o f ET-1 receptor antagonists, suggesting a role for E T - 1 (Nava et al. 1997). In addition, Verma et al have demonstrated that the M A B from fructose hypertensive rats contained greater absolute amounts o f E T - 1 than the control rats (Verma et al. 1995). They also reported that chronic E T - 1 receptor blockade completely prevented the rise in B P in these rats and proposed the possibility that hyperinsulinemia may serve as a continual stimulus for E T - 1 synthesis, leading to increased peripheral resistance and raised B P . Furthermore, insulin may also exert a vascular action through regulating production o f C O X pathway metabolites (Axelrod and Levine 1983; Axelrod et al. 1986; K e e n et al. 1997; Rebolledo et al. 1998; van Veen and Chang 1997). It has been reported that inhibition o f T x A  2  synthesis attenuated hypertension induced by  chronic insulin infusion in S D rats (Keen et al. 1997). Thus, interplay between E D C F and insulin may be important in regulating vascular reactivity, and thereby peripheral resistance and blood pressure. In addition to its pressor action, insulin at physiological concentration has been shown to produce vasodilation o f skeletal muscle vascular beds in humans (Laakso et al. 1990). This vasodilatory action has been shown to be impaired in states o f insulin resistance, such as obesity and type 2 diabetes (Laakso et al. 1990; Laakso et al. 1992). It has been suggested that impairment o f insulin-mediated vasodilation may contribute to the increase in peripheral resistance, the characteristic o f hypertension (Feldman and Bierbrier 1993). The insulin-induced increase in blood flow could be abolished by L - N M M A (Scherrer et al. 1994) and this in vivo observation is supported by a study in isolated skeletal muscle arterioles (Chen and Messina 1996). Insulin attenuation o f NE-induced vasoconstriction by stimulation o f N O release was also observed in isolated rat mesenteric resistance arteries  62  (Walker et al. 1997b). Recently, insulin was reported to directly increase N O production in cultured human umbilical vein endothelial cells (Zeng and Quon 1996). Insulin may also regulate N O production by increasing availability of the cofactor BH4 for activation of N O synthas (Verma et al. 1998). Insulin may also exert a modulatory effect on local vasodilator responses by increasing N a - K +  +  ATPase and Ca -ATPase activity (Sowers et al. 1991; 2+  Tirupattur etal. 1993). Taken together, these data suggested that the link between hyperinsulinemia/ insulin resistance and hypertension is likely to be complex and multifactorial. However, it has been suggested that interplay between insulin and endothelial factors may be an important factor in regulating vascular reactivity, and thereby peripheral resistance and blood pressure (Baron 1999; Brands et al. 1998; Nava et al. 1999; Yki-Jarvinen, Utriainen 1998). Hence in hyperinsulinemia/insulin-resistant states a blunted vasodilator and/or an exaggerated pressor effect of insulin may cause an increase in peripheral resistance leading to hypertension.  2.3.  An Animal Model of Hypertension with Insulin Resistance and Hyperinsulinemia: the Zucker Obese Rat  2.3.1. Characteristics of Zucker obese rats The Zucker strain of obese rats represents an animal model that combines obesity heredity with insulin resistance, hyperinsulinemia and hyperlipidemia. By contrast, lean Zucker rats are normal in this regard. Zucker obese rats were first described by Zucker and Zucker (Zucker and Zucker 1961). The obesity is transmitted through an autosomal recessive gene. By 5 weeks of age, animals that are homozygous for this trait (fa/fa) show visible differences in body fat content and in body shape. Hyperinsulinemia appears as early as 3 to 4 weeks of age (Bray  63  1977). Circulating insulin levels have been shown to be 3 to 10 fold higher, while concentrations o f plasma glucose are normal as compared with age-matched lean Zucker rats (Bray 1970; Ionescu et al. 1985; Y o r k et al. 1972). The major site o f insulin resistance in obese Zucker rats appears to be skeletal muscle (Crettaz et al. 1980; Kemmer et al. 1979; Smith and Czech 1983) and involves both receptor and post-receptor abnormalities (Slieker et al. 1990; van de Werve et al. 1987). Hyperlipidemia is uniformly present in obese Zucker rats (Witztum and Schonfeld 1979). Although total cholesterol levels were higher in obese than in lean Zucker rats, the most striking abnormality was found in triglycerides ( T G ) . The <  abnormalities in lipids occur at an early age and the levels o f T G and cholesterol increase with age (Bray 1977; Kasiske et al. 1988; Kasiske et al. 1991). There is evidence that obese Zucker rats develop a modest hypertension at an older age than the metabolic changes (Cox and K i k t a 1992; Kurtz et al. 1989). C o x and K i k t a (Cox and K i k t a 1992) measured systolic B P o f Zucker rats indirectly on a weekly basis from the age o f 6 weeks up to 36 weeks. They found a significantly higher arterial pressure developing in the obese group between 24 and 36 weeks than lean littermates. However, the data in the literature in this regard are not consistent. Some studies were unable to find a difference in blood pressure in obese Zucker rats relative to lean littermates (Auguet et al. 1989; Bunag and Barringer 1988; Pawloski et al. 1992). The variable findings could result from different measurement techniques (Bunag 1983), from differences in the age and or sex o f rats studied, or from differences in the animal colonies. Recently Alonso-Galicia et al measured B P continuously 24 h per day in conscious chronically instrumented, age-matched lean and obese Zucker rats, with carefully controlled N a C l intake, and found that the obese Zucker rats were hypertensive at age 13 to 14 weeks o f age (Alonso-Galicia et al. 1996).  64  Zemel and coworkers also reported an increased blood pressure (both systolic and diastolic pressure) by direct measurement in 10-week old obese Zucker rats (Zemel et al. 1990). The increase in M A P was about 14 to 20mmHg in the studies that reported a greater B P in obese Zucker rats as compared with lean Zuckers (Alonso-Galicia et al. 1996; Bohlen and Lash 1995; K a m et al. 1996; Paulson and Tahiliani 1992; W u et al. 1996). It has been suggested that the mild hypertension that develops in the obese rats is not dependent on increased body weight per se since moderate caloric restriction, achieved by pair-feeding with lean rats, decreased weight gain but did not attenuate hypertension (Kurtz et al. 1989). Thus, the obese Zucker rat may be a useful animal model for detailed and controlled investigation into the abnormalities o f smooth muscle and endothelium function in hypertension associated with hyperinsulinemia/insulin resistance, and how hyperinsulinemia /insulin resistance may be linked to the pathogenesis o f hypertension.  2.3.2. Changes in vascular reactivity The evidence that the hypertension observed in obese Zucker rats may be dependent on exaggerated vascular reactivity comes from the observation that the obese Zucker rats exhibited greater pressor sensitivity to both A n g II and N E during ganglionic blockade (Zemel et al. 1992). Studies with the  conduit vessel,  aorta,  revealed  an  enhanced  sensitivity to  vasoconstrictors, which was independent o f endothelium function and structural changes (Cox and K i k t a 1992; Ouchi et al. 1996; Turner et al. 1995; Zemel et al. 1991; [Hopfner, 1998 #319]). In contrast, K a m et al reported that there was no significant difference in the sensitivity and maximum response to N E , methoxamine or serotonin in isolated small  65  mesenteric arteries from obese and lean Zucker rats at age o f 22 weeks, when the obese rats were hypertensive (Kam et al. 1996). In agreement with this, W u et al did not find a difference in the contractile sensitivity to N E between the isolated perfused  mesenteric  arterial beds from 25 week-old hypertensive obese Zucker rats and lean controls ( W u et al. 1996). A n unchanged reactivity to P E or N E was also observed in isolated small mesenteric arteries from young (12 week-old) pre-hypertensive (Walker et al. 1997a) and in perfused mesenteric arterial beds from older (32 week-old) hypertensive (Turner et al. 1995) obese Zucker rats. Interestly, it has been reported that the maximum tension development for E T 1- and methoxamine-evoked vasoconstrction in perfused M A B was slightly but not significantly lower in 12-week old hypertensive obese compared with lean Zucker rats (Hopfner et al. 1999). The results o f the studies o f the endothelium function in mesenteric vasculature are not consistent. Endothelium-dependent relaxation in response to A C h , A D P or methacholine were reported to be normal in intestinal arteriole, small mesenteric arteries and mesenteric arterial bed o f obese Zucker rats as compared to lean Zucker rats (Bohlen and Lash 1995; K a m et al. 1996; Turner et al. 1995; (Hopfner et al. 1999). In other studies, the relaxation-induced by A C h was attenuated in obese mesenteric resistant arteries (Walker et al. 1997a; W u et al. 1996; Zanchi et al. 1995), while the responses to A D P ( W u et al. 1996) and A23187 (Walker et al. 1997a) were not significantly different from those in lean Zucker  rats. Endothelium-independent  relaxation o f mesenteric  arteries  to  sodium  nitroprusside (SNP) was not impaired in obese Zucker rats as compared with lean Zucker rats (Kam et al. 1996; Turner et al. 1995). Furthermore, there were no significant structural changes in the resistance vessels from obese Zucker rats when the passive tension-  66  circumference relationships and morphological characteristics were evaluated (Bohlen and Lash 1995; K a m etal. 1996). The effects o f insulin on the reactivity o f mesenteric vasculature from Zucker obese rats have not been well characterized. T w o studies examined the influence o f exogenous insulin on vasoconstriction induced by ct-adrenoceptor agonists. Insulin was reported to have no effect in either lean or hypertensive obese Zucker rats in one study (Turner et al. 1995), and to have a small inhibitory effect (8 to 13% inhibition in the presence o f 50 to 5000 mU/1 insulin) in lean but not pre-hypertensive obese Zucker rats in the other (Walker et al. 1997a). The  latter  observation  suggested  that  insulin-induced attenuation  o f NE-mediated  vasoconstriction is impaired in the obese Zucker rat, and that this defect precedes, and therefore could contribute to, the development o f hypertension in this insulin-resistant animal model (Walker et al. 1997a).  VI.  SUMMARY  The mesenteric arterial bed plays an important role in the maintenance and control o f peripheral resistance. The sympathetic neuronal control o f mesenteric vascular tone appears to predominate along the mesenteric arterial tree, and is mainly  mediated by the  neurotransmitter N E acting on ai-adrenoceptors. A particular feature o f the excitationcoupling properties o f the mesenteric artery smooth muscle, especially the smooth muscle o f small arteries, appears to be the dependence o f tone on voltage-operated C a  2 +  channels and,  in turn on the membrane potential. Endothelium-derived factors interact with neuronal, humoral and myogenic determinants to help maintain the normal resistance, or change it in response to metabolic demand.  67  Further investigation into the factors determining these characteristics and the mechanisms that regulate smooth muscle tone, and how they are altered in hypertension may be expected to provide important information as regards our understanding o f the control o f the cardiovascular system in health and disease. This dissertation work examined some cellular mechanisms that regulate smooth muscle reactivity and endothelium functionality in rat mesenteric vasculature, as well as the abnormalities in hypertensive states. The topics o f the three parts o f the study focus on: 1) whether agonist-induced CI" current contributes functionally to the V S M response to a i adreoceptor activation, and the possible functional changes in CI" channels in renovascular hypertensive rats; 2) the role o f CI" and K channels in ACh-induced endothelium-dependent +  vasorelaxation and the factors that mediate the responses; and 3) how endothelium-derived relaxing and contracting factors regulate vascular reactivity to catecholamines and how abnormal release o f these vasoactive factors contributes to the vascular abnormalities in hypertensive rats with hyperinsulinemia and insulin resistance. The hypotheses and specific research objectives for each part o f the study are described in the respective Chapters.  68  PART 1.  THE CONTRIBUTION OF CHLORIDE CHANNELS TO ALPHAi-ADRENOCEPTOR MEDIATED VASOCONSTRICTION IN RAT MESENTERIC ARTERY  I.  RATIONALE  cti-adrenergic receptors play an important role in the control o f vascular smooth muscle contraction and thereby, in regulation o f peripheral resistance, blood flow and blood pressure. The contraction mediated by the ai-adrenoceptor depends mainly on an increase o f free intracellular calcium concentration that results from C a  2 +  release from intracellular  organelles (i.e. the sarcoplasmic reticulum) and/or influx from extracellular fluid. It is clear that in smooth muscle cells, activation o f ai-adrenoceptors causes formation o f inositol 1,4,5,-triphosphate which promotes C a  2 +  release from intracellular stores.  by which the receptor activation opens cell surface C a  2 +  The mechanism  channels is still an interesting topic  attracting many researchers' attention (Minneman 1988; Clapham 1995; Fasolato et al. 1994; Mironneau and Macrez-Lepretre 1995) In  vascular  smooth  muscle,  oti-adrenoceptor-mediated  contraction  is usually  accompanied by a depolarization and an increase in membrane conductance (Bolton et al. 1984; Byrne and Large 1987; Casteels et al. 1977; Mekata and N i u 1972; Takata 1980). Since it is well known that CI" is concentrated inside the smooth muscle cell ( A i c k i n and Brading 1982; Chipperfield et al. 1993; Davis 1992; Davis et al. 1991; Gerstheimer et al. 1987) and its equilibrium potential is more positive than the resting membrane potential (see Introduction), the CI" conductance must represent a potentially important depolarizing  69  mechanism. In rat portal vein, it was demonstrated that N E greatly increased CI" efflux with a smaller effect on K efflux and no influence on N a flux. This indicates that ai-adrenoceptor +  +  activation increased CI" permeability (Wahlstrom 1973b). The involvement o f CI" ions in N E induced depolarization was also confirmed in rat mesenteric arteries where N E increased CI" efflux when producing depolarization without altering the rate o f K  +  efflux or N a  +  influx  (Videbaek et al. 1990). In addition, microelectrode recording from guinea pig mesenteric veins showed that the reversal potential o f NE-stimulated current is the same as E c i . Lowering external CI" concentration suppressed the rapid depolarization produced by N E . This implies that an increased  CI" conductance  is responsible  for the  NE-induced  depolarization (Van Helden 1988). Although membrane depolarization may result from either an influx o f cation or efflux o f anion, current evidence in the literature favors the latter mechanism for a-adrenoceptor-mediated depolarization. A calcium activated chloride channel (Ici(ca)) has now been identified in several types of blood vessels, and can be activated by a number o f vasoconstrictor agonists (Amedee et al. 1990b; Byrne and Large 1988b; Droogmans et al. 1991; Klockner 1993; Pacaud et al. 1989a; V a n Renterghem and Lazdunski 1993; Wang and Large 1993). The properties o f Ici(Ca) have been intensively studied in single V S M cells.  The whole cell patch pipette recording  technique has given the most convincing data, which demonstrated that pharmacological agonists utilize intracellular C a  2 +  stores to evoke Ici(ca), while extracellular C a  2 +  is not  essential for activation o f Ici(ca). (Amedee et al. 1990b; Droogmans et al. 1991; Pacaud et al. 1992; Pacaud et al. 1989b; Wang and Large 1993). In addition, it was shown that activation of the CI" channels by N E could depolarize the membrane (see above references) and that the depolarization brought the membrane potential to between -20 and -30 m V . A t these values,  70  the open-state probability o f Ca  z+  channels is high (Pacaud et al. 1989b).  Based on the  evidence obtained from electrophysiological studies, it was suggested that in vascular smooth muscle, ai-adrenoceptor-mediated calcium release from intracellular stores activates the CI" channels leading to changes in membrane potential. The resulting depolarization could then stimulate calcium entry through voltage-dependent calcium channels (Amedee et al. 1990b; H o g g et al. 1993; Pacaud et al. 1991; Pacaud et al. 1992; Pacaud et al. 1989b).  Thus, the  most likely role o f Ici(Ca) in vascular smooth muscles is to produce membrane depolarization and subsequently C a  2 +  entry and sustained vasoconstriction, especially in response  to  excitatory agonists. The lack o f a potent selective antagonist has been an obstacle in evaluating the physiological role o f Ici(Ca) (Doughty et al. 1998; Large and Wang 1996). Recently, electrophysiological studies have demonstrated that N F A , a nonsteroidal anti-inflammatory agent, is a potent reversible blocker o f Ici(Ca) (White and A y l w i n 1990). In some vascular smooth muscle cells, N F A seemed to block Ici(Ca) when the channels were open (Hogg et al. 1994a). Unlike other CI" channel blockers, it inhibits agonist-evoked Ici(Ca) at concentrations in the micromolar range (Hogg et al. 1994a; Lamb et al. 1994; Pacaud et al. 1989b). A t concentrations up to 5xl0" M , N F A did not (1) reduce the NE-evoked non-specific cation 5  current (Hogg et al. 1994a), (2) inhibit voltage-dependent C a Lamb et al. 1994); or (3) evoke a K  +  2 +  channels (Hogg et al. 1994a;  current (Greenwood and Large 1995; Ottolia, Toro  1994; X u et al. 1994). It was also suggested that N F A did not inhibit at the oti-adrenoceptor recognition site or NE-induced release o f C a not inhibit NE-evoked Ca -activated K 2+  +  2 +  from the intracellular stores, since N F A did  current (Greenwood and Large 1995; H o g g et al.  71  1994a).  Therefore, N F A seems to be a potentially useful tool for evaluation o f the role o f  Ici(Ca) in ai-adrenoceptor-induced contraction. A t the time this project was started, no studies on the functional role o f CI" channels in  ai-adrenoceptor-mediated  vasoconstriction  in  mesenteric  arteries  were  available.  However, during the course o f the investigation, Criddle et al (Criddle et al. 1996) demonstrated that at a concentration o f l O u M , N F A produced a comparable attenuation o f a component o f the NE-evoked contraction when compared with the C a  2 +  channel antagonist  nifedipine in rat isolated aorta. Later, they also reported that N F A (30 u M ) could reduce a component o f NE-induced pressor responses in rat mesenteric arteries (Criddle et al. 1997). However, they did not examine in either study whether the reduction o f the mechanical response in blood vessels by N F A could be attributed to the specific blockade o f Ici(ca> Moreover, there is no evidence available in the literature to indicate whether N F A can affect ai-adrenoceptor-mediated  vasoconstriction in vivo,  nor  any study  on its effects  in  hypertension. The 2 K 1 C renovascular hypertensive rat has been widely used in investigations o f the mechanisms producing and maintaining hypertension. Hemodynamic studies in the 2 K 1 C rat indicate that the 2 K 1 C hypertensive rat is associated with increased peripheral resistance and the main increase in resistance lies in resistance vessels, especially in the mesenteric vascular bed (Meininger et al. 1984; Russell et al. 1983; Teranishi and Iriuchijima 1985). Experiments in 2 K 1 C rats showed that the sensitivity o f mesenteric vasculature to N E is increased (Carvalho et al. 1997; Fortes et al. 1990; McGregor and Smirk 1968). There is no unequivocal explanation o f the molecular mechanism(s) that mediate the increased sensitivity to N E in 2 K 1 C hypertensive rats, and little is known about changes in the signal transduction  72  pathways o f the oci-adrenoceptor that are responsible for membrane depolarization, and thereby C a  2 +  influx in this type o f hypertension (see Introduction for more details).  73  n.  W O R K I N G HYPOTHESES AND SPECIFIC R E S E A R C H OBJECTIVES  The major aim o f this part o f the study was to obtain further information about the possible physiological role o f the Ici(Ca) in the process o f ai-adrenoceptor-mediated vasoconstriction, and therefore i n regulation o f blood flow and blood pressure. In these experiments, we have used N F A as a tool to analyze the functional role o f the Ici(ca) i n ctiadrenoceptor-mediated vasoconstriction both in vitro and in vivo i n mesenteric resistance arteries. The change in the function o f Ici(Ca) that mediates  ai-adrenoceptor-mediated  contraction in hypertensive rats was also examined. The following working hypotheses and specific objectives were addressed.  Working Hypotheses A.  Blockade o f calcium-activated chloride channels with niflumic acid ( N F A ) inhibits oti-adrenoceptor-induced vasoconstriction i n rat mesenteric artery both in vitro and in vivo. The inhibitory effect o f N F A may be greater in hypertensive rats due to an increased functional contribution by CI" channels.  B.  The decrease  in ai-adrenoceptor-induced  contraction due to chloride channel  inhibition with N F A , i n rat mesenteric artery, results from an indirect inhibition o f voltage-gated nifedipine-sensitive C a  2 +  channels.  In other words: A.  Niflumic  acid ( N F A ) ,  a putative selective calcium-activated chloride channel  antagonist, inhibits ai-adrenoceptor-induced vasoconstriction in rat mesenteric artery both in vitro and in vivo. The inhibitory effect o f N F A may be greater in hypertensive rats than that in normotensive rats.  74  B.  N F A , in rat mesenteric artery, inhibits cti-adrenoceptor-induced contraction by blocking a chloride channel, leading to an indirect inhibition o f voltage-gated nifedipine-sensitive C a  2 +  channels.  Specific Objectives: Functional studies. 1)  T o examine the influence o f N F A and CI" free solution (propionate ions as substitute) on the vasopressor response to cirazoline, a selective a 1-adrenoceptor agonist, in rat isolated perfused mesenteric arterial beds ( M A B ) .  2)  T o investigate the effects o f N F A on cirazoline-induced changes in vascular conductance in the superior mesenteric artery in pentobarbital-anaesthetized rats.  3)  To  compare  the  vascular  effects  of  vasoconstriction in two kidney one-clip  N F A on (2K1C)  oti-adrenoceptor-stimulated  hypertensive rats to those in  normotensive rats both in vitro and in vivo.  To rule out that N F A has a direct effect on cirazoline-evoked C a entry and to confirm that N F A indirectly blocks cirazoline-induced C a  2 +  2 +  release or C a  2 +  influx in smooth  muscle o f M A B , the following specific objectives were proposed: 4)  T o examine the effects o f N F A on cirazoline-induced vasoconstriction in l o w C a  2 +  and Ca -free, EGTA-containing solution in rat isolated perfused M A B . 2+  5)  To investigate the influence o f nifedipine, an L-type calcium channel blocker, alone and in combination with N F A on cirazoline-induced vasoconstriction in rat isolated MAB.  75  Ion efflux study: 1)  T o assess the action o f cirazoline on C f ion efflux (  125  I efflux was measured as an  index o f membrane CI" conductance) in absence or presence o f prazosin in rat isolated small mesenteric arteries ( 2  nd  or 3  r d  order branches o f the superior mesenteric artery)  Only preliminary experiments were done. 2)  T o examine the effect o f N F A on cirazoline-induce CI" ion efflux (using  1 2 5  I as  substitute) in rat isolated small mesenteric arteries. Only preliminary experiments were done.  76  m.  M E T H O D S AND MATERIALS  1.  Surgical Preparation of Hypertensive Rats Goldblatt hypertension (2K1C) was induced as described previously by Goldblatt  (1934). Briefly, male Sprague-Dawley rats (180 - 230g) were anaesthetized with halothane (5% in 100% oxygen for induction; 1% in 100% oxygen for maintenance).  After a  retroperitoneal flank incision, the left renal artery was dissected free, and a U-shape silver clip with an internal diameter o f 0.22 ± 0.01 mm was placed around the renal artery, close to its junction with the aorta. The wound was closed and bupivacaine (1%) and Cicatrin were applied topically to the site o f incision. Sham-operated rats underwent renal artery isolation but no clip was placed on the renal artery.  Animals were housed individually with 12 h  light/dark cycle and free access to normal food (Purina rat chow) and tap water.  Animals  were then randomly selected for experiments. Four weeks after renal artery clipping or sham operation, animals were anaesthetized with halothane (5% mixed with 100% oxygen for induction; 1% mixed 100% oxygen for maintenance), and catheters (Polyethylene tubing I D . 0.58 mm, O . D . 0.965 mm) were inserted into the left femoral artery for measurement o f arterial blood pressure and removal of blood samples, and the left femoral vein for administration o f drugs. The catheters were filled with heparinized saline (25 I U / m l in 0.9% N a C l ) and tunneled subcutaneously to the back o f the neck, exteriorized and secured. Bupivacaine (1%) was applied topically to the site o f incision and animals were allowed to recover for 24 hr. O n the following day, blood pressure was recorded using a pressure transducer (PD23ID Gould Statham, C A , USA) and Grass polygraph (Model 79D Grass Instruments, M A , USA) and the heart rate was measured using a tachograph (Model 7 P 4 G Grass Instruments, M A , USA) continuously for 30-45 minutes in free-moving conscious rats. After 30-45 minutes, a blood sample was taken for  77  measurement o f renin activity. 2 K 1 C rats with diastolic blood pressure o f > 100 m m H g were used, and animals with malignant phase hypertension, as evidenced by the onset o f weight loss, were excluded from the study.  2.  Measurement of Plasma Renin Activity Renin-dependent hypertension was verified by determination o f plasma renin activity.  B l o o d (1 ml) was collected into a pre-chilled syringe containing E D T A to yield a final concentration o f 1 mg/ml. After centrifugation, the plasma was frozen and stored at -20° C until it was assayed. Plasma renin activity was determined as angiotensin I generated under control conditions in which converting enzyme and angiotensinase activities were inhibited by use o f E D T A , dimercaprol and 8-hydroxyquinoline. The amount o f generated angiotensin I was measured by radioimmunoassay using a commercial polyclonal antiserum against angiotensin I (Du Pont, Ont., Canada) and a double antibody determination system.  Function Study: 3.  Perfused Isolated Mesenteric Artery Preparation Each animal was anaesthetized with sodium pentobarbital (35 mg/kg, iv).  The  abdominal cavity was opened and mesenteric artery was cannulated through an incision at the confluence with the dorsal aorta and then isolated as previously described by McGregor (McGregor 1965). The mesenteric artery and its branches were flushed with heparinized physiological salt solution, and then the M A B was transferred into a warmed organ chamber, and perfused with Krebs-bicarbonate (normal Krebs) buffer maintained at 37° C and gassed with 95% O2: 5% CO2. The Krebs-bicarbonate buffer used was o f the following composition (in m M ) : N a C l 120, K C 1 4.6, glucose 11, M g S 0  4  1.2, C a C l  2  2.5, K H 2 P 0 4 1.2, N a H C 0  3  78  25.3. The p H o f the buffer following saturation with a 9 5 % 0 : 5 % C 0 gas mixture was 7.4. 2  2  The other perfusion buffers used in the experiments were: 1) Cl'-free buffer o f the following composition (in m M ) : C H C O O N a 120, C H C O O K 3.5, glucose 11, M g S 0 2  ( C 6 H n 0 ) 2.5, K H P 0 7  2  2  4  5  2  1.2, N a H C 0  3  5  25; 2) L o w C a  2 +  buffer: C a  m M in normal Krebs; 3) Ca -free E G T A containing buffer: C a 2+  2 +  2 +  4  1.2, C a  was decreased to 0.5  was omitted from and  I m M E G T A was added to normal Krebs. The perfusion rate was kept constant at 5 ml/min using a polystaltic peristaltic pump (Buchler Instruments, Buchler Fort Lee, N J , U S A ) . Changes in perfusion pressure were measured and recorded using a pressure transducer (PD23ID Gould Statham, C A , U S A ) and Grass polygraph (Model 79D Grass Instruments, M A , U S A ) . The perfused blood vessels were allowed to stabilize for 1 hr before the start o f each experiment.  4.  Experimental Protocols in Perfused M A B  4.1.  Effects of Vehicle or NFA on the Vasoconstrictor Responses to Cirazoline in Perfused MAB  Series 1. This procedure was performed using normal Krebs buffer. The M A B s from 2 K 1 C and sham rats were initially exposed to a submaximal dose o f cirazoline (9 nmol) to check the viability and responsiveness o f the preparations, and then were allowed to further equilibrate for 1 hr.  A control dose-response curve for cirazoline was constructed by  injection o f 6 separate bolus doses o f cirazoline (0.09 - 30 nmol). Perfusion pressure was allowed to return to baseline between each injection o f agonist. The second and the third dose-response curve to cirazoline were determined in the presence o f vehicle (0.03 or 0.1% alcohol), or N F A (3 or 10 p M ) in the perfusion media. B l o o d vessels were perfused with buffer containing either vehicle or N F A for 20 m i n and thereafter dose-response curves for  79  the agonist were determined.  After the completion o f each dose-response  curve for  cirazoline, a single bolus injection o f K C 1 (60 jumol) was also made. Series 2. Effects o f vehicle and N F A were also evaluated in CT-free buffer. A control dose-response curve to cirazoline in mesenteric arteries perfused with normal Krebs was obtained as described before.  The tissues were then allowed to stabilize for 40 min. while  being perfused with normal Krebs solution. The solution was then changed to Cl"-free buffer and 20 min. was allowed to elapse before a dose-response determined.  curve to cirazoline was  After the completion o f the second dose-response curve, the tissues were  perfused again with normal Krebs for 40 minutes. The perfusion solution was then changed to Cr-free buffer containing vehicle (0.1% alcohol) or N F A (3 or 10 u M ) , with which blood vessels were perfused for 20 min and thereafter the determination o f the final dose-response curve to cirazoline. The perfusion time is long enough to greatly decrease [Cl"]i in smooth muscle cells ( A i c k i n and Brading 1982). Separate tissues were used for each concentration o f NFA.  4.2.  Effects of NFA on Cirazoline-Induced Vasoconstriction in MAB Perfused with Low Ca * and C^-free Solution, and Compared with Effect ofNifedipine. 2  In the presence of nifedipine The M A B from 2 K 1 C and sham rats were initially exposed to a submaximal dose o f cirazoline (9 nmol), and then were allowed to further equilibrate for 1 hr. Three consecutive dose-response curves for cirazoline were determined by injection o f 6 separate bolus doses o f cirazoline (0.09 - 30 nmol). Perfusion pressure was allowed to return to baseline between each injection o f agonist. The first dose-response curve served as control. The second dose-response curve to cirazoline was performed with nifedipine ( 3 u M ) in the perfusion media, while the third dose response curve was determined in the presence o f  80  nifedipine ( 3 p M ) plus N F A (3 or 10 p M ) in the perfusion media. Inhibitors were added 20 min before and until dose-response curves for the agonist were determined.  After the  completion o f each dose-response curve for cirazoline, a single bolus injection o f KC1 (60 pmol) was also made. Low C a  solution The protocol was the same as the above except that the second  dose-response curve to cirazoline was obtained in a perfusion medium containing 0.5 m M C a , while the third dose response curve was determined in the presence o f 0.5 m M C a 2 +  2 +  plus N F A (3 or 10 p M ) in the perfusion buffer. Ca  free-EGTA-containing solution Since reproducible dose-response curves for  cirazoline could not be obtained in C a -free, EGTA-containing solution (preliminary experiments, data not shown), only a single bolus dose o f cirazoline was applied each time. In addition, a single bolus injection o f KC1 (30 pmol) was made before each cirazoline dose was given. A total o f 5 sets o f injections o f KC1 and cirazoline were given in each M A B under different perfusion buffer conditions. The perfusing sequence o f the different perfusion buffers was as follows: normal Krebs, Ca -free solution, Ca -free solution in the presence 2+  2+  2+  of N F A , C a -free solution again, and Ca-free solution in the presence o f nifedipine. After each injection o f cirazoline, tissue was allowed to equilibrate for 40 m i n by perfusing with normal Krebs to refill the internal C a  2 +  stores. Antagonists were added i n the perfusion buffer  20 m i n before injection o f KC1 and were present until the pressor response to cirazoline returned to baseline. The C a - f r e e - E G T A containing buffer was perfused for 10 m i n before 2+  KC1 was applied and thereafter until vasoconstriction to cirazoline was measured.  81  5.  In vivo Measurement of Blood Flow and Vascular Conductance Surgical preparation. Each animal was anaesthetized with sodium pentobarbital (35  mg/kg, iv), and an additional catheter (Polyethylene tubing I D . 0.58 mm, O . D . 0.965 mm) was inserted into the right femoral vein for administration o f cirazoline.  The abdominal  cavity was opened through a ventral midline incision, and the superior mesenteric artery was exposed and dissected free. A transonic flow probe (Model 1RB630, Transonic System Inc. N Y , U . S . A . ) was placed on the mesenteric artery. B l o o d flow was measured using the flowmeter (Model T206, Transonic Systems Inc. N Y , U . S . A ) and displayed on a Grass polygraph (Model 79D Grass Instruments, M A , U . S . A . ) . B l o o d pressure and heart rate were continuously monitored.  B o d y temperature i n these animals was maintained at 36 + 1°C  using a heating lamp and monitored by a rectal mercury thermometer. After completion o f the surgery, each animal was allowed to stabilize for a period o f 60 min..  6.  Experimental Protocols for in vivo Experiments Effects o f N F A on blood pressure, blood flow and mesenteric vascular conductance  were examined in four groups o f rats.  Each animal initially received a cumulative  continuous infusion o f cirazoline (0.13, 0.34, 1.00 and 2.77 mg/kg/min), and each dose was infused for 6 min.  After the completion o f the first dose-response curve, animals were  allowed to recover for 50 min. This period was sufficient to allow blood pressure, heart rate and mesenteric blood flow to return to the baseline. Each animal then received either vehicle (0.3 ml/kg; N a H C 0 3 in glucose solution) or N F A (3 mg/kg) as a bolus iv injection, and 10 min. was allowed to elapse before the second cumulative doses-response curve to cirazoline was determined.  82  Ion Efflux Studies: 7.  Isolation of Small Mesenteric Arteries M a l e Sprague-Dawley rats (300-400g) were anaesthetized with sodium pentobarbital  (65 mg/Kg) i.p. and the mesenteric arterial bed was removed and placed in the Krebs bicarbonate buffer. The second- or third-order branches from the superior mesenteric artery were dissected free from surrounding tissue and cleaned. The small arteries were cut 0.5 cm in length and mounted on a single stainless steel wire holder.  8.  Experimental Protocols for Measurement of  125  I Efflux in Small Mesenteric  Arteries 1 2 5  I was chosen as a marker o f CI" channel activity because: 1) it has higher specific  activity than  36  C 1 and it is transported poorly by the N a / K / C l " cotransporter in vascular +  +  smooth muscle cells and by anion exchangers (O'Donnell and Owen 1986; Dalmark and Wieth 1972); 2) the permeability o f I" through calcium-activated CI" channels is greater than that o f CI" (Amedee et al. 1990b); 3) measurement o f  1 2 5  I efflux from cells and tissues has  been used by other investigators as an index o f membrane CI" conductance (White et al. 1995). "l efflux was measured using a washout method (McMahon and Jones 1988; Smith L  and Jones 1985). Briefly, isolated arteriolar segments were allowed to equilibrate for 2 hours in Krebs buffer at 37 °C, p H 7.4. Following equilibration, tissues were transferred to fresh Krebs (2 ml) containing 6 p C i  1 2 5  I to load tissues for 1 hour. F o r each experiment, four  loaded arteriolar segments from the same mesenteric vascular bed were tested in parallel. After a 2-second rinse, each loaded tissue was transferred at 1 min intervals through a series o f vigorously gassed (with 95% 0 : 5% C 0 ) tubes containing 1 ml non-radioactive Krebs in 2  2  the absence or presence o f cirazoline (1 or 3 or 10 u M ) or prazosin (0.3 p M ) or N F A (10  83  u M ) or cirazoline (3 u M ) plus either o f the antagonists. The total washout time was 3 2 min. Antagonists were added at the beginning (t = 0) and were present throughout. Cirazoline stimulation started at 21 min and was present for the last 12 min. A t the end o f the washout, tissues were blotted and then the radioactivity i n each tissue and each washout tube was counted using a gamma counter. 9.  Chemicals A l l chemicals were purchased from Fisher Scientific (Richmond, B . C . , Canada),  Sigma Chemical C o (St. Louis, M O , U S A ) or Research Biochemical International (Natick, M A , U S A ) . Angiotensin I [  125  I] radioimmunoassay kits and carrier- free  1 2 5  I were purchased  from D u Pont Company (Mississauga, Ont., Canada). A stock solution o f N F A (10" M ) was 1  prepared in 100% ethanol ( E T O H ) and diluted to the required concentration in perfusate reservoir for experiments in isolated mesenteric vascular beds, or in Krebs buffer for the ion efflux assays. N F A was dissolved i n N a H C 0 3 with 5% glucose (4 x 10" M , p H 8.5) and 1  prepared as a stock solution (10 mg/ml) for in vivo studies. Cirazoline and prazosin were dissolved in normal saline (0.9% N a C l ) or twice distilled water for both in vivo and in vitro studies. Nifedipine was dissolved in ethanol and the experiments with nifedipine were performed in tissue baths protected from light. A l l solutions were made freshly each day. 10.  Data and Statistical Analysis For in vitro studies, the absolute increases i n perfusion pressure following bolus  injection o f each dose o f cirazoline were plotted.  Vascular conductance in vivo was  calculated as flow divided by mean blood pressure ( M A P ) . Conductance was calculated i n order to assess active changes in vascular tone (Lautt  1999;  Tabrizchi and Pang  1993).  MAP  was calculated as diastolic blood pressure + 1/3 (systolic blood pressure - diastolic blood  84  pressure).  The decreases in conductance were expressed as decreases in percentage o f the  control conductance obtained just before infusion o f cirazoline. Ion efflux is characterized by a simple elimination model. The elimination rate constant equation (Wahlstrom 1973a) is as follows:  , C = Co e*  where C is radioactivity in the tissue at time t; C was calculated by sequentially back-adding the radioactive counts in each tube to the radioactive counts remaining in the tissue at the end of the experiment; Co is the initial radioactivity in the tissue at t=0; k is elimination constant per min (efflux rate). In the ion efflux study, each washout curve was computed by the equation and then the k values in the cirazoline-stimulated portion o f the efflux curve were averaged and compared with the averaged k o f the control in the absence o f any drugs, and the averaged k obtained in the presence o f antagonist and cirazoline plus antagonist for the same period, respectively. The effects o f drugs on  1 2 5  I efflux were plotted as percentage o f  control k. A l l data are presented as mean ± S E M .  Student's unpaired t test was used for  comparisons between two means, and two-way A N O V A was used for multiple comparisons between the two groups o f rats (i.e. 2 K 1 C and sham). One-way A N O V A was used for multiple comparisons in one group o f rats (normal rats). Duncan's multiple range test was used to compare between multiple means. analysis.  P < 0.05 was considered as significant in the  85  IV.  RESULTS  1.  Characteristics of 2K1C Hypertensive Rats Systolic and diastolic blood pressure and heart rate o f conscious 2 K 1 C rats were  significantly (n= 42; P < 0.05) higher than those o f sham rats (Table 1.1). Furthermore, the plasma renin activity was significantly (n = 42; P < 0.05) elevated in 2 K 1 C hypertensive rats when compared to that o f sham normotensive rats (Table 1.1).  2.  Effect of N F A on Cirazoline-Induced  Vasoconstriction in Isolated Mesenteric  Arteries Perfused with Normal Krebs. There was no significant difference in the basal perfusion pressures in isolated M A B perfused with normal Krebs between 2 K 1 C hypertensive and sham normotensive rats, (27.4 ± 0.9 and 27.9 ± 0.9 mmHg, 2 K 1 C vs. sham rats mean ± S E M n = 12 P > 0.05). Bolus injections o f cirazoline (0.09 - 30 nmol) evoked dose-dependent pressor responses i n isolated M A B from 2 K 1 C hypertensive and sham normotensive rats. Cirazoline-evoked increases in perfusion pressure in mesenteric arteries obtained from 2 K 1 C hypertensive rats were significantly higher than those in sham normotensive rats (Fig. 1.1 - 1.2).  The presence o f  vehicle (0.03% & 0.1% ethanol) did not influence the dose-response curve to cirazoline (Fig 1.1). While 3 u M N F A inhibited the response at only 0.9 nmol cirazoline, cirazolinemediated vasoconstriction was significantly (n = 6; P < 0.05) inhibited at all doses (0.09-30 nmol) in presence o f the higher concentration o f N F A (10 u M ) in M A B from both 2 K 1 C and sham rats (Fig. 1.2).  There were no differences in the magnitude o f the inhibition o f the  cirazoline responses by N F A between 2 K 1 C hypertensive and sham normotensive rats. O n the other hand, vasoconstriction evoked by bolus injection o f KC1 (60 u.mol) in isolated mesenteric arterial beds perfused with normal Krebs were not affected by the presence o f  86  TABLE 1.1 Characteristics o f 2 K 1 C and Sham rats: B l o o d pressure (mmHg), heart rate (beats/min), plasma renin activity (mg ml" h" ) and body weight (g) o f 2 K T C hypertensive and sham 1  1  normotensive rats.  2K1C  Sham  Arterial pressure Systolic  244±5  a  134±2  Diastolic  166±4  a  94±2  Heart rate  417±8  a  370±5  Plasma renin activity  18.37±2.10  B o d y weight  367±6  a  3.03±0.28 392±6  Values are pooled and shown as mean ± S E M n = 42 for each group o f rats. a  Significantly different from sham, P < 0.05 (unpaired t-test).  87  FIGURE 1.1  Effect o f vehicle ( E T O H , 0.03% and 0.1%) on vasoconstrictor responses to bolus injection o f cirazoline in isolated M A B from either hypertensive (2K1C) or normotensive (Sham) rats perfused with normal Krebs at constant flow. Data are shown as mean ± S E M , n = 6. P < 0.05 vs. sham (two-way A N O V A followed by a  Duncan's test).  2K1C o5 x E E CD i_ ~j co co CD  250 • 200  Control  0ETOH(O.O3%) •  150  ETOH(0.1%)  h  c o CO  =>  100  Q_ 50  CD co 03 CD i_  o c  h  0 0.03  0.09  0.3  0.9  Cirazoline (nmol)  Sham _  250  X  •  E  f rj co CO CD  2  0  0  Control  •  ETOH(0.03%)  •  ETOH(0.1%)  150  c g  CO  tr. Q_ 0) CO 03 CD  100  50  o c 0 0.03  0.09  0.9 Cirazoline (nmol)  89  FIGURE 1.2  Effect o f N F A (3 u M and 10 u M ) on pressor responses to cirazoline in M A B from hypertensive (2K1C) and normotensive (Sham) rats perfused with normal Krebs. Data are shown as mean ± S E M , n = 6 . P < 0.05 vs. s h a m , P < 0.05 vs. c o n t r o l , P < 0.05 a  b  vs. N F A (3 p M ) (two-way A N O V A followed by Duncan's test).  0  2K1C  X  200  r  E 180 E 0 CO CO CD  160 140 -  u. 120 D_  c 100 o CO  3  80 -  Q.  60 -  ase  tCD  CD  b  40 20 0 0.09  0.30  0.90  3.00  9.00  30.00  9.00  30.00  Cirazoline (nmol) Sham ' 3 200 X  r  E 180 E, o 160 CO CO  140 -  0 120 1_ 0_  Control WZtt N F A (3uM) H i NFA(10uM)  c  o 100 CO  t: 0  Q.  c 0 CO CD  0  b c  80 60 40 20 0 -  be 0.09  0.30  0.90  3.00  Cirazoline (nmol)  91  N F A in the perfusion medium (Fig. 1.3).  There was also no difference in the response to  KC1 between 2 K 1 C hypertensive and sham normotensive rats (87.5 ± 8 . 8 rnmFfg and 71.1 ± 9.8 mmHg, respectively).  3.  Effect of N F A on Cirazoline-Induced Vasoconstriction in Isolated Mesenteric Arteries Perfused with Cl"-Free Buffer. When the perfusion buffer was changed from normal Krebs to the Cf-free buffer,  there was a transient increase in perfusion pressure with the peak being reached in 2 to 3 min, and then the perfusion pressure stabilized again at 28.7 ± 1 . 1 and 27.3 ± 1 . 0 m m H g for 2 K 1 C and sham rats respectively. The stabilized basal perfusion pressure obtained in Cf-free solution did not significantly differ from control value achieved in normal Krebs for 2 K 1 C and sham rats (see above). The transient increase in perfusion pressure in Cf-free buffer was 18.4 ± 4.4 and 4.9 ± 0.5 m m H g for 2 K 1 C hypertensive and sham normotensive rats, respectively. This increase in perfusion pressure was significantly (n = 18; P < 0.05) greater in  2 K 1 C hypertensive  than  that  in  sham  normotensive  rats.  Cirazoline-induced  vasoconstriction in isolated mesenteric beds obtained from 2 K 1 C hypertensive and sham normotensive rats was impaired following perfusion with Cf-free buffer when compared to normal Krebs (Fig. 1.4, 1.5 & 1.6). The inhibition was significant (P < 0.05) at cirazoline doses o f 3, 9 and 30 nmol. Perfusion o f mesenteric blood vessels with Cf-free buffer resulted in a significantly (P < 0.05) greater inhibition o f cirazoline-mediated vasoconstriction in sham normotensive rats than in 2 K 1 C hypertensive rats (Fig. 1.5 & 1.6 insert). W e did find that in Cf-free buffer, cirazoline-mediated vasoconstriction was further inhibited by the presence o f N F A (Fig. 1.5, 1.6), but not vehicle (Fig. 1.4) in the perfusion media. N F A (3 u M ) significantly (P < 0.05) inhibited cirazoline-mediated vasoconstriction at doses o f 0.9, 3,  92  FIGURE 1.3  Effect o f N F A (3 u M and 10 u M ) on KCl-evoked vasoconstriction in M A B from hypertensive (2K1C) or normotensive (Sham) rats perfused with normal Krebs. Data are shown as mean ± S E M , n = 6. N o difference was found in this experiment, (twoway A N O V A )  93  2K1C  100  r  Control  3MMNFA  10  u-MNFA  KCI (60umol )  Sham  100  r  Control  3 uM N F A K C I (60  nmol)  10 u M N F A  94  FIGURE 1.4  Effects of Cl'-free buffer and vehicle (0.1% E T O H ) on pressor responses to bolus injection of cirazoline. Control (in normal Krebs, open bar), Cl"-free buffer alone (hatched bar), vehicle in Cf-free buffer (solid bar). Data are shown as mean ± S E M , n = 6 . P < 0.05 vs. sham, a  A N O V A followed by Duncan's test).  b  P < 0.05 vs. control, (two-way  2K1C  250  r  200  h  CD X E E,  •  Control  CD  0  Cl-free  Zi CO CO  •  ETOH/CI-free  i—  2?  150  Q. o CO  t  100 h  CD  Q_  50 h  CD CO  CD CD  i_  o c  JX  0  0.09  0.03  0.3  0.9 Cirazoline (nmol)  Sham oS  180  x E E  160 h  •  Control  2 140  0  Cl-free  CO  •  ETOH/CI-free  s c g  'co  1  _  _  2  0  100 80  t  CD  CL  CD CO CD CD  i_  o c  60 40 20 0  Cirazoline (nmol)  3  96  FIGURE 1.5  Effect o f N F A (3 p M ) on pressor responses to cirazoline i n M A B from 2 K 1 C and sham rats perfused with CI" -free buffer. Insert: shows % change in perfusion pressure corresponding to the data in ( 2 K 1 C ) and (Sham). Control (normal Krebs, opened columns); Cl"-free buffer alone (hatched columns); N F A (3 p M ) in C f - free buffer (solid columns). Data are shown as mean ± S E M , n = 6. a  P < 0.05 vs. sham.  b  P < 0.05 vs. control,  0  P < 0.05 vs. Cl"-free buffer alone (two way  A N O V A followed by Duncan's test). e  P < 0.05 vs. Cl'-free buffer in 2 K 1 C rats, P < 0.05 vs. 3 u M N F A + Cl"-free buffer in 2 K 1 C  rats (unpaired student's t-test).  d  97  2K1C  0.09  0.30  0.90  3.00  9.00  30.00  9.00  30.00  Cirazoline(nmol)  Sham  0.09  0.30  0.90  3.00  C i r a z o l i n e (nmol)  98  FIGURE 1.6  Effect o f N F A (10 u M ) on pressor responses to cirazoline in M A B from 2 K 1 C and sham rats perfused with C f -free buffer. Insert: shows % change in perfusion pressure corresponding to the data in ( 2 K 1 C ) and (Sham). Control (normal Krebs, opened columns); Cl'-free buffer alone (hatched columns); N F A (10 p M ) in Cf-free buffer (solid columns). Data are shown as mean ± S E M , n = 6. a  P < 0.05 vs. sham.  b  P < 0.05 vs. control,  0  P < 0.05 vs. CT-free buffer alone (two way  A N O V A followed by Duncan's test). e  P < 0.05 vs. Cl"-free buffer alone in 2 K 1 C rats,  2 K 1 C rats (unpaired student's t-test).  d  P < 0.05 vs. 10 u M N F A + CI" free in  99  0.09  0.30  0.90  3.00  9.00  30.00  C i r a z o l i n e (nmol)  Sham  2! = 100  0.09  0.30  0.90  3.00  C i r a z o l i n e (nmol)  9.00  30.00  100  9 and 30 nmol in 2K1C hypertensive rats and sham normotensive rats. The magnitude o f blockade produced by N F A o f cirazoline-mediated vasoconstriction was significantly (n = 6; P < 0.05) greater in sham rats than that in 2K1C rats (Fig. 1.5 insert). N F A (10 u M ) suppressed responses to cirazoline in Cf-free buffer in a similar manner (Fig 1.6). The presence o f a higher concentration o f N F A significantly (n = 6; P < 0.05)  inhibited  cirazoline-mediated vasoconstriction at doses o f 0.9, 3, 9 and 30 nmol in 2K1C hypertensive rats and in normotensive rats. The magnitude o f the reduction in the vasoconstrictor response to cirazoline again was significantly (n = 6; P < 0.05) greater in sham normotensive rats than that in 2K1C hypertensive rats (Fig. 1.6 insert).  4.  Influence of N F A on Cirazoline-Induced  Change  in Mesenteric  Vascular  Conductance in Anaesthetized 2K1C Hypertensive and Sham Normotensive Rats The effect o f N F A was tested in vivo in anaesthetized rats. There was no significant difference in the baseline values o f the superior mesenteric blood flow between 2K1C hypertensive and sham normotensive rats. However, the basal vascular conductance in superior mesenteric artery was significantly (n = 5; P < 0.05) lower in 2K1C hypertensive rats in comparison to sham normotensive rats (Table 1.2). Administration o f either N F A or vehicle did not alter the baseline values o f M A P , superior mesenteric flow or conductance in anaesthetized rats (Table 1.2).  Intravenous  infusion o f cumulative doses o f cirazoline caused dose-dependent increases in M A P (Table 1.3), and decreases in superior mesenteric vascular conductance in 2K1C hypertensive and sham normotensive rats (Table 1.4). However, the degree o f reduction in the conductance induced by cirazoline in 2K1C hypertensive rats was similar to that in sham normotensive  101  TABLE 1.2 Mean blood pressure ( M A P ; mmHg), superior mesenteric artery blood flow ( S M A F ; ml/min) and conductance ( S M A C ; ml mmHg'Wn" ) values before and after injection o f vehicle 1  ( N a H C 0 3 , 0.3 ml/kg) or N F A (3 mg/kg) in 2 K 1 C hypertensive and sham normotensive rats.  MAP  2K1C  Pre-vehicle  164±14  Post-vehicle 1 4 8 ± 8  a  a  SMAF  SMAC  Sham  2K1C  Sham  98±3  13.9±0.9  14.1±2.5  0.088±0.012  a  0.140±0.028  92±3  14.2±0.9  14.5±1.9  0.097±0.009  a  0.158±0.021  2K1C  Sham  Pre-NFA  162±13  a  97±5  11.9±1.3  15.5±1.8  0.076±0.012  a  0.165±0.027  Post-NFA  153±16  a  95±7  12.4±2.8  16.6*2.4  0.086±0.025  a  0.180±0.032  Each value represents the mean ± S E M , n = 5 a  Significantly different from sham rats, p < 0.05 (two-way A N O V A followed by Duncan's  test).  102  TABLE 1. 3. Effects  o f cirazoline on mean arterial pressure ( M A P , mmHg)  in anesthetized 2 K 1 C  hypertensive and sham normotensive rats before (control) and after treatment with either vehicle ( N a H C 0 , 0.3 ml/kg) or N F A (3 mg/kg). 3  Cirazoline (u.g kg" min" ) 1  0.13  0.34  1  1.00  2.77  2K1C Control  171±16  Vehicle-treated  160±9  Control  170±13  NFA-treated  154±15  163±14  188±14  237±5  Control  104±3  108±2  127±4  172±3  Vehicle-treated  96±4  103±3  118±4  173±5  Control  104±7  109±4  126±5  167±3  NFA-treated  99±7  106±6  125±5  165±2  a  181±17  a  168±10 a  183±15  200±14  a  188±13 a  215±14  250±19 243±15  a  253±5  a  Sham  Each value represents the mean ± S E M , n = 5 a  Significantly different from sham rats, P < 0.05 (two-way A N O V A followed by Duncan's  test).  103  TABLE 1.4 Effects of cirazoline  on decrease in vascular conductance (% o f control) in superior  mesenteric artery ( S M A C ; ml mmHg" min" , control conductances were shown in Table 1.2) 1  1  in anesthetized 2 K 1 C hypertensive and sham normotensive rats before (control) and after treatment with either vehicle ( N a H C 0 , 0.3 ml/kg) or N F A (3 mg/kg), 3  Cirazoline (pg kg" min" ) 1  1  0.13  0.34  1.00  2.77  Control  8.9±2.6  20.1±3.8  39.8±5.0  65.3±3.7  Vehicle-treated  9.2±3.8  17.6±3.8  36.2±5.0  64.4±3.9  Control  10.8±2.0  22.4±3.2  46.8±3.5  68.4±3.2  NFA-treated  1.3±2.0  2.0±6.0  24.3±8.0  2K1C  a  a  a  61.0±6.5  Sham Control  12.4±1.3  23.2±3.1  42.6±4.4  69.3±3.1  Vehicle-treated  10.4±2.2  19.8±3.9  34.6±4.9  69.1±3.1  Control  12.5±1.6  26.4±3.6  47.7±4.1  72.1±4.0  NFA-treated  3.0±3.0  13.6±4.1  a  a  34.3±6.2  a  67.0±5.0  Each value represents the mean ± S E M , n = 5 Significantly different from control (before treatment with N F A ) , P < 0.05 (two-way A N O V A followed by Duncan's post test).  104  rats (Table 1.4). Pretreatment with vehicle did not affect cirazoline-induced changes in M A P or conductance when compared to the absence o f vehicle (Table 1.3 and 1.4). In addition, i n animals that were treated with N F A , cirazoline-mediated pressor responses were  not  significantly affected when compared to control (Table 1.3). However, pretreatment with N F A significantly (n= 5; P <0.05) impaired cirazoline-mediated decreases in vascular conductance at doses o f 0.13 to 1.00 mg/kg/min in 2 K 1 C hypertensive and normotensive rats (Table 1.4). There was no significant difference in the magnitude o f the attenuation o f the decrease in vascular conductance between 2 K 1 C and sham rats (P > 0.05)  5.  Effect of Nifedipine and Nifedipine Plus N F A on Cirazoline- and KCl-Induced Vasoconstriction in Isolated M A B Perfused with Normal Krebs The effect o f the voltage-dependent C a  2 +  channel blocker, nifedipine and the effect o f  nifedipine plus N F A were evaluated in isolated M A B from normal S D rats perfused with normal Krebs. The presence o f nifedipine (3 u M ) in the perfusion media significantly inhibited the vasoconstrictor action o f all doses o f cirazoline (0.03-9 nmol) (Fig. 1.7A). Nifedipine also markedly decreased K C I (60 u.mol)-evoked vasoconstriction (Fig. 1.7B). However, in the presence o f nifedipine plus N F A (10 u M ) , the inhibitory effect on pressor responses o f M A B to cirazoline did not statistically differ from that in the presence o f nifedipine alone (Fig. 1.7A). The combination o f nifedipine and N F A also had no additive inhibitory effect on contractile response to K C I (Fig. 1.7B).  105  FIGURE 1.7  Effect o f nifedipine ( N F D P , 3 p M ) and N F D P ( 3 p M ) plus N F A (10 p M ) on vasoconstriction induced by bolus injection o f cirazoline (A) or KC1 (B) in M A B from normal S D rats perfused with normal Krebs. Data are shown as mean ± S E M , n = 6.  b  P < 0.05 vs. control (normal Krebs, in absence o f  N F D P or N F A ) (one-way A N O V A followed by Duncan's test)  106  140  r  120 100  -  80  -  60  -  40  -  20  -  0  •  Control  •  NFDP  •  b  0.03  (3pM)  NFDP+NFA(10uJV1)  b  b b  0,09  mm. 0.3  0.9  9  Cirazoline(nmol)  Normal Krebs  NFDP(3uM) K C I (60  pmol)  N F D P + NFA(10uM)  107  6.  Effect of N F A on Cirazoline- and KCl-Induced Vasoconstriction in Isolated  M A B Perfused with Low C a Lowering C a  2 +  2 +  Solution  concentration from 2.5 to 0.5 m M in perfusion buffer did not  significantly affect cirazoline-induced vasoconstriction in isolated M A B from normal S D rats (Fig 1.8A & F i g . 1.9 A B ) , whereas the pressor response to K C I was significantly reduced (Fig, 1.8B & F i g . 1.10A B ) . Addition o f N F A (either 3 or 10 u M ) into low C a  2 +  perfusion  buffer had no further inhibitory effect on the response to K C I as compared to perfusion with low C a  2 +  buffer alone (Fig 1.10A.B). In contrast, the presence o f 10 u M , but not 3 u M , N F A ,  decreased the responses to cirazoline significantly (Fig. 1.9A B ) . In low C a  2 +  solution,  repetition o f the dose-response curve in the presence o f vehicle did not affect the pressor responses to cirazoline (Fig. 1.8A). Similarly, the second response evoked by repeated application o f K C I , in the presence o f vehicle, was not significantly different from the first in isolated M A B perfused with low C a s o l u t i o n alone (Fig. 1.8B). 2+  7.  Effect  of N F A on  Cirazoline-Induced  Vasoconstriction in Isolated M A B  Perfused with C a - f r e e - E G T A Solution 2+  Cirazoline, at 0.3 nmol, induced an initial transient peak followed by a sustained increase in perfusion pressure in isolated M A B perfused with normal Krebs containing 2.5 mM Ca  2 +  (Fig l . H A : a ) . Perfusion with Ca -ffee solution containing 1 m M E G T A abolished 2+  the sustained plateau o f pressor response, while leaving the initial transient peak intact (Fig. l . H A : b ) . The amplitude o f the initial transient peak response to cirazoline with Ca -free 2+  solution did not differ from that obtained with normal Krebs (Fig. 1.1 I B ) . In contrast, perfusing  with  Ca -free 2+  EGTA-containing  solution  totally  abolished  KCl-evoked  vasoconstriction (Fig. 1.11 A ) . Thus, the initial contractile response to 0.3 nmol cirazoline  108  FIGURE 1.8  Effect o f low Ca  z+  (0.5 m M ) buffer and vehicle (0.1% E T O H ) on the pressor response to  bolus injection o f cirazoline ( A ) and KC1 (B) in M A B from normal S D rats. Data are shown as mean ± S E M , n = 6. followed by Duncan's test)  b  P < 0.05 vs. normal Krebs. (one-way A N O V A  109  A x  160  r  c  0.03  0.09  0.3  0.9  3  9  Cirazoline (nmol)  B D5 1 2 0  r  X  E £  100  -  Normal Krebs  Ca(0.5mM) K C I (60 Li m o l )  Ca(0.5mM)+ETOH  110  FIGURE 1.9  Effect o f N F A (3 u M in A , 10 u M in B ) on pressor response to bolus injection o f cirazoline in M A B from S D rats perfused with low C a  2 +  buffer (0.5 m M ) .  Data are shown as mean ± S E M , n = 6. P < 0.05 vs. normal Krebs , b  (0.5 m M ) buffer (one-way A N O V A followed by Duncan's test)  0  P < 0.05 vs. low C a  2 +  Ill  -j?  180 r  0.03  0.09  0.3  0.9  3  9  3  9  Cirazoline (nmol)  0.03  0.09  0.3  0.9  Cirazoline (nmol)  112  FIGURE 1.10  Effect o f N F A (3 u M i n A , 10 u M i n B ) on KC1 evoked contraction i n M A B from normal S D rats perfused with low C a ( 0 . 5 m M ) buffer. 2+  Data are shown as mean ± S E M , n = 6. followed by Duncan's test)  b  P < 0.05 vs. normal Krebs. (one-way A N O V A  120  r  100  h  Control  Ca(0.5mM)  NFA(3uM)/ Ca(0.5mM)  K C I (60 u m o l )  B 120  r  Control  Ca(0.5mM)  NFA(10uiVI)/ Ca(0.5mM)  KCI(60umol)  114  FIGURE 1.11  Effect o f N F A (10 p M ) on the pressor response to bolus injection o f cirazoline (0.3 nmol) in M A B from S D rats perfused with C a  2 +  free-EGTA (1 m M ) solution (B). Upper panel ( A ) is a  representative trace obtained from one o f the four M A B tested. Data are shown as mean ± S E M , n=4. (one-way A N O V A )  115  K (30umol )  Cirazoline(0.3nmol)  +  B • Control (Krebs) • oS30 x E E  Cafree+EGTA(1mM)  • C a free+EGTA (1mM)+NFA •  (10uM)  Cafree+EGTA(1mM)  mCa  free+EGTA (1mM)+NFDP (3uM)  C i r a z o l i n e (0.3  nmol)  116  seems not to include the component that induced by C a  2 +  influx from extracellular space.  Therefore, it allows assessing the effect o f N F A on the cirazoline-induced vasoconstrictor response that mediated by C a influence o f C a  2 +  2 +  released from intracellular store independently  o f the  influx via V O C s . To confirm this, nifedipine was used as a positive control.  Neither niflumic acid (10 u M ) nor nifedipine (3 u M ) had a significant inhibitory effect on the vasoconstrictor response to cirazoline in M A B perfused with C a - f r e e - E G T A containing 2+  solution (Fig 1.11 A : c . d & B )  8.  125  1 Efflux from Small Mesenteric Arteries  The effect o f cirazoline on  1 2 5  I efflux was evaluated, and preliminary experiments on  the effects o f prazosin and N F A were carried out. A s illustrated in F i g . 1.12, cirazoline caused an increase in  1 2 5  I efflux. F i g . 1.12A shows the  1 2 5  I efflux curve from an individual  vessel; the effect o f cirazoline took 2-3 min to reach its peak, and then fell rapidly. However, it took another 2-3 min to return from the peak to basal value. The effect o f cirazoline was concentration-dependent, 1, 3, 10 u M cirazoline increasing  1 2 5  I efflux by 141 ± 2%, 187 ±  20% and 168 ± 49%, respectively (Fig. 1.12 B ) . Prazosin (0.3 u M ) as well as N F A (10 u M ) did not affect the basal (Table 1.5).  1 2 5  I efflux, but inhibited cirazoline-induced increase in  1 2 5  I efflux  117  FIGURE 1.12  125  A. Representative  I efflux curve in isolated small mesenteric arteries of SD rats. Cirazoline  (3 pM) was added at 21 min. B. Effects of cirazoline on  125  I efflux in isolated small mesenteric arteries of SD rats (n = 4).  Data are shown as mean ± SEM, n = 4  118  A  0 I  '  1  1  i  i  i  i  0  5  10  15  20  25  30  35  Time  (min)  B  250 ,  Control  1 M-V1  3|iv1 Cirazoline  10  pM  119  T A B L E  A . Effect of Prazosin on cirazoline-induced  Control  kfmin" ) 1  %Control  0.098±0.0016 100  kCmin" ) 1  %Control  0.096±0.00076 100  I efflux (n=2)  Cirazoline (3uM) 0.129±0.0095 145.1±12.1  B . Effect o f N F A on cirazoline induced  Control  1 2 5  1.5  Prazosin (0.3uM) 0.095±0.00115 101.2±4,1  Cirazoline+Prazosin (3uM) (0.3uM) 0.072±0.0010 75.6±1.8  I efflux (n=2)  Cirazoline (3uM)  NFA (lOuM)  0.154±0.0020  0.076±0.0037  160.1±16.4  88.3±2.3  Cirazoline+NFA (3uM) (lOuM) 0.085±0.0082 95.9±8.6  120  V.  DISCUSSION The main objective of this part o f the work was to examine the importance o f CI" ions  in ai-adrenoceptor-mediated vasoconstriction in rat mesenteric arteries. T o test this we employed N F A , an anti-inflammatory agent that has been characterized as a potent Ici(Ca) blocker with fewer nonselective actions in rabbit portal vein as compared with other known Ici(ca) blockers, such as D I D S , A - 9 - C I A A (Hogg et al. 1994a; Large and Wang 1996). W e found that N F A was capable o f inhibiting cirazoline-induced vasoconstriction both in vitro as well as in vivo. It failed to produce additive inhibition i n the presence o f the C a  2 +  channel  inhibitor nifedipine, which also attenuated the cirazoline-induced contraction significantly. We also found that removal o f C f could suppress the cirazoline-induced contraction. In addition, we showed that cirazoline induced  1 2 5  I efflux from rat small mesenteric arteries.  This effect o f cirazoline was inhibited by both prazosin and N F A . These data suggest that chloride ions play an important role in vasoconstrictor responses that are mediated v i a the stimulation o f cti-adrenoceptor in rat mesenteric arteries. Based on our results, it also seems that the role o f CI" in cirazoline-mediated vasoconstriction is less important in blood vessels obtained from 2 K 1 C hypertensive compared to normotensive rats.  The Role of CT Channels in ai-Adrenoceptor-Induced Vasoconstriction. Opening o f CI" channels depolarizes V S M . This is because CI" is transported into V S M cells against its electrochemical gradient, resulting in an intracellular CI" concentration that is much higher than that predicted by passive distribution (Davis 1992). Based on the evidence obtained from electrophysiological studies, a role for agonist-induced Ici(Ca) has been identified in a number o f blood vessels, such as rabbit and rat portal veins (Byrne and Large  121  1988a; Pacaud et al. 1989b), rabbit ear artery (Amedee et al. 1990b), human mesenteric artery (Klockner  1993; Klockner and Isenberg  1991) and rat renal resistance  artery  (Gordienko et al. 1994). In rat portal veins as well as in rabbit ear arteries, it has been repeatedly reported that NE-mediated calcium release from intracellular stores preferentially produces an increase in CI" conductance leading to changes in membrane potential (Amedee et al. 1990a; Pacaud et al. 1991; Pacaud et al. 1989b) and the opening o f voltage-gated calcium channels (Pacaud et al. 1991; Pacaud et al. 1989b). Calcium activated CI" current in some vascular smooth muscles has been reported to be blocked by drugs such as 4',4'diisothiocyanostilbene-2,2-disulfonic acid (DJDS) and N F A (Hogg et al. 1994a; H o g g et al. 1994b; K i r k u p et al. 1996a; Lamb et al. 1994; Large and Wang 1996; Pacaud et al. 1989a). Our current findings demonstrated  that both N F A and removal o f CI" inhibited c i i -  adrenoceptor-mediated vasoconstriction i n perfused mesenteric blood vessels. In addition, as expected, nifedipine, a specific blocker o f voltage-gated C a  2 +  channels (Kuriyama et al.  1995), also significantly inhibited cirazoline-induced contraction; there was however no additional reduction in the contraction o f mesenteric arteries by the combination o f N F A and nifedipine, a phenomenon that was also observed on NE-induced contraction in rat aorta (Criddle et al. 1996).  Norepinephrine has been reported to increase CI" efflux while  producing depolarization in rat mesenteric arteries without altering either the rate o f K  +  efflux or N a influx (Videbaek et al. 1990). Moreover, responses that are mediated v i a the +  cti-adrenoceptors have been shown to be sensitive to the actions o f calcium channel antagonists (Chen and Rembold 1995). In rat mesenteric microvessels, the calcium-entry blocker nitrendipine was found to reduce NE-mediated constriction (Chen et al. 1996). Therefore, the most likely mechanism that mediates the relaxant action o f N F A in rat  122  mesenteric arteries is an inhibition o f C f current evoked by cirazoline, thereby indirectly preventing C a  2 +  from entering v i a voltage-gated C a  2 +  channels.  The role o f CI" channels in mediating cti-adrenoceptor-induced contraction was further confirmed by experiments in which extracellular CI" was removed. It has been shown that reduction o f extracellular CI" concentration decreases [Cl"]i in smooth muscle cells ( A i c k i n and Brading 1982). In addition, the probability o f opening o f certain CI" channels is dependent on the intracellular and extracellular concentrations o f CI", and the CI" current is much more affected by changes o f the intracellular CI" concentration than predicted simply from the change in CI" driving force (Chesnoy-Marchais 1983; Dinudom et al. 1993; Jackson et al. 1996). Furthermore, it has been demonstrated that removal o f CI" or lowering the extracellular CI" concentration ([Cl"]o) suppresses cti-adrenoceptor-induced CI" currents and membrane depolarization in several types o f smooth muscles including V S M (Bulbring and Tomita 1987; Large 1984; V a n Helden 1988; V a n Renterghem and Lazdunski 1993). V a n Helden has studied the kinetics o f the effect o f low-Cl" on changes in CI" conductance induced by N E in smooth muscle o f guinea pig mesenteric veins (Van Helden 1988). H e observed that the currents increased in amplitude during about the first 40s exposure to l o w C l " solution and decreased afterward. However, complete suppression was not always observed even after a 15min exposure to low-Cl" solution. H e also found that the initial increase in amplitude o f the CI" current was consistent with an increase in the driving force for CI". H e suggested that the decrease in the response must be related to an inactivation o f the CI" conductance  mechanism itself, because when the response was significantly  suppressed i n low CI" solution, the reversal membrane potential remained more positive than the control values. Thus, we expected that prolonged perfusion with Cl"-free buffer would  123  decrease the level o f intracellular CI", thereby inhibiting agonist-activated CI" channels and attenuating cirazoline-induced vasoconstriction in the mesenteric arterial bed. Our results confirmed this notion and showed that the pressor responses to cirazoline were significantly inhibited after perfusing with Cl"-free buffer for 20 min. It was also found that i n Cl"-free solution not only 10 u M but also 3 u M N F A significantly inhibited cirazoline-induced vasoconstriction. There can be a number o f explanations for this observation, which could account for the further inhibitory actions o f N F A in Cf-free buffer. First, it is possible that N F A inhibited the efflux o f residual CI" that remained inside the vascular smooth muscle cells in Cf-free buffer. This speculation is supported by observations in a number o f articles. M c M a h o n and Jones demonstrated that in Cf-free propionate substituted solution, the reduction o f [Cl"]i was slower than the loss o f CI" from the extracellular site in rat aorta strips ( M c M a h o n and Jones 1988). Others showed that the relationship o f [Cl"]i to [Cl"]o in smooth muscle cells measured with a Cl'-sensitive microelectrode was hyperbolic (Aickin and Brading 1982). Total removal o f extracellular CI" (gluconate as substitution) caused a rapid fall in [Cl"]i, but measurement o f [Cl"]i in Cf-free solution one hour later still showed a positive value. The value o f [ C f ]i agrees well with [Cl"]i estimated from the CI" efflux study with three other CI" substitutes (Aickin and Brading 1982; Casteels 1971). A n alternative explanation is that either propionate or bicarbonate anions made a small contribution to the process o f agonist-induced depolarization via efflux through the CI" channels, while in the presence o f N F A this effect was blocked. It has been shown that CI" channels are rather unselective for many anions, although with varying permeability (Amedee et al. 1990b; Franciolini and Petris 1990; Large and Wang 1996).  124  1-  " I efflux from vascular smooth muscle cells has been used as an indicator o f CI"  movements to study the properties o f agonist-induced CI" channels, owing to its high selectivity for conductive channels (White et al. 1995). It has known that I" has a higher permeability than CI" v i a agonist-activated CI" channels in blood vessels (Amedee et al. 1990b; Large and Wang 1996). In addition, it has been demonstrated that  1 2 5  I is transported  poorly by various anion carriers, such as N a / K / C l " cotransporters and C1"/HC03 exchangers +  +  (Dalmark and Wieth 1972; O'Donnell and Owen 1986), and that these transporters do not affect agonist-stimulated results o f  1 2 5  1 2 5  I efflux in vascular smooth muscle cells (White et al. 1995). The  I efflux measurement in the present study suggest that rat small mesenteric  arteries contain conductive CI" channels that are activated in response to cirazoline. The cirazoline-induced increase in  1 2 5  I efflux was antagonized by prazosin in a concentration-  dependent manner, which suggests that the CI" channels are opened by cirazoline through stimulation o f cti-adrenoceptors. Moreover, like prazosin, N F A also inhibited the  1 2 5  I efflux  induced by cirazoline. This result, together with the data from contractile experiments in perfused mesenteric arterial bed, suggests that CI"  ions play a direct role in the cti-  adrenoceptor-mediated excitation-contraction coupling. Current evidence in the literature supports the view that vasoconstrictor responses arising from the activation o f cti-adrenoceptor in rat mesenteric arterial bed are mediated by the activation o f ctiA-adrenoceptor sub-types (Chen et al. 1996; K o n g et al. 1994; Williams and Clarke 1995). Studies using rat aorta indicate that cirazoline has a higher affinity for the ctiA and a m subtypes than for the a m sub-type (Buckner et al. 1996). However, a previous study using human cloned ai-adrenoceptors had indicated that cirazoline had a higher affinity for the a i  a  subtype rather than the aib and a i d subtypes (Horie et al. 1995).  125  Contraction in vascular smooth muscle mediated via the activation of a 1-adrenoceptors is dependent upon both an influx of C a  2 +  and C a  2 +  release from intracellular stores (Cauvin and  Malik 1984; Minneman 1988; Chen and Rembold 1995). An elevation in the concentration of intracellular C a  2 +  is believed to produce an increase in CI" conductance (Amedee et al.  1990b; Pacaud et al. 1992; Large and Wang, 1996). Based on our present findings, it would seem that vasoconstriction mediated via the stimulation of ctiA-adrenoceptors in the mesenteric arterial bed depends in part on the presence of intracellular CI".  The Selectivity of NFA Because of the relatively low degree of selectivity of most CI" channel blockers, alternative explanations, i.e. actions unrelated to inhibition of CI" channels, for the ability of N F A to inhibit the contractile responses to cirazoline in mesenteric arteries must be considered. N F A did not affect KCl-evoked contraction in normal C a Second, N F A remained ineffective in low C a  2 +  2 +  containing buffer.  solution in which the response to KCI was  impaired. However, N F A significantly inhibited pressor responses to cirazoline under both conditions. These observations suggest that the inhibitory effect of N F A on cirazolineinduced vasoconstriction is not due to a direct blockade of voltage-gated Ca channels, or to 2+  non-specific effects on the contractile apparatus. Additionally, N F A did not affect cirazolineinduced vasoconstriction in the absence of extracellular Ca , so it is unlikely that N F A interfered with the contraction induced by C a  2 +  release from SR or had an inhibitory effect  on the contractile proteins. It has been demonstrated that N F A has no effect on spontaneous transient current produced by lK(Ca), but it can enhance NE-induced iK(Ca) in rabbit portal vein.  126  It was thus suggested that N F A increases C a  2 +  release from S R (Hogg et al. 1994a). In our  experiments, in which rat mesenteric arteries were employed, this seems not to be the case. W e found that N F A alone neither affected basal perfusion pressure in the  perfused  mesenteric arterial bed nor inhibited mesenteric blood flow or vascular conductance in anaesthetized rats. Therefore, it seems unlikely that the inhibitory effect o f N F A on cirazoline-induced vasoconstriction is due to an activation o f K  +  channels.  Altered Function of Ct Channels in mediating cci-Adrenoceptor-Induced Vasoconstriction in MAB from 2K1C Hypertensive Rats. In a previous study, M c G r e g o r and Smirk (McGregor and Smirk 1968) reported that mesenteric arteries from renal hypertensive rats (2K1C) exhibited a higher vasoconstrictor response to N E . In the present study, it was found that the cirazoline-induced increase in perfusion pressure was greater in blood vessels from hypertensive rats.  The higher  vasoconstrictor responses observed in renal hypertensive tissues have been suggested to be the result o f increased resistance to flow (Russell et al. 1983). Significant reductions in external diameter and increased media-to-lumen ratio have been reported to be responsible for increased vascular reactivity in 2 K 1 C hypertensive rats (Deng and Schiffrin 1991). In the present study, we also found that basal vascular conductance in situ was lower in 2 K 1 C hypertensive rats when compared to sham normotensive rats, which is consistent with the proposal that morphological changes may account for changes in the function o f blood vessels in 2 K 1 C hypertensive rats (Bennett and Thurston 1996; L i et al. 1996). However, this may not entirely account for the altered behavior o f blood vessels in 2 K 1 C hypertensive versus normotensive rats, since no difference in vasoconstrictor responses evoked by K C 1 was observed in the present study.  127  The results o f our in vitro and in vivo studies indicate that N F A had a similar efficacy at inhibiting cirazoline-mediated vasoconstriction in both normotensive and hypertensive rats. However, this was not the case when CI" ions were replaced with propionate ions. In Cf-free solution, N F A was more effective at inhibiting cirazoline-mediated vasoconstriction in normotensive than i n hypertensive rats. Moreover, removal o f CI" affected cirazoline-induced vasoconstriction to a greater extent in sham than in hypertensive rats. This may reflect a diminished role o f CI" channels in 2 K 1 C hypertensive rats, which could be caused by a decreased channel number or impaired channel activity. However, the interpretation o f these results may not be straightforward. Differences in membrane potential between blood vessels from 2 K 1 C been  hypertensive and those o f sham normotensive rats in Cf-free buffer may have  responsible  for  the  increased  ability o f N F A to  inhibit cirazoline-induced  vasoconstriction i n sham when compared with 2 K 1 C hypertensive rats. The possibility exists that adaptive changes in ion content and /or permeability o f vascular smooth muscle in 2 K 1 C hypertensive rats may have occurred. Certainly, lowering CI" concentration o f physiological salt solution has been found to result in changes in resting membrane potential in vascular smooth muscle secondary to changes in Eci (Davis et al. 1991; Harder and Sperelakis 1978). It has been reported that removal o f [Cf ]o accompanied an initial transient depolarization o f smooth muscle membrane due to initial net efflux C f from cells (Aickin and Brading 1982; Harder and Sperelakis 1978). In agreement with this, we found that a small transient contraction developed shortly after changing to Cf-free buffer in mesenteric arterial beds from both groups o f rats, suggesting that removal o f [Cl"]o produces a small depolarization sufficient to reach the threshold for activation o f V O C s and thereby smooth muscle contraction. W e also found that the magnitude o f the spontaneous contraction was greater i n  128  2 K 1 C hypertensive rats than that in sham rats, implying that the depolarization produced upon removal o f CI" is greater in M A B from 2 K 1 C hypertensive rats than normotensive rats. On the other hand, the greater spontaneous response could also be due to a decreased resting membrane potential ( V ) in M A B from 2 K 1 C rats. It has been shown that in one-kidney, m  one-clip (1K1C) hypertension, the resting tail artery was depolarized by about 7 m V . This depolarization may be caused by a humoral substance since plasma supernatants  from  hypertensive rats also depolarized the muscle cells i n control animals (Pamnani et al. 1985). However, V under  m  o f mesenteric smooth muscle in 2 K 1 C hypertensive rats may be not changed  control conditions as compared with normotensive  control rats because  the  vasoconstrictor responses to KC1 in M A B were similar between the two groups o f rats in our experiments. Thus, the greater transient contraction induced by initial removal o f CI" may be due to a higher [Cl"]i that could shift E c i to more positive direction, and/or a greater permeability in hypertensive mesenteric smooth muscle than normotensive ones, leading to more depolarization as the net efflux CI" contributes more to V . It has been demonstrated m  that deoxycorticosterone acetate (DOCA)/salt-induced hypertension in the rat is associated with a significant rise in intracellular CI" in arterial smooth  muscle and this difference in  [Cl"]i can be attributed to an increase in activity o f the N a / K / C l " cotransporter (Davis et al. +  +  1993). Data regarding the changes in CI" handling and membrane properties in 2 K 1 C hypertensive rats are limited. N o t until quite recently was there direct evidence to support our speculation. However, recently Goecke et al reported that there was a significant increment in the N a / K pump and N a / K / C l " cotransporter in aortic rings from two kidney-Goldblatt +  +  +  +  hypertensive rats (Goecke et al. 1998). This evidence supports our assumptions. Since the N a / K pump is electrogenic, activation o f N a / K pump would hyperpolarize the membrane +  +  +  +  129  (Cheung 1989). O n the other hand, the N a / K / C l " cotransporter accumulates intracellular CI" +  +  above equilibrium. Although it is electroneutral ( A i c k i n and Brading 1990; Chipperfield 1986), by raising [Cl"]i, it has a depolarizing influence on V Thus, the V  m  m  (Davis 1992; Davis et al. 1991).  o f mesenteric smooth muscle in 2 K 1 C hypertensive rats may not change under  control conditions, although there are increased activities o f the N a / K pump and N a / K / C l " +  cotransporter.  +  +  +  However, the levels o f [Cl"]i may be higher in smooth muscle o f the  hypertensive M A B perfused with physiological salt solution than that o f normotensive ones due to the high activity o f N a / K / C l " cotransporter. I f this is true, the greater spontaneous +  +  contraction shown in the present study in 2 K 1 C M A B upon changing the perfusion solution to Cf-free was most likely due to an elevated [Cl"]i (see above discussion). The elevated [Cf]i could also explain why an impaired function o f C f channels in M A B from 2 K 1 C was only shown in Cf-free buffer. It is known that the magnitude o f ion currents flowing through open channels is determined by the number o f ion channels, the single channel current and the channel activity (or P en) (Nelson and Quayle 1995). The single channel current depends op  on the ion concentration and the driving force for the ions (Aidley 1998). Therefore, in normal physiological buffer, the elevated [Cl"]i could enhance the efficacy o f CI" channels when they are open, owing to the enhanced driving force for C f in M A B from 2 K 1 C hypertensive rats. Thus, the effect caused by decreased channel number or/and impaired channel activity could be masked by an increased single channel current. However, in C f free buffer, the fall in [Cl"]i due to removal o f C f would be greater in 2 K 1 C M A B than in sham control rats because o f the greater driving force and increased N a / K / C l " cotransporter +  +  activity. Prolonged perfusion with Cf-free buffer could eventually abolish or greatly decrease the differences in [Cl"]i between mesenteric smooth muscles from 2 K 1 C hypertensive and  130  normotensive rats, thereby revealing a decreased function o f CI" channels. The diminished function o f CI" channels could account for the smaller reduction in contractile responses and the lesser inhibitory effect o f N F A in hypertensive M A B when challenged with cirazoline in Cl'-free buffer. Whether the above explanations are correct can be clarified with the help o f studies that combine measurement o f membrane potential, [Cl"]i and channel activity. In addition, a future study involving blockade o f N a / K / C l " cotransport may help to resolve the +  +  involvement o f the cotransporter in the changes observed in the present study. The impaired CI" function might reflect an adaptive change due to the enhanced reactivity to ctiadrenoceptor stimulation in 2 K 1 C hypertension. Yoshida et al (Yoshida et al. 1989) had previously reported that an increase in intracellular calcium content o f vascular smooth muscle in 2 K 1 C hypertensive rabbits did occur. However, the possibility that an increase in the availability o f intracellular calcium may be responsible for a diminished role for CI" during agonist-mediated pharmacomechanical processes in 2 K 1 C hypertensive rats cannot be determined from our present study. Nevertheless, the results presented here are the first indication o f altered function o f CI" channels in blood vessels in experimental hypertension.  131  VI.  1.  SUMMARY  In vivo, cirazoline induced a dose-dependent reduction in superior mesentery vascular conductance. The extent of the reduction in the conductance in 2 K 1 C hypertensive rats was similar to that in normotensive rats. N F A attenuated the reduction in the vascular conductance induced by cirazoline to a similar degree in both normotensive and hypertensive rats.  2.  In vitro, cirazoline induced a concentration-dependent increase in perfusion pressure in rat isolated perfused mesenteric arterial bed. The pressor responses to cirazoline in M A B from 2 K 1 C hypertensive rats were significantly greater than those in sham rats. N F A suppressed the cirazoline-induced vasoconstriction in sham and 2 K 1 C M A B , but had no effect on KCl-evoked pressor responses.  3.  Removal of C f from the perfusion buffer also impaired the pressor responses to cirazoline. The inhibition was greater in normotensive rats than that in hypertensive rats. N F A caused a further inhibition of cirazoline-mediated vasoconstriction in C f free buffer. The inhibitory effect of N F A was smaller in hypertensive rats than that in normotensive rats.  4.  Nifedipine also inhibited cirazoline-induced vasoconstriction. The magnitude of the inhibitory effect of N F A plus nifedipine on cirazoline-induced contraction in perfused M A B was similar to that seen with nifedipine alone. In the presence of nifedipine, N F A had no additive effect on pressor responses to KCI.  5.  In low C a  2+  solution, in which the response to K C I was already attenuated, N F A  reduced cirazoline-induced contraction with no effect on KCl-evoked contraction.  132  6.  Cirazoline  elicited  contraction  in  Ca -free, 2+  EGTA-containing solution.  The  contraction was transient in response to 3 nmol o f cirazoline, and was not inhibited by either 10 p M N F A or 3 p M nifedipine. However, in the Ca -free, EGTA-containing 2+  solution, responses to KC1 were abolished. 7.  In small mesenteric arteries, cirazoline caused a dose-dependent increase in efflux. The increased  1 2 5  I efflux was inhibited by prazosin and blocked by N F A .  1 2 5  I  133  VII  CONCLUSIONS  Overall, our results demonstrated that N F A is capable o f inhibiting cti-adrenoceptormediated vasoconstriction o f rat mesenteric artery both in vitro and in vivo. The mechanism o f this action o f N F A in our experimental system appears to involve a decreased CI" efflux, but not direct inhibition o f C a  2 +  influx or release from intracellular stores. Our observations  suggest that CI" plays an important role in oti- adrenoceptor-mediated vasoconstriction in rat mesenteric vessels, probably by producing membrane depolarization that leads to opening o f voltage-gated C a though C a  2 +  2 +  channels, and consequently, a sustained contraction w i l l be maintained  influx. This contribution o f CI" in blood vessels from hypertensive rats appears  to be reduced.  134  PART 2.  THE MECHANISMS OF THAT ACETYLCHOLINEINDUCED RELAXATION IN RAT MESENTERIC ARTERY: A COMPARISON WITH AORTA  I.  RATIONALE  In rat mesenteric  arteries,  endothelium-dependent  relaxation to A C h has  been  demonstrated to be mediated by N O , and an unidentified E D H F that elicits NO/PGI2independent hyperpolarization by activation o f K channels (Chen and Cheung 1997; Fukao +  et al. 1995). However, there are conflicting results regarding the relative contribution o f these E D R F s and the mechanisms that mediate ACh-induced relaxation by these factors in rat mesenteric arteries. The types o f K  +  channels activated by E D H F have not yet been definitely  identified. In addition, the role o f PGI2 in ACh-induced relaxation in mesenteric arteries is not clear (Adeagbo and Triggle 1993; Chen and Cheung 1997; Garland and Plane 1996; Hansen and Olesen 1997; H w a et al. 1994; Waldron and Garland 1994; Weidelt et al. 1997; White and H i l e y 1997; W u et al. 1997) (and please see Introduction). The release o f N O , E D H F and P G I by A C h acting at muscarinic receptors has been 2  studied (Fukao et al. 1997c; Luckhoff and Busse 1990). There is evidence that an agonistinduced increase C a  2 +  influx from the extracellular space is essential for the maintained  production o f E D R F s , and consequently, the full magnitude o f agonist-evoked  smooth  muscle relaxation (Fukao et al. 1997c; Kruse et al. 1994; Luckhoff and Busse 1990). However, the dependence on extracellular C a  2 +  for their synthesis seems to be different  among these E D R F s (Luckhoff 1988; Luckhoff and Busse 1990; White and Martin 1989). In endothelial cells, opening o f C a  2 +  entry channels is believed to require agonist binding to  membrane receptor and / or depletion o f intracellular C a  2 +  stores (Putney 1991; W a n g and  135  van Breemen 1997). Unlike in smooth muscle cells, opening C a  entry channels in  endothelial cells is not voltage-dependent (Colden-Stanfield et al. 1987; Johns et al. 1987). However, the driving force for C a  2 +  entry depends on the transmembrane potential, which is  controlled by a variety o f ion channels. electrochemical driving force promoting C a  Membrane hyperpolarization augments the 2 +  entry (Adams et al. 1989; Johns et al. 1987;  Laskey et al. 1990; Luckhoff and Busse 1990). It has been reported that in freshly isolated rabbit aortic endothelial cells and in native endothelium from intact rat aorta, stimulation o f muscarinic receptors with A C h activates Ca -dependent K 2+  +  channels leading to transient membrane hyperpolarization, which is  sensitive to tetraethylammonium ( T E A ) and/or charybdotoxin ( C T X ) (Busse et al. 1988; Marchenko and Sage 1994; Sakai 1990; Wang et al. 1995a; Wang et al. 1996). A C h activation o f K channels leading to endothelium hyperpolarization was also demonstrated in +  freshly isolated guinea pig coronary artery (Chen and Cheung 1992) and intact guinea pig carotid artery (Quignard et al. 2000). Thus, K channels present on endothelium seem to have +  an important role in hyperpolarizing endothelium, and facilitating C a  2 +  entry (Usachev et al.  1995). Indeed, in native freshly isolated rat aortic endothelial cells, high K  +  blocked the  increase in [ C a ] ; and greatly inhibited N O release in response to A C h (Luckhoff and Busse 2+  1990), while in intact rabbit aorta, T E A was capable o f inhibiting ACh-induced  Ca  2 +  elevation and E D R F synthesis/release as well as vasorelaxation based on results o f bioassay and fura-2 spectrofluorimetry techniques (Demirel et al. 1994). These results suggest that endothelial K  +  channels can modulate ACh-induced relaxation by controlling membrane  potential, and subsequent C a  2 +  influx and E D R F synthesis/release.  136  Recently, CI" channels have been identified in various endothelial cells (Groschner and Kukovetz 1992; Nilius et al. 1996; N i l i u s et al. 1997a; White et al. 1995). Some researchers have demonstrated that lowering the extracellular CI" concentration or exposing cells to CI" channel antagonists ( N F A or N-phenylanthranilic acid) inhibits sustained C a signaling stimulated by agonists such as A T P and histamine and triggered by C a  2 +  2 +  store  depletion with thapsigargin and cyclopiazonic acid in human aortic endothelial cells (Hosoki and Iijima 1994; Hosoki and Iijima 1995; Yumoto et al. 1995). Wang and van Breemen (Wang and van Breemen 1999) have reported that in freshly isolated rabbit aortic endothelial cells, A C h activated a slowly developing C f current, which was blocked by C f channel antagonists.  In addition, removal o f extracellular C f ions abolished the ACh-induced  sustained C a  2 +  signal as well as divalent cation entry. After clamping the membrane potential  at a hyperpolarizing level close to K A C h - induced C a  2 +  equilibrium potential, C f removal had no effect on  entry, indicating that C f current modulates C a  polarized membrane conjunction with K  +  +  potential after  2 +  influx by maintaining a  A C h activation. Thus, C f channels  may act in  channels on regulating ACh-induced E D R F synthesis, thereby causing  vasorelaxation. However, there is no evidence yet for a functional role o f C f channels in contributing to agonist-induced relaxation. Based on the evidence mentioned above, we conducted a functional study to investigate the mechanisms mediating ACh-induced relaxation, especially to see whether besides K  +  channels, C f channels in vascular endothelial cells could also modulate A C h -  induced relaxation o f underlying smooth muscle; i f so, whether the relative contribution o f CI" as well as K channels is different in elastic vs. muscular arteries. W e also looked at how +  N O , P G I 2 and E D H F differentially contribute to ACh-induced relaxation in the different  137  arteries. These experimentally observed differences in the relative contribution o f E D R F s and the mechanisms that mediate endothelium-dependent relaxation between large and small arteries may have some physiological significance; in particular, since it may be clinically important for vascular diseases, such as atherosclerosis and hypertension. To test this, we compared the effects o f N F A , which is a potent calcium-activated C f channel blocker in endothelium (Nilius et al. 1997a), T E A , and the combination o f these two drugs on ACh-induced relaxation in isolated rings o f rat aorta and superior mesenteric artery with intact endothelium. L - N M M A , the N O synthase inhibitor, and indomethacin, the C O X inhibitor, were used alone or in combination to functionally separate the relaxation mediated by different factors, namely, N O , PGI2 and E D H F . In addition, to better understand the mechanisms o f ACh-mediated relaxation in the rat mesenteric vascular bed, we compared the characteristics o f the muscarinic receptor-induced and the receptor-independent calcium ionophore A23187-induced relaxation in mesenteric arteries to that in rat aorta. W e also further  analyzed EDHF-mediated ACh-induced relaxation using different  K  +  channel  blockers in mesenteric arteries. The following research hypotheses and specific experimental objectives were addressed:  138  n.  W O R K I N G HYPOTHESES A N D SPECIFIC R E S E A R C H OBJECTIVES:  Working Hypotheses: A.  The relative importance o f endothelial CI" channels and K  +  channels in modulating  ACh-induced relaxation is different in muscular mesenteric arteries vs. elastic aorta. B.  The relative contributions o f N O , E D H F , and PGI2 to ACh-induced relaxation are different in rat mesenteric artery and aorta.  Specific Objectives: 1) .  T o examine the effects o f niflumic acid ( N F A ) , tetraethylammonium ( T E A ) , a relatively selective large conductance Ca -activated K 2+  channel antagonist and the  +  combination o f these two drugs on A C h - induced relaxation in intact rat aortic and mesenteric rings. 2) .  T o test the influence o f L - N M M A , a N O synthase inhibitor, on ACh-induced relaxation in the absence or presence o f T E A or N F A plus T E A in isolated rat aorta and mesenteric arteries.  3)  T o examine the effects o f N F A , T E A , and the combination o f these two drugs on Ca  4)  2 +  ionophore A23187-induced relaxation in intact rat aortic and mesenteric rings.  T o compare the influence o f L - N M M A in combination with indomethacin on A C h induced relaxation in the absence and presence o f T E A and the combination o f N F A plus T E A in isolated rat aorta and mesenteric arteries.  5)  T o examine the effects o f L - N M M A and isotonic high K buffer on A23187- induced +  relaxation in isolated rat aorta and mesenteric arteries.  139  To investigate the effects o f isotonic high K buffer, K +  +  channel blockers and their  combinations {including charybdotoxin ( C T X ) , a intermediate and large conductance Ca -activated K 2+  +  channel blocker, apamin ( A M P ) , a small conductance  Ca 2 +  activated K channel blocker, the combination o f C T X and A M P , the combination o f +  T E A and A M P } on ACh-induced relaxation resistant to L - N M M A / i n d o m e t h a c i n i n isolated rat mesenteric arteries.  140  ELL  METHODS AND MATERIALS  1.  Isolated Artery Ring Preparation for Isometric Tension Measurement M a l e Wistar rats (250-350g) were anaesthetized by intraperitoneal injections o f  sodium pentobarbitone (65mg/kg). The thoracic aorta and superior mesenteric arteries were removed and placed in Krebs solution o f composition (in m M ) : N a C l 113, K C I 4.7, CaCl2 2.5, KH2PO4 1.2, M g S 0  4  1.2 N a H C O s 35 and dextrose 11.5, at room temperature. The  arteries were carefully cleaned o f connective tissues and fat, and were then cut into rings o f 4 mm and 3 mm in length for aorta and mesenteric arteries, respectively. In some rings, the endothelium was removed by inserting a wire or the tip o f a forceps and gently rolling the rings back and forth on a finger moistened with Krebs buffer. Each ring was suspended horizontally between two triangular-shaped stainless steel hooks in individual organ baths containing 20 m l Krebs solution maintained at 37° C and gassed with 95% O2 - 5 % C 0  2  resulting in p H 7.4. Rings o f aorta and mesenteric arteries were placed under resting tension o f 2.0 and 1.0 g, respectively. These tensions were determined in preliminary studies to be optimal.  Isometric tension was measured and recorded using a Grass F T 0.3 force  displacement transducer and a Grass 7 E polygraph (Grass Instrument Quincy Mass). Rings were allowed to stabilize for 90 min, during which the bathing solution was changed every 20 min, before the start o f each experiment.  2.  Experimental Protocols The rings were initially stimulated with a submaximal concentration (EDgo) o f 7  phenylephrine (PE, 10" M and 3x 10  6  M for aorta and mesenteric arteries, respectively,  except where stated). After the responses to P E had stabilized, tissues were relaxed with A C h  141  (10 u M ) to ascertain endothelial integrity. The tissue then was allowed to re-equilibrate for 60 min with washing every 20 min. The rings with intact endothelium were then contracted with P E (10" M and 3 x 10" M for aorta and mesenteric arteries, respectively) again. W h e n a 7  6  stable contraction was obtained, A C h (10" to 10" M ) or A23187 (3 xlO" to 3 x l O ^ M ) was 8  4  9  added to the bath cumulatively. In experiments in which the inhibitors were used, they were added to the tissue bath before the tone was raised with P E . The pre-incubation time was 30 min with N F A (30 p M ) , T E A (3 m M ) and indomethacin (20 p M ) , 15 min with C T X (0.1 p M ) and apamin (0.3 p M ) , and l h with L - N M M A (300pM). Control experiments with vehicle were performed in the same manner. The effects o f each inhibitor alone and in combination were examined in separate tissues. When the effect o f K (30 m M ) was tested, +  the bath solution was exchanged with Krebs solution containing 30 m M KC1 immediately before addition of P E . The high K solution was prepared by isotonic substitution of N a C l by +  KC1. In some experiments, indomethacin (10 p M ) was present throughout to prevent formation o f prostanoid by C O X .  3.  Chemicals Acetylcholine  chloride,  the  calcium  ionophore  A23187,  (^-phenylephrine  hydrochloride, N F A , T E A , C T X , apamin, and indomethacin were purchased from Sigma (St. Louis, M O , U S A ) . L - N M M A monoacetate was obtained from Calbiochem (San Diego, C A , U S A ) . Stock solutions o f N F A (0.1 M ) and indomethacin (0.1 M ) were prepared in ethanol. A23187 (10~ M ) was dissolved in dimethyl sulphoxide ( D M S O ) and diluted with ethanol. 2  The final concentration in the bath was < 0.06 (vol)% for ethanol, < 0.03 (vol)% for D M S O . A C h , P E , T E A , L - N M M A , C T X and apamin were dissolved in twice-distilled water. A l l solutions were made fresh every day.  142  4.  Statistical Analysis Relaxation is expressed as the percent decrease in PE-induced tone. p D values 2  {defined as -log (ED50)} and percentage o f maximum relaxation (Rmax) as determined from individual curves were fitted according to the following logistic equation:  ED "  H  5Q  +A " n  where R is the relaxation (%), A is the concentration o f vasorelaxant, n H is the H i l l coefficient and ED50 is the molar concentration of vasorelaxant which causes 50% of the maximum relaxation. A l l data are presented as mean ± S E M . One-way A N O V A was used for multiple comparisons between control and treated tissue values for p D and Rmax- Two-way 2  A N O V A was used to compare data between two groups (as stated). Duncan's multiple range post test was used to compare between multiple means. Unpaired Student's t-test was used for comparison between two means. P < 0.05 was considered significant.  s  143  IV.  RESULTS  1.  A C h - Induced Relaxation  1.1.  The Effect of NFA and TEA on ACh-induced Relaxation in Rat Aorta and Mesenteric arteries. A C h induced a concentration-dependent  relaxation o f intact rings o f aorta and  mesenteric arteries precontracted with P E (Fig. 2.1). This relaxation effect was endotheliumdependent since removal o f endothelial cells abolished the relaxation in both aorta and mesenteric arteries (data not shown). Addition o f 30 p M N F A did not affect baseline or P E induced tension in either arteries (legend to Fig. 2.1). Incubation o f aorta or mesenteric rings with 3 m M T E A also had no effect on the basal tone, but potentiated PE-evoked tone to a similar degree in the two arterial rings in terms o f percentage o f their respective control (165 ± 13%, n = 6 and 150 ± 14%, n = 5 for aorta and mesenteric arteries, respectively, P > 0.05). In intact aorta, neither N F A nor T E A alone had a significant effect on the response to A C h ; however, in the presence o f N F A (30 p M ) plus T E A (3mM), the A C h concentrationrelaxation curve was shifted to the right (Fig. 2.1 A , Table 2.1). Combined use o f N F A and T E A significantly decreased the sensitivity ( p D ) to A C h without changing the maximum 2  relaxation (Table 2.1). In intact mesenteric arteries, as in aorta, N F A alone had no effect on the response to A C h (Fig. 2. I B , Table 2.1). In contrast to aorta, T E A alone resulted in a rightward displacement o f concentration-response curve to A C h in mesenteric arteries (Fig. 2. I B ) . T E A significantly decreased the A C h p D value without affecting the maximum 2  relaxation (Table 2.1). The concomitant application o f N F A and T E A did not further inhibit the relaxation to A C h as compared to T E A alone (Table 2.1).  144  FIGURE 2.1  A : Effect o f N F A (30 u M ) and T E A (3 m M ) or N F A (30 u M ) plus T E A (3 m M ) on relaxation response to A C h in intact rat aorta rings. Corresponding PE-induced maximum tensions were 1.02 ± O . l l g (Control), 1.03 ± 0.10g ( N F A ) , 1.62 0.10g  ab  ± 0.11g  ab  ( T E A ) , 1.51 ±  ( N F A + T E A ) ( P < 0.05 vs. control, P < 0.05 vs. N F A ) . n= 6. a  b  B: Effect o f N F A (30 u M ) , T E A (3 m M ) or N F A (30 u M ) plus T E A (3 m M ) on A C h induced relaxation in intact rat mesenteric artery rings. Corresponding PE-induced maximum tensions were 0.61 ± 0.10g (Control), 0.62 ± 0.09g ( N F A ) , 0.88 ± 0.15g 0.10g  ab  ( N F A + T E A ) ( P < 0.05 vs. control, P < 0.05 vs. N F A ) . n = 5. a  b  ab  ( T E A ) , 0.85 ±  145  146  TABLE 2.1 Potency ( p D ) and maximum relaxation ( R m 2  ax  ,  % loss o f P E tone) to A C h or A23187 (aorta  only) in the absence (Control) and in the presence o f N F A (30 p M ) , T E A (3 m M ) or N F A (30 p M ) plus T E A (3 m M ) in isolated aortic and mesenteric artery rings with intact endothelium. Arteries were precontracted with P E (10" M and 3 x 1 0 " M for aorta and ?  6  mesenteries, respectively).  Aorta  Mesentery  ACh pD  2  A23187 pD  Rmax (%)  2  Rmax (%)  ACh pD  Rmax(%)  2  Control  7.41±0.13  102±1  7.88±0.06  86±4  7.94 ± 0.09  102±2  NFA  7.11±0.14  105±1  7.55±0.13  80±4  7.84 ± 0 . 5 9  107±5  TEA  7.06±0.41  102±1  7.63±0.10  72±12  7.07±0.16  a b  111±6  NFA+TEA  6.11±0.29  83±5  6.94±0.17  a b  101±1  a b c  lOOtfclO 7.73±0.15  Each value represents the mean o f six (aorta) and five (mesentery) experiments ± S E M . ( p < a  0.05 vs. control, p < 0.05 vs. N F A , p < 0.05 vs. T E A ) . b  c  147  1.2.  , Effect of L-NMMA on ACh-induced Relaxation of PE-Evoked Tension In order to elucidate the nature o f the inhibition by N F A in combination with T E A o f  the response to A C h in rat aorta, and to further explore the different mechanisms that may be responsible for A C h mediated endothelium-dependent relaxation in aorta and mesenteric arteries, the effect o f a N O synthase inhibitor, L - N M M A , on ACh-induced relaxation under the experimental conditions tested above (excepted for N F A alone, because o f it's lack o f effect in both arteries) was investigated. 300 u M L - N M M A alone slightly raised the basal tone in aorta (by 0.17 ± 0.03g tension, n = 7), but not in mesenteric arteries (n = 9). Precontractions produced by EDgo o f P E were augmented by L - N M M A (n = 6 and 5, for aorta and mesenteric arteries, respectively, P < 0.05-see legend to Fig. 2.2). Neither T E A (3 m M ) alone nor N F A (30 u M ) plus T E A (3 m M ) further increased the P E tone in the presence o f L N M M A in either artery (Fig. 2.2 legend). In aorta, L - N M M A significantly attenuated the relaxation response induced by A C h (Fig. 2.2A). It decreased the pD2 value and also reduced the maximum relaxation (Table 2.2). In the presence o f L - N M M A , T E A alone, as well as N F A plus T E A , further inhibited ACh-induced relaxation; they caused a greater reduction in both p D value and maximum relaxation in comparison with L - N M M A alone (n=6, P < 0.05) 2  (Fig 2.2A, Table 2.2). However, there was no difference between the inhibitory effect o f T E A alone and N F A plus T E A in the presence o f L - N M M A (n .= 6, P > 0.05) (Table 2.2). In mesenteric arteries, the A C h concentration-response curve was shifted to the right in the presence o f L - N M M A (300 u M ) (Fig. 2.2B). L - N M M A significantly decreased the A C h p D  2  value (n = 5, P < 0.05), but the maximum relaxation to A C h was not affected (Table 2.2). Pretreatment with L - N M M A plus T E A (3 m M ) further reduced the A C h p D value (n = 5, P 2  148  FIGURE 2.2  A : Effect o f L - N M M A (300 p M ) on the relaxation response to A C h in intact rat aorta, i n the absence and presence o f T E A (3 m M ) or N F A (30 p M ) plus T E A (3 m M ) . The initial tensions induced by P E were 0.90 ± 0.19g (Control); 1.66 ± 0.38g ( L - N M M A ) ; a  1.74 ± 0.29g  a  (TEA+L-NMMA);  1.75± 0.21g  (NFA+TEA+L-NMMA)  a  ( P < 0.05 vs. a  control), n = 6  B: Effect o f L - N M M A (300 p M ) on the relaxation response to A C h in intact mesenteric artery rings, in the absence and presence o f T E A (3 m M ) or N F A (30 p M ) plus T E A (3 m M ) . The initial tensions induced by P E were 0.62 ± 0.14g (Control); 1.11 ± 0.12g ( L - N M M A ) ; a  1.15 ± 0.07g ( T E A + L - N M M A ) ; 1.12 ± 0.1 l g ( N F A + T E A + L - N M M A ) a  control), n = 5.  a  ( P < 0.05 vs. a  149  150  TABLE 2.2 Effects o f L - N M M A (300 u M ) (A) and L - N M M A plus indomethacin (Indo, 2 0 u M ) (B) on potency ( p D ) and maximum relaxation (Rmax) to A C h in the absence and in the presence o f 3 2  m M T E A or 30 u M N F A plus 3 m M T E A in intact rat aortic and mesenteric arterial rings precontracted with P E (10" M and 3 x l 0 7  - 6  M , respectively). The A C h curve without L -  N M M A ( A ) or L - N M M A + I n d o (B) served as a control.  A C h (Aorta) pD  A C h (Mesentery) pD  Rmax (%)  2  Rmax (%)  2  A. Control  7.44 + 0.12  L-NMMA  6.40 ± 0.25  a  40 + 9  TEA+L-NMMA  5.67 ± 0.28  a b  21 ± 13  NFA+TEA+L-NMMA  5.64 ± 0.30  a b  26+  98 + 3  7.57 + 0.04 a  a b  9  a b  111 + 5  7.09 ± 0 . 1 4  a  103 + 6  5.97 + 0.23  a b  101 + 2  6.20 ± 0.06  a b  90+14  B.  Control  7.47 ± 0.22  L - N M M A ± Indo  6.83 ± 0.24  a  T E A + L - N M M A ± Indo  6.06 ± 0.26  a b  N F A + T E A + L - N M M A ± Indo Values represent  106 + 7  /  the mean ± S E M . from  34 + 7 23 ± 4  7.75 + 0.12 a  a  / six (aorta)  101+2  6.99 +0.15  a  5.71 + 0.19  a b  5.85 + 0.21 "  91 + 5  b  102 + 2 85+14  and five (mesenteric  arteries)  experiments. ( P<0.05 vs. control, P<0.05 vs. L - N M M A in A or vs. L - N M M A + I n d o i n B ; a  b  there are no significant differences between A and B , P > 0.05; two-way A N O V A )  151  < 0.05). However, the maximum response to A C h was still unchanged (Table 2.2). In the presence o f L - N M M A , N F A plus T E A shifted the concentration-relaxation curve for A C h to the right to a similar degree as T E A alone did.  1.3.  Effect of Indomethacin on ACh-induced Relaxation of PE-Evoked Tension The above experiments were repeated in the presence o f indomethacin. Application o f  indomethacin in combination with other inhibitors namely L - N M M A , T E A or T E A plus N F A had no additional effect on basal tone as compared with the corresponding controls in either mesenteric arteries or aorta (data not shown) There was no significant difference in p D values or the maximum relaxations to A C h (n = 5 for each artery, P > 0.05) between the 2  curves obtained in the absence or in the presence o f indomethacin under each o f the conditions (Table 2.2). Indomethacin alone had also no effect on the response o f control to A C h obtained in absence o f any inhibitor (pD : control, 7.47 ± 0.22, indomethacin, 7.40 ± 2  0.11; Rmax: control, 106 ± 7%, indomethacin, 107 ± 6% n = 5 for aorta, P > 0.05; p D : 2  control, 7.63 ± 0.08, indomethacin: 7.58 ± 0.02; Rmax: control: 102 ± 3%, indomethacin: 105 ± 6% n = 5, for mesenteric arteries, P > 0.05). Thus, the contribution o f P G I to ACh-induced 2  relaxation in both arteries seems to be negligible.  2.  A23187-Induced Relaxation  2.1.  The Effect of NFA and TEA on A23187-Induced Relaxation in Rat Aorta and Mesenteric Arteries. Relaxation to A23187 was examined as a comparison with A C h . A23187 induced a  concentration-dependent relaxation o f endothelium-intact rings o f rat aorta and mesenteric arteries precontracted with P E (Fig. 2.3A, B ) . In aorta, pretreatments with N F A (30 p M ) or  152  T E A (3 m M ) , or a combination o f N F A (30 u M ) and T E A (3 m M ) had no effect on the A23187-induced relaxation (Table 2.1). (3mM)  In mesenteric arteries, pretreatment with T E A  (n = 5, F i g . 2.3B) reduced the maximum relaxation o f P E tone to 38 ± 11 %. Since  N F A had no effect on ACh-induced relaxation in mesenteric arteries, we did not further test the effect of the C f channel blocker on responses to A23187.  2.2.  Effect of L-NMMA and JT" on A23187-Induced Relaxation of PE-Evoked Tension When the tissues were incubated with 300 u M L - N M M A , the responses to A23187  were almost abolished in both arteries. The maximum relaxations to A23187 in the presence of L - N M M A were 12.3±7.3 % (n = 6) and 6.0 ± 6.0 % (n = 5) for aorta and mesenteric artery, respectively (Fig. 2.3A, B ) . In addition, pretreatment with 30 m M K  +  also greatly  inhibited the relaxation induced by A23187 in both arteries (Fig. 2.3A, B ) . The maximum relaxations o f P E tension were 10.7 ± 6.6 % (n = 6) and 21 ± 12 % (n = 5), respectively. Interestingly, as the representative traces in Fig. 2.4 show, application o f A C h ( 3 0 u M ) did not further relax the aorta in the presence o f either L - N M M A (Fig. 2.4A(b)) or K (Fig. +  2.4A(c)), while when no further relaxation to A23187 was observed, subsequent addition o f A C h (30 u M ) caused full relaxation in the presence o f L - N M M A (Fig. 2.4 B (b)) and a small further decrease (to 41 ± 12 %, n = 5) in tension in the presence o f K (Fig. 2.4 B(c)) in the +  mesenteric arteries.  3.  Effect of K C I and K+ Channel Blockade with Apamin, C T X , T E A and Their Combinations on ACh-induced N O - Independent Relaxation The ACh-induced relaxation o f rat mesenteric arteries that was resistant to a L -  N M M A and T E A was further investigated in mesenteric arteries (Fig. 2.5). In the presence  153  FIGURE 2.3  A : Effects o f L - N M M A (300 u M ) and KC1 (30 m M ) on A23187-induced relaxation in intact rat aorta. The initial maximum tensions induced by P E were 1.18 ± 0.29g (Control), 1.72 ± 0 . 1 4 g ( L a  N M M A ) and 1.70 ± 0.18g ( K ) ( P < 0.05 vs. control), n = 6 a  +  a  B : Effect o f T E A (3 m M ) , L - N M M A (300 u M ) and K C 1 (30 m M ) on A23187-induced relaxation in intact rat mesenteric arteries. The initial maximum tensions induced by P E were 0.49 ± 0.09g (Control), 0.88 ± 0.09g ( L a  N M M A ) , 0.89 ± 0 . 0 9 ( K ) , and 0.90 ± 0 . 1 2 ( T E A ) (B) ( P < 0.05 vs. control), n = 5 a  +  a  a  154  B  - l o o  1  9  1  1  8  1  1  1  7 -Log [A23187]M  1  —  6  •  •  —  '  1  5  155  FIGURE 2.4 Representative traces showing the relaxation responses to A23187 in intact rings from aorta (A) and mesenteric artery (B).  157  of L - N M M A (300 u M ) , A P M (0.3 u M ) , a small conductance C a -dependent K channel blocker, and C T X (0.1 u M ) , a large- and intermediate- conductance Ca -dependent 2+  K  channel blocker had no effect on baseline tension or P E (3xl0" M ) tone (Fig. 2.5, legend). 6  A P M (n = 5) alone did not affect the response to A C h (P > 0.05, Table 2.3). C T X (n = 6) shifted the A C h concentration-response curve to the right without altering the maximum relaxation o f P E tension (Fig. 2.5). The A C h p D value was significantly decreased by C T X 2  as compared to control, but the magnitude o f the reduction was smaller (P < 0.05) than that obtained in the presence o f T E A (3 m M , n = 5) alone (Table 2.3). When A P M (0.3 p M ) was applied in combination with C T X (0.1 p M , n = 7) or T E A (3 m M , n = 5), the relaxations induced by A C h were further attenuated. The maximum relaxation was significantly (P < 0.05) decreased to 64 ± 8% and 69 ± 5%, respectively. The degree o f reduction in p D value 2  and maximum relaxation was similar (P > 0.05) between the two combinations (Table 2.3). Changing Krebs buffer to high K  +  (30 m M ) solution caused a small contraction o f artery  rings (0.40 ± 0.06g, n = 5). However, the initial maximum tension, after addition o f P E ( 3 x l 0 M ) , was not significantly (P < 0.05) different from L - N M M A alone (Fig. 2.5 legend). 6  30 m M K  +  further reduced the maximum relaxation to 38 ± 4 %. The blockade was greater  than that o f co-application o f T E A and A P M (P < 0.05), as well as C T X plus A P M (P < 0.05).  158  FIGURE 2. 5  Effects o f K C I (30 m M ) and K  + ( c a )  channel blockers on L-NMMA/indomethacin-resistant  response to A C h in intact rat mesenteric artery rings. Corresponding PE-induced maximum tensions were 0.96 ± 0.09g (Control), 1.04 ± 0.12g ( A P M ) , 1.01 ± 0.07g ( C T X ) , 1.31 ± 0.27g ( T E A ) , 1.08 ± 0.14g ( C T X + A P M ) , 1.38 ± 0 . 0 9 g ( T E A + A P M ) , .1.1.1 ± 0.15g (K+) (P > a  0.05, one-way A N O V A ) . n = 5-7.  159  - L o g [ACh]M  160  TABLE 2.3 Potency (pD ) and maximum relaxation (R^x, % loss o f P E tone) to A C h in the absence 2  (Control) and in the presence o f A P M (0.3 u M ) , C T X (0.1 u M ) , T E A (3 m M ) , A P M (0.3 u M ) plus C T X (0.1 u M ) , A P M (0.3 u M ) plus T E A (3 m M ) or K C I ( K , 30 m M ) in isolated +  mesenteric artery rings with intact endothelium and precontracted with P E (3x10 M ) . The -6  experiments were performed in the presence o f L - N M M A (300 u M ) plus indomethacin (10 uM).  ACh PD  Rmax (%)  2  Control  7.27 ± 0 . 1 1  100 ± 1  APM  6.98 ± 0.07  96 ± 5  CTX  6.37 ± 0.07  a  91 ± 9  TEA  5.85 ± 0.24  a b  95 ± 4  CTX +APM  5.88 ± 0 . 3 3  a b  64±8  TEA + APM  5.48 ± 0.25  a b  69 ± 5  K  5.60±0.15  +  a b  38±4  a c  a  c  a  Each value represents the mean o f five to seven experiments ± S E M . ( P<0.05 vs. Control; a  P<0.05 vs. C T X ; P<0.05 vs. K ) . C  +  b  161  V.  DISCUSSION The aim o f the second part o f the thesis was to understand how the endothelium  differentially regulates smooth muscle relaxation in rat mesenteric arteries as compared to aorta.  Using isometric tension measurements, we examined the function o f CI" and K  +  channels in muscarinic receptor-mediated relaxation in both arteries, and compared the relative importance o f N O and  P G I 2  as well as E D H F i n endothelium-dependent  smooth  muscle relaxation. W e also examined the component o f ACh-induced relaxation that is resistant to inhibition o f N O and  P G I 2 ,  in mesenteric artery. The effects o f A23187 were  examined as a comparison.  Aorta Effect of NFA and TEA Addition o f either 30 p M N F A or 3 m M T E A alone to aorta did not significantly affect ACh-induced endothelium-dependent  vasorelaxation. However, pretreatment with a  combination o f the two compounds decreased the potency o f A C h approximately 10 fold, although it had no effect on the magnitude o f the maximal relaxation. N F A is a potent calcium-activated C I - channel blocker in both endothelium and smooth muscle (Hogg et al. 1994a; Nilius et al. 1997a). T E A at the concentration used in the study has been shown to selectively inhibit large conductance calcium-activated K  +  channels in arteries (Beech and  Bolton 1989; Farley and Rudy 1988; Langton et al. 1991; Nelson and Quayle 1995).  Thus  the results suggest that in rat aorta both calcium-activated CI" and K channels are involved +  in the response to A C h , and activation o f either channel is sufficient to produce a full relaxation. In contrast to A C h , we found that N F A plus T E A did not affect the endotheliumdependent relaxation induced by A23187. L i k e A C h , A23187 evokes relaxation by releasing  162  N O (White and Martin 1989) and also induces endothelium-dependent (Chen and Suzuki 1990). A23187 is a receptor-independent C a [Ca ]i by causing C a 2+  Ca  2 +  2 +  2 +  hyperpolarization  ionophore, which increases  entry from the extracellular space v i a electroneutral exchange o f one  for two Ff" ions (Reed and Lardy 1972). O n the other hand, A C h activates the  muscarinic receptor, and elevates [ C a ] i by releasing C a 2+  2 +  from D?3-sensitive intracellular  stores (Wang et al. 1995b) and stimulating transmembrane C a and/or store-operated  Ca  2 +  2 +  influx through receptor-  channels (Putney 1991; W a n g and van Breemen 1997). The  elevation o f [ C a ] i in endothelial cells is believed to trigger the release o f N O , PGI2 and 2+  E D H F that evoke endothelium-dependent  smooth muscle relaxation (Busse et al. 1989;  Fukao et al. 1997c; Whorton et al. 1984). Activation o f CI" or K channels by A C h in aortic +  endothelial cells has been shown to facilitate C a force for C a  2 +  2 +  influx by providing a constant driving  entry, as well as by preventing the depolarization-mediated inactivation o f  R O C (Usachev et al. 1995; Wang and van Breemen 1999). Blocking endothelial CI" and K channels would inhibit C a  2 +  +  influx (Demirel et al. 1994; Hosoki and Iijima 1994; H o s o k i and  Iijima 1995; Luckhoff and Busse 1990; Wang and van Breemen 1999; Yumoto et al. 1995), whereas C a  2 +  entry produced by A23187 should not be affected. These data suggest that the  inhibitory effect o f the combination o f N F A and T E A on A C h -induced relaxation in aorta is due to an action o f the two channel blockers on endothelial cells, rather than an action o f blocking the activity o f these E D R F s on smooth muscle cells. Considering the fact that the presence o f either o f the channel blockers alone had no effect on the A C h relaxant response, we speculate that when CI" channels are inhibited, E D R F release by A C h may elicit vasodilation through a compensatory pathway involving activation o f K versa in aorta.  +  channels and vise  163  Activation o f agonist-induced CI" channels on smooth muscle evokes smooth muscle depolarization (Amedee and Large 1989; Byrne and Large 1988a; Pacaud et al. 1989b; V a n Helden 1988), which is excitatory. W e have reported that N F A inhibited oti-adrenoceptorinduced vasoconstriction in isolated perfused rat mesenteric arterial beds (He and Tabrizchi 1997). However, N F A did not inhibit PE-induced contraction in this study since the initial PE-induced tension in the presence o f N F A was not different from that in the absence o f N F A in either aorta or superior mesenteric artery. In preliminary experiments (data not shown), we did find that N F A , at the concentration used in the present study, significantly inhibited cirazoline-induced tone in superior mesenteric artery but had little effect on P E induced tension. In some preparations, we also found that the efficacy o f cirazoline, a specific ctiA-adrenoceptor agonist, is apparently less than that o f P E , a nonselective ctiadrenoceptor agonist. Therefore, we chose P E to precontract the arterial segments i n these experiments. A m o n g the E D R F s , N O is the one whose synthesis most critically relies on extracellular C a  2 +  (Fukao et al. 1997c; Luckhoff and Busse 1990; White and Martin 1989).  We assessed whether activation o f CI" and K channels could contribute specifically to N O +  synthesis/release by employing 300 p M L - N M M A . L - N M M A , an L-arginine analogue, is a specific N O synthase inhibitor that causes reversible inhibition o f N O synthesis due to the competitive inhibition o f L-arginine metabolism (Mayer et al. 1989; Moncada et al. 1991; Rees et al. 1989). It has been reported that 300 p M L - N M M A functioned the same as 1000 p M L - N M M A , causing maximum inhibition o f endothelium-dependent relaxation to A C h i n rat aorta (Rees et al. 1990). Furthermore, 100 p M L - N M M A could completely inhibit A C h induced N O release measured by chemiluminescence in a bioassay system (Rees et al. 1989).  164  In the preliminary experiments, we confirmed that application o f a higher concentration o f L N M M A had no further effect on relaxation in response to A C h as compared with 300 u M L - N M M A when incubated for 60 min before segments were subjected to agonist challenge. W e assumed that using this protocol N O synthesis would be blocked, and that i f the C f and K  channels do specifically contribute to stimulation o f N O synthesis, the combination o f  +  N F A and T E A would no longer have any inhibitory effect on ACh-induced relaxation. In the presence o f L - N M M A , the maximum relaxation to A C h was decreased nearly 60% and the p D was reduced by approximately 10 fold, and T E A caused a further significant reduction in 2  maximum relaxation and sensitivity to A C h , whereas N F A plus T E A had no further inhibitory effect as compared to T E A alone. These results imply that the inhibitory effect o f N F A plus T E A on ACh-induced relaxation seen in the absence of L - N M M A is in part due to an inhibition o f N O synthesis. Since the results also revealed that the inhibition o f N O synthesis unmasked a relaxation component sensitive to T E A , our observations thus suggest that C a  2 +  activated C f and K channels are involved, at least in part, in ACh-induced N O +  dependent relaxation, while K c channels, but not C f channels mediate the NO-independent a  relaxation response to A C h in rat aorta.  NO-Mediated and NO-Independent Relaxation In the presence o f L - N M M A , A C h - induced endothelium-dependent relaxation was greatly but not completely inhibited, suggesting that vasorelaxation to muscarinic agonist in aorta has at least two components; one o f which is mediated via N O , while the other is probably mediated via E D H F because it is sensitive to K c blocker T E A . Although the a  chemical nature o f E D H F has not been defined, it has been suggested that E D H F relaxes  165  vascular smooth muscle cells through hyperpolarization v i a opening o f K  +  channels. That  A C h could induce an endothelium dependent hyperpolarization o f smooth muscle owing to an increase in K  +  conductance has been found in rat aorta (Taylor and Weston 1988). N O  may stimulate smooth muscle hyperpolarization in some vessels (Bolotina et al. 1994; Mistry and Garland 1998), but ACh-induced vasorelaxation may also be associated with N O independent hyperpolarization (Garland et al. 1995; K o m o r i and Vanhoutte 1990). The existence o f an E D H F distinct from N O , which may also contribute to ACh-induced relaxation in aorta, was first tested by Chen and Suzuki (Chen and Suzuki 1989; Chen et al. 1988). They found that in rat aorta A C h caused an endothelium dependent relaxation that was reduced but not blocked by methylene blue. They also found that A C h produced an endothelium-dependent hyperpolarization o f smooth muscle cells that was not blocked by methylene blue but could be abolished by raising the external K  +  concentration. These  observations have been confirmed in later studies using N O synthase inhibitors. It was shown that in the rat aorta, application o f A C h or carbachol evoked an endothelium-dependent hyperpolarization that contains an initial peak component and is followed by a sustained component. N O S inhibitor had no effect on the magnitude o f the first transient peak although it diminished the second component o f the endothelium-dependent  hyperpolarization  (Vanheel et al. 1994). ACh-induced endothelium-dependent relaxation was only partially reduced by N O S inhibitor, and this NOS-resistant relaxation was blocked by high K solution +  in the aorta (Hatake et al. 1995; Zygmunt et al. 1994a; Zygmunt et al. 1995). Our data confirm and extend the results o f those functional studies, by suggesting that K c is, at least a  in part, responsible for the relaxation mediated by the E D H F .  166  Previous studies have reported that the N O S inhibitor-resistant relaxation in rat aorta amounted to 30 % to 40 % o f the unblocked response to A C h (Chen and Suzuki 1989; Zygmunt et al. 1995) and appeared only when carefully titrated the precontactile response to a certain lower level (Hatake et al. 1995; Zygmunt et al. 1994a). Therefore, it has been suggested that in rat aorta E D H F may play a minor role in the relaxation response to A C h in the absence o f N O S inhibitor (Chen and Suzuki 1989; Hatake et al. 1995). The different effects  o f precontractile responses on ACh-induced N O S inhibitor-resistant relaxation  observed by Zygmunt (Zygmunt et al. 1994a) and Hatake (Hatake et al. 1995) may also reconcile with earlier reports that i n the presence o f a N O S inhibitor the ACh-induced relaxation was completely abolished in aorta (Nagao et al. 1992; Rees et al. 1990; Thomas and Ramwell 1991; Vargas et al. 1991). Other factors may also account for the different degree o f relaxation response to A C h i n the presence o f N O S inhibitor, such as the strains o f rats and the anatomical location o f the vessel segments used by these researchers. It has been reported that endothelium-derived nitric oxide (NO)-dependent relaxation to A C h in the thoracic aorta precontracted with N E was significantly greater in the middle and distal segments than in the proximal segments, suggesting that there are regional variations in the ACh-induced release o f endothelium-derived N O in the rat thoracic aorta (Honda et al. 1997). Nevertheless, evidence in the literature is basically consistent, i.e. in rat aorta N O may be a main E D R F mediating ACh-induced relaxation. In our study, the L - N M M A - r e s i s t a n t , T E A sensitive component was revealed only after N O synthesis was inhibited since T E A had no effect on ACh-induced relaxation in the absence o f L - N M M A , and it was responsible for only a small part o f ACh-induced relaxation (in the presence o f L - N M M A T E A reduced the maximum relaxation to A C h from 40% to 20%), consistent with reports that N O is a major  167  mediator of ACh-induced relaxation, while EDHF may be a back up mechanism when NO pathway is impaired in rat aorta.  Mesenteric Artery Effect of NFA and TEA  Most electrophysiological data on endothelium hyperpolarization in the literature were obtained from large conduit blood vessels due to the difficulties in isolating the endothelial cells from small vessels. Recently, endothelium membrane potentials have been recorded in isolated intact smaller arteries with intracellular microelectrodes. It was reported that stimulation with ACh induced endothelium hyperpolarization which was also reduced by K channel blockers in resistance arteries from hamster gracilis muscles (Bolz et al. 1999). +  ACh hyperpolarization of endothelial cells by activating Ca -activated K channels which 2+  +  are sensitive to CTX was also recorded in endothelial cells using patch clamp technique in multicellular preparations from guinea pig mesenteric arterioles (Yamamoto et al. 1999). However, to our knowledge there are no electrophysiological data available so far for the effect of Cf channels on endothelium membrane potential and Ca  2+  handling in muscular and  resistance vessels. In contrast to the large elastic aorta, superior mesenteric artery is a small muscular conduit artery that directly transfers blood flow to the resistance vascular bed. In this artery, NFA alone, as in aorta, did not alter the relaxation caused by ACh, but in contrast to aorta, TEA alone decreased ACh pD2 by approximately 8.7 fold without reducing the maximum relaxation. Furthermore, NFA plus TEA had no further inhibitory effect as compared to T E A alone. These observations suggest that in contrast to aorta, Cf channels are not involved in mediating endothelium-dependent relaxation to ACh in mesenteric arteries. The lack of the  168  involvement o f C f channels has been further confirmed by the observation that there was no difference between the inhibitory effects o f N F A plus T E A and T E A alone on responses to A C h when L - N M M A was present. The existence o f an inhibitory effect o f T E A in both the absence and presence o f L - N M M A implicates K  +  channels in ACh-induced relaxation in  superior mesenteric arteries.  NO-Mediated  and NO-Independent Relaxation  Heterogeneous distribution o f endothelium-dependent relaxations resistant to N O S in rats has been reported (Nagao et al. 1992). In contrast to aorta, we found that inhibition o f N O synthase with L - N M M A decreased the potency o f A C h by 4.8 fold, but did not affect the maximal relaxation to A C h in mesenteric arteries. This suggests that the contribution o f N O is less, while the NO-independent relaxation in response to A C h is greater in mesenteric arteries than that in aorta. Our results are similar to previous reports o f a large N O S inhibitorresistant component o f ACh-induced relaxation in Wistar rat superior mesenteric arteries (Chen and Cheung 1997; Fukao et al. 1995). In contrast, two other studies, one using Sprague-Dawley ( H w a et al. 1994), and the other using young female Wistar (Van de Voorde and Vanheel 1997) rats showed a greater inhibition o f ACh-induced relaxation o f mesenteric arteries by N O S blockers. The difference may be attributed to the different strain, age or sex of rats used. A s in aorta, it has been suggested that the NO-independent relaxation to A C h is mediated by E D H F , which hyperpolarizes the smooth muscle through K channel activation, +  in the superior mesenteric arterial circulation (Adeagbo and Triggle 1993; Chen and Cheung 1997; Fukao et al. 1997a; Garland and McPherson 1992). From the data with T E A alone, we could not distinguish whether T E A had an inhibitory effect on K c  a  channels in the  169  endothelium, which would interfere with N O / E D H F synthesis, and/or in the smooth muscle cells, which would directly inhibit N O (see discussion below)/EDHF action. Nevertheless, the fact that the inhibitory effect o f T E A alone was greater than that o f L - N M M A alone, and that in the presence o f L - N M M A , T E A further decreased potency o f A C h by about 10 fold, suggests that besides N O , E D H F is also involved in ACh-induced relaxation, and that its effect is greater than that o f N O in the mesenteric arteries. In addition, since L - N M M A and T E A alone produce significant inhibition of the A C h response and their effect in combination is additive, it can be postulated that N O and E D H F may be released at the same time and act in parallel to cause relaxation in response to A C h in mesenteric artery v i a different mechanisms. Although T E A significantly decreased the sensitivity o f the NO-independent response to A C h , it had no effect on the maximal relaxation. Thus, other K  +  channels present in  arteries both in smooth muscle (Nelson and Quayle 1995) and endothelial cells (Marchenko and Sage 1996) may also be involved. U s i n g specific K<ca) channel blockers, we found that in the presence o f L - N M M A and indomethacin, apamin alone did not significantly affect relaxation to A C h ; C T X , like T E A , attenuated the response to A C h , but did not reduce the maximal relaxation; a combination o f C T X and apamin significantly inhibited the L NMMA/indomethacin-resistant relaxation induced by A C h , and the maximal relaxation was reduced to an extent similar as that following pretreatment with the combination o f T E A and apamin. Similar results were reported by Chen and Cheung (Chen and Cheung 1997). These investigators simultaneously measured smooth muscle membrane potential and tension in rat superior mesenteric arteries and found that in the presence o f a N O S inhibitor, apamin was effective in inhibiting ACh-induced hyperpolarization in resting arteries, but less effective in  170  NE-contracted arteries. Furthermore, T E A significantly inhibited the hyperpolarization to A C h to a similar extent in both the resting and NE-stimulated arteries, as did C T X , although the effect o f C T X was smaller. However, in their study, the combination o f apamin and C T X completely abolished the both hyperpolarization and relaxation in response to A C h , while in our study the maximal relaxation A C h was reduced only to 57% in the presence o f these two toxins. Nevertheless, these results  suggest  that in superior mesenteric  arteries A C h  simultaneously activates both S K c and B K c , and that combined inhibition o f both channels a  a  is necessary to inhibit E D H F . Elevation o f the extracellular K  +  concentration [K ]o to above 25 m M (25, 30 or 60 +  m M ) abolishes the NO/PGl2-independent hyperpolarization and relaxation induced by A C h in rat mesenteric arteries (Adeagbo and Triggle 1993; Fukao et al. 1995; Garland and McPherson 1992; M c C u l l o c h et al. 1997; Randall et al. 1997; Waldron and Garland 1994). Generally, increasing the [K ]o above 20 m M w i l l decrease the K +  the extent low enough to prevent hyperpolarization to K  +  +  equilibrium potential to  channel activation (Nelson and  Quayle 1995), thereby preventing relaxation to E D H F . In the present study, application o f 30 m M K C I with P E together further reduced the maximal relaxation to A C h to 38% o f the control, indicating that besides K c channels other K a  +  channels may be also involved.  However, ACh-induced vasorelaxation was not completely inhibited by the combination o f L - N M M A and K C I . Therefore, it would appear that A C h may produce another E D R F (or more than one) in mesenteric artery that mediates ACh-induced vasorelaxation independent of N O and K  +  channel activation. Previous studies have suggested that ACh-induced  endothelium-dependent relaxation is mediated by a relaxing factor that is not N O , PGI2 or E D H F in rat superior mesenteric artery (Shimokawa et al. 1996; W u et al. 1993). In addition,  171  cyclopiazonic acid, a C a  i +  endothelium-dependent  relaxation that was affected  combination o f K  +  mobilizing compound like A C h , as well as A23187, induced an by neither  60 m M K  +  nor  the  and N O pathway inhibitors in the rat mesenteric arterial bed (Kamata et  al. 1996b). However, the actual characteristics o f the novel relaxing factor(s) need to be further investigated.  Aorta and Mesenteric Arteries  Effect of PGh in ACh- Induced Relaxation in Aorta and Mesenteric Arteries Prostacyclin (PGI2), the principal metabolite o f arachidonic acid, is produced by C O X in endothelium o f most blood vessels including aorta and mesenteric arteries (Moncada et al. 1977; Peredo et al. 1997). It mediates endothelium-dependent relaxation, probably v i a the c A M P pathway and evokes membrane hyperpolarization o f smooth muscle sensitive to glibenclamide in some blood vessels and species (Gryglewski et al. 1991; Jackson et al. 1993;  Moncada and Vane 1978a; Murphy and Brayden 1995b; Parkington et al. 1995;  Triggle et al. 1999; Zygmunt et al. 1998). Data available in the literature have demonstrated that inhibition o f C O X with indomethacin either alone or in the presence o f a N O S inhibitor does not interfere with the relaxing effect o f A C h on rat aorta and mesenteric  arteries,  suggesting that PGI2 is not involved (Adeagbo and Triggle 1993; Chen et al. 1988; Hatake et al. 1995; Shimokawa et al. 1996). However, recent studies showed that indomethacin-sensitive  relaxation and hyperpolarization to A C h were  COX-dependent, revealed  after  inhibition o f both N O and E D H F pathway in rat hepatic and rabbit mesenteric arteries, and raised concerns that the role o f P G I  2  may have been overlooked (Murphy and Brayden  1995b; Zygmunt et al. 1998). In this study, we systemically compared the effects o f indomethacin on ACh-induced relaxations. Indomethacin alone or in combination with L -  172  N M M A or L - N M M A plus T E A did not alter either the sensitivity or the magnitude o f the maximum relaxation to A C h obtained in the absence o f indomethacin. Our results confirmed the previous observations in the aorta and mesenteric arteries and further excluded the role for a C O X product after inhibition with L - N M M A and T E A in these two arteries.  Endothelium-dependent relaxation to A23187 in aorta and mesenteric arteries In the present study, A23187 also induced an endothelium- concentration-dependent relaxation in both aorta and mesenteric arteries. W e found that in the presence o f L - N M M A the relaxations induced by A23187 were completely inhibited in both aorta and mesenteric arteries, suggesting the relaxations were exclusively N O dependent. It was surprising that increasing K  +  to 30 m M in the bathing solution also totally abolished the relaxation by  A23187 in aorta as well as in mesenteric arteries. This indicates that activation o f K  +  conductance(s) in smooth muscle was responsible for the full relaxation o f the arteries, since responses to E D R F release by calcium ionophore w i l l be insensitive to K channel blockade +  at the level of the endothelium. Thus, it appears that NO-mediated endothelium-dependent relaxation to A23187 was mediated via K channel on smooth muscle in both aorta and mesenteric arteries. The results +  with A23187 are apparently different from those obtained with A C h (discussed above), which induced both L - N M M A - s e n s i t i v e and insensitive relaxations although the latter was less prominent in aorta. The lack o f L - N M M A - i n s e n s i t i v e response to A23187 could be due to the influence o f the degree o f precontraction with P E . It has been shown that the degree o f inhibition o f ACh-induced  relaxation by N O S inhibitors depends on the  level  of  precontraction in rat aorta (Hatake et al. 1995; Zygmunt et al. 1994a) (see above discussion). However, under the same conditions as the A23187 response was obtained, A C h did induce a  173  NO-independent relaxation in both aorta and mesenteric arteries. Therefore, the initial tension seems not to be a factor that would affect only the A23187-induced relaxation. This was further supported by experiments in mesenteric arteries, where application o f A C h in the presence o f A23187 fully reversed the inhibitory effect o f L - N M M A , but had only small relaxant effect on the P E tone in the arterial rings challenged with K . Addition o f A C h did +  not stimulate further relaxation in the presence o f L - N M M A or K G in aorta. The effects o f A C h in these experiments were consistent with those obtained in the absence o f A23187 in mesenteric arteries. The lack o f further relaxation to A C h in the presence o f L - N M M A in aorta is perhaps due to the minimal contribution o f E D H F in this vessel. In this situation, the presence o f A23187, which could interfere with the initial C a  2 +  profile,- may have an effect  on the synthesis/release o f E D H F . The results supported our contention that: 1) N O accounts fully for A23187-induced relaxation in both aorta and mesenteric arteries, 2) N O is also a major mediator in ACh-induced relaxation in aorta, 3) E D H F plays a predominant role in mesenteric artery, and 4) activation o f K  +  conductance is involved in regulating both N O -  dependent and NO-independent relaxation. That N O mediated vasorelaxation may involve hyperpolarization o f vascular smooth muscle by activation o f K  +  channels has been proposed. The contribution o f N O to A C h - or  carbachol-induced hyperpolarization o f smooth muscle has been observed in guinea pig uterine artery (Tare et al. 1990), rat aorta (Vanheel et al. 1994), rabbit carotid artery (Cohen et al. 1997) and rat small mesenteric arteries (Weidelt et al. 1997). N O has been reported to directly stimulate CTX-sensitive K  +  channels in the rabbit aorta, rat mesenteric artery and  rabbit carotid artery (Bolotina et al. 1994; Mistry and Garland 1998; Plane et al. 1998; Weidelt et al. 1997). N O has also been reported to activate  K  A  T P  channels in rabbit and rat  174  mesenteric arteries (Murphy and Brayden 1995a; Weidelt et al. 1997), K c channels in rabbit a  middle cerebral arteries (Dong et al. 1998) and voltage-gated K channels in rat pulmonary +  artery (Yuan et al. 1996) through either guanylate cyclase- cGMP-dependent or independent pathways. In addition, N O donor SESf-evoked relaxation can be fully accounted for by activation o f a CTX-sensitive pathway with little or no contribution from a pathway activated by increased levels o f cyclic G M P in rat mesenteric arteries (Plane et al. 1996). In this study we did not attempt to characterize the specific type o f K channels implicated in N O action on smooth muscle cells. W e did find that in the absence o f L N M M A , T E A did not affect A23187-induced relaxation in aorta, but greatly inhibited the response in mesenteric arteries. This result at least indicates that: 1) there are different K  +  channels that mediate NO-dependent response to A23187 in mesenteric artery and aorta, and 2) Ca -activated K channels contribute to the process i n mesenteric arteries but not i n aorta. 2+  +  In addition, the differential effects o f T E A in aorta and mesenteric arteries also eliminated the possibility that T E A could exert a nonspecific effect on membrane potential rather than a specific effect on K c channels. a  Different abilities o f A23187 and A C h to release N O and/or E D H F in the same preparations have been suggested i n other studies. It has been suggested that A23187 only evokes the release o f N O from the endothelium in rabbit carotid artery preconstricted with P E , whereas A C h can induce release o f both N O and E D H F (Dong et al. 1997). In rabbit femoral artery, the relaxation to A23187 o f NE-induced tension seems to be mediated predominantly via E D H F , while ACh-induced relaxation has been explained solely in terms o f N O release (Plane et al. 1995). Based on our observations and those o f others, we speculate that A23187 acting as an ionophore, and A C h , releasing endoplasmic reticulum  175  (ER) Ca.  z+  and opening C a  2 +  channels, might facilitate an increase [Ca ]i into different 2+  regions o f endothelial cells, leading to activation o f different enzymes that are responsible for synthesis o f N O and EDFfF. The question o f cellular compartmentalization with respect to the synthesis o f N O , PGI2 and E D H F , and also colocalization o f ion channels and kinases has been raised by Triggle et al (Triggle et al. 1999) based on the high variability o f the cellular mechanisms that mediate vasodilation i n response to these factors i n different tissues and  species  (Gambone et al. 1997; Garland and McPherson 1992; Triggle et al. 1999; Waldron et al. 1999). It has also been suggested that the nature o f the contractile agonist can determine the release and/or effects o f N O and/or other endothelial-derived mediators (Plane and Garland 1996). Although there is very little in the literature that addresses this question, it was reported recently that the synthesis o f PGI2 versus 6 - o x o - P G F i was different i n porcine a  endothelial cells stimulated  chemically with A23187 compared  with cell  stimulated  mechanically (Erdbugger et al. 1997). Our study did not explore the chemical nature o f the E D H F that mediated N O independent relaxation in response to A C h . It is possible that either a diffusible factor(s) (Campbell et al. 1996; Chen et al. 1991; Popp et al. 1996) or direct electric connection between V S M and endothelium (Chaytor et al. 1998; Edwards et al. 1999; Yamamoto et al. 1999) is involved. Endothelium-dependent  hyperpolarization is produced by a humoral  substance, E D H F , in the coronary arteries o f guinea pig (Chen et al. 1991). In porcine coronary arteries, E D H F may be eicosatrienoic acids metabolized from arachidonic acid (Campbell et al. 1996; Popp et al. 1996). However, electrical coupling between endothelial and smooth muscle cells through gap junctions has also been demonstrated in the rat aorta  176  (Marchenko and Sage 1996), porcine coronary arteries (Beny 1997; von der W e i d and Beny 1993) and guinea pig submucosal arterioles (Iwase et al. 1998). Recently, it has been reported that blocking o f the myoendothelial junction with a specific inhibitory gap junction peptide abolished ACh-induced hyperpolarization i n guinea pig internal carotid artery (Edwards et al. 1999) and mesenteric arterioles (Yamamoto et al. 1999), and inhibited A C h induced, NO/PGb-independent relaxation in rabbit aorta and superior mesenteric artery (Chaytor et al. 1998). These findings suggest that endothelium-dependent  hyperpolarization  o f smooth muscle is produced by an electrotonic spread o f potentials from the endothelial cells. It was also reported that inhibiting the gap junction blocked A C h - , but not A23187evoked hyperpolarization o f the rabbit mesenteric artery, and it was concluded that A23187mediated endothelium-dependent  relaxation requires chemical transmission through  the  extracellular space, whereas relaxation to A C h involves gap junction communication (Hutcheson et al. 1999). I f the NO-independent relaxation to A C h was mediated through myoendothelial gap junction in our preparations, that A23187 failed to conduct endothelial hyperpolarization response to smooth muscle cells through myoendothelial junctions may also account for its differential ability to release N O and E D H F .  177  VI.  SUMMARY  1.  A C h induced a concentration- and endothelium-dependent relaxation o f PE-induced tone in both isolated aorta and mesenteric arteries.  2.  In intact aorta, neither N F A nor T E A alone had a significant effect on the response to A C h . However, in the presence o f N F A plus T E A , the concentration-relaxation curve ( C R C ) to A C h was shifted to the right without a change in maximum relaxation (Rmax). In intact mesenteric arteries, the presence o f T E A alone resulted in a rightward displacement o f the C R C to A C h . The combination o f N F A and T E A did not further inhibit the relaxation to A C h as compared to T E A alone. N F A alone also had no effect on response to A C h .  3.  In aorta, L - N M M A greatly attenuated the relaxation response induced by A C h , decreasing both the p D value and Rma . In the presence o f L - N M M A , T E A further 2  X  shifted the C R C for A C h to the right without change in Rma as compared to L X  N M M A alone. In the presence o f L - N M M A , N F A plus T E A had no additive effect as compared to T E A alone. In contrast, in mesenteric arteries, L - N M M A displaced the C R C for A C h to the right without altering the R ^ x . L - N M M A plus T E A further shifted the C R C to the right as compared to L - N M M A alone, but the Rmax to A C h was still unchanged. In the presence o f L - N M M A , N F A plus T E A had no additional effect as compared with T E A alone. 4.  When indomethacin was used in combination with L - N M M A or with L - N M M A plus T E A , the relaxation responses to A C h were not different as compared to L - N M M A alone or L - N M M A plus T E A only in either aorta or mesenteric arteries.  178  5.  A23187 also induced an endothelium-dependent  relaxation in both aorta and  mesenteric arteries. In intact aorta, the pretreatment with N F A or T E A or N F A plus TEA  had no effect on the response to A23187. In intact mesenteric arteries,  pretreatment with T E A reduced the Rmax to A23187. In the presence o f L - N M M A or K C I (30 m M ) the response to A23187 was abolished in both arteries. The P E response resistant to A23187 in the presence o f L - N M M A was completely reversed by subsequent addition o f A C h in mesenteric arteries but not in aorta. A C h had no further effect on the tension remaining in the presence o f K C I (30 m M ) in aorta, but slightly decreased it in mesenteric arteries 6.  Apamin  alone had no effect  on ACh-evoked,  NO-independent relaxation in  mesenteric arteries. C T X and T E A displaced the A C h C R C to the right without altering the Rmax- Apamin plus C T X or T E A further inhibited the relaxation to A C h with a reduction in the R x , while high K ma  +  (30mM) buffer had a greater inhibitory  effect on the Rmax than the combinations in mesenteric arteries.  179  vn.  CONCLUSIONS  Our results indicate that both N O and E D H F - l i k e factors mediated ACh-induced endothelium-dependent relaxation in both aorta and mesenteric arteries. However, the mechanisms by which ACh-induces endothelium-dependent relaxation are different in these two arteries. ACh-induced relaxation appears to be primarily mediated by N O in aorta, and CI" channels and K  +  channels together may regulate the NO-dependent, ACh-induced  relaxation in this artery. In contrast, in mesenteric artery, E D H F played a more important role in ACh-mediated relaxation. K channels, but not CI" channels, contributed to ACh-induced +  relaxation. Other endothelium-dependent relaxing factor(s) may also be involved.  Ca  ionophore A23187-induced relaxation is solely N O dependent, which is mediated by K  2 +  +  conductance in both aorta and mesenteric artery. Blockade o f CI" channels had no effect on A23187-mediated relaxation in rat aorta. The results suggest that K conductance regulates +  both NO-dependent and -independent relaxation in both aorta and mesenteric arteries. Both small- and large- conductance K (Ca) channels play a role in ACh-induced NO-independent +  relaxation in mesenteric artery. Other K channels may be also involved. CI" channels in the +  endothelium are only involved in NO-dependent ACh-induced relaxation in rat aorta, probably by participating in maintaining endothelial membrane potential compatible for C a influx, thus ensuring a sustained N O synthesis and release.  2 +  180  vm  PHYSIOLOGICAL  SIGNIFICANCE  The physiological importance o f the relative contribution by N O and E D H F released by a variety o f stimuli in vasculature is still unclear. From our study and many others (Garland et al. 1995) (Clark and Fuchs 1997; Triggle et al. 1999; Woodman et al. 2000), it seems that the relative contribution depends on the function o f blood vessels. In some arteries, mainly large conducting arteries such as aorta, and also some resistance arteries such as coronary beds (Clark and Fuchs 1997), which assume primary importance in some disease states such as atherosclerosis, N O is the major mediator under normal conditions, while E D H F may be o f a secondary importance. However, in the majority o f small arteries, such as mesenteric arteries, skeletal beds (Clark and Fuchs 1997; Woodman et al. 2000), which are mainly responsible for regulating peripheral resistance, E D H F appears to be a major determinant  o f vascular caliber, while N O and possibly other  endothelium-dependent  relaxing factors may act together with E D H F to achieve the optimal relaxation. However, many questions still need be answered in mesenteric artery and other blood vessels, including the nature o f E D H F and other E D R F ( s ) , the cellular target o f the E D H F , and the interaction between these endothelium-dependent relaxing factors.  181  PART 3.  NOREPINEPHRINE-INDTJCED VASOCONSTRICTION IN ISOLATED PERFUSED MESENTERIC ARTERIAL BED FROM OBESE ZUCKER RATS: THE EFFECT OF INSULIN  L  RATIONALE  The hemodynamic hallmark o f most forms o f hypertension is an increase in peripheral vascular resistance, which is largely ascribed to abnormalities in the reactivity o f small resistance vessels to neurotransmitters  and circulating hormones, such as N E . In  addition, altered regulation o f vascular tone by endothelium-derived vasoactive products has been implicated. A n abnormal release o f endothelial-derived relaxing factors such as N O and also contracting factors such as endothelin (ET) and C O X pathway metabolites have been observed in several types o f hypertension (Luscher et al. 1993b; Mistry and Nasjletti 1988; Purkerson et al. 1986; W i l c o x et al. 1996). In states o f insulin resistance, hyperinsulinemia has been found to be associated independently with hypertension (Modan et al. 1985; Salonen et al. 1998). However, whether there is a causal relationship between hyperinsulinemia/insulin resistance and hypertension remains controversial (Brands et al. 1998; Yki-Jarvinen and Utriainen 1998). Insulin is known to exert many actions that may directly affect vascular reactivity at the levels o f both the  endothelium and  smooth muscle. These  include, on one hand,  increasing N O  synthesis/release (Chen and Messina 1996; Steinberg et al. 1994; Zeng and Quon 1996), and enhancing N a - K - A T P a s e and C a - A T P a s e gene expression and activity (Sowers et al. +  +  2+  1991; Tirupattur et al. 1993), and on the other, promoting E T - 1 gene expression (Oliver et al. 1991) and increasing E T - 1 release (Hu et al. 1993; N a v a et al. 1997), elevating sympathetic  182  activity and increasing N E release (Lembo et al. 1992; Liang et al. 1982) and promoting vascular smooth muscle cell growth (Ridray 1995). In addition, the effects o f insulin on vascular reactivity may also involve modulation o f C O X pathway metabolism (Axelrod 1991; Keen etal. 1997; Rebolledo et al. 1998; van Veen and Chang 1997; Yanagisawa-Miwa et al. 1990). Studies in vitro in intact resistance vessels from control animals have revealed both inhibitory and potentiating effects o f insulin on the pressor responses to vasoactive substances (Alexander and Oake 1977; Townsend et al. 1992; Walker et al. 1997b; W u et al. 1994). Therefore, either impaired vasodilator and/or exaggerated vasoconstrictor effects o f insulin could contribute to an increased vascular reactivity leading to hypertension in insulinresistant states. The genetically obese Zucker rat (fa/fa) is an insulin-resistant animal model with early onset severe hyperinsulinemia, hyperlipidemia and normal plasma glucose ( Y o r k et al. 1972). These rats usually develop a modest hypertension at an older age (Cox and K i k t a 1992). The obese Zucker rat thus represents a model in which the effects o f insulin resistance/hyperinsulinemia associated with hypertension on vascular reactivity can be examined. However, although the reactivity o f arteries from obese Zucker rats has been investigated in a number o f studies (e.g. Bohlen and L a s h 1995; K a m et al. 1996; Turner et al. 1995; Walker et al. 1997a; W u et al. 1996; Zanchi et al. 1995), the results are not all in agreement. Furthermore, the effect o f insulin on vascular reactivity in Zucker rats has not yet been defined (Turner et al. 1995; Walker et al. 1997a). Thus, the purpose o f the present study was to investigate whether altered vascular reactivity to N E could be detected, in the absence and/or presence o f a pathophysiologically relevant concentration o f insulin, in obese Zucker rats with established hypertension. In  183  addition, the contribution o f endogenous vasoactive substances including N O , prostanoids and E T - 1 to N E responses and to the vascular actions o f insulin was investigated. The whole mesenteric arterial bed ( M A B ) was chosen for this study. The following research hypotheses were proposed and specific experimental objectives were undertaken.  184  II.  W O R K I N G HYPOTHESIS A N D SPECIFIC R E S E A R C H OBJECTIVES  Working Hypotheses: A.  NE-induced vasoconstriction is altered in Zucker obese rat M A B compared to their lean littermate controls.  B.  Insulin, at a concentration similar to that to which obese Zucker rats are exposed in vivo, alters pressor responses to N E to a different extent in M A B from obese Zucker rats compared to their lean littermates.  C.  Release o f endothelium-derived vasoconstrictors (such as E T - 1 or PGH2/TXA2) and vasodilators (such as N O ) and their interaction influence the pressor responses to N E as well as the effects o f insulin i n M A B from Zucker rat. The impact o f these endothelial factors is different in Zucker obese rat M A B than in their lean littermates  Specific Objectives: 1) .  T o compare the vasoconstrictor responses to N E in isolated perfused M A B from obese Zucker rats and their lean littermates.  2 ).  T o compare the effect o f insulin, at a concentration similar to the circulating level o f insulin in obese Zucker rats, on the pressor response to N E in isolated perfused M A B from obese Zucker rats and their lean littermates.  3) .  T o investigate the effects o f N ° - monomethyl-L-arginine ( L - N M M A ) , a nitric oxide synthase inhibitor, alone and in the presence o f insulin on the vasoconstriction to N E in M A B from Zucker rats.  4) .  T o examine the effects o f indomethacin, a C O X inhibitor, alone and in the presence o f insulin, on NE-induced contraction in M A B from Zucker rats.  185  To test the effect o f S Q 29,548, a TXA2/PGH2 receptor antagonist, alone and i n the presence o f insulin, on pressor response to N E in M A B from Zucker rats. To  evaluate the effects o f L - N M M A  plus indomethacin and L - N M M A  plus  indomethacin plus insulin on pressor responses to N E in M A B from Zucker rats. To investigate the effects o f bosentan, a non-selective endothelin receptor (both E T B , and E T ) antagonist, alone and in the presence o f insulin on vasoconstriction to N E in A  M A B from Zucker rats. To examine the effect o f B Q 788, a selective E T B receptor antagonist, or B Q 123, a selective E T receptor antagonist, alone and B Q 788 or B Q 123 in the presence o f A  insulin on pressor responses to N E in M A B from Zucker rats  186  III  M E T H O D S AND MATERIALS  1.  General Methodology  Animals M a l e obese (fa/fa) Zucker rats and their lean (Fa/?) littermate controls were obtained at age 8 to 10 weeks from the Department o f Physiology, University o f British Columbia (Vancouver, Canada). They were treated according to the guidelines o f the Canadian Council on Animal Care. Animals were pair-housed under a 12 h light/dark regime and given free access to normal food (Purina rat chow) and tap water, until they were 25 weeks old.  Blood pressure measurement Systolic blood pressure ( S B P ) was measured by the tail-cuff method in animals randomly selected from those used in in vitro studies, one week before the animals were used in experiments. Rats were placed in restrainers and pre-warmed for 30 min at 27°C. S B P was measured with an inflatable cuff and a sensor placed around the tail and  coupled to a blood pressure analyzer (IITC model 179, IITC Inc./Life Science  Instruments, Woodland Hills, C A , U . S . A ) . The inflated cuff pressure was 250 m m H g and pressure was released by 500 m m H g min" . T o accustom them to the setting, rats were placed 1  in the apparatus once each day for 3 days prior to the actual day o f measurement.  Blood  pressure was recorded and calculated as the mean o f five to six measurements.  Biochemical analysis of blood samples B l o o d samples from a tail tip cut were collected into heparinized capillary tubes. The blood was centrifuged at 10,000 x g for 15 min. and the plasma was collected immediately, frozen and stored at - 7 0 ° C until it was assayed. Plasma glucose and triglyceride levels were determined by enzymatic colorimetric methods using  187  commercial kits obtained from Boehringer Mannheim (Laval, Quebec, Canada). Plasma insulin levels were determined by radioimmunoassay using kits obtained from L i n c o Research, Inc. (St. Charles, Missouri, U . S . A . )  Perfused isolated M A B preparation O n each day o f experiments, one obese rat and a lean littermate were anaesthetized with sodium pentobarbital (120 mg kg" , subcutaneously over 1  the back and thighs in four equivalent dosages). The abdominal cavity was opened, the mesenteric artery was cannulated through an incision at the confluence with the dorsal aorta and then the M A B was isolated as described by M c G r e g o r (McGregor 1965). The M A B was flushed with heparinized physiological salt solution (25 IU/ml), transferred into a warmed organ chamber and perfused with Krebs-bicarbonate (normal Krebs) buffer maintained at 37° C and gassed with 95% 0 : 5% C 0 . The Krebs-bicarbonate buffer was o f the following 2  2  composition (in m M ) : N a C l 113, K C I 4.7, glucose 11.5, M g S 0 NaHC0  3  25.0.  4  1.2, C a C l 2 2 . 5 , K H P 0 2  The p H o f the buffer following saturation with a 95% 0 : 5% C 0 2  4  2  1.2, gas  mixture was 7.4. The perfusion rate was kept constant at 3 ml/min using a polystaltic peristaltic pump (Buchler Instruments, Buchler Fort Lee, N J , U S A ) .  Changes in perfusion  pressure were measured and recorded using a pressure transducer (PD23 I D Gould Statham, C A , U . S . A ) and Grass polygraph (Model 79D Grass Instruments, M A , U S A ) . The perfused M A B was allowed to stabilize for 1 hr before the start o f the experiment.  2.  Experiment Protocols The tissues were initially treated with a maximal concentration o f K C I (120 urnol) by  bolus injection 4 times. Perfusion pressure was allowed to return to baseline between each  188  injection o f KC1. The M A B s were then allowed to equilibrate for a further 40 min following which two or three consecutive dose-response curves ( D R C s ) to N E were constructed from 5 separate bolus injections o f N E (0.9-90 nmol). Perfusion pressure was allowed to return to baseline between the injections o f each dose o f agonist. The first N E D R C served as a control. The second D R C for N E was constructed in the presence o f insulin (200 mU/f) or indomethacin (20 p M ) or SQ 29,548 (0.3 p M ) or L - N M M A (300 p M ) or L - N M M A plus indomethacin or bosentan (3 p M ) or B Q 788 (0.3 p M ) or B Q 123 (0.3 p M ) in the perfusion buffer. The third curve was constructed in the presence o f a combination o f insulin plus the inhibitor(s) used in the 2  n d  D R C . The M A B was pre-perfused with insulin for 2 hrs.  Bosentan, B Q 788, B Q 123, L - N M M A , indomethacin and SQ 29,548 were added into perfusion buffer 5 min, 15 min, 15 min, 30 min, 30min and 30min, respectively, before the D R C was constructed or before insulin was added. After the completion o f each D R C for N E , a single bolus injection o f KC1 (60 pmol) was made. The three D R C s were constructed at fixed time intervals.  A time control experiment that consisted o f three D R C s for N E  without the addition o f insulin or any inhibitors was also done.  To confirm the effect o f  insulin on the N E response, another set o f experiments was performed, in which the first two N E D R C s were obtained in untreated tissues, followed by insulin infusion for 2 hrs and thereafter the 3 D R C was constructed. rd  3.  Chemicals (-)-Norepinephrine hydrochloride and indomethacin were obtained  from  Sigma  Chemical C o . (St. Louis M O , U . S . A . ) . L - N M M A and B Q 788 were purchased  from  Calbiochem Corporation ( L a Jolla, C A , U . S . A . ) . Bosentan (R047-0203) was a gift  from  189  Hoffmann-La Roche Ltd. (Bazel Switzerland). B Q 123 and SQ 29,548 were purchased from Research Biochemical International (Natick, M A , USA). Insulin (Humulin R, Eli Lily Co., St. Louis) was purchased from a local pharmacy. A stock solution of N E was prepared daily in distilled water containing 1 mg ml" ascorbic acid. The volume of N E for each injection 1  was 30 ul. Indomethacin and bosentan were dissolved in 100% ethanol and prepared as stock solutions of 0.1 M and 0.01 M , respectively. The solutions were made fresh each day. L - N M M A (0.1 M) was made in distilled water. All inhibitors and insulin were diluted to the required concentration in the perfusate reservoir. The final ethanol concentrations (0.03% and 0 . 0 2 % , v/v) in the perfusion buffer were without effect on contractile responses.  4.  Statistical Analysis To compare the reactivity of M A B from lean and obese rats to N E and KCI,  vasoconstrictor responses were expressed as the absolute increase in perfusion pressure. In control experiments, NE-induced pressor responses were found to increase on the second and third exposure to 3 to 90 nmol N E in untreated M A B from both obese and lean rats (Fig. 3.1). Therefore, to allow evaluation of the effects of the inhibitors, alone and in the presence of insulin, N E responses in the second and third DRC were expressed as a percent of the maximum response of the initial N E DRC. The responses to N E at each time point in the control experiments served as the control for the responses in the presence of the inhibitors, alone or in combination with insulin. All data are presented as mean ± SEM. Student's unpaired t-test was used for comparisons between two means. Two-way A N O V A using the general linear model approach (repeated measurements) followed by Newman-Keul's test was used for multiple  190  comparisons between obese and lean rats. One-way A N O V A followed by the Bonferroni post-hoc test was used for within-group comparison o f multiple means. P < 0.05 was considered statistically significant.  191  FIGURE 3.1  Control experiments for responses to N E in isolated mesenteric arterial bed obtained from lean or obese Zucker rats, perfused with normal Krebs at constant flow. The responses at each concentration o f N E (control-1, control-2 and control-3, respectively) were obtained 2.5 h apart. Data represent the mean ± S E M o f seven (lean) and six (obese) experiments. 0.05 vs. control-1; P<0.05 vs. control-2. b  a  P <  192  Lean  _250 cn  n  X  E  £200  •  Control-1  CD  i—  0  Control-2  CO  •  Control-3  2 150 c g CO  100 H  t CD  CL CD co ro CD  o c: 0.9  3.0  9.0  30.0  90.0  30.0  90.0  NE(nmol)  Obese  o>  250 -,  x E  f  200  H  150  A  100  H  L—  CO CO  0)  c g 'co  t  CD  Q_ CD CO  ro CD  i_  o c  0.9  3.0  9.0 N E (nmol)  193  IV.  RESULTS  1.  General Characteristics of Zucker Rats A t 25 weeks o f age, systolic blood pressure was significantly higher and body weight  was significantly greater in obese rats than in their lean littermates (Table 3.1). Plasma insulin and triglyceride levels were also significantly elevated in obese as compared to lean rats. However, plasma glucose concentrations were not significantly different between the two phenotypes (Table 3.1).  2.  NE-Induced Vasoconstriction in Isolated Perfused M A B from Obese and Lean Zucker Rats The basal perfusion pressures in isolated M A B o f obese and lean rats were 7.1 ± 0.9  and 6.6 ± 0.7 m m H g (mean ± S E M , n = 28, P > 0.05), respectively. Bolus injection o f N E (0.9 to 90 nmol) produced a concentration-dependent increase in perfusion pressure that was significantly lower at 90 nmol N E in M A B from obese than from lean rats (Fig. 3.2A). Vasoconstrictor responses to K C I were also significantly smaller in M A B from obese than from lean rats (Fig. 3.2B).  3.  Effect of NOS and/or C O X Inhibition on NE-Induced Responses Infusion of L - N M M A (300uM) or indomethacin (20 u M ) alone or in combination for  30 min did not alter the basal perfusion pressure of M A B from either obese or lean rats (data not shown). However, L - N M M A significantly potentiated vasoconstrictor responses to N E in M A B from both groups o f animals (Fig 3.3). L - N M M A appeared to produce a leftward shift in the N E dose-response curve, since there was no increase in the maximum response to N E in M A B from lean rats (Fig. 3.3). In contrast, responses of M A B from obese  194  TABLE 3.1 Physiological characteristics o f lean and obese Zucker rats  Lean  Obese  B o d y weight (g)  418 ± 5 (50)  592 ± 7 (45)  a  Systolic B P (mmHg)  128 ± 2 (16)  157 ± 1 (15)  a  Plasma insulin (mU/1)  64 ± 6 (32)  267 ± 2 7 (31)  Plasma triglyceride (mmol/1)  1.48 ± 0 . 1 4 (25)  36.93 ± 0.20 (27)  Plasma glucose (mmol/1)  7.34 ± 0 . 2 7 ( 5 )  6.92 ± 0:31 (7)  Values are shown as mean ± S E M (number o f rats in parentheses). a  P < 0.05, vs. lean (Student unpaired t-test).  a  a  195  FIGURE 3.2  Initial concentration-response curve to N E (A) and responses to K C I (B) in isolated perfused M A B obtained from lean ( • ) and obese ( • ) Zucker rats, perfused with normal Krebs at constant flow. Data represent the mean ± S E M . pooled from 48 experiments. P < 0.05 vs. a  lean (Student unpaired t-test).  KCI (umbl)  197  rats to all concentrations of N E , including the maximal, were significantly increased in the presence of L - N M M A (Fig. 3.3). O n the other hand, the effects o f indomethacin o n N E responses were opposite to those of L - N M M A , in that it inhibited pressor responses to N E in M A B from both lean and obese rats (Fig. 3.3). Interestingly, in the presence o f the combination o f indomethacin plus L - N M M A , responses o f M A B from lean and obese rats to N E were similar to those in the absence o f inhibitors (Fig. 3.3). The only difference noted was that the response o f obese M A B to 0.9 nmol N E was significantly potentiated i n the presence o f L - N M M A and indomethacin compared to in their absence (Fig. 3.3). To elucidate whether the inhibitory effect o f indomethacin alone on responses o f lean and obese rats to N E is due to reduction in the release o f the C O X pathway contracting factor P G H / T x A , the effect o f SQ 29,548, a P G H / T x A 2  2  2  2  receptor antagonist was examined.  However, although SQ 29,548 (0.3 u M ) tended to inhibit the responses to 9, 30 and 90 nmol N E in both obese and lean M A B , the inhibition was significant only at 30 nmol N E in the obese M A B (113 ± 4% in the absence vs. 92 ± 8% in the presence o f SQ 29,548, P < 0.05).  4.  Effect of Insulin on NE-Induced Vasoconstriction in Isolated Perfused M A B . To investigate the influence o f hyperinsulinemia on reactivity o f the M A B to N E ,  tissues were pre-perfused with 200 mU/1 insulin, a concentration close to that which the obese rats were exposed to in vivo (Table 3.1). Perfusion with insulin for two hours had no detectable effect on responses o f M A B from lean rats to any concentration o f N E , when compared to N E responses obtained at the same time in the absence o f insulin (Fig. 3.4). Similarly, insulin had no significant effect on the maximum pressor responses to N E o f M A B  198  FIGURE 3.3  Contractile response to NE in the absence (control, n=6) (•) and presence of 300 pM L NMMA (n=6) (B ), or 20 pM indomethacin (n=6) ( 0 ) or 300 pM L-NMMA plus 20 pM indomethacin (n=5) (•) in isolated MAB from lean or obese Zucker rats, perfused with Krebs solution at constant flow, n represents the number of the experiments. Data are expressed as mean ± SEM. P < 0.05 vs. control (one-way ANOVA followed by Bonferroni post test: a  compare all column vs. control column).  Lean  250 -| • Control-2 0  B L - N M M A (300uM)  c 200 o CO  • Indomethacin (20nM)  Q .  •  CO  0  L-NMMA+lndo  E 150 E X  ro  100 -  ro  '_c  50 -  JUL  a 0-  0.9  NE  (nmol)  Obese  250 n  • Control-2 B L - N M M A (300nM)  CO  § 200 Q . CO  0  • Indomethacin (20nM) •  L-NMMA+lndo  % 150 H E  J  100  ro  50 H  a  ==J a A  0  0.9 NE  (nmol)  200  FIGURE 3.4  Concentration-response curves to N E in the absence (control, • ) and presence o f 200 mU/1 insulin ( • ) in isolated M A B from lean or obese Zucker rats, perfused with normal Krebs solution at constant flow. Data represent the mean ± SEM from 8 obese, 9 lean and 10 control experiments. P < 0.05 vs. lean; P < 0.05 vs. control (two-way A N O V A followed by a  Newman Keuls post test)  b  Lean 180 -.  • Control • Insulin (200mU/i)  CD C O  c o  C L CO CD  or E E x TO C O  0.9  3  9 NE (nmol)  Obese  180 -| 160 CD CO  c  • Control • Insulin (200mU/i)  140 H  NE (nmol)  202  from  obese  rats. However, responses  o f the  latter preparation to the two lowest  concentrations of N E tested (0.9 and 3 nmol) were significantly enhanced in the presence o f insulin (Fig. 3.4). Insulin had no effect on either basal perfusion pressure or on the KCI response in M A B from lean or obese rats (data not shown).  5.  Influence of NOS, C O X ,  PGH2/TXA2  Receptor and E T Receptor Inhibition on  Insulin-Potentiation of N E Responses To investigate the pathway mediating insulin potentiation o f the responses o f M A B from obese rats to 0.9 and 3 nmol N E , the influence o f various inhibitors on responses to 0.9, 3.0 and 9 nmol N E in the absence and presence o f insulin were determined. None o f the inhibitors tested ( L - N M M A , indomethacin, SQ 29,548, the selective E T  B  antagonist, B Q  788, the selective E T A receptor antagonist, B Q 123, and the non-selective ET-1 receptor antagonist, bosentan) had any effect on the basal perfusion pressure, either alone or in combination with insulin. A s shown in F i g 3.5, insulin further enhanced the L - N M M A induced potentiation o f responses to 0.9, 3 but also 9 nmol N E . In contrast, insulin no longer had effect on responses to N E in the presence o f indomethacin. In addition, indomethacin prevented insulin from further increasing the responses to N E in the presence o f L - N M M A . Although SQ 29,548 alone did not significantly alter responses to N E , like indomethacin, it blocked the potentiation by insulin of the N E responses. The effects o f bosentan were similar to those o f S Q 29,548, in that it blocked the potentiating effect o f insulin on the responses o f the obese preparations to 0.9 and 3 nmol N E , although it had no effect on N E responses in the absence o f insulin. In contrast, insulin still produced significant potentiation o f the N E responses in the presence o f the selective E T receptor antagonists B Q 788 and B Q 123.  203  FIGURE 3.5  Influence o f various inhibitors on potentiating effect o f insulin on N E responses in isolated M A B from obese Zucker rats. Responses to 0.9, 3.0 and 9.0 nmol N E were compared i n the absence ( • ) and presence o f 200 mU/1 insulin ( • ) under control condition without any inhibitors (n=8) or in the presence o f L - N M M A (300pM) (n=6); 20 p M indomethacin (Indo, n=6); 3 0 0 p M L - N M M A plus 20 p M indomethacin (n=6); 0.3 p M B Q 788 (n=5); 0.3 p M B Q 123 (n=5); 3 p M bosentan (BST, n=5); or 0.3 p M SQ 29,548 (n=6). n represents the number of the experiments. Data are expressed as mean ± S E M .  a  P< 0.05 with insulin vs. paired  without insulin (one-way A N O V A followed by Bonferroni comparing selected pairs o f columns).  0.9 nmol NE • NE • NE+lnsulin  3.0 nmol NE • NE • NE+lnsulin  CD CO  § Q .  9.0 nmol NE 180-,  • NE • NE+lnsulin  205  V.  DISCUSSION  There are two main findings in this part o f the thesis work. The first is that N E induced vasoconstriction is markedly influenced by both N O release and vasoconstrictor C O X pathway products in M A B from both obese and lean Zucker rats. A balance between the suppressive effect o f N O and the potentiating effect o f vasoconstrictor C O X product(s) apparently contributes to the net pressor responses to N E . The second finding is that insulin, at a pathophysiological concentration that obese Zucker rats are exposed to in vivo, had a potentiating effect on pressor responses to low concentrations o f N E in obese M A B , which was mediated by C O X metabolites and was enhanced after inhibition o f N O synthesis and release.  Characteristics of Zucker Obese Rats The Zucker obese rats used in this study were moderately hypertensive at 25 weeks o f age; the S B P o f these animals was on average 29 m m H g greater than that o f their lean littermates. The obese rats also had severe hyperinsulinemia, with a plasma insulin levels about 4 times higher than those in the lean rats. The mechanism(s) o f hypertension development  in Zucker obese  rats  are  not  clear.  It  has  been  hypothesized that  hyperinsulinemia in the insulin resistance state may contribute to hypertension through stimulating sympathetic activity (Anderson et al. 1991; Dornfeld et al. 1987; Sowers et al. 1982) and renal sodium retention (Baum 1987; DeFronzo 1981). However, obese Zucker rats were shown to retain less sodium than lean rats (Kurtz et al. 1989). In addition, renal injury has been found in obese Zucker rats, but it has been reported that mild hypertension preceded the development o f progressive focal glomerulosclerosis (Kasiske et al. 1985). Furthermore,  206  ganglion blockade did not lower in vivo blood pressure of conscious obese rats and during the ganglion blockade the obese rats still exhibited greater pressor sensitivity to Ang II and N E (Zemel et al. 1992). Based on these results, it has been proposed that an enhanced pressor sensitivity, independent  of sympathetic neural activity, appears to  supports  hypertension in Zucker obese rats (Zemel et al. 1992). Thus, local regulatory mechanisms including smooth muscle contractility and endothelial function, especially in resistance vessels, may be important determinants of elevated blood pressure in these animals. In addition, in view of the vascular actions of insulin as described in the introduction, hyperinsulinemia itself is still a factor in need of considerable research.  NE-Induced Vasoconstriction in MAB of Zucker Rats 1.  Reactivity to NE and KCI in isolated perfused MAB Previous studies of vascular reactivity in mesenteric resistance vessels have found no  marked differences in pressor responses to a-adrenoceptor agonists such as P E and N E (Kam et al. 1996; Turner et al. 1995; Walker et al. 1997a; Wu et al. 1996) in obese versus lean rats. Endothelium-dependent relaxations in mesenteric resistance vessels from obese rats were reported to be either impaired (Walker et al. 1997a; Wu et al. 1996; Zanchi et al. 1995) or preserved (Bohlen and Lash 1995; Kam et al. 1996; Turner et al. 1995) as compared to agematched lean rats. In agreement with those contractile studies, we did not find any major differences in vasoconstrictor responses to N E rats in perfused mesenteric arterial beds from obese compared to lean rats, except for a small decrease in responsiveness to the highest concentration of N E tested (90 nmol). However, we did find that pressor responses to K C I were attenuated in obese M A B . In these experiments, we did not further explore the  207  mechanisms o f the decreased responsiveness to KC1 in M A B from obese rats. However, it is known that challenge with K C 1 stimulates  endogenous  N E release  from  peripheral  sympathetic nerve endings (Vanhoutte et al. 1981) and it has been shown that inhibition o f the endogenous N E with phentolamine decreases K C l - e v o k e d tension. Therefore, it is possible that the reduced response to K C 1 in M A B reflects the diminished sympathetic nervous system activity that has been reported in obese Zucker rats (Levin et al. 1980).  2.  Blockade of the NO synthesis enhanced vasoconstrictor responses to NE. The augmentation o f pressor responses to N E in both obese and lean M A B by L -  N M M A suggests a modulatory role for N O in NE-mediated vasoconstriction.  Since we  found L - N M M A had no effect on basal perfusion pressures, N O may not be spontaneously released or the release may be too l o w to attenuate perfusion pressure under the basal perfusion conditions (perfusion rate o f 3ml/min). Thus, the enhancement o f NE-induced vasoconstriction by L - N M M A could be due to an agonist-stimulated N O release. This finding is consistent with those reported by most authors using rat isolated M A B perfused at rates under lOml/min. In these studies, it has been shown that N O S inhibition augmented ctadrenoceptor mediated vasoconstriction in an endothelium-dependent manner (Adeagbo et al. 1994; Amerini et al. 1995; Tatchum-Talom and Atkinson 1997), but lacked an effect on the basal tone (Adeagbo et al. 1994; Amerini et al. 1995; Baisch et al. 1994; Ebeigbe et al. 1990; Tatchum-Talom and Atkinson 1997). In contrast, in in vivo studies in intact (Gardiner et al. 1990) or in ganglion-blocked (Fozard and Part 1991) rats, infusion o f L - N M M A induced a 50% reduction o f mesenteric vascular conductance, indicating a physiological role o f N O in the control o f the tone in the mesenteric vascular bed and even in the absence o f functional  208  sympathetic activity. Recently, H o r i et al (Hori et al. 1998) have directly measured changes in N O metabolite ( N O x ) concentration in the perfusate outflow during changes in flow and shear stress in isolated rat mesenteric arterial beds. Their data showed that basal N O x concentration (at perfusion rate o f 4ml/min) in control rats was very l o w as compared with background values and did not significantly increase until the perfusion rate reached 48 ml/min. In addition, after treatment with L - N M M A , the amount o f N O x released only decreased significantly at a flow rate o f 48 ml/min. W e have demonstrated in another study that in vivo, the blood flow through the superior mesentery is approximately 14 ml/min in control rats, much higher than the rate we used in this study (He and Tabrizchi 1997). Thus, the lack o f effect o f L - N M M A on basal perfusion pressure observed in our study is probably due to the l o w perfusion rate plus the very low viscosity o f the Krebs solution so that the shear stress is too l o w to evoke a significant N O release. The mechanisms that mediate the release o f N O in the presence o f N E in blood vessels are not clear. It may be shear stress-dependent and/or adrenoceptor-coupled. It has been reported that an increase in shear stress by a vasoconstriction at constant flow enhances the release of N O , as well as P G ^ f r o m perfused rabbit femoral arteries (Hecker et al. 1993); the N O release in response to short shear exposure due to decrease in vessel diameter by vasoconstrictors was C a / camodulin-dependent (Busse et al. 1993; Kuchan and Frangos 2 +  1994). This concept was supported by a study in Wistar rat perfused mesenteric arterial bed, in which the inhibition o f N O synthesis or endothelium denudation was able to comparably potentiate the response to either receptor-mediated ( N E ) or receptor-independent (KCI) vasoconstriction, but had no effect on basal tone, suggesting that N O release can be triggered by active tone (Amerini et al. 1995). This shear stress/active tone-induced N O release is  209  unlikely in the present study since N O S inhibition dramatically enhanced the  pressor  response to N E , but had little effect on contraction evoked by K C I in mesenteric preparations from either obese or lean Zucker rats (n= 6 for each group o f rats, data not shown). cti and ©^-adrenoceptors have been implicated in the endothelium-dependent depression  o f N E contraction in rat aorta (Kaneko and  Sunano  Both  NO-mediated  1993). NE-induced  endothelium-dependent relaxation was first shown in pig and dog isolated coronary arteries (Cocks and Angus 1983) and was suggested to be mediated by a v ©^-adrenoceptor on the endothelium (Angus et al. 1986; Vanhoutte and M i l l e r 1989). The partial ct2-adrenoceptor agonist clonidine induced relaxation v i a N O release in porcine coronary resistance artery (Tschudi et al. 1991), rat aorta (Kaneko and Sunano 1993) and perfused mesenteric arterial bed (Kamata et al. 1994). However, a -adrenergic, endothelium-dependent 2  responses are  much less pronounced in mesenteric arteries compared to femoral, carotid, and coronary arteries o f dog (Angus et al. 1986). This may reflect a lower density o f endothelial 0C2adrenoceptors in this vascular bed. In addition, there still is an uncertainty with regard to 012adrenoceptor mechanisms in rat mesenteric arteries since clonidine does not increase the c G M P level in rat mesenteric arteries with intact endothelium (MacLeod et al. 1987). Recently, N O release as an endothelial response secondary to vasoconstriction evoked by sympathetic nerve stimulation has been demonstrated in perfused rat M A B (Boric et al. 1999). Both the electrically evoked vasoconstriction and N O release were abolished by prazosin, supporting the involvement o f cti-adrenoceptors and making any possible direct effect o f ©^-adrenoceptor unlikely. It is known that in rat mesenteric vasculature, N E causes contraction predominantly, i f not exclusively, v i a ai-adrenoceptors  (Chen et al. 1996;  Colucci et al. 1980; McPherson et al. 1984; Nielsen et al. 1991; P i p i l i 1986). However, there  210  is no evidence so far to demonstrate the presence  o f ai-adrenoceptors  on vascular  endothelium o f rat mesenteric arteries. O n the other hand, it has been demonstrated that endothelial cells and smooth muscles are electrochemically coupled through myoendothelial junctions (Chaytor et al. 1998; Yamamoto et al. 1999), and the existence o f bi-directional communication between endothelial and smooth muscle cells has been reported (Beny and Pacicca 1994). Recently, Dora et al (Dora et al. 1997) have shown that during PE-induced vasoconstriction, a signal can originate in smooth muscle cells and act on the endothelium to cause synthesis o f N O in arterioles from hamster cheek pouch. Thus, cti-adrenoceptor stimulation o f vascular smooth muscle may result in activation o f e N O S via intercellular communication, and it may explain the N E responses to L - N M M A observed in the present study.  3.  Blockade of COX pathway suppresses pressor responses to NE. In contrast to effect o f L - N M M A , the application o f the C O X inhibitor indomethacin  to mesenteric arterial beds induced a significant suppression o f pressor responses to N E in both lean and obese rats. Because indomethacin did not affect the responses to K C 1 in preparations  from  either group o f rats (data not shown), the  suppressive  effect  of  indomethacin on N E responses is unlikely to be non-specific. Thus, our observations strongly suggest that activation o f the C O X pathway is necessary  for N E to exert its full  vasoconstrictor effect in rat mesenteric vasculature from Zucker rats. Cyclooxygenase pathway metabolites o f arachidonic acid ( A A ) have previously been found to be released under basal and NE-stimulated conditions in rat perfused M A B (Desjardins-Giasson et al. 1982; Pipili et al. 1988), and to modulate the N E response (Coupar 1980; M a l i k et al. 1976). The mechanisms that mediate prostanoid release by N E are not known but could include  211  either a transient increase in shear stress/active tone (Hecker et al. 1993) and/or activation o f adrenergic receptors (Pipili et al. 1988). In the present study, we found that indomethacin had no effect on basal perfusion pressure i n Zucker mesenteric arterial bed. This may be due to the removal o f a balanced basal release o f vasodilator and vasoconstrictor prostaglandins (PGs). Indeed, unbalanced release o f vasodilator and vasoconstrictor P G s in rat mesenteric resistance vessels in S H R rats has been reported (Matrougui et al. 1997; Soma et al. 1985). Prostanoid modulation o f the N E response in rat mesenteric vascular beds was reported repeatedly in the late 1970s and early 1980s. It has been shown that the structurally different C O X inhibitors indomethacin (Coupar 1980; Coupar and McLennan 1978; M a l i k et al. 1976; M a n k u and Horrobin 1976), aspirin, mefenamic acid (Manku and Horrobin 1976) and 5, 8, 11, 14-eicosatetraynoic acid (Coupar 1980) caused a significant depression o f pressor responses to N E in rat isolated perfused mesenteric blood vessels. In addition, indomethacin reduced an increase in release o f a P G E - l i k e activity stimulated by N E to below resting 2  values (Coupar 1980), and P G E , as well as other prostaglandins restored the indomethacin2  depressed response to N E (Coupar 1980; Coupar and McLennan 1978; M a l i k et al. 1976; Manku  and Horrobin 1976). Furthermore,  arachidonic acid  potentiated  NE-induced  vasoconstriction, which was abolished by simultaneous infusion o f indomethacin (Malik et al. 1976). These results provided strong support to our observations in Zucker rats. Since indomethacin had a pronounced inhibitory effect on the response to N E (the maximum inhibition was around 4 5 % and 63% for lean and obese, respectively) in the present study, N E may have a greater propensity to stimulate the release o f contracting factor(s) than relaxing factors such as P G I via the C O X pathway in Zucker mesenteric vessels. In addition, 2  since indomethacin blocks all pathways o f C O X , the pronounced decrease in response to N E  212  seen in Zucker mesenteric vascular beds may be due not only to the removal o f the contracting prostanoid(s), but also to an intact vasodilator effect o f N O protected from chemical inactivation by superoxide anion. It has long been known that O2" is a byproduct o f C O X pathway metabolism (Katusic and Vanhoutte 1989; Kukreja et al. 1986; Y o k o t a and Yamazaki 1977) and that indomethacin can inhibit O2" release arising from this pathway (Holland et al. 1990; Kontos et al. 1985). O2" is known to react with and inactivate N O (Gryglewski et al. 1986; Rubanyi and Vanhoutte 1986). Therefore, blockade o f C O X with indomethacin may not only block release o f prostanoids, but also inhibit O2" production, and thus enhance N O activity (Cosentino et al. 1994), resulting in an additional inhibition o f N E induced contractions. Metabolites o f the C O X pathway known to produce vascular contractions include P G H , T x A , PGF2«, and in rat, P G E . O f these, PGH2 and T x A , which interact with a 2  2  2  2  common receptor, have been proposed to be endothelium-derived contracting  factors  (Luscher et al. 1992; Vanhoutte 1996) and therefore, may be responsible for indomethacininduced depressor effect in this study. However, the selective PGH2/TXA2 receptor antagonist SQ 29,548 only minimally inhibited NE-induced vasoconstriction in both lean and obese M A B . A possible explanation for the smaller inhibitory effect o f SQ 29,548 is that besides PGH2 and/or TxA2, other vasoconstrictor prostanoids may be also involved (Quilley et al. 1989)(also as mentioned above). Alternatively, the possibility can not be excluded that an increased O2" generated via activation o f the C O X pathway by N E , that was not inhibited by SQ 29,548, may reduce the biological activity o f N O and indirectly enhance the NE-induced tone, resulting in a smaller effect o f SQ 29,548 than o f indomethacin.  213  4.  Effect of COX inhibition on pressor responses to NE after blocking of NO synthesis We observed an opposing effect on responses to N E after combined C O X and N O S  blockade in mesenteric arterial beds from both obese and lean Zucker rats. It then should be considered that the potentiation o f responses to N E by L - N M M A alone is the result o f the concomitant  activation o f C O X leading to release  o f predominantly  vasoconstrictor  prostanoids, while the attenuation o f responses to N E with indomethacin alone is due to the concomitant release of N O . In the presence o f both indomethacin and L - N M M A , the pressor response to N E was essentially the same as the control response, suggesting in mesenteric arterial bed vasculature, N O and COX-derived contracting factors released by N E stimulation almost completely counteract each other. In obese mesenteric arterial beds, after blockade o f NOS  and C O X , we found that the pressor responses tended to be greater at most  concentrations o f N E , but it was only statistically significant at 0.9 nmol N E . Thus, a slight imbalance in the release o f these factors, favoring vasodilation, may account for the decreased maximum response of M A B from obese rats to N E in the absence o f insulin.  5.  Lack of influence of endothelin on responses to NE. Pretreatment with bosentan, a non-selective E T - 1 receptor antagonist, or blocking o f  either the E T  A  or the E T  B  receptor with selective antagonists  B Q 123 or B Q 788,  respectively, had no effects on N E induced vasoconstriction. Thus, E T - 1 does not seem to be involved in modulating the pressor response to N E in Zucker M A B .  214  Insulin Effect on Vasoconstrictor Responses to NE in MAB of Zucker Rats 1.  Hyperinsulinemia elevated pressor responses to NE in MAB from obese rats T w o studies have previously addressed the effects o f insulin on reactivity o f the M A B  to N E , with differing results. Walker et al (Walker et al. 1997a) showed that insulin (50 and 500 mU/1) slightly attenuated the maximum response to N E in isolated small mesenteric arteries from lean rats, while the action o f insulin was impaired in tissues from prehypertensive obese rats, suggesting obese mesenteric arteries are resistant to a vasodilator action o f insulin. The significance o f this observation is uncertain, since the attenuation produced by 50 mU/1 insulin was only on the order o f 8%, and 500 m U / L insulin is much higher than obese rats would be exposed to in vivo. O n the other hand, Turner et al (Turner et al. 1995) demonstrated that 100 mU/1 insulin had no effect on pressor responses to P E or depressor responses to A C h in isolated perfused mesenteric arterial beds from either lean or hypertensive obese rats at 12 months o f age. However, this concentration o f insulin is lower than that which obese rats are exposed to in vivo. In contrast with these reports, we found potentiation o f pressor responses to 0.9 and 3.0 nmol N E on M A B from obese rats by a concentration o f insulin (200 pU/ml) close to that which obese rats were exposed to in vivo. This suggests that exposure to chronic hyperinsulinemia enhances vascular reactivity to concentrations o f N E that are within the physiological range for circulating N E i n rats (Dargie et al. 1977; Katholi et al. 1982). In contrast, perfusion with the same concentration o f insulin had no effect on lean tissues, indicating that acutely raising the insulin level does not affect the reactivity o f the M A B under normal conditions. Verma and M c N e i l l demonstrated that in fructose-induced hypertensive (FH) rats, which  are  insulin-resistant  and  hyperinsulinemic,  insulin  at  a  pathophysiological  215  concentration potentiated N E responses in M A B to a greater extent as compared to control rats (Verma and M c N e i l l 1999). In addition, this altered M A B response to insulin was evident prior to the development o f hypertension i n these rats, which were already hyperinsulinemic (Verma and M c N e i l l 1999). Other in vivo studies reported that the mean blood pressure and total peripheral resistance measured during chronic insulin infusion in rats is elevated (independent o f changes in cardiac output and heart rate) (Brands et al. 1991; Brands  et  al  1996).  Furthermore  administration  of  an  insulin-sensitizing  drug  bis(maltolato)oxovanadium, restored plasma insulin levels in the obese Zucker rats tolevels in lean rat and ameliorated the age-dependent increase in blood pressure observed in obese Zucker rats (Yuen et al. 1996). These observations support our results suggesting chronic hyperinsulinemia may be physiologically relevant in promoting hypertension by increasing peripheral vascular resistance via exaggeration of M A B responses in Zucker obese rats.  2.  Blockade of NO synthesis enhanced the vascular effect of Insulin in obese rats Previous studies have suggested that insulin can exhibit both vasodilator and  vasoconstrictor effects, and that its overall effect in a given vascular bed may depend on the balance o f vasodilators and vasoconstrictors it releases. For instance, Baron and Brechtel (Baron  and Brechtel 1993) reported  that in lean human  subjects,  a physiological  concentrations o f insulin caused an approximately 5-fold greater fall in muscle vascular resistance than in systemic vascular resistance. They suggested that insulin preferentially reduced vascular resistance in skeletal muscle beds but may actually increase vascular resistance in other vascular beds (e.g. splanchnic circulation). In vitro studies i n rats have demonstrated that insulin administration attenuates contractile reactivity to N E and dilates isolated resistance vessels (Alexander and Oake 1977; Chen and Messina 1996; Walker et al.  216  1997b). However, under physiological conditions, insulin has been repeatedly reported to potentiate the vasoconstriction elicited by several agonists in isolated perfused M A B (Townsend et al. 1992; W u et al. 1994; Verma and M c N e i l l 1999) and in femoral arteries (Nava et al. 1997). In these studies, the vasodilator effect o f insulin appeared to be mediated by the release o f N O (Chen and Messina 1996; Steinberg et al. 1994; Walker et al. 1997b), while the vasoconstrictor effect o f insulin may be linked to changes in production o f C O X pathway metabolites ( W u et al. 1994) or increased release o f endothelin (Nava et al. 1997). Recently, Schroeder et al. (Schroeder et al. 1999) demonstrated that insulin induced a concentration-dependent increase in diameter o f endothelium-intact arterioles isolated from male Wistar rat gastrocnemius muscle. However, inhibition o f N O synthesis or removal o f the endothelium inhibited the insulin-induced arteriolar dilation and revealed an insulininduced vasoconstriction. Consistent with this, in the present investigation L - N M M A further enhanced the insulin-mediated potentiation o f vasoconstriction to N E in M A B from obese rats. The amplification by L - N M M A o f the potentiating effect o f insulin on vasoconstrictor responses to N E in M A B from Zucker obese rats suggests that insulin-induced potentiation is normally suppressed to some extent by concomitant release of N O .  3.  Inhibition of COX blocked insulin effect in MAB Involvement o f prostaglandins in the mechanisms o f insulin action has been reported.  It was demonstrated that indomethacin treatment caused a metabolic state o f insulin resistance in rats (Wasner et al. 1994). In addition, indomethacin markedly decreased the insulin-induced increase in forearm blood flow o f healthy humans (van Veen and Chang 1997) and prevented the relaxant effects o f insulin on ET-1 and A V P contractions after N O S inhibition in male Wistar rat aorta (Rebolledo et al. 1998). Therefore, it has been suggested  217  that insulin stimulates production/release o f vasodilating prostaglandins.  Furthermore,  Axelrod and coworkers (Axelrod and Levine 1982; Axelrod and Levine 1983; Axelrod et al. 1986) reported that insulin  in physiological concentrations  inhibited catecholamine-  stimulated PGI2 and PGE2 production by adipose tissue both in vitro and in vivo, and hypothesized (Axelrod  1991) that hyperinsulinemia may increase peripheral vascular  resistance and blood pressure by inhibiting the stimulatory effect o f adrenergic agonists on the production o f these vasodilative eicosanoids in adipose tissue (and perhaps other tissues). Insulin has also been shown to specifically enhance T x A - i n d u c e d vasoconstriction i n 2  porcine coronary vascular beds (Yanagisawa-Miwa et al. 1990). Moreover, Keen et al. (Keen et al. 1997) recently reported that inhibition o f TxA2 synthase markedly attenuated mean blood pressure increased by chronic insulin infusion i n rats, suggesting that TXA2 is a key component o f the chronic hypertensive effect o f insulin. In this study, we demonstrated that application o f indomethacin and SQ 29, 548 completely inhibited the potentiating effect o f insulin on pressor responses to N E i n M A B from obese Zucker rats, suggesting that the potentiating effect o f insulin is mediated by release o f vasoconstrictor C O X metabolites, possibly PGH2 and/or T x A . In addition, the lack o f further effect o f insulin in the presence 2  o f indomethacin plus L - N M M A on responses to N E i n M A B from obese Zucker rats further confirms the notion in M A B from obese Zucker rats.  4.  ET-1 contributing to potentiating effect of insulin on responses to NE in obese rats In this study, we also examined the role o f endothelin in the effect o f insulin on  vasoconstrictor responses to N E in obese Zucker rats. W e found that the non-selective E T receptor antagonist bosentan prevented the potentiating effect o f insulin on the pressor responses to N E in obese M A B . The result suggests that insulin-induced, prostanoid-  218  mediated enhancement  o f vasoconstrictor responses to N E in obese Zucker M A B also  involves endothelin release, at least in part. This result is consistent with those o f previous studies, which have implicated the vasoconstrictor E T in the vascular actions o f insulin. F o r instance, insulin was reported to increase E T - 1 gene expression (Oliver et al. 1991) and induce ET-1 release (Hattori et al. 1991; H u et al. 1993) in cultured endothelial cells, while an increase in contractile response induced by insulin in rat femoral arteries has been found to be partially mediated by endothelin (Nava et al. 1997). In addition, in fructose hypertensive rats, insulin-induced exaggerated M A B responses to N E were completely abrogated in the presence o f both indomethacin and bosentan, B y contrast indomethacin completely prevent the insulin response in M A B from control rats, suggesting a role o f ET-1 for the F H rats (Verma and M c N e i l l 1999). Furthermore, insulin has been shown to selectively increase E T receptor expression in V S M cells (Hopfner et al. 1998) and both A  E T A and E T B receptor expression have been found to be increased in mesenteric arteries and aorta from obese Zucker rats with hypertension although local E T - 1 production is decreased (Wu et al. 2000). Moreover, endothelin activated P L A  2  in blood vessels, leading to the  release A A (Resink et al. 1989; Reynolds et al. 1989), and release o f both vasoconstrictor and vasodilator prostanoids has also been demonstrated (Matsuda et al. 1993; Tabuchi et al. 1989a; Taddei and Vanhoutte 1993). It has also been shown that subpressor doses o f endothelin enhanced pressor responses to N E in human coronary arteries, rabbit aorta and perfused rat mesenteric arterial beds (Henrion and Laher 1993; K i t a et al. 1998; Tabuchi et al. 1989b; Yang et al. 1990). The failure o f either o f the selective E T receptor antagonists to mimic the effects o f bosentan means that the effect o f endothelin cannot be attributed to its actions on either o f the  219  receptor subtypes alone. Although the effects o f bosentan might have resulted from an action unrelated to antagonism o f E T receptors, blockade o f both receptor subtypes has been previously shown to be required for antagonism o f some actions o f E T (Fukuroda et al. 1996; M i c k l e y et al. 1997).  220  VI.  SUMMARY  1.  Zucker obese rats were moderately hypertensive and severely hyperinsulinemic at 25 weeks o f age.  2.  In perfused M A B from both obese and lean Zucker rats, N E induced a concentrationdependent increase in perfusion pressure that was significantly lower at maximum response to N E in M A B from the obese than from the lean rats.  3.  Insulin perfusion had no effect on N E responses o f the lean M A B , but potentiated the responses o f the obese M A B to 0.9 and 3 nmol N E .  4.  The N O S inhibitor L - N M M A enhanced the responses o f both the obese and the lean M A B to N E . In the presence o f L - N M M A , insulin further increased the N E response of M A B from the obese rats.  5.  Perfusion with indomethacin alone inhibited the pressor responses to N E in M A B from both the lean and the obese rats In the presence o f indomethacin, insulin no longer had any effect on the N E responses in the obese M A B .  6.  The presence o f indomethacin inhibited the potentiating effect o f L - N M M A on the responses to N E in M A B from both the obese and the lean rats. Indomethacin also prevented insulin from further increasing the responses to N E i n the presence o f L N M M A in the obese M A B .  7.  B Q 123, a selective E T receptor antagonist, and B Q 788, a selective antagonist for A  ETB, alone had no effect on pressor responses to concentrations o f N E in both lean and obese M A B . However, in the presence o f either B Q 123 or B Q 788, insulin still potentiated the response of M A B to 0.9 and 3 nmol N E .  221  Bosentan, a non-selective E T - 1 receptor inhibitor, alone had no effect on pressor responses to concentrations o f N E in both lean and obese M A B . The presence o f bosentan abolished the potentiating effect o f insulin on vasoconstrictor responses to the lower concentration o f N E .  222  VII.  CONCLUSIONS  W e present evidence that NE-induced vasoconstriction is normally regulated by release o f both N O and vasoconstrictor C O X product(s) in isolated perfused M A B from both obese and lean Zucker rats. Insulin, at a concentration close to that obese rats are exposed to in vivo, increased the release o f contracting C O X product(s) and enhanced contractile responses to physiological concentrations o f N E in M A B from obese, but not from the lean rats. The effects o f insulin in obese rats may be partially mediated by E T - 1 and are suppressed to some extent by concomitant release o f N O . Taken together, our results suggest that chronic hyperinsulinemia may elevate reactivity o f mesenteric resistance arteries and serve to increase peripheral resistance in vivo. Thus, hyperinsulinemia could play a role in development  and/or maintenance  o f hypertension in obese Zucker rats. Whether  the  coexistent hyperlipidemia w i l l interfere with the bioavailability o f N O and exaggerate the insulin effects needs to be further investigated. To our knowledge, the present study is the first time an altered action o f insulin leading to the release o f contracting factor (s) in insulinresistant animals has been demonstrated.  223  CONCLUDING REMARKS  Mesenteric arteries and arterioles play an important role in the maintenance and control o f peripheral resistance, thereby regulating blood flow and blood pressure. A particular feature o f ai-adrenoceptor-mediated excitation-contraction coupling o f mesenteric arterial smooth muscle appears to be the dependence o f the contractile response on V O C s , and thus on the membrane potential. Information on the role o f CI" channels in the membrane  depolarization  to  agonist  activation  has  been  obtained  largely  from  electrophysiological and ion efflux studies. In this dissertation, functional evidence is presented that CI" channels mediate cti-adrenoceptor-induced contraction v i a opening o f nifedipine-sensitive C a  2 +  channels. This contribution o f CI" channels in mesenteric arteries  from 2K1C hypertensive rats appears to be reduced. The diminished role o f CI" may reflect an adaptive change in response to an enhanced reactivity in hypertensive mesenteric arterial bed. The endothelium also has an active role in regulating local tone by integrating diverse biochemical and mechanical signals, and by responding to them through the release o f vasoactive substances. In the present study, N O and E D H F were demonstrated to be released in response to A C h and to contribute to different extents to the relaxation o f the muscular mesenteric artery compared to the elastic aorta. Endothelial-dependent smooth muscle relaxation in the mesenteric arteries appears mainly to reflect the action o f E D H F , with N O playing a minor role. In the aorta, N O is the primary relaxant, with E D H F contributing to lesser extent. The underlying mechanisms o f ion channel regulation are also different between the two vessels. K channels, but not CI" channels mediate the function o f A C h in +  224  mesenteric  arteries.  In contrast,  both channels are important for releasing N O from  endothelium in aorta. The differences in relative contributions and regulatory mechanisms o f these E D R F s may prove to have physiological and/or pathophysiological significance in disease states such as atherosclerosis and hypertension. The interplay among E D C F s and E D R F s and among these endothelial-derived factors and neurotransmitters and hormones may have a profound impact on vascular homeostasis. I report in this dissertation that N O and vasoconstrictor C O X products modulate NE-induced vasoconstriction in M A B o f lean and obese Zucker rats. Insulin, at a concentration close to which obese rats are exposed in vivo, increases the release o f contracting C O X product(s) and enhances contractile responses to physiological concentrations o f N E in M A B from hypertensive obese, but not from the normotensive lean rats. Another E D C F , E T - 1 , and E D R F , N O are also involved. The altered insulin action and the imbalanced interaction among the endothelium-derived substances may elevate reactivity o f mesenteric resistance arteries and could play a role in hypertension in Zucker obese rats. Further investigation into the intracellular signaling, which leads to C f channel activation in response to oti-adrenoceptor stimulation in mesenteric smooth muscle, would necessarily address the interrelationship between C a the regulation o f contractile mechanisms by C a  2 +  2 +  and membrane potential and in turn  and membrane potential in these blood  vessels. In addition, further research on identification o f the E D H F and other substances that released from endothelium would advance the study o f mechanisms that mediate relaxation o f smooth muscle in the mesenteric arteries. Finally, elucidation o f these mechanisms w i l l lead to a greater understanding o f the regulation o f peripheral resistance under normal and pathological conditions and may have important therapeutic implications.  225  BIBLIOGRAPHY Abdel-Latif, A . A . : Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol Rev 38: 227-272, 1986 Adams, D . J., Barakeh, J., Laskey, R . , and V a n Breemen, C : Ion channels and regulation o f intracellular calcium in vascular endothelial cells. FASEB J 3: 2389-2400, 1989 Adeagbo, A . S. and Henzel, M . K . : Calcium-dependent phospholipase A 2 mediates the production o f endothelium-derived hyperpolarizing factor i n perfused rat mesenteric prearteriolar bed. J Vase Res 35: 27-35, 1998 Adeagbo, A . S. and M a l i k , K . U . : Endothelium-dependent and B R L 34915-induced vasodilatation in rat isolated perfused mesenteric arteries: role o f G-proteins, K + and calcium channels. Br J Pharmacol 100: 427-434, 1990 Adeagbo, A . S., Tabrizchi, R., and Triggle, C. R.: The effects o f perfusion rate and NG-nitroL-arginine methyl ester on cirazoline- and KCl-induced responses i n the perfused mesenteric arterial bed o f rats. Br J Pharmacol 111: 13-20, 1994 Adeagbo, A . S. and Triggle, C . R . : Varying extracellular [ K ] : a functional approach to separating E D H F - and EDNO-related mechanisms i n perfused rat mesenteric arterial bed. J Cardiovasc Pharmacol 21: 423-429, 1993 +  A i c k i n , C. C. and Brading, A . F . : Measurement o f intracellular chloride i n guinea-pig vas deferens by ion analysis, chloride efflux and micro-electrodes. J Physiol 326: 139154,1982 36  A i c k i n , C . C . and Brading, A . F.: Towards an estimate o f chloride permeability in the smooth muscle o f guinea-pig vas deferens. J Physiol 336: 179-197, 1983 A i c k i n , C . C . and Brading, A . F . : Effect o f N a and K on CI" distribution in guinea-pig vas deferens smooth muscle: evidence for N a , K , CI" co-transport. J Physiol 421: 13-32, 1990 +  +  +  +  Aidley, D . J.: The physiology o f excitable cells. Cambridge University Press, Cambridge, 1998 Alexander, W D . and Oake, R . J.: The effect o f insulin on vascular reactivity to norepinephrine. Diabetes 26: 611-614, 1977 Alonso-Galicia, M . , Brands, M . W . , Zappe, D . H . , and Hall, J. E . : Hypertension i n obese Zucker rats. Role o f angiotensin II and adrenergic activity. Hypertension 28: 10471054, 1996  226  Altiere, R . J., Kiritsy-Roy, J. A . , and Catravas, J. D . : Acetylcholine-induced contractions i n isolated rabbit pulmonary arteries: role o f thromboxane A . J Pharmacol Exp Ther 236: 535-541, 1986 2  Altura, B . M . : Evaluation o f neurohumoral substances i n local regulation o f blood flow. Am J Physiol 2 J2: 1447-1454, 1967 Amedee, T., Benham, C . D . , Bolton, T. B . , Byrne, N . G . , and Large, W . A . : Potassium, chloride and non-selective cation conductances opened by noradrenaline i n rabbit ear artery cells. J Physiol 423: 551-568, 1990a Amedee, T. and Large, W . A . : Microelectrode study on the ionic mechanisms which contribute to the noradrenaline-induced depolarization in isolated cells o f the rabbit portal vein. Br J Pharmacol 97: 1331-1337, 1989 Amedee, T., Large, W . A . , and Wang, Q.: Characteristics o f chloride currents activated by noradrenaline i n rabbit ear artery cells. J Physiol 428: 501-516, 1990b Amerini, S., Mantelli, L . , and Ledda, F.: Enhancement o f the vasoconstrictor response to K C I by nitric oxide synthesis inhibition: a comparison with noradrenaline. Pharmacol Res 31: 175-181, 1995 Anderson, E . A . , Balon, T. W . , Hoffman, R . P., Sinkey, C . A . , and Mark, A . L . : Insulin increases sympathetic activity but not blood pressure i n borderline hypertensive humans. Hypertension 19: 621-627, 1992 Anderson, E . A . , Hoffman, R . P., Balon, T. W . , Sinkey, C . A . , and Mark, A . L . : Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans. J Clin Invest 87: 2246-2252, 1991 Angus, J. A . , Cocks, T. M . , and Satoh, K . : Alpha 2-adrenoceptors and endotheliumdependent relaxation i n canine large arteries. Br J Pharmacol 88: 767-777, 1986 Arai, H , Hori, S., Aramori, I., Ohkubo, H . , and Nakanishi, S.: Cloning and expression o f a c D N A encoding an endothelin receptor. Nature 348: 730-732, 1990 Archer, S. L . , Huang, J. M . , Hampl, V . , Nelson, D . P., Shultz, P. J., and Weir, E . K . : Nitric oxide and c G M P cause vasorelaxation by activation o f a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 75837587, 1994 Ashida, T., Schaeffer, J., Goldman, W . F . , Wade, J. B . , and Blaustein, M . P.: Role o f sarcoplasmic reticulum in arterial contraction: comparison o f ryanodines's effect i n a conduit and a muscular artery. Circ Res 62: 854-863, 1988  227  Auch-Schwelk, W . , Katusic, Z . S., and Vanhoutte, P. M . : Thromboxane A 2 receptor antagonists inhibit endothelium-dependent contractions. Hypertension 15: 699-703, 1990 Auguet, M . , Delaflotte, S., and Braquet, P.: Increased influence o f endothelium in obese Zucker rat aorta. Journal of Pharmacy & Pharmacology 41: 861-864, 1989 Averill, D . B . , Ferrario, C. M . , Tarazi, R. C , Sen, S., and Bajbus, R : Cardiac performance in rats with renal hypertension. Circ Res 38: 280-288, 1976 Axelrod, L . : Insulin, prostaglandins, and the pathogenesis o f hypertension. Diabetes 40: 1223-1227, 1991 Axelrod, L . and Levine, L . : Plasma prostaglandin levels in rats with diabetes mellitus and diabetic ketoacidosis. Diabetes 31: 994-1001, 1982 Axelrod, L . and Levine, L . : Inhibitory effect o f insulin on prostacyclin production by isolated rat adipocytes. Prostaglandins 25: 571-579, 1983 Axelrod, L . , Ryan, C. A . , Shaw, J. L . , Kieffer, J. D . , and Ausiello, D . A . : Prostacyclin production by isolated rat adipocytes: evidence for cyclic adenosine 3',5'monophosphate-dependent and independent mechanisms and for a selective effect o f insulin. Endocrinology 119: 2233-2239, 1986 Baisch, A . L . , Larrue, J., and Freslon, J. L . : Involvement o f endothelium-derived N O in the basal tone and in the vasodilator responses to muscarinic agonists in the rat isolated mesenteric arterial bed. Fundam Clin Pharmacol 8: 54-63, 1994 Baro, I. and Eisner, D . A . : Factors controlling changes in intracellular C a concentration produced by noradrenaline in rat mesenteric artery smooth muscle cells. J Physiol 482: 247-258, 1995 2 +  Baron, A . , Pacaud, P., Loirand, G . , Mironneau, C , and Mironneau, J.: Pharmacological block of Ca( )-activated C f current i n rat vascular smooth muscle cells in short-term primary culture. Pflugers Arch 419: 553-558, 1991 2+  Baron, A . D . : Vascular reactivity. [Review]. Am J Cardiol 84: 25J-27J, 1999 Baron, A . D . and Brechtel, G . : Insulin differentially regulates systemic and skeletal muscle vascular resistance. Am J Physiol 265: E61-67, 1993 Batra, V . K . , M c N e i l l , J. R., X u , Y . , Wilson, T. W . , and Gopalakrishnan, V . : E T B receptors on aortic smooth muscle cells o f spontaneously hypertensive rats. Am J Physiol 264: C479-484, 1993  228  Baum, M . : Insulin stimulates volume absorption in the rabbit proximal convoluted tubule. J Clin Invest 79: 1104-1109, 1987 Baum, T. and Shropshire, A . T.: Vasoconstriction induced by sympathetic stimulation during development o f hypertension. Am J Physiol 212: 1020-1024, 1967 Beckman, J. S., Beckman, T. W . , Chen, J., Marshall, P . A . , and Freeman, B . A . : Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990 Beech, D . J. and Bolton, T. B . : T w o components o f potassium current activated by depolarization o f single smooth muscle cells from the rabbit portal vein. J Physiol 418: 293-309, 1989 Benedetti, R. G . and Linas, S. L . : Mechanism o f decreased vascular response to angiotensin II in renal vascular hypertension. Kidney Int 31: 906-912, 1987 Benetos, A . , Gavras, H . , Stewart, J. M . , Vavrek, R . J., Hatinoglou, S., and Gavras, I.: Vasodepressor role o f endogenous bradykinin assessed by a bradykinin antagonist. Hypertension 8: 971-974, 1986 Bennett, M . A . and Thurston, H . : Effect o f angiotensin-converting enzyme inhibitors on resistance artery structure and endothelium-dependent relaxation i n two-kidney, oneclip Goldblatt hypertensive and sham-operated rats. Clin Sci 90: 21-29, 1996 Bennett, M . A . , Watt, P . A . , and Thurston, H . : Impaired endothelium-dependent relaxation i n two-kidney, one clip Goldblatt hypertension: effect o f vasoconstrictor prostanoids. J Hypertens ll(Suppl5): S134, 1993 Beny, J.: Electrical coupling between smooth muscle cells and endothelial cells i n p i g coronary arteries. Pflugers Arch 433: 364-367, 1997 Beny, J. L . and Pacicca, C : Bidirectional electrical communication between smooth muscle and endothelial cells in the pig coronary artery. Am J Physiol 266: H1465-1472, 1994 Berridge, M . J.: Phosphatidylinositol hydrolysis: a multifunctional transducing mechanism. Mol Cell Endocrinol 24: 115-140, 1981 Berridge, M . J.: Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993 Bevan, J. A . , Bevan, R . D . , and Duckies, S.: Adrenergic regulation o f vascular smooth muscle. In D . F . Bpohr, A . P. Somlyo, and H . V . Sparks (eds.): Handbook of Physiology, Section 2: The cardiovascular System,, pp. 515-566, A m . Physiol. S o c , B e t h e s d a M D , 1980  229  Bevan, J. A . and Laher, I.: Pressure and flow-dependent vascular tone. [Review]. 2267-2273, 1991  FASEBJ5:  Bevan, J. A . and Osher, J. V . : A direct method for recording tension changes in the wall o f small blood vessels in vitro. Agents Actions 2: 257-260, 1972 Bhagyalakshmi, A . and Frangos, J. A . : Mechanism o f shear-induced prostacyclin production in endothelial cells. Biochem BiophysRes Commun 158: 31-37, 1989 Bhardwaj, R. and Moore, P. K . : Endothelium-derived relaxing factor and the effects o f acetylcholine and histamine on resistance blood vessels. Br J Pharmacol 95: 835843, 1988 Bing, R. F., Russell, G . I., Swales, J. D . , and Thurston, H . : Effect o f 12-hour infusions o f saralasin or captopril on blood pressure in hypertensive conscious rats. Relationship to plasma renin, duration o f hypertension, and effect o f unclipping. J Lab Clin Med 95: 302-310,1981 Blair, I. A . , Barrow, S. E . , Waddell, K . A . , Lewis, P . J., and Dollery, C . T.: Prostacyclin is not a circulating hormone in man. Prostaglandins 23: 579-589, 1982 Blue, D . R., Jr., Vimont, R. L . , and Clarke, D . E . : Evidence for a noradrenergic innervation to alpha lA-adrenoceptors in rat kidney. Br J Pharmacol 107: 414-417, 1992 Bohlen, H . G . : Intestinal microvascular adaptation during maturation o f spontaneously hypertensive rats. Hypertension 5: 739-745, 1983 Bohlen, H . G : Regional vascular behavior in the gastrointestinal wall. Fed Proc 43: 7-15, 1984 Bohlen, H . G . and Lash, J. M . : Endothelial-dependent vasodilation is preserved in noninsulin-dependent Zucker fatty diabetic rats. Am J Physiol 268: Ul^-l^lA, 1995 Bolotina, V . M . , Najibi, S., Palacino, J. J., Pagano, P. J., and Cohen, R. A . : Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994 Bolton, T. B . : Mechanisms o f action o f transmitters and other substances on smooth muscle. Physiol Rev 59: 606-718, 1979 Bolton, T. B . , Lang, R. J., and Takewaki, T.: Mechanisms o f action o f noradrenaline and carbachol on smooth muscle o f guinea-pig anterior mesenteric artery. J Physiol 351: 549-572, 1984  230  Bolz, S. S., de Wit, C , and Pohl, U . : Endothelium-derived hyperpolarizing factor but not N O reduces smooth muscle C a during acetylcholine-induced dilation o f microvessels. Br J Pharmacol 128: 124-134, 1999 2 +  Boric, M . P., Figueroa, X . F., Donoso, M . V . , Paredes, A . , Poblete, I., and Ffuidobro-Toro, J. P.: Rise in endothelium-derived N O after stimulation o f rat perivascular sympathetic mesenteric nerves. Am J Physiol 277: H I 027-103 5, 1999 Bowman, W . C. and Rand, M . J.: Textbook o f Phamacology. Blackwell, London, 1980 Boyer, J. L . , Waldo, G . L . , and Harden, T. K . : Beta gamma-subunit activation o f G-proteinregulated phospholipase C. J Biol Chem 267: 25451-25456, 1992 Brands, M . W . , Hall, J. E . , and Keen, H . L . : Is insulin resistance linked to hypertension? Clin Exp Pharmacol Physiol 25: 70-76, 1998 Bray, G . A . : Metabolic and regulatory obesity in rats and man. Horm Metab Res 2: 175-180, 1970 Bray, G . A . : The Zucker-fatty rat: a review. Fed Proc 36: 148-153, 1977 Brunner, H . R., Kirshman, J. D . , Sealey, J. E . , and Laragh, J. H . : Hypertension o f renal origin: evidence for two different mechanisms. Science 174: 1344-1346, 1971 Buckner, S. A . , Oheim, K . W . , Morse, P . A . , Knepper, S. M . , and Hancock, A . A . : Alpha 1adrenoceptor-induced contractility in rat aorta is mediated by the alpha I D subtype. Eur J Pharmacol 297: 241-248, 1996 Bulbring, E . and Tomita, T.: Catecholamine action on smooth muscle. Pharmacol Rev 39: 49-96, 1987 Bunag, R . D . : Facts and fallacies about measuring blood pressure in rats. Clin Exp Hypertens [A] 5: 1659-1681, 1983 Bunag, R. D . and Barringer, D . L . : Obese Zucker rats, though still normotensive, already have impaired chronotropic baroreflexes. Clinical & Experimental Hypertension Part A, Theory & Practice 1: 257-262, 1988 Busse, R . , Fichtner, H . , Luckhoff, A . , and Kohlhardt, M . : Hyperpolarization and increased free calcium i n acetylcholine-stimulated endothelial cells. Am J Physiol 255: H 9 6 5 969, 1988 Busse, R . and Fleming, I.: Pulsatile stretch and shear stress: physical stimuli determining the production o f endothelium-derived relaxing factors. [Review]. J Vase Res 35: 73-84, 1998  231  Busse, R., Luckhoff, A . , and Pohl, U . : Generation and transmission o f endotheliumdependent vasodilator signals. In J. D . Catravas, C . N. Gillis, and U . S. Ryan (eds.): Vascular endothelium: receptors and transduction mechanisms, pp. 225-236, Plenum Press, N e w Y o r k /London, 1989 Busse, R., Mulsch, A . , Fleming, I., and Hecker, M . : Mechanism o f nitric oxide release from the vascular endothelium. Circulation 87 (suppl V): V 1 8 - V 2 5 , 1993 Buus, C. L . , Aalkjar, C , Nilsson, H . , Juul, B . , Moller, J. V . , and Mulvany, M . J.: Mechanisms o f C a sensitization o f force production by noradrenaline in rat mesenteric small arteries. J Physiol 510: 577-590, 1998 2 +  Bychkov, R., Gollasch, M . , Steinke, T., Ried, C , Luft, F. C , and Haller, H . : Calciumactivated potassium channels and nitrate-induced vasodilation in human coronary arteries. J Pharmacol Exp Ther 285: 293-298, 1998 Bylund, D . B . , Bond, R. A . , Clarke, D . E . , Eikenburg, D . C , Hieble, J. P., Langer, A . Z . , Lfkowitz, R. I , Minneman, K . P., Molinoff, P. B . , Ruffolo, R . R , Strosberg , A . D . , and Trendelenburg, U . G : Adrenoceptors. The IUPHAR Compendium of receptor characterization and classification, pp 58—74, IUPELAR M e d i a L t d , London, 1998 Bylund, D . B . , Eikenberg, D . C , Hieble, J. P., Langer, S. Z . , Lefkowitz, R. J., Minneman, K . P., Molinoff, P. B . , Ruffolo, R. R., Jr., and Trendelenburg, U . : International U n i o n o f Pharmacology nomenclature o f adrenoceptors. Pharmacol Rev 46: 121-136, 1994 Byrne, N . G . and Large, W . A . : The action o f noradrenaline on single smooth muscle cells freshly dispersed from the guinea-pig pulmonary artery. Br J Pharmacol 91: 89-94, 1987 Byrne, N . G . and Large, W . A . : Mechanism o f action o f alpha-adrenoceptor activation i n single cells freshly dissociated from the rabbit portal vein. Br J Pharmacol 94: 475482, 1988a Byrne, N . G . and Large, W . A . : Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. J Physiol 404: 557-573, 1988b Campbell, W . B . , Gebremedhin, D . , Pratt, P . F., Harder, D . R.: Identification o f epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415-423, 1996 Carmines, P. K . : Segment-specific effect o f chloride channel blockade on rat renal arteriolar contractile responses to angiotensin II. Am JHypertens 8: 90-94, 1995 Carretero, O. A . and Gulati, O. P.: Effects o f angiotensen antagonist in rats with acute, subacute, and chronic two-kidney renal hypertension. J Lab Clin Med 91: 264-271, 1978  232  Carrier, G . O., Fuchs, L . C , Winecoff, A . P., Giulumian, A . D . , and White, R. E . : Nitrovasodilators relax mesenteric microvessels by cGMP-induced stimulation o f C a activated K channels. Am J Physiol 273: H76-84, 1997 Carter, T. D . and Pearson, J. D . : Regulation o f prostacyclin synthesis i n endothelial cells. News Physiol. Sci. 7: 64-69, 1992 Carvalho, M . H . , Fortes, Z . B . , Nigra, D . , Oliveira, M . A . , and Scivoletto, R . : The role o f thromboxane A 2 in the altered microvascular reactivity i n two-kidney, one-clip hypertension. Endothelium 5: 167-178, 1997 Casteels, R.: The distribution o f chloride ions i n the smooth muscle cells o f the guinea-pig's taenia coli. J Physiol 214: 225-243, 1971 Casteels, R.: Membrane potential in smooth muscl cells. In E . Bulbring, A . F . Brading, A . W . Jones, and T. Tomita (eds.): Smooth Muscle: An Assessment of Current Knowledge., pp. 105-126, Edward Arnold, London, 1981 Casteels, R . , Kitamura, K . , Kuriyama, H . , and Suzuki, H . : The membrane properties o f the smooth muscle cells o f the rabbit main pulmonary artery. J Physiol 271: 41-61, 1977 Cauvin, C . and M a l i k , S.: Induction o f C a influx and intracellular C a release in isolated rat aorta and mesenteric resistance vessels by norepinephrine activation o f alpha-1 receptors. J Pharmacol Exp Ther 230: 413-418, 1984 2 +  2 +  Cauvin, C . and Pegram, B . : Decreased relaxation o f isolated mesenteric resistance vessels from 2-kidney, 1 clip Goldblatt hypertensive rats. Clinical & Experimental Hypertension - Part A, Theory & Practice 5: 383-400, 1983 Cauvin, C , Saida, K . , and van Breemen, C : Extracellular C a dependence and diltiazem inhibition o f contraction in rabbit conduit arteries and mesenteric resistance vessels. Blood Vessels 21: 23-31, 1984 2 +  Chataigneau, T., Feletou, M . , Duhault, J., and Vanhoutte, P. M . : Epoxyeicosatrienoic acids, potassium channel blockers and endothelium-dependent hyperpolarization i n the guinea-pig carotid artery. Br J Pharmacol 123: 574-580, 1998a Chataigneau, T., Feletou, M . , Thollon, C , Villeneuve, N , Vilaine, J. P., Duhault, J., Vanhoutte, P . M . : Cannabinoid C B 1 receptor and endothelium-dependent hyperpolarization i n guinea-pig carotid, rat mesenteric and porcine coronary arteries. Br J Pharmacol 123: 968-974, 1998b Chaytor, A . T., Evans, W . H . , and Griffith, T. M . : Central role o f heterocellular gap junctional communication i n endothelium-dependent relaxations o f rabbit arteries. J Physiol 508: 561-573, 1998  233  Chen, G . and Cheung, D . W . : Modulation o f endothelium-dependent hyperpolarization and relaxation to acetylcholine in rat mesenteric artery by cytochrome P450 enzyme activity. Circ Res 79: 827-833, 1996 Chen,  G . and Cheung, D . W . : Effect o f K(+)-channel blockers on ACh-induced hyperpolarization and relaxation in mesenteric arteries. Am J Physiol 272: H23062312, 1997  Chen, G . and Suzuki, H . : Some electrical properties o f the endothelium-dependent hyperpolarization recorded from rat arterial smooth muscle cells. J Physiol 410: 9 1 106, 1989 Chen, G . , Suzuki, H . , and Weston, A . H . : Acetylcholine releases endothelium-derived hyperpolarizing factor and E D R F from rat blood vessels. Br J Pharmacol 95: 11651174, 1988 Chen, G . , Yamamoto, Y . , M i w a , K . , and Suzuki, H . : Hyperpolarization o f arterial smooth muscle induced by endothelial humoral substances. Am J Physiol 260: H1888-1892, 1991 Chen, G . F . and Cheung, D . W . : Characterization o f acetylcholine-induced hyperpolarization in endothelial cells. Circ Res 70: 257-263, 1992 Chen,  membrane  G . F . and Suzuki, H . : Calcium dependency o f the endothelium-dependent hyperpolarization in smooth muscle cells o f the rabbit carotid artery. J Physiol 421: 521-534, 1990  Chen, H . , Fetscher, C , Schafers, R . F . , Wambach, G , Philipp, T., and M i c h e l , M . C : Effects o f noradrenaline and neuropeptide Y on rat mesenteric microvessel contraction. Naunyn Schmiedebergs Arch Pharmacol 353: 314-323, 1996 Chen, X . L . and Rembold, C. M . : Phenylephrine contracts rat tail artery by one electromechanical and three pharmacomechanical mechanisms. Am J Physiol 268: H74-81, 1995 Chen, Y . L . and Messina, E . J.: Dilation o f isolated skeletal muscle arterioles by insulin is endothelium dependent and nitric oxide mediated. Am J Physiol 270: H2120-2124, 1996 Chesnoy-Marchais, D . : Characterization o f a chloride conductance hyperpolarization in Aplysia neurones. J Physiol 342: 277-308, 1983  activated  by  Cheung, D . W . : Electrophysiological properties o f vascular smooth muscle in hypertension. In C. Y . K w a n (ed.): Membrane abnormalities in hypertension, pp 1-14, C R C Press, B o c a Raton, Fla, 1989  234  Cheung, D . W . , Chen, G . , M a c K a y , M . J., and Bumette, E . : Regulation o f vascular tone by endothelium-derived hyperpolarizing factor. Clin Exp Pharmacol Physiol 26: 172175, 1999 Chipperfield, A . R.: The ( N a - K - C f ) co-transport system. Clin Sci 71: 465-476, 1986 +  +  Chipperfield, A . R., Davis, J. P., and Harper, A . A . : A n acetazolamide-sensitive inward chloride pump in vascular smooth muscle. Biochem Biophys Res Commun 194: 407412, 1993 Chlopicki, S., Nilsson, H . , and Mulvanny, M . J.: Initial and sustained myogenic response o f rat mesenteric small arteries: role o f potassium channels and cytochrome P-450 metaboletes. J. Microcirc. Clin. Exp. 16: 87, 1996 Clapham, D . E . . Calcium signaling. Cell 80: 259-268, 1995 Clark, S. G . and Fuchs, L . C : Role o f nitric oxide and Ca -dependent K channels i n mediating heterogeneous microvascular responses to acetylcholine in different vascular beds. J Pharmacol Exp Ther 282: 1473-1479, 1997 2+  +  Clozel, M . and Gray, G . A . : A r e there different E T B receptors mediating constriction and relaxation? JCardiovasc Pharmacol 26: S262-264, 1995 Cocks, T. M . and Angus, J. A . : Endothelium-dependent relaxation o f coronary arteries by noradrenaline and serotonin. Nature 305: 627-630, 1983 Cohen, R. A . , Plane, F., Najibi, S., Huk, I., Malinski, T., Garland, C . J.: Nitric oxide is the mediator o f both endothelium-dependent relaxation and hyperpolarization o f the rabbit carotid artery. Proc Natl Acad Sci USA 94: 4193-4198, 1997 Colden-Stanfield, M . , Schilling, W . P., Ritchie, A . K . , Eskin, S. G , Navarro, L . T., and Kunze, D . L . : Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ Res 61: 632-640, 1987 Collis, M . G . and Alps, B . J.: Vascular reactivity to noradrenaline, potassium chloride, and angiotensin II in the rat perfused mesenteric vasculature preparation, during the development o f renal hypertension. Cardiovasc Res 9: 118-126, 1975 Collis, M . G . and Vanhoutte, P. M . : Increased renal vascular reactivity to angiotensin II but not to nerve stimulation or exogenous norepinephrine in renal hypertensive rats. Circ Res 43: 544-552, 1978 Colucci, W . S., Gimbrone, M . A . , Jr., and Alexander, R. W . : Characterization o f postsynaptic alpha-adrenergic receptors by [3H]-dihydroergocryptine binding in muscular arteries from the rat mesentery. Hypertension 2: 149-155, 1980  235  Colucci, W . S., Gimbrone, M . A . , Jr., and Alexander, R. W . : Regulation o f the postsynaptic alpha-adrenergic receptor in rat mesenteric artery. Effects o f chemical sympathectomy and epinephrine treatment. Circ Res 48: 104-111, 1981 Conway, J.: Hemodynamic aspects o f essential hypertension in humans. Physiol Rev 64: 617-660, 1984 Coombes, J. E . , Hughes, A . D . , and Thorn, S. A . : Intravascular pressure-evoked changes i n intracellular calcium [ C a ] ; and tone in rat mesenteric and rabbit cerebral arteries i n vitro. J Hum Hypertens 13: 855-858, 1999 2+  Cornwell, T. L . , Pryzwansky, K . B . , Wyatt, T. A . , and Lincoln, T. M . : Regulation o f sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40: 923-931, 1991 Corriu, C , Feletou, M . , Canet, E . , Vanhoutte, P. M . : Inhibitors o f the cytochrome P450mono-oxygenase and endothelium-dependent hyperpolarizations i n the guinea-pig isolated carotid artery. Br J Pharmacol 117: 607-610, 1996 Cosentino, F., Sill, J. C , and Katusic, Z . S.: Role o f superoxide anions i n the mediation o f endothelium-dependent contractions. Hypertension 23: 229-235, 1994 Cotecchia, S., Kobilka, B . K . , Daniel, K . W . , Nolan, R . D . , Lapetina, E . Y , Caron, M . G . , Lefkowitz, R . J., and Regan, J. W . : Multiple second messenger pathways o f alphaadrenergic receptor subtypes expressed in eukaryotic cells. J Biol Chem 265: 63-69, 1990 Coupar, I. M . : Prostaglandin action, release and inactivation by rat isolated mesenteric blood vessels. Br J Pharmacol 68: 757-763, 1980  perfused  Coupar, I. M . and McLennan, P . L . : The influence o f prostaglandins on noradrenalineinduced vasoconstriction isolated perfused mesenteric blood vessels o f the rat. Br J Pharmacol 62: 51-59, 1978 Cox, R. H . and Kikta, D . C : Age-related changes in thoracic aorta o f obese Zucker rats. Am J Physiol 262:m54%-\556, 1992 Crettaz, M . , Prentki, M . , Zaninetti, D . , and Jeanrenaud, B . : Insulin resistance i n soleus muscle from obese Zucker rats. Involvement o f several defective sites. Biochem J 186: 525-534, 1980 Criddle, D . N , de Moura, R . S., Greenwood, I. A . , and Large, W . A . : Effect o f N F A on noradrenaline-induced contractions o f the rat aorta. Br J Pharmacol 118: 1065-1071, 1996  236  Criddle, D . N . , de Moura, R. S., Greenwood, I. A . , and Large, W . A . : Inhibitory action o f N F A on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed o f the rat. Br J Pharmacol 120: 813-818, 1997 Dalmark, M . and Wieth, J. O.: Temperature dependence o f chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells. J Physiol 224: 583-610, 1972 Dargie, H . J., Franklin, S. S., and Reid, J. L . : Central and peripheral noradrenaline in the two kidney model o f renovascular hypertension in the rat. Br J Pharmacol 61: 213-215, 1977 Davis, J. P.: The effects o f Na(+)-K(+)-Cl" co-transport and Cl(-)-HC03-exchange blockade on the membrane potential and intracellular chloride levels o f rat arterial smooth muscle, in vitro. Exp Physiol 77: 857-862, 1992 Davis, J. P.: Evidence against a contribution by Na(+)-Cl(-) cotransport to chloride accumulation in rat arterial smooth muscle. J Physiol 491: 61-68, 1996 Davis, J. P., Chipperfield, A . R., and Harper, A . A . : Comparison o f the electrical properties o f arterial smooth muscle in normotensive rats and rats with deoxycorticosterone acetate-salt-induced hypertension: possible involvement o f (Na(+)-K(+)-Cl(-) cotransport. Clin Sci 81: 73-78, 1991 Davis, J. P., Chipperfield, A . R., and Harper, A . A . : Accumulation o f intracellular chloride by ( N a - K - C l ) co-transport in rat arterial smooth muscle is enhanced in deoxycorticosterone acetate (DOCA)/salt hypertension. J Mol Cell Cardiol 25: 233237, 1993 Davis, M . J., Ferrer, P. N . , and Gore, R. W . : Vascular anatomy and hydrostatic pressure profile in the hamster cheek pouch. Am J Physiol 250: H291-303, 1986 D e M e y , J. G . and Vanhoutte, P. M . : Heterogeneous behavior o f the canine arterial and venous wall. Importance o f the endothelium. Circ Res 51: 439-447, 1982 DeFronzo, R . A . : The effect o f insulin on renal sodium metabolism. A review with clinical implications. Diabetologia 21: 165-171, 1981 DeFronzo, R. A . , Cooke, C . R., Andres, R . , Faloona, G . R , and Davis, P. J.: The effect o f insulin on renal handling o f sodium, potassium, calcium, and phosphate in man. J Clin Invest 55: 845-855, 1975 Demirel, E . , Rusko, J., Laskey, R. E . , Adams, D . I , and van Breemen, C : T E A inhibits ACh-induced E D R F release: endothelial Ca(2+)-dependent K channels contribute to vascular tone. Am J Physiol 267: H I 135-1141, 1994 +  237  Deng, L . Y . , L i , J. S., Schiffrin, E . L . : Endothelin receptor subtypes i n resistance arteries from humans and rats. Cardiovasc Res 29: 532-535, 1995 Deng, L . Y . and Schiffrin, E . L . : Morphological and functional alterations o f mesenteric small resistance arteries in early renal hypertension in rats. Am J Physiol 261: H I 1711177, 1991 Denker, P . S. and Pollock, V . E . : Fasting serum insulin levels i n essential hypertension. A meta-analysis. Arch Intern Med 152: 1649-1651, 1992 Desjardins-Giasson, S., Gutkowska, J., Garcia, R., and Genest, J.: Effect o f angiotensin i i and norepinephrine on release o f prostaglandins E 2 and 12 by the perfused rat mesenteric artery. Prostaglandins 24: 105-114, 1982 D i Wang, H . , Hope, S., D u , Y . , Quinn, M . T., Cayatte, A . , Pagano, P . J., and Cohen, R . A . : Paracrine role o f adventitial superoxide anion i n mediating spontaneous tone o f the isolated rat aorta in angiotensin H-induced hypertension. Hypertension 33: 12251232, 1999 Dickinson, C . J. and Y u , R . : Mechanisms involved in the progressive pressor response to very small amounts o f angiotensin i n conscious rabbits. Circ Res 21: Suppl 2:157, 1967 Diederich, D . , Yang, Z . H . , Buhler, F . R., and Luscher, T. F . : Impaired endotheliumdependent relaxations i n hypertensive resistance arteries involve cyclooxygenase pathway. Am J Physiol 258: H445-451, 1990 Dinudom, A . , Young, J. A . , and Cook, D . I.: N a and CI" conductances are controlled by cytosolic CI" concentration i n the intralobular duct cells o f mouse mandibular glands. JMembr Biol 135: 289-295, 1993 +  Dohi, Y . , Thiel, M . A . , Buhler, F. R., and Luscher, T. F . : Activation o f endothelial L-arginine pathway i n resistance arteries. Effect o f age and hypertension. Hypertension 16: 170179, 1990 Dong, H , Waldron, G . I , Cole, W . C , Triggle, C . R . : Roles o f calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation o f the rabbit middle cerebral artery. Br J Pharmacol 123: 821-832, 1998 Dong, H . , Waldron, G . J., Galipeau, D . , Cole, W . C , Triggle, C . R.: NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery. Br J Pharmacol 120: 695-701, 1997  238  Dora, K . A . , Doyle, M . P., and Duling, B . R.: Elevation o f intracellular calcium in smooth muscle causes endothelial cell generation o f N O in arterioles. Proc Natl Acad Sci U S A 94: 6529-6534, 1997 D'Orleans-Juste, P., Claing, A . , Warner, T. D . , Yano, M . , and Telemaque, S.: Characterization o f receptors for endothelins in the perfused arterial and venous mesenteric vasculatures o f the rat. Br J Pharmacol 110: 687-692, 1993 . Dornfeld, L . P., Maxwell, M . H . , Waks, A . , and Tuck, M . : Mechanisms o f hypertension in obesity. Kidney Int Suppl 22: S254-258, 1987 Doughty, J. M . , Miller, A . L . , and Langton, P. D . : Non-specificity o f chloride channel blockers in rat cerebral arteries: block o f the L-type calcium channel. J Physiol 507: 433-439, 1998 Doughty, J. M . , Plane, F., and Langton, P. D . : Charybdotoxin and apamin block E D H F in rat mesenteric artery i f selectively applied to the endothelium. Am J Physiol 276: H I 1071112, 1999 Drenth, J. P. H . , Nishimura, J., Nouaihetas, V . L . A . , and van Breemen, C : Receptormediated C-kinase activation contributes to alpha-adrenergic tone in rat mesenteric resistance artery. J. Hypertens. 7: S41-S45, 1989 Droogmans, G . , Callewaert, G . , Declerck, I., and Casteels, R.: ATP-induced C a release and CI" current in cultured smooth muscle cells from pig aorta. J Physiol 440: 623-634, 1991 2 +  Droogmans, G . , Declerck, I., and Casteels, R . : Effect o f adrenergic agonists on Ca -channel currents in single vascular smooth muscle cells. Pflugers Arch 409: 7-12, 1987 2+  Drummond, G . R. and Cocks, T. M . : Evidence for mediation by endothelium-derived hyperpolarizing factor o f relaxation to bradykinin in the bovine isolated coronary artery independently o f voltage-operated C a channels. Br J Pharmacol 117: 10351040, 1996 2 +  Duling, B . R , Gore, R. W . , Dacey, R. G , Jr., and Damon, D . N . : Methods for isolation, cannulation, and in vitro study o f single microvessels. Am J Physiol 241: H I 08-116, 1981 Ebeigbe, A . B . , Cressier, F., Konneh, M . K . , L u u , T. D . , and Criscione, L . : Influence o f N G monomethyl-L-arginine on endothelium-dependent relaxations in the perfused mesenteric vascular bed o f the rat. Biochem Biophys Res Commun 169: 873-879, 1990 Edwards, D . H . , Griffith, T. M . , Ryley, H . C , and Henderson, A . H . : Haptoglobinhaemoglobin complex in human plasma inhibits endothelium dependent relaxation:  239  evidence that endothelium derived relaxing factor acts as a local autocoid. Cardiovasc Res 20: 549-556, 1986 Edwards, G . , Dora, K . A . , Gardener, M . J . , Garland, C . J . , and Weston, A . H . : K is an endothelium-derived hyperpolarizing factor i n rat arteries. Nature 396: 269-272, 1998 +  Edwards, G . , Feletou, M . , Gardener, M . J . , Thollon, C , Vanhoutte, P. M . , and Weston, A . H : Role o f gap junctions in the responses to EDFLF in rat and guinea-pig small arteries. Br JPharmacol 128: 1788-1794, 1999 Edwards, G . , Thollon, C , Gardener, M . J . , Feletou, M . , Vilaine, J . , Vanhoutte, P. M . , and Weston, A . H . . Role o f gap junctions and E E T s in endothelium-dependent hyperpolarization o f porcine coronary artery. Br J Pharmacol 129: 1145-1154, 2000 Eltze, M . , Boer, R., Sanders, K . H . , and Kolassa, N . : Vasodilatation elicited by 5 - H T 1 A receptor agonists in constant-pressure-perfused rat kidney is mediated by blockade o f alpha lA-adrenoceptors. Eur J Pharmacol 202: 33-44, 1991 Erdbugger, W . , Vischer, P., and Bauch, H . . Prostaglandin synthesis in endothelial- and vascular smooth muscle cells upon mechanical stimulation and stimulation with calcium ionophore A23187. Pharmacol. Rev. Commun 9: 107-111, 1997 Faber, J . E . and Brody, M . J . : Neural contribution to renal hypertension following acute renal artery stenosis in conscious rats. Hypertension 5: 1155-164, 1983 Farley, J . and Rudy, B . : Multiple types o f voltage-dependent Ca -activated K + channels o f large conductance in rat brain synaptosomal membranes. Biophys J 53: 919-934, 1988 2+  Fasolato, C , Innocenti, B . , and Pozzan, T.: Receptor-activated C a influx: how many mechanisms for how many channels? Trends Pharmacol Sci 15: 77-83, 1994 2 +  Feldman, R . D . and Bierbrier, G . S.: Insulin-mediated vasodilation: impairment with increased blood pressure and body mass. Lancet 342: 707-709, 1993 Feletou, M . and Vanhoutte, P. M . : The alternative: E D H F . J Mol Cell Cardiol 31: 15-22, 1999 Fenger-Gron, J . , Mulvany, M . J . , and Christensen, K . L . : Mesenteric blood pressure profile o f conscious, freely moving rats. J Physiol 488: 753-760, 1995 Fenger-Gron, J . , Mulvany, M . J . , and Christensen, K . L . : Intestinal blood flow is controlled by both feed arteries and rnicrocirculatory resistance vessels i n freely moving rats. J Physiol 498: 215-224, 1997  240  Ferrannini, E . , Buzzigoli, G . , Bonadonna, R . , Giorico, M . A . , Oleggini, M . , Graziadei, L . , Pedrinelli, R . , Brandi, L . , and Bevilacqua, S.: Insulin resistance in essential hypertension. NEnglJMed317: 350-357, 1987 Ferrari, P., Weidmann, P., Shaw, S., Giachino, D . , Riesen, W . , Allemann, Y . , and Heynen, G. : Altered insulin sensitivity, hyperinsulinemia, and dyslipidemia in individuals with a hypertensive parent. Am JMed 91: 589-596, 1991 Ferrario, C M . : Contribution o f cardiac output and peripheral resistance to experimental renal hypertension. Am J Physiol 226: 711-717, 1974 Fleischmann, B . K . , Murray, R . K . , and Kotlikoff, M . I.: Voltage window for sustained elevation o f cytosolic calcium in smooth muscle cells. Proc Natl Acad Sci USA 91: 11914-11918, 1994 Fleming, I., Bauersachs, J., and Busse, R . : Calcium-dependent and calcium-independent activation o f the endothelial N O synthase. [Review]. J Vase Res 34: 165-174, 1997 Fleming, I., Bauersachs, J., Schafer, A , Scholz, D . , Aldershvile, J., and Busse, R.: Isometric contraction induces the Ca -independent activation o f the endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96: 1123-1128, 1999 2+  Folkow, B . . Physiological aspects o f primary hypertension. Physiol Rev 62: 347-504, 1982 Folkow, B . and N e i l , E . : Circulation. Oxford University Press, N e w York, 1971 Ford, A . P., Daniels, D . V . , Chang, D . J., Gever, J. R . , Jasper, J. R., Lesnick, J. D . , and Clarke, D . E . : Pharmacological pleiotropism o f the human recombinant a l p h a l A adrenoceptor: implications for alpha 1-adrenoceptor classification. Br J Pharmacol 121: 1127-1135, 1997 Forstermann, U . , Closs, E . I , Pollock, J. S., Nakane, M . , Schwarz, P., Gath, I., and Kleinert, H . : Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23: 1121-1131, 1994 Forstermann, U . , Hertting, G . , and Neufang, B . : The role o f endothelial and non-endothelial prostaglandins i n the relaxation o f isolated blood vessels o f the rabbit induced by acetylcholine and bradykinin. Br J Pharmacol 87: 521-532, 1986 Fortes, Z . B . , Costa, S. G , Nigro, D . , Scivoletto, R , de Oliveira, M . A , and de Carvalho, M . H . : Effect o f indomethacin on the microvessel reactivity o f two-kidney, one-clip hypertensive rats. Arch Int Pharmacodyn Ther 316: 75-89, 1992 Fortes, Z . B . , Costa, S. G , Nucci, G , Nigro, D . , Scivoletto, R., and Carvalho, M . H . : Comparison o f the reactivity o f micro-and macrovessels to noradrenaline and  241  endothelin i n rats with renal ( 2 K 1 C ) hypertension. Clinical & Experimental Hypertension - Part A, Theory & Practice 12: 47-61, 1990 Fozard, J. R . and Part, M . L . : Haemodynamic responses to NG-monomethyl-L-arginine i n spontaneously hypertensive and normotensive Wistar-Kyoto rats. Br J Pharmacol 102: 823-826, 1991 Franciolini, F . and Petris, A . : Chloride channels o f biological membranes. Biochim Biophys Acta 1031: 247-259, 1990 Freay, A . , Johns, A . , Adams, D . J., Ryan, U . S., and V a n Breemen, C : Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells. Pflugers Arch 414: 377-384, 1989 Fukao, M . , Hattori, Y . , Kanno, M . , Sakuma, I., and Kitabatake, A . : Evidence for selective inhibition by lysophosphatidylcholine o f acetylcholine-induced endotheliumdependent hyperpolarization and relaxation in rat mesenteric artery. Br J Pharmacol 116: 1541-1543, 1995 Fukao, M . , Hattori, Y . , Kanno, M . , Sakuma, I., and Kitabatake, A . : Alterations i n endothelium-dependent hyperpolarization and relaxation i n mesenteric arteries from streptozotocin-induced diabetic rats. Br J Pharmacol 121: 1383-1391, 1997a Fukao, M . , Hattori, Y . , Kanno, M . , Sakuma, I., and Kitabatake, A . : Evidence against a role o f cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery. Br J Pharmacol 120: 439-446, 1997b Fukao, M . , Hattori, Y . , Kanno, M . , Sakuma, I., and Kitabatake, A . : Sources o f C a i n relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br J Pharmacol 120: 1328-1334, 1997c 2 +  Fukuroda, T., Ozaki, S., Ihara, M . , Ishikawa, K . , Yano, M . , Miyauchi, T., Ishikawa, S., Onizuka, M . , Goto, K . , and Nishikibe, M . : Necessity o f dual blockade o f endothelin E T A and E T B receptor subtypes for antagonism o f endothelin-1-induced contraction in human bronchi. Br J Pharmacol 117: 995-999, 1996 Fukuroda, T., Ozaki, S., Ihara, M . , Ishikawa, K . , Yano, M . , and Nishikibe, M . : Synergistic inhibition by BQ-123 and B Q - 7 8 8 o f endothelin-1-induced contractions o f the rabbit pulmonary artery. Br J Pharmacol 113: 336-338, 1994 Furchgott, R . F . , Carvalho, M . H , Khan, M . T., and Matsunaga, K . : Evidence for endothelium-dependent vasodilation o f resistance vessels by acetylcholine. Blood Vessels 24: 145-149, 1987  242  Furchgott, R . F . and Vanhoutte, P. M . : Endothelium-derived relaxing and contracting factors. 2007-2018, 1989 Furchgott, R . F . and Zawadzki, J. V . : The obligatory role o f endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373-376, 1980 Furness, J. B . : Arrangement o f blood vessels and their relation with adrenergic nerves i n the rat mesentery. JAnat 115: 347-364, 1973 Furness, J. B . and Marshall, J. M . : Correlation o f the directly observed responses o f mesenteric vessles o f the rat to nerve stimulation and noradrenaline with the distribution o f adrenergic nerves. J Physiol 239: 75-88, 1974 Gambone, L . M . , Murray, P. A . , and Flavahan, N . A . : Synergistic interaction between endothelium-derived N O and prostacyclin i n pulmonary artery: potential role for K A T P channels. Br J Pharmacol 121: 271-279, 1997 +  Ganitkevich, V . and Isenberg, G : Contribution o f two types o f calcium channels to membrane conductance o f single myocytes from guinea-pig coronary artery. J Physiol 426: 19-42, 1990 Gardiner, S. M . , Compton, A . M . , Bennett, T., Palmer, R . M . , and Moncada, S.: Control o f regional blood flow by endothelium-derived nitric oxide. Hypertension 15: 486-492, 1990 Garland, C. J. and Plane, F . : Relative importance o f endothelium-derived hyperpolarizing factor for the relaxation o f vascular smooth muscle i n different arterial beds. In P . M . Vanhoutte (ed.): Endothelium-derived hyperpolarizing factor, pp. p 173-179, Harwood Academic Publishers, Amsterdam, 1996 Garland, C . I , Plane, F . , Kemp, B . K . , and Cocks, T. M . : Endothelium-dependent hyperpolarization: a role i n the control o f vascular tone. Trends Pharmacol Sci 16: 23-30, 1995 Garland, J. G . and McPherson, G . A . : Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J Pharmacol 105: 429-435, 1992 George, M . J. and Shibata, E . F.: Regulation o f calcium-activated potassium channels by Snitrosothiol compounds and cyclic guanosine monophosphate i n rabbit coronary artery myocytes. JInvestigMed 43: 451-458, 1995 Gerstheimer, F . P., Muhleisen, M . , Nehring, D . , and Kreye, V . A . : A chloride-bicarbonate exchanging anion carrier i n vascular smooth muscle o f the rabbit. Pflugers Arch 409: 60-66, 1987  243  Ghisdal, P., Gomez, J. P., and Morel, N . : Action o f a N O donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery. J Physiol 1: 8396, 2000 Gibson, A . , McFadzean, I., Wallace, P., and Wayman, C . P.: Capacitative C a entry and the regulation o f smooth muscle tone. Trends Pharmacol Sci 19: 266-269, 1998 2 +  Godfraind, T.: Calcium entry blockade and excitation contraction coupling i n the cardiovascular system (with an attempt o f pharmacological classification). Acta Pharmacol Toxicol 58: 5-30, 1986 Goecke, A , Kusanovic, J. P., Serrano, M . , Charlin, T., Zuniga, A , and Marusic, E . T.: Increased N a , K , C l cotransporter and N a , K - A T P a s e activity o f vascular tissue in twokidney Goldblatt hypertension. Biol Res 31: 263-271, 1998 Goldblatt, H . , Lynch, J., Hanzal, R. F., and Summerville, W . W . : Studies on experimental hypertension. 1: The production o f persistent elevation o f systolic blood pressure by means o f renal ischaemia. J Exp Med Sci 9: 347-378, 1934 Gordienko, D . V . , Clausen, C , and Goligorsky, M . S.: Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. Am J Physiol 266: F325-341, 1994 Gore, R. W . and Bohlen, H . G . : M c r o v a s c u l a r pressures in rat intestinal muscle and mucosal villi. Am J Physiol 233: H685-693, 1977 Graham, R. M . , Perez, D . M . , H w a , J., and Piascik, M . T.: alpha i-adrenergic receptor subtypes. Molecular structure, function, and signaling. Circ Res 78: 737-749, 1996 Greenwood, I. A . , Hogg, R. C , and Large, W . A . : Effect o f frusemide, ethacrynic acid and indanyloxyacetic acid on spontaneous Ca-activated currents in rabbit portal vein smooth muscle cells. Br J Pharmacol 115: 733-738, 1995 Greenwood, I. A . and Large, W . A . : Comparison o f the effects o f fenamates on Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle cells. Br J Pharmacol 116: 2939-2948, 1995 Gregoire, G . , Loirand, G . , and Pacaud, P.: C a and Sr * entry induced C a release from the intracellular C a store in smooth muscle cells o f rat portal vein. J Physiol 472: 483500, 1993 2 +  2  2 +  2 +  Griendling, K . K . , Minieri, C . A . , Ollerenshaw, J. D . , and Alexander, R. W . : Angiotensin U stimulates N A D H and N A D P H oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141 -1148, 1994  244  Griffith, T. M . , Edwards, D . H . , Newby, A . C , Lewis, M . I , and Henderson, A . H : Production o f endothelium derived relaxant factor is dependent on oxidative phosphorylation and extracellular calcium. Cardiovasc Res 20: 7-12, 1986 Groschner, K . and Kukovetz, W . R . : Voltage-sensitive chloride channels o f large conductance in the membrane o f pig aortic endothelial cells. Pflugers Arch 421: 209217, 1992 Gruetter, C. A . , Gruetter, D . Y . , Lyon, J. E . , Kadowitz, P. J., and Ignarro, L . J.: Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation o f coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects o f methylene blue and methemoglobin. J Pharmacol Exp Ther 219: 181-186, 1981 Grunfeld, B . , Balzareti, M . , Romo, M . , Gimenez, M . , and Gutman, R . : Hyperinsulinemia i n normotensive offspring o f hypertensive parents. Hypertension 23: 112-15, 1994 Gryglewski, R . J., Botting, R . M . , and Vane, J. R . : Prostacyclin: from discovery to clinic application. In G . M . Rubanti (ed.): Cardiovascular Significance of EndotheliumDerived Vasoactive Factors, pp 3-37, Future Publishing, Company, Inc., Mount K i s c o , N Y , 1991 Gryglewski, R . J., Palmer, R . M . , and Moncada, S.: Superoxide anion is involved i n the breakdown o f endothelium-derived vascular relaxing factor. Nature 320: 454-456, 1986 Guibert, C , Marthan, R . , and Savineau, J. P.: Oscillatory CI" current induced by angiotensin II i n rat pulmonary arterial myocytes: C a dependence and physiological implication. Cell Calcium 21: 421-429, 1997 2 +  Gustafsson, H . , Mulvany, M . J., and Nilsson, H . : Rhythmic contractions o f isolated small arteries from rat: influence o f the endothelium. Acta Physiol Scand 148: 153-163, 1993 Haeusler, G . and Haefely, W . : Pre- and postjunctional supersensitivity o f the mesenteric artery preparation from normotensive and hypertensive rats. Naunyn Schmiedebergs Arch Pharmacol 266: 18-33, 1970 Hallam, T. I , Jacob, R . , and Merritt, J. E . : Influx o f bivalent cations can be independent o f receptor stimulation in human endothelial cells. BiochemJ 259: 125-129, 1989 Hallam, T. J., Pearson, J. D . , and Needham, L . A . : Thrombin-stimulated elevation o f human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production. BiochemJ 251: 243-249, 1988  245  Hallback-Nordlander, M . , Noresson, E . , and Lundgren, Y . : Haemodynamic alterations after reversal o f renal hypertension in rats. Clin Sci 57 Suppl 5: 15 s-17s, 1979 Halpern, W . , Osol, G . , and Coy, G . S.: Mechanical behavior o f pressurized in vitro prearteriolar vessels determined with a video system. Ann Biomed Eng 12: 463-479, 1984 Halushka, P. V . , Mais, D . E . , and Mayeus, P. R . : Thromboxane, prostaglandin leukotriene receptors. Annu Rev Pharm Tox 10: 213-239, 1989  and  Hansen, P. R. and Olesen, S. P.: Relaxation o f rat resistance arteries by acetylcholine involves a dual mechanism: activation o f K channels and formation o f nitric oxide. Pharmacol Toxicol 80: 280-285, 1997 +  Harder, D . R. and Sperelakis, N . : Membrane electrical properties o f vascular smooth muscle from the guinea pig superior mesenteric artery. PflugersArch 378: 111-119, 1978 Hatake, K . , Wakabayashi, I., Hishida, S.: Endothelium-dependent relaxation resistant to N G nitro-L-arginine in rat aorta. Eur J Pharmacol 274: 25-32, 1995 Hattori, Y . , Kasai, K . , Nakamura, T., Emoto, T., and Shimoda, S.: Effect o f glucose and insulin on immunoreactive endothelin-1 release from cultured porcine aortic endothelial cells. Metabolism: Clinical & Experimental 40: 165-169, 1991 He, J., Klag, M . J., Caballero, B . , Appel, L . J., Charleston, J., and Whelton, P . K . : Plasma insulin levels and incidence o f hypertension in African Americans and whites. Arch Intern Med 159: 498-503,1999 He, Y . and Tabrizchi, R : Effects o f N F A on alphal-adrenoceptor-induced vasoconstriction in mesenteric artery in vitro and in vivo in two-kidney one-clip hypertensive rats. Eur JPharmacol 328: 191-199, 1997 Hebel, R. and Stromberg, M . : Anatomy o f the laboratory rat. Williams & Wilkins,, Baltimore, 1976 Hecker, M . , Bara, A . T., Bauersachs, J., and Busse, R . : Characterization o f endotheliumderived hyperpolarizing factor as a cytochrome P450-derived arachidonic acid metabolite in mammals. J Physiol 481: 407-414, 1994 Hecker, M . , Mulsch, A . , Bassenge, E . , and Busse, R.: Vasoconstriction and increased flow: two principal mechanisms o f shear stress-dependent endothelial autacoid release. Am JPhysiol 265: H828-833, 1993 Heitzer, T., Wenzel, U . , Hink, TJ., Krollner, D . , Skatchkov, M . , Stahl, R. A . , MacHarzina, R , Brasen, J. H . , Meinertz, T., and Munzel, T.: Increased N A D ( P ) H oxidase-mediated  246  superoxide production in renovascular hypertension: evidence for an involvement o f protein kinase C . Kidney Int 55: 252-260, 1999 Henrion, D . and Laher, I.: Potentiation o f norepinephrine-induced endothelin-1 in the rabbit aorta. Hypertension 22: 78-83, 1993  contractions by  Hirakawa, Y . , Gericke, M . , Cohen, R . A . , and Bolotina V . M.:Ca -dependent CI" channels i n mouse and rabbit aortic smooth muscle: regulation by intracellular C a and N O . Am J Physiol 46: H1732-H1744, 1999 2+  2 +  Hirst, G . D . and Edwards, F . R.: Sympathetic neuroeffector transmission i n arteries and arterioles. Physiol Rev 69: 546-604, 1989 Hofmann, F . and Klugbauer, N : Molecular biology and expression o f smooth muscle L-type calciun channels. In M . Barany (ed.): Biochemistry of Smooth Muscle Contraction, pp. 221-226, Academic Press, N e w York, 1996 Hogg, R . C , Wang, Q., and Large, W . A . : Time course o f spontaneous calcium-activated chloride currents i n smooth muscle cells from the rabbit portal vein. J Physiol 464: 15-31, 1993 Hogg, R . C , Wang, Q., and Large, W . A . : Action o f N F A on evoked and spontaneous calcium-activated chloride and potassium currents i n smooth muscle cells from rabbit portal vein. Br J Pharmacol 112: 977-984, 1994a Hogg, R . C , Wang, Q., and Large, W . A . : Effects o f CI channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein. Br J Pharmacol 111: 1333-1341, 1994b Holland, J. A . , Pritchard, K . A . , Pappolla, M . A . , Wolin, M . S., Rogers, N . J., and Stemerman, M . B . : Bradykinin induces superoxide anion release from human endothelial cells. J Cell Physiol 143: 21-25, 1990 Holtz, J., Forstermann, U . , Pohl, U . , Giesler, M . , and Bassenge, E . : Flow-dependent, endothelium-mediated dilation o f epicardial coronary arteries i n conscious dogs: effects o f cyclooxygenase inhibition. J Cardiovasc Pharmacol 6: 1161-1169, 1984 Honda, H . , Ushijima, D . , Ishihara, H . , Yanase, M . , and K o g o , H . : A regional variation o f acetylcholine-induced relaxation i n different segments o f rat aorta. Physiology & Behavior 63: 55-58, 1997 Hopfner, R . L . , Hasnadka, R . V . M c N e i l l , J, R . , Wilson, T. W . Gopalakrishnan, V . : insulin increases endothelin-1-evoked intracellular free calcium responses by increased E T ( A ) receptor expression in rat aortic smooth muscle cells. Diabetes 47: 937-9'44, 1998  247  Hopfner, R. L . , Misurski, D . A . , M c N e i l l , J. R., and Gopalakrishnan, V . : Effect o f sodium orthovanadate treatment on cardiovascular function in the hyperinsulinemic, insulinresistant obese Zucker rat. J Cardiovasc Pharmacol 34: 811 -817, 1999 . Hori, N , Wiest, R., and Groszmann, R. J.: Enhanced release o f nitric oxide in response to changes in flow and shear stress in the superior mesenteric arteries o f portal hypertensive rats. Hepatology 28: 1467-1473, 1998 Horie, K . , Obika, K . , Foglar, R., and Tsujimoto, G . : Selectivity o f the imidazoline alphaadrenoceptor agonists (oxymetazoline and cirazoline) for human cloned alpha 1adrenoceptor subtypes. Br J Pharmacol 116: 1611-1618, 1995 Horowitz, A . , Menice, C. B . , Laporte, R., and Morgan, K . G . : Mechanisms o f smooth muscle contraction. Physiol Rev 76: 967-1003, 1996 Hosoki, E . and Iijima, T.: Chloride-sensitive C a entry by histamine and A T P in human aortic endothelial cells. Eur J Pharmacol 266: 213-218, 1994 2 +  Hosoki, E . and Iijima, T.: Modulation o f cytosolic C a concentration by thapsigargin and cyclopiazonic acid in human aortic endothelial cells. Eur J Pharmacol 288: 131-137, 1995 2 +  Hrometz, S. L . , Edelmann, S. E . , McCune, D . F . , Olges, J. R., Hadley, R. W . , Perez, D . M . , and Piascik, M . T.: Expression o f multiple alphai-adrenoceptors on vascular smooth muscle: correlation with the regulation o f contraction. J Pharmacol Expe Ther 290: 452-463, 1999 H u , R. M . , Levin, E . R., Pedram, A . , and Frank, H . J.: Insulin stimulates production and secretion o f endothelin from bovine endothelial cells. Diabetes 42: 351-358, 1993 Huang, W . C , Tsai, R. Y . , and Fang, T. C : Nitric oxide modulates the development and surgical reversal o f renovascular hypertension in rats. JHypertens 18: 601-613, 2000 Hussain, M . B . and Marshall, I.: Characterization o f alphai-adrenoceptor subtypes mediating contractions to phenylephrine in rat thoracic aorta, mesenteric artery and pulmonary artery. Br J Pharmacol 122: 849-858, 1997 Hutcheson, I. R., Chaytor, A . T., Evans, W . H . , and Griffith, T. M . : Nitric oxide-independent relaxations to acetylcholine and A23187 involve different routes o f heterocellular communication. Role o f Gap junctions and phospholipase A 2 . Circ Res 84: 53-63, 1999 Hutcheson, I. R. and Griffith, T. M . : Mechanotransduction through the endothelial cytoskeleton: mediation o f flow- but not agonist-induced E D R F release. Br J Pharmacol 118: 720-726, 1996  248  H w a , J., Graham, R . M . , and Perez, D . M . : Identification o f critical determinants o f alpha 1adrenergic receptor subtype selective agonist binding. J Biol Chem 270: 2318923195, 1995 H w a , J. J., Ghibaudi, L . , Williams, P., and Chatterjee, M . : Comparison o f acetylcholinedependent relaxation i n large and small arteries o f rat mesenteric vascular bed. Am J Physiol 266: H952-958, 1994 Hwang, I. S., H o , H . , Hoffman, B . B . , and Reaven, G . ML: Fructose-induced insulin resistance and hypertension in rats. Hypertension 10: 512-516, 1987 Hyvelin, J. M . , Guibert, C , Marthan, R . , and Savineau, J. P.: Cellular mechanisms and role o f endothelin-1-induced calcium oscillations in pulmonary arterial myocytes. Am J Physiol 275: L269-282, 1998 Ignarro, L . J.: Heme-dependent activation o f soluble guanylate cyclase by nitric oxide: regulation o f enzyme activity by porphyrins and metalloporphyrins. Semin Hematol 26: 63-76, 1989 Ignarro, L . J.: Biosynthesis and metabolism o f endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol 30: 535-560, 1990a Ignarro, L . J.: Haem-dependent activation o f guanylate cyclase and cyclic G M P formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling. Pharmacol Toxicol 67: 1-7, 1990b Ignarro, L . J., Buga, G . M . , Wood, K . S., Byrns, R . E . , and Chaudhuri, G : Endotheliumderived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84: 9265-9269, 1987 Ignarro, L . J., Harbison, R . G , Wood, K . S., and Kadowitz, P. J.: Dissimilarities between methylene blue and cyanide on relaxation and cyclic G M P formation i n endotheliumintact intrapulmonary artery caused by nitrogen oxide-containing vasodilators and acetylcholine. J Pharmacol Exp Ther 236: 30-36, 1986 Iijima, K . , L i n , L . , Nasjletti, A . , and Goligorsky, M . S.: Intracellular signaling pathway o f endothelin-1. J Cardiovasc Pharmacol 7 7: S146-149, 1991 lino, M . : Calcium release mechanisms i n smooth muscle. Jpn J Pharmacol 54: 345-354, 1990 lino, M . , Kobayashi, T., and Endo, M . : U s e o f ryanodine for functional removal o f the calcium store i n smooth muscle cells o f the guinea-pig. Biochem Biophys Res Commun 152: All-All, 1988  249  Imaizumi, Y . , Takeda, M . , Muraki, K . , and Watanabe, M . : Mechanisms o f NE-induced reduction o f C a current i n single smooth muscle cells from guinea pig vas deferens. Am J Physiol 260: C17-25, 1991 Insel, P . A . , Balboa, M . A . , Mochizuki, N , Post, S. R . , Urasawa, K . , and X i n g , M . : Mechanisms for activation o f multiple effectors by alpha 1-adrenergic receptors. Adv Pharmacol 42: 451-453, 1998 Ionescu, E . , Sauter, J. F., and Jeanrenaud, B . : Abnormal oral glucose tolerance in genetically obese (fa/fa) rats. Am J Physiol 248: E500-506, 1985 Ito, K . , Ikemoto, T., and Takakura, S.: Involvement o f C a influx-induced C a release i n contractions o f intact vascular smooth muscles. Am J Physiol 261: H1464-1470, 1991 2 +  2 +  Iwase, M . , Yamamoto, M . , lino, K . , Ichikawa, K . , Shinohara, N , Yoshinari, M . , and Fujishima, M . : Obesity induced by neonatal monosodium glutamate treatment i n spontaneously hypertensive rats: an animal model o f multiple risk factors. Hypertens Res 21: 1-6, 1998 Jackson, P. S., Churchwell, K . , Ballatori, N , Boyer, J. L . , and Strange, K . : Swellingactivated anion conductance in skate hepatocytes: regulation by cell CI" and A T P . Am J Physiol 270: C57-66, 1996 Jackson, W . F., K o n i g , A . , Dambacher, T., and Busse, R.: Prostacyclin-induced vasodilation in rabbit heart is mediated by ATP-sensitive potassium channels. Am J Physiol 264: H238-243, 1993 Jaffe, E . A . , Grulich, J., Weksler, B . B . , Hampel, G . , and Watanabe, K . : Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels i n cultured human endothelial cells. J Biol Chem 262: 8557-8565, 1987 Jameson, M . , D a i , F . X . , Luscher, T., Skopec, J., Diederich, A . , and Diederich, D . : Endothelium-derived contracting factors in resistance arteries o f young spontaneously hypertensive rats before development o f overt hypertension. Hypertension 21: 280288, 1993 Jensen, P . E . , Mulvany, M . J., and Aalkjaer, C : Endogenous and exogenous agonist-induced changes in the coupling between [ C a ] i and force in rat resistance arteries. Pflugers Arch 420: 536-543, 1992 2+  Johns, A . , Lategan, T. W . , Lodge, N . J., Ryan, U . S., V a n Breemen, C , and Adams, D . J.: Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells. Tissue Cell 19: 733-745, 1987  250  Julou-Schaeffer, G . and Freslon, J. L . : Effects o f ryanodine on tension development in rat aorta and mesenteric resistance vessels. Br J Pharmacol 95: 605-613, 1988 K a m , K . L . , Pfaffendorf, M . , and van Zwieten, P. A . : Pharmacodynamic behaviour o f isolated resistance vessels obtained from hypertensive-diabetic rats. Fundamental & Clinical Pharmacology 10: 329-336, 1996 Kamata, K . , Numazawa, T., and Kasuya, Y . : Vasodilator effects o f cionidine on the mesenteric arterial beds in normotensive and spontaneously hypertensive rats. Res Commun Chem Pathol Pharmacol 84: 371-374, 1994 Kamata, K . , Numazawa, T., and Kasuya, Y . : Characteristics o f vasodilatation induced by acetylcholine and platelet-activating factor in the rat mesenteric arterial bed. Eur J Pharmacol 298: 129-136, 1996a Kamata, K . , Umeda, F., and Kasuya, Y . : Possible existence o f novel endothelium-derived relaxing factor in the endothelium o f rat mesenteric arterial bed. J Cardiovasc Pharmacol 27:601-606, 1996b Kaneko, K . and Sunano, S.: Involvement o f alpha-adrenoceptors in the endotheliumdependent depression o f noradrenaline-induced contraction in rat aorta. Eur J Pharmacol 240: 195-200, 1993 Kanmura, Y . , Missiaen, L . , Raeymaekers, L . , and Casteels, R.: Ryanodine reduces the amount o f calcium in intracellular stores o f smooth-muscle cells o f the rabbit ear artery. PflugersArch 413: 153-159, 1988 Karaki, H . , Ozaki, H . , Hori, M . , Mitsui-Saito, M . , Amano, K . , Harada, K . , Miyamoto, S., Nakazawa, H . , W o n , K . J., and Sato, K . : Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev 49: 157-230, 1997 Kasiske, B . L . , Cleary, M . P., O'Donnell, M . P., and Keane, W . F.: Effects o f genetic obesity on renal structure and function in the Zucker rat. Journal of Laboratory & Clinical Medicine 106: 598-604, 1985 Kasiske, B . L . , O'Donnell, M . P., Cleary, M . P., and Keane, W . F.: Treatment o f hyperlipidemia reduces glomerular injury in obese Zucker rats. Kidney Int 33: 667672, 1988 Kasiske, B . L . , O'Donnell, M . P., Lee, H . , K i m , Y . , and Keane, W . F.: Impact o f dietary fatty acid supplementation on renal injury in obese Zucker rats. Kidney Int 39: 1125-1134, 1991 Katholi, R. E . , Winternitz, S. R , and Oparil, S.: Decrease in peripheral sympathetic nervous system activity following renal denervation or unclipping in the one-kidney one-clip Goldblatt hypertensive rat. JClin Invest 69: 55-62, 1982  251  Kato, T., Iwama, Y . , Okumura, K., Hashimoto, H , Ito, T., and Satake, T.: Prostaglandin H2 may be the endothelium-derived contracting factor released by acetylcholine in the aorta of the rat. Hypertension 15: 475-481, 1990 Katusic, Z. S.: Superoxide anion and endothelial regulation of arterial tone. Free Radio Biol Med 20: 443-448, 1996 Katusic, Z. S., Schugel, J., Cosentino, F., and Vanhoutte, P. M . : Endothelium-dependent contractions to oxygen-derived free radicals in the canine basilar artery. Am J Physiol 264: H859-864, 1993 Katusic, Z. S., Shepherd, J. T., and Vanhoutte, P. M . : Endothelium-dependent contractions to calcium ionophore A23187, arachidonic acid, and acetylcholine in canine basilar arteries. Stroke 19: 476-479, 1988 Katusic, Z. S. and Vanhoutte, P. M . : Superoxide anion is an endothelium-derived contracting factor. Am J Physiol 257: H33-37, 1989 Kawasaki, H., Takasaki, K., Saito, A., and Goto, K.: Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature 335: 164-167, 1988 Kawazoe, T., Kosaka, H., Yoneyama, H., and Hata, Y : Involvement of superoxide in acute reaction of angiotensin II in mesenteric microcirculation. Jpn J Physiol 49: 437-443, 1999 Kawazoe, T., Kosaka, H . , Yoneyama, H . , and Hata, Y : Acute production of vascular superoxide by angiotensin II but not by catecholamines. J Hypertens 18: 179-185, 2000 Keen, H . L . , Brands, M . W., Smith, M . J., Jr., Shek, E . W., and Hall, J. E.: Inhibition of thromboxane synthesis attenuates insulin hypertension in rats. Am J Hypertens 10: 1125-1131, 1997 Kemmer, F. W., Berger, M . , Herberg, L., Gries, F. A., Wirdeier, A., and Becker, K.: Glucose metabolism in perfused skeletal muscle. Demonstration of insulin resistance in the obese Zucker rat. BiochemJ 178:12>3-1A\, 1979 Kemp, B. K., Smolich, J. J., Ritchie, B. C , Cocks, T. M . : Endothelium-dependent relaxations in sheep pulmonary arteries and veins: resistance to block by NG-nitro-L-arginine in pulmonary hypertension. Br J Pharmacol 116: 2457-2467, 1995 Khan, M . T., Jothianandan, D., Matsunaga, K., and Furchgott, R. F.: Vasodilation induced by acetylcholine and by glyceryl trinitrate in rat aortic and mesenteric vasculature. J Vase Res 29: 20-28, 1992  252  Kilpatrick, E . V . and Cocks, T. M . : Evidence for differential roles o f nitric oxide ( N O ) and hyperpolarization in endothelium-dependent relaxation o f pig isolated coronary artery. Br J Pharmacol 112: 557-565, 1994 Kirkup, A . J., Edwards, G . , Green, M . E . , Miller, M . , Walker, S. D . , Weston, A . H . : Modulation o f membrane currents and mechanical activity by N F A in rat vascular smooth muscle. Eur J Pharmacol 317: 165-174, 1996a Kirkup, A . J., Edwards, G , and Weston, A . H . : Investigation o f the effects o f 5-nitro-2-(3phenylpropylamino)-benzoic acid ( N P P B ) on membrane currents in rat portal vein. Br J Pharmacol 117: 175-183, 1996b Kita, S., Taguchi, Y . , and Matsumura, Y . : Endothelin-1 enhances pressor responses to norepinephrine: involvement o f endothelin-B receptor. J Cardiovasc Pharmacol 31: S119-121, 1998 Klockner, U . : Intracellular calcium ions activate a low-conductance chloride channel in smooth-muscle cells isolated from human mesenteric artery. Pflugers Arch 424: 231237, 1993 Klockner, U . and Isenberg, G . : Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current. Pflugers Arch 418: 168-175, 1991 Knowles, R. G . and Moncada, S.: Nitric oxide synthases in mammals. Biochem J 298: 249258, 1994 K o m o r i , K . and Vanhoutte, P. M . : Endothelium-derived hyperpolarizing factor. Blood Vessels 27: 238-245, 1990 Koncz, C . and Daugirdas, J. T.: Use o f M Q A E for measurement o f intracellular [Cf] in cultured aortic smooth muscle cells. Am J Physiol 267: H2114-2123, 1994 K o n g , J. Q., Taylor, D . A . , and Fleming, W . W . : Functional distribution and role o f alpha-1 adrenoceptor subtypes in the mesenteric vasculature o f the rat. J Pharmacol Exp Ther 268: 1153-1159, 1994 Kontos, H . A . : Oxygen radicals in cerebral vascular injury. [Review]. Circ Res 57: 508-516, 1985 Kontos, H . A . , W e i , E . P., Ellis, E . F., Jenkins, L . W . , Povlishock, J. T., Rowe, G . T., and Hess, M . L . : Appearance o f superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ Res 57: 142-151, 1985  253  Kreye, V . A . , Kern, R., and Schleich, I.: ^Chloride efflux from noradrenline-stimulated rabbit aorta inhibited by sodium nitroprusside and nitroglycerine. In R. Casteels (ed.): Excitation Contraction Coupling in Smooth Muscle, pp. 145-150, Elsevier/NorthHolland, Amsterdam,The Netherlands, 1977 Krippeit-Drews, P., Morel, N , and Godfraind, T.: Effect o f nitric oxide on membrane potential and contraction o f rat aorta. J Cardiovasc Pharmacol 20: S72-75, 1992 Kruse, H . J., Grunberg, B . , Siess, W . , and Weber, P. C : Formation o f biologically active autacoids is regulated by calcium influx in endothelial cells. Arterioscler Thromb 14: 1821-1828, 1994 Kruszyna, R., Kruszyna, H . , Smith, R . P., Thron, C . D . , and Wilcox, D . E . : Nitrite conversion to nitric oxide in red cells and its stabilization as a nitrosylated valency hybrid o f hemoglobin. J Pharmacol Exp iher 241: 307-313, 1987 Kuchan, M . J. and Frangos, J. A . : Role o f calcium and calmodulin in flow-induced nitric oxide production i n endothelial cells. Am J Physiol 266: C628-636, 1994 Kukreja, R. C , Kontos, H . A . , Hess, M . L . , and Ellis, E . F.: P G H synthase and lipoxygenase generate superoxide in the presence o f N A D H or N A D P H . Circ Res 59: 612-619, 1986 Kung, C. F. and Luscher, T. F.: Different mechanisms o f endothelial dysfunction with aging and hypertension in rat aorta. Hypertension 25: 194-200, 1995 Kuriyama, H . , Kitamura, K . , and Nabata, H . : Pharmacological and physiological significance o f ion channels and factors that modulate them in vascular tissues. Pharmacol Rev 47: 387-573, 1995 Kurtz, T. W . , Morris, R. C , Pershadsingh, H . A . : The Zucker fatty rat as a genetic model o f obesity and hypertension. Hypertension 13: 896-901, 1989 Laakso, M . , Edelman, S. V . , Brechtel, G . , and Baron, A . D . : Decreased effect o f insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J Clin Invest 85: 1844-1852, 1990 Laakso, M . , Edelman, S. V . , Brechtel, G . , and Baron, A . D . : Impaired insulin-mediated skeletal muscle blood flow in patients w i t h N L D D M . Diabetes 41: 1076-1083, 1992 Lagaud, G . J., Skarsgard, P. L . , Laher, I., and van Breemen, C : Heterogeneity o f endothelium-dependent vasodilation in pressurized cerebral and small mesenteric resistance arteries o f the rat. J Pharmacol Exp Ther 290: 832-839, 1999  254  Laight, D . W . , K a w , A . V . , Carrier, M . J., and Anggard, E . E . : Interaction between superoxide anion and nitric oxide i n the regulation o f vascular endothelial function. Br J Pharmacol 124: 238-244, 1998 Lamb, F. S. and Barna, T. J.: Chloride ion currents contribute functionally to norepinephrineinduced vascular contraction. Am J Physiol 275: H I 51-160, 1998 Lamb, F. S., Volk, K . A . , and Shibata, E . F . : Calcium-activated chloride current i n rabbit coronary artery myocytes. Circ Res 75: 742-750, 1994 Lamb, F. S., Kooy, N . W . and Lewis, S. J. : Role o f CI" Channels in a-adrenoceptormediated vasoconstriction in the anesthetized rat. Eur J Pharmacol 40: 403-412, 2000 Lamontagne, D . , Konig, A . , Bassenge, E . , and Busse, R . : Prostacyclin and nitric oxide contribute to the vasodilator action o f acetylcholine and bradykinin in the intact rabbit coronary bed. J Cardiovasc Pharmacol 20: 652-657, 1992 Land, E . J. and Swallow, A . J.: One-electron reactions i n biochemical systems as studied by pulse radiolysis. I V . Oxidation o f dihydronicotinamide-adenine dinucleotide. Biochim Biophys Acta 234: 34-42, 1971 Landsberg, L . : Diet, obesity and hypertension: an hypothesis involving insulin, the sympathetic nervous system, and adaptive thermogenesis. Q J Med 61: 1081-1090, 1986 Lang, M . G . , N o l l , G , and Luscher, T. F . : Effect o f aging and hypertension on contractility o f resistance arteries: modulation by endothelial factors. Am J Physiol 269: H 8 3 7 844, 1995 Langton, P . D . , Nelson, M . T., Huang, Y . , and Standen, N . B . : B l o c k o f calcium-activated potassium channels i n mammalian arterial myocytes by tetraethylammonium ions. Am J Physiol 260: mil-914, 1991 Large, W . A . : The effect o f chloride removal on the responses o f the isolated rat anococcygeus muscle to alpha 1-adrenoceptor stimulation. J Physiol 352: 17-29, 1984 Large, W . A . and Wang, Q.: Characteristics and physiological role o f the Ca(2+)-activated CI- conductance i n smooth muscle. Am J Physiol 271: C435-454, 1996 Laskey, R . E . , Adams, D . J., Johns, A , Rubanyi, G . M . , and van Breemen, C : Membrane potential and N a - K pump activity modulate resting and bradykinin-stimulated changes i n cytosolic free calcium i n cultured endothelial cells from bovine atria. J Biol Chem 265: 2613-2619, 1990 +  +  255  Lautt, W. W.: Should clinical cardiologists report total peripheral resistance or total peripheral conductance? Can J Cardiol 15: 45-47, 1999 Le Marquer-Domagala, F. and Finet, M . : Comparison of the nitric oxide and cyclooxygenase pathway in mesenteric resistance vessels of normotensive and spontaneously hypertensive rats. Br J Pharmacol 121: 588-594, 1997 Lee, M . W. and Severson, D. L . : Signal transduction in vascular smooth muscle: diacylglycerol second messengers and P K C action. Am J Physiol 267: C659-678, 1994  Lee, R. M . , Forrest, J. B . , Garfield, R. E., and Daniel, E. E.: Ultrastructural changes in mesenteric arteries from spontaneously hypertensive rats. A morphometric study. Blood Vessels 20: 72-91, 1983a Lee, R. M . , Garfield, R. E., Forrest, J. B., and Daniel, E. E.: Morphometric study of structural changes in the mesenteric blood vessels of spontaneously hypertensive rats. Blood Vessels 20: 57-71,1983b Lembo, G , Napoli, R., Capaldo, B., Rendina, V., Iaccarino, G., Volpe, M . , Trimarco, B., and Sacca, L . : Abnormal sympathetic overactivity evoked by insulin in the skeletal muscle of patients with essential hypertension. J Clin Invest 90: 24-29, 1992 Lepretre, N . , Mironneau, J., Arnaudeau, S., Tanfin, Z., Ffarbon, S., Guillon, G., and Ibarrondo, J.: Activation of alpha-1A adrenoceptors mobilizes calcium from the intracellular stores in myocytes from rat portal vein. J Pharmacol Exp Ther 268: 167174, 1994 Levin, B . E., Triscari, J., and Sullivan, A. C : Abnormal sympatho- adrenal function and plasma catecholamines in obese Zucker rats. Pharmacology, Biochemistry & Behavior 13: 107-113, 1980 L i , J. and Bukoski, R. D.: Endothelium-dependent relaxation of hypertensive resistance arteries is not impaired under all conditions. Circ Res 72: 290-296, 1993 L i , J. S., Knafo, L . , Turgeon, A . , Garcia, R., and Schiffrin, E. L . : Effect of endothelin antagonism on blood pressure and vascular structure in renovascular hypertensive rats. Am J Physiol 271: H88-93, 1996 Liang, C , Doherty, J. U . , Faillace, R., Maekawa, K . , Arnold, S., Gavras, H . , and Hood, W. B., Jr.: Insulin infusion in conscious dogs. Effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J Clin Invest 69: 1321-1336, 1982  256  L i n , L . , Balazy, ML, Pagano, P . J., and Nasjletti, A . : Expression o f prostaglandin H2mediated mechanism o f vascular contraction i n hypertensive rats. Relation to lipoxygenase and prostacyclin synthase activities. Circ Res 74: 197-205, 1994 Lincoln, T. M . and Cornwell, T. L . : Towards an understanding o f the mechanism o f action o f cyclic A M P and cyclic G M P in smooth muscle relaxation. Blood Vessels 28: 129137, 1991 Lindsey, C . J., de Paula, U . M . , and Paiva, A . C : Protracted effect o f converting-enzyme inhibition on the rat's response to intraarterial bradykinin. Hypertension 5: V134-137, 1983 Lissner, L . , Bengtsson, C , Lapidus, L . , Kristjansson, K . , and Wedel, H . : Fasting insulin i n relation to subsequent blood pressure changes and hypertension i n women. Hypertension 20: 797-801, 1992 Llahi, S. and Fain, J. N . : Alpha 1-adrenergic receptor-mediated activation o f phospholipase D i n rat cerebral cortex. JBiol Chem 267: 3679-3685, 1992 Loirand, G , Pacaud, P., Baron, A . , Mironneau, C , and Mironneau, J.: Large conductance calcium-activated non-selective cation channel i n smooth muscle cells isolated from rat portal vein. J Physiol 437: 461-475, 1991 Loirand, G , Pacaud, P., Mironneau, C , and Mironneau, J.: GTP-binding proteins mediate noradrenaline effects on calcium and chloride currents i n rat portal vein myocytes. J Physiol 428: 517-529, 1990 Long, C . J. and Stone, T. W . : The release o f endothelium-derived relaxant factor is calcium dependent. Blood Vessels 22: 205-208, 1985 Lopez-Jaramillo, P., Gonzalez, M . C , Palmer, R . M . , and Moncada, S.: The crucial role o f physiological C a concentrations in the production o f endothelial nitric oxide and the control o f vascular tone. Br J Pharmacol 101: 489-493, 1990 2 +  L o w , A . M . , Gaspar, V . , Kwan, C . Y . , Darby, P . I , Bourreau, J. P., and Daniel, E . E . : Thapsigargin inhibits repletion o f phenylephrine-sensitive intracellular C a pool i n vascular smooth muscles. J Pharmacol Expe Ther 258: 1105-1113, 1991 2 +  L o w , A . M . , Kotecha, N . , Neild, T. O., Kwan, C . Y . , and Daniel, E . E . : Relative contributions o f extracellular C a and C a stores to smooth muscle contraction in arteries and arterioles o f rat, guinea-pig, dog and rabbit. Clin Exp Pharmacol Physiol 23:310-316,1996 2 +  2 +  Luckhoff, A . : Release o f prostacyclin and E D R F from endothelial cells is differentially controlled by extra- and intracellular calcium. Eicosanoids 1: 5-11, 1988  257  Luckhoff, A . and Busse, R.: Increased free calcium in endothelial cells under stimulation with adenine nucleotides. J Cell Physiol 126: 414-420, 1986 Luckhoff, A . and Busse, R.: Calcium influx into endothelial cells and formation o f endothelium-derived relaxing factor is controlled by the membrane potential. Pflugers Arch 416: 305-311,1990 Luckhoff, A . and Clapham, D . E . : Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca( )-permeable channel. Nature 355: 356-358, 1992 2+  Luckhoff, A . , Pohl, U . , Mulsch, A . , and Busse, R.: Differential role o f extra- and intracellular calcium in the release o f E D R F and prostacyclin from cultured endothelial cells. Br J Pharmacol 95: 189-196, 1988 Lundgren, O.: Role o f splanchnic resistance vessels in overall cardiovascular homeostasis. Fed Proc 42: 1673-1677, 1983 Lundgren, Y . and Weiss, L . : Cardiovascular design after 'reversal' o f long-standing renal hypertension in rats. Clin Sci 57 Suppl 5: 19s^21s, 1979 Luscher, T. F.: Endothelial control o f vascular tone and growth. [Review]. Clinical & Experimental Hypertension - Part A, Theory & Practice 12: 897-902, 1990 Luscher, T. F., Aarhus, L . L . , and Vanhoutte, P. M . : Indomethacin improves the impaired endothelium-dependent relaxations in small mesenteric arteries o f the spontaneously hypertensive rat. Am J Hypertens 3: 55-58, 1990 Luscher, T. F., Boulanger, C . M . , Dohi, Y . , and Yang, Z . H . : Endothelium-derived contracting factors. i^/?erte«5/'on 7P: 117-130, 1992 Luscher, T. F., Oemar, B . S., Boulanger, C . M . , and Ffahn, A . W . : Molecular and cellular biology o f endothelin and its receptors-Part H J Hypertens 11: 121-126, 1993a Luscher, T. F., Oemar, B . S., Boulanger, C . M . , and Hahn, A . W . A . : Molecular and cellular biology o f endothelin and its receptors. In K . Lindpainter and D . Ganten (eds.): Molecular review in cardiovascular medicine, Chapman and H a l l , London, 1996 Luscher, T. F., Seo, B . G , and Buhler, F. R.: Potential role o f endothelin in hypertension. Controversy on endothelin in hypertension. [Review]. Hypertension 21: 752-757, 1993b Luscher, T. F. and Vanhoutte, P. M . : Endothelium-dependent contractions to acetylcholine in the aorta o f the spontaneously hypertensive rat. Hypertension 8: 344-348, 1986 Luscher, T. F. and Vanhoutte, P. M . : The endothelium: Modulator o f cardiovascular function. C R C Inc, ( U S A ) , 1990  258  M a c L e o d , K . M . , N g , D . D . , Harris, K . H . , and Diamond, J.: Evidence that c G M P is the mediator o f endothelium-dependent inhibition o f contractile responses o f rat arteries to alpha-adrenoceptor stimulation. Mol Pharmacol 32: 59-64, 1987 Mais, D . E . , Saussy, D . L . , Jr., Chaikhouni, A . , Kochel, P . J., Knapp, D . R , Hamanaka, N . , and Halushka, P . V . : Pharmacologic characterization o f human and canine thromboxane A2/prostaglandin H 2 receptors i n platelets and blood vessels: evidence for different receptors. J Pharmacol Exp Ther 233: 418-424, 1985 M a l i k , K . TJ., Ryan, P., and M c G i f f , J. C . : Modification by prostaglandins E l and E 2 , indomethacin, and arachidonic acid o f the vasoconstrictor responses o f the isolated perfused rabbit and rat mesenteric arteries to adrenergic stimuli. Circ Res 39: 163168, 1976 Manicardi, V . , Camellini, L . , Bellodi, G , Coscelli, C , and Ferrannini, E . : Evidence for an association o f high blood pressure and hyperinsulinemia in obese man. Journal of Clinical Endocrinology & Metabolism 62: 1302-1304, 1986 Manku, M . S. and Horrobin, D . F.: Indomethacin inhibits responses to all vasoconstrictors i n the rat mesenteric vascular bed: restoration o f responses by prostaglandin E 2 . Prostaglandins 12: 369-376, 1976 Marchenko, S. M . and Sage, S. O.: Mechanism o f acetylcholine action on membrane potential o f endothelium o f intact rat aorta. Am J Physiol 266: H23 88-2395, 1994 Marchenko, S. M . and Sage, S. O.: Calcium-activated potassium channels i n the endothelium o f intact rat aorta. J Physiol 492: 53-60, 1996 Marks, E . S., Thurston, H . , Bing, R . F . , and Swales, J. D . : Pressor responsiveness to angiotensin i n renovascular and steroid hypertension. Clin Sci 57 Suppl 5: 47s-50s, 1979 Marsden, P. A . , Schappert, K . T., Chen, H . S., Flowers, M . , Sundell, C . L . , Wilcox, J. N . , Lamas, S., and Michel, T.: Molecular cloning and characterization o f human endothelial nitric oxide synthase. FEBSLett 307: 287-293, 1992 Martin, W . , Smith, J. A . , and White, D . G . : The mechanisms by which haemoglobin inhibit the relaxation o f rabbit aorta induced by nitrovasodilators, nitric oxide, or bovine retractor penis inhibitory factor. Br J Pharmacol 89: 563-571, 1986 Martin, W . , Villani, G . M . , Jothianandan, D . , and Furchgott, R . F.: Blockade o f endotheliumdependent and glyceryl trinitrate-induced relaxation o f rabbit aorta by certain ferrous hemoproteins. J Pharmacol Exp Ther 233: 679-685, 1985  259  Martinez-Maldonado, M . : Pathophysiology o f renovascular hypertension. Hypertension 17: 101-119,  1991  Masaki, T.: Possible role o f endothelin in endothelial regulation o f vascular tone. Annu Rev Pharmacol Toxicol 35: 235-255, 1995 Masuo, K . , M i k a m i , H . , Ogihara, T., and Tuck, M . L . : Prevalence o f hyperinsulinemia i n young, nonobese Japanese men. J Hypertens 15: 157-165, 1997 Matrougui, K . , Maclouf, J., Levy, B . I., and Henrion, D . : Impaired nitric oxide- and prostaglandin-mediated responses to flow i n resistance arteries o f hypertensive rats. Hypertension 30: 942-947, 1997 Matsuda, H . , Beppu, S., Ohmori, F., Yamada, M . , and Miyatake, K . : Involvement o f cyclooxygenase-generated vasodilating eicosanoid(s) i n addition to nitric oxide i n endothelin-1-induced endothelium-dependent vasorelaxation i n guinea pig aorta. Heart & Vessels 8: 121-127, 1993 Mayer, B . , Schmidt, K . , Humbert, P., and Bohme, E . : Biosynthesis o f endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells C a dependently converts L-arginine into an activator o f soluble guanylyl cyclase. Biochem Biophys Res Commun 164: 678-685,1989 2 +  Mayer, B . and Werner, E . R . : In search o f a function for tetrahydrobiopterin i n the biosynthesis o f nitric oxide. Naunyn Schmiedebergs Arch Pharmacol 351: 453-463, 1995 M c C u l l o c h , A . I., Bottrill, F . E . , Randall, M . D . , and Hiley, C . R . : Characterization and modulation o f EDHF-mediated relaxations i n the rat isolated superior mesenteric arterial bed. Br J Pharmacol 120: 1431-1438, 1997 McGregor, D . D . : The effect o f sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels o f the rat. J Physiol 177: 21-30, 1965 McGregor, D . D . and Smirk, F . H . : Vascular responses i n mesenteric arteries from genetic and renal hypertensive rats. Am J Physiol 214: 1429-1433, 1968 M c M a h o n , E . G . and Jones, A . W . : Altered chloride transport in arteries from aldosterone salt-hypertensive rats. J Hypertens 6: 593-599, 1988 McPherson, G . A . , Coupar, I. M . , and Taylor, D . A . : Competitive antagonism o f alpha 1adrenoceptor mediated pressor responses i n the rat mesenteric artery. Journal of Pharmacy & Pharmacology 36: 338-340, 1984 McQueen, D . D . : The effect o f control o f blood pressure on vascular reactivity i n experimental renal hypertension. Clinic Science 21: 133-140, 1961  260  Mehta, J. L . , Lawson, D . L . , Yang, B . C , Mehta, P., and Nichols, W . W . : Modulation o f vascular tone by endothelin-1: role o f preload, endothelial integrity and concentration of endothelin-1.5r J Pharmacol 106: 127-132, 1992 Meininger, G . A . , Fehr, K . L . , Yates, M . B . , Borders, J. L . , and Granger, H . J.: Hemodynamic characteristics o f the intestinal microcirculation i n renal hypertension. Hypertension 8: 66-75, 1986 Meininger, G . A , Nyhof, R . A . , and Granger, H . J.: Central and regional hemodynamics during the acute onset o f renal hypertension i n rats. Clinical & Experimental Hypertension - Part A, Theory & Practice 6: 2173-2196, 1984 Meininger, G . A . , Routh, L . K . , and Granger, H . J.: Autoregulation and vasoconstriction i n the intestine during acute renal hypertension. Hypertension 7: 364-373, 1985 Mekata, F . and N i u , H . : Biophysical effects o f adrenaline on the smooth muscle o f the rabbit common carotid artery. J Gen Physiol 59: 92-102, 1972 Melaragno, M . G . and Fink, G . D . : Enhanced slow pressor effect o f angiotensin U i n twokidney, one clip rats. Hypertension 25: 288-293, 1995 Mezzano, V . , Donoso, V . , Capurro, D . , and Huidobro-Toro, J. P.: Increased neuropeptide Y pressor activity in Goldblatt hypertensive rats: i n vivo studies with BTJ3P 3226. Peptides 19: 1227-1232, 1998 Mickley, E . J., Gray, G . A . , and Webb, D . J.: Activation o f endothelin E T A receptors masks the constrictor role o f endothelin E T B receptors in rat isolated small mesenteric arteries. Br J Pharmacol 120: 1376-13 82, 1997 Miller, B . G , Connors, B . A . , Bohlen, H . G , and Evan, A . P.: Cell and wall morphology o f intestinal arterioles from 4- to 6- and 17- to 19-week-old Wistar-Kyoto and spontaneously hypertensive rats. Hypertension 9: 59-68, 1987 Miller, V . M . and Vanhoutte, P . M . : Endothelium-dependent contractions to arachidonic acid are mediated by products o f cyclooxygenase. Am J Physiol 248: H432-437, 1985 M i n , S. A . , Stapleton, M . P., and Tabrizchi, R . : Influence o f chloride ions on alphaladrenoceptor mediated contraction and C a influx i n rat caudal artery. Life Sci 64: 1631-1641, 1999 2 +  Minneman, K . P.: A l p h a 1-adrenergic receptor subtypes, inositol phosphates, and sources o f cell C a . Pharmacol Rev 40: 87-119, 1988 2 +  261  Mironneau, C , Rakotoarisoa, L . , Sayet, I , and Mironneau, J.: Modulation o f [3H]dihydropyridine binding by activation o f protein kinase C in vascular smooth muscle. Eur JPharmacol 208: 223-230, 1991 Mironneau, J. and Macrez-Lepretre, N . : Modulation o f C a channels by alpha 1A- and alpha 2A-adrenoceptors in vascular myocytes: involvement o f different transduction pathways. [Review]. Cell Signal 7: 471-479, 1995 2 +  Mistry, D . K . and Garland, C . J.: Nitric oxide (NO)-induced activation o f large conductance Ca -dependent K channels (BK(c )) in smooth muscle cells isolated from the rat mesenteric artery. Br J Pharmacol 124: 1131-1140, 1998 2+  +  a  Mistry, M . , Bing, R. F., Swales, J. D . , and Thurston, H . : The role o f vascular hypertrophy in early and chronic renovascular hypertension. J Hypertens Suppl 1: 79-81, 1983 Mistry, M . and Nasjletti, A . : Role o f pressor prostanoids in rats with angiotensin IJ-saltinduced hypertension. Hypertension 11: 758-762, 1988 Mitchell, J. H . and Blomqvist, G . : M a x i m a l oxygen uptake. N Engl J Med 284: 1018-1022, 1971 Modan, M . , Ffalkin, EL, Almog, S., Lusky, A . , Eshkol, A . , Shefi, M . , Shitrit, A . , and Fuchs, Z.: Hyperinsulinemia. A link between hypertension obesity and glucose intolerance. J Clin Invest 75: 809-817, 1985 Mohazzab, K . M . , Kaminski, P. M . , and W o l i n , M . S.: N A D H oxidoreductase is a major source o f superoxide anion in bovine coronary artery endothelium. Am J Physiol 266: H2568-2572, 1994 Mombouli, J. V . , Vanhoutte, P. M . : Endothelium-derived hyperpolarizing factor(s): updating the unknown. [Review]. Trends Pharmacol Sci 18: 252-256, 1997 Moncada, S., Gryglewski, R., Bunting, S., and Vane, J. R.: A n enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263: 663-665, 1976a Moncada, S., Gryglewski, R. J., Bunting, S., and Vane, J. R.: A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X ) which prevents platelet aggregation. Prostaglandins 12: 715-737, 1976b Moncada, S., Herman, A . G . , Higgs, E . A . , and Vane, J. R.: Differential formation o f prostacyclin ( P G X or PGI2) by layers o f the arterial wall. A n explanation for the antithrombotic properties o f vascular endothelium. Thromb Res 11: 323-344, 1977  262  Moncada, S., Palmer, R. M . , and Higgs, E . A . : Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991 Moncada, S. and Vane, J. R.: Pharmacology and endogenous roles o f prostaglandin endoperoxides, thromboxane A 2 , and prostacyclin. Pharmacol Rev 30: 293-331, 1978a Moncada, S. and Vane, J. R : Prostacyclin (PGI2), the vascular wall and vasodilatation. In P . M . Vanhoutte and I. Leusen (eds.): Mechanisms of vasodilatation., pp. pp. 107-121, Basel, Karger, 1978b Mondon, C. E . and Reaven, G . M . : Evidence o f abnormalities o f insulin metabolism in rats with spontaneous hypertension. Metabolism 37: 303-305, 1988 Moreland, S., M c M u l l e n , D . M . , Delaney, C . L . , Lee, V . G , and Hunt, J. T.: Venous smooth muscle contains vasoconstrictor E T B - l i k e receptors. Biochem Biophys Res Commun 184: 100-106, 1992 Morton, J. J. and Wallace, E . C : The importance o f the renin-angiotensin system i n the development and maintenance o f hypertension in the two-kidney one-clip hypertensive rat. Clin Sci 64: 359-370, 1983 Mulvany, M . J.: Resistance Vessels in Hypertension. In J. D . Swales (ed.): Textbook of Hyprtension, pp 103, Blackwell Scientific Publications, Oxford, 1994 Mulvany, M . J. and Aalkjaer, C : Structure and function o f small arteries. Physiol Rev 70: 921-961, 1990 Mulvany, M . J. and Halpern, W . : Mechanical properties o f vascular smooth muscle cells in situ. Nature 260: 617-619, 1976 Mulvany, M . J., Hansen, O. K . , and Aalkjaer, C . : Direct evidence that the greater contractility o f resistance vessels in spontaneously hypertensive rats is associated with a narrowed lumen, a thickened media, and an increased number o f smooth muscle cell layers. Circ Res 43: 854-864, 1978 Mulvany, M . J. and Korsgaard, N . : Correlations and otherwise between blood pressure, cardiac mass and resistance vessel characteristics in hypertensive, normotensive and hypertensive/normotensive hybrid rats. JHypertens 1: 235-244, 1983 Mulvany, M . J., Nilsson, H . , and Flatman, J. A . : Role o f membrane potential in the response of rat small mesenteric arteries to exogenous noradrenaline stimulation. J Physiol 332: 363-373, 1982 Munzel, T., Hink, TJ., Heitzer, T., and Meinertz, T.: Role for N A D P H / N A D H oxidase in the modulation o f vascular tone. [Review]. Ann Ny Acad Sci 874: 386-400, 1999  263  Murphy, M . E . and Brayden, J. E . : Nitric oxide hyperpolarizes rabbit mesenteric arteries v i a ATP-sensitive potassium channels. J Physiol 486: 47-58, 1995a Murphy, M . E . and Brayden, J. E . : Apamin-sensitive K channels mediate an endotheliumdependent hyperpolarization i n rabbit mesenteric arteries. J Physiol 489: 723-734, 1995b +  Nagao, T., Illiano, S., and Vanhoutte, P . M . : Heterogeneous distribution o f endotheliumdependent relaxations resistant to NG-nitro-L-arginine i n rats. Am J Physiol 263: H1090-1094, 1992 Nagao, T. and Vanhoutte, P. M . : Hyperpolarization as a mechanism for endotheliumdependent relaxations in the porcine coronary artery. J Physiol 445: 355-367, 1992 Nakane, H . , Miller, F . J., Jr., Faraci, F . M . , Toyoda, K . , and Heistad, D . D . : Gene transfer o f endothelial nitric oxide synthase reduces angiotensin II-induced endothelial dysfunction. Hypertension 35: 595-601, 2000 Nakashima, M . and Vanhoutte, P . M . : Endothelin-1 and -3 cause endothelium-dependent hyperpolarization in the rat mesenteric artery. Am J Physiol 265: H2137-2141, 1993 Nakayama, S., Smith, L . M . , Tomita, T., and Brading, A . F.: Multiple open states o f calcium channels and their possible kinetic schemes,. In T. a. T. Bolton, T. (ed.): Smooth Muscle Excitation, pp. 13-25, Academic Press, London, 1996 Nanjo, T.: Effects o f noradrenaline and acetylcholine on electro-mechanical properties o f the guinea-pig portal vein. Br J Pharmacol 81: 427-440, 1984 Nava, P., Collados, M . T., Masso, F., and Guarner, V . : Endothelin mediation o f insulin and glucose-induced changes in vascular contractility. Hypertension 30: 825-829, 1997 Nava, P., Guarner, V . , Posadas, R . , Perez, I., and Banos, G . : Insulin-induced endothelin release and vasoreactivity in hypertriglyceridemic and hypertensive rats. Am J Physiol 277: H399-404, 1999 Nelson, M . T., Huang, Y . , Brayden, J. E . , Hescheler, J., Standen, N . B.:Arterial dilations i n reponse to calcitonin gene-related peptide involve activaton o f K channels. Nature 344: 110-113. 1990a +  Nelson, M . T., Patlak, J. B . , Worley, J. F., and Standen, N . B . : Calcium channels, potassium channels, and voltage dependence o f arterial smooth muscle tone. Am J Physiol 259: C3-18, 1990b Nelson, M . T. and Quayle, J. M . : Physiological roles and properties o f potassium channels i n arterial smooth muscle. Am J Physiol 268: C799-822, 1995  264  Nelson, M . T., Standen, N . B . , Brayden, J. E . , and Worley, J. F . d.: Noradrenaline contracts arteries by activating voltage-dependent calcium channels. Nature 336: 382-385, 1988 Nielsen, H . , Pilegaard, H . K . , Hasenkam, J. M . , Mortensen, F . V . , and Mulvany, M . J.: Heterogeneity o f postjunctional alpha-adrenoceptors i n isolated mesenteric resistance arteries from rats, rabbits, pigs, and humans. J Cardiovasc Pharmacol 18: 4-10, 1991 Nilius, B . , Eggermont, J., Voets, T., and Droogmans, G . : Volume-activated C I - channels. Gen Pharmacol 27: 1131-1140, 1996 Nilius, B . , Szucs, G , Heinke, S., Voets, T., and Droogmans, G : Multiple types o f chloride channels in bovine pulmonary artery endothelial cells. J Vase Res 34: 220-228, 1997a Nilius, B . , Viana, F . , and Droogmans, G : Ion channels in vascular endothelium. Annu Rev Physiol 59: 145-170, 1997b Nilsson, H . : Different nerve responses i n consecutive sections o f the arterial system. Acta PhysiolScand 121: 353-361, 1984 Nilsson, H . : Adrenergic nervous control o f resistance and capacitance vessels. Studies on isolated blood vessels from the rat. Acta Physiol Scand Suppl 541: 1-34, 1985 Nilsson, H . , Goldstein, M . , and Nilsson, O.: Adrenergic innervation and neurogenic response in large and small arteries and veins from the rat. Acta Physiol Scand 126: 121-133, 1986 Nilsson, H . , Jensen, P. E . , and Mulvany, M . J.: M i n o r role for direct adrenoceptor-mediated calcium entry in rat mesenteric small arteries. J Vase Res 31: 314-321, 1994 Nishida, K . , Harrison, D . G . , Navas, J. P., Fisher, A . A . , Dockery, S. P., Uematsu, M . , Nerem, R . M . , Alexander, R . W . , and Murphy, T. J.: Molecular cloning and characterization o f the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 90: 2092-2096, 1992 Nishio, E . , Nakata, H . , Arimura, S., and Watanabe, Y . : alpha-1-Adrenergic receptor stimulation causes arachidonic acid release through pertussis toxin-sensitive G T P binding protein and J N K activation in rabbit aortic smooth muscle cells. Biochemical & Biophysical Research Communications 219: 277-282, 1996 Nishizuka, Y . : Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9: 484-496, 1995  265  N o l l , G , Lang, M . G , Tschudi, M . R . , Ganten, D . , and Luscher, T. F . : Endothelial vasoconstrictor prostanoids modulate contractions to acetylcholine and A N G II i n Ren-2 rats. Am J Physiol 272: H493-500, 1997 O'Donnell, M . E . and Owen, N . E . : Atrial natriuretic factor stimulates N a / K / C l cotransport i n vascular smooth muscle cells. Proc Natl Acad Sci U SA 83: 6132-6136, 1986 Oliver, F . J., de la Rubia, G , Feener, E . P., Lee, M . E . , Loeken, M . R . , Shiba, T., Quertermous, T., and K i n g , G . L . : Stimulation o f endothelin-1 gene expression by insulin in endothelial cells. J Biol Chem 266: 23251-23256, 1991 Oshita, M . , Kigoshi, S., and Muramatsu, I.: Three distinct binding sites for [3H]-prazosin i n the rat cerebral cortex. Br J Pharmacol 104: 961-965, 1991 Osol, G , Laher, I., and Cipolla, M . : Protein kinase C modulates basal myogenic tone i n resistance arteries from the cerebral circulation. Circ Res 68: 359-367, 1991 Ottolia, M . and Toro, L . : Potentiation o f large conductance K C a channels by niflumic, flufenamic, and mefenamic acids. BiophysJ 67: 2272-2279, 1994 Ouchi, Y . , Han, S. Z . , K i m , S., Akishita, M . , K o z a k i , K . , Toba, K . , Orimo, H . : Augmented contractile function and abnormal C a handling i n the aorta o f Zucker obese rats with insulin resistance. Diabetes 45: s55-58, 1996 2 +  Pacaud, P., Loirand, G . , Baron, A . , Mironneau, C , and Mironneau, J.: C a channel activation and membrane depolarization mediated by C I - channels i n response to noradrenaline in vascular myocytes. Br J Pharmacol 104: 1000-1006, 1991 2 +  Pacaud, P., Loirand, G . , Gregoire, G . , Mironneau, C , and Mironneau, J.: Calciumdependence o f the calcium-activated chloride current in smooth muscle cells o f rat portal vein. Pjlugers Arch 421: 125-130, 1992 Pacaud, P., Loirand, G , Gregoire, G , Mironneau, C , and Mironneau, J.: Noradrenalineactivated heparin-sensitive C a entry after depletion o f intracellular C a store in portal vein smooth muscle cells. J Biol Chem 268: 3866-3872, 1993 2 +  2 +  Pacaud, P., Loirand, G , Lavie, J. L . , Mironneau, C , and Mironneau, J.: Calcium-activated chloride current i n rat vascular smooth muscle cells in short-term primary culture. PflugersArch 413: 629-636, 1989a Pacaud, P., Loirand, G , Mironneau, C , and Mironneau, J.: Noradrenaline activates a calcium-activated chloride conductance and increases the voltage-dependent calcium current in cultured single cells o f rat portal vein. Br J Pharmacol 97: 139-146, 1989b Palmer, R . M . , Ashton, D . S., and Moncada, S.: Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664-666, 1988  266  Palmer, R . M . , Ferrige, A . G . , and Moncada, S.: Nitric oxide release accounts for the biological activity o f endothelium-derived relaxing factor. Nature 327: 524-526, 1987 Pals, D . T., Masucci, F . D . , Denning, G . J., Sipos, F., and Fessler, D . C : Role o f the pressor action o f angiotensin TJ i n experimental hypertension. Circ Res 29: 673-681, 1971 Pamnani, M . B . , Bryant, H . J., Harder, D . R . , and Haddy, F . J.: Vascular smooth muscle membrane potentials in rats with one-kidney, one clip and reduced renal mass-saline hypertension: the influence o f a humoral sodium pump inhibitor. JHypertens 3(Supp 3): S29-S31, 1985 Parkington, H . C , Tare, M . , Tonta, M . A . , and Coleman, H . A . : Stretch revealed three components in the hyperpolarization o f guinea-pig coronary artery i n response to acetylcholine. J Physiol 465: 459-476, 1993 Parkington, H . C , Tonta, M . A . , Coleman, H . A . , and Tare, M . : Role o f membrane potential in endothelium-dependent relaxation o f guinea-pig coronary arterial smooth muscle. J Physiol 484: 469-480, 1995 Parsaee, H . , M c E w a n , J. R , Joseph, S., and MacDermot, J.: Differential sensitivities o f the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium i n bovine aortic endothelial cells. Br J Pharmacol 107: 1013-1019, 1992 Parsons, S. J., H i l l , A , Waldron, G . J., Plane, F . , and Garland, C . J.: The relative importance o f nitric oxide and nitric oxide-independent mechanisms i n acetylcholine-evoked dilatation o f the rat mesenteric bed. Br J Pharmacol 113: 1275-1280, 1994 Paulson, D . J. and Tahiliani, A . G . : Cardiovascular abnormalities associated with human and rodent obesity. Life Sci 51: 1557-1569, 1992 Pawloski, C . M . , Kanagy, N . L . , Mortensen, L . H . , and Fink, G . D . : Obese Zucker rats are normotensive on normal and increased sodium intake. Hypertension 19:190-95, 1992 Peach, M . J., Singer, H . A . , Izzo, N . J., Jr., and Loeb, A . L . : Role o f calcium in endotheliumdependent relaxation o f arterial smooth muscle. Am J Cardiol 59: 35A-43A, 1987 Peng, W . , Hoidal, J. R . , and Farrukh, I S . : Regulation o f Ca(2+)-activated K channels in pulmonary vascular smooth muscle cells: role o f nitric oxide. J Appl Physiol 81: 1264-1272, 1996 +  Peredo, H . A . , Feleder, E . C , and Adler-Graschinsky, E . : Differential effects o f acetylcholine and bradykinin on prostanoid release from the rat mesenteric bed: role o f endothelium and o f nitric oxide. Prostaglandins Leukot Essent Fatty Acids 56: 253-258, 1997  267  Perez, D . M , DeYoung, M . B . , and Graham, R. M . : Coupling o f expressed alpha I B - and alpha lD-adrenergic receptor to multiple signaling pathways is both G protein and cell type specific. Mol Pharmacol 44: 784-795, 1993 Piascik, M . T., Smith, M . S., Soltis, E . E . , and Perez, D . M . : Identification o f the m R N A for the novel alpha lD-adrenoceptor and two other alpha 1-adrenoceptors in vascular smooth muscle. Mol Pharmacol 46: 30-40, 1994 Piper, P. and Vane, J.: The release o f prostaglandins from lung and other tissues. Ann N y Acad Sci 180: 363-385, 1971 Pipili, E . : A study on the postjunctional excitatory alpha-adrenoreceptor subtypes in the mesenteric arterial bed of the rat. JAuton Pharmacol 6: 125-132, 1986 Pipili, E . , Zoumboulis, G , and Maragoudakis, M . E . : Prostaglandin 12 and thromboxane A 2 production in relation to alpha 1 and alpha 2-adrenoreceptor activation in the normotensive and hypertensive rat. JAuton Pharmacol 8: 333-342, 1988 Pirotton, S., Raspe, E . , Demolle, D . , Erneux, C , and Boeynaems, J. M . : Involvement o f inositol 1,4,5-trisphosphate and calcium in the action o f adenine nucleotides on aortic endothelial cells. J Biol Chem 262: 17461-17466, 1987 Plane, F., Garland, C . J.: Influence o f contractile agonists on the mechanism o f endotheliumdependent relaxation in rat isolated mesenteric artery. Br J Pharmacol 119: 191-193, 1996 Plane, F . , Holland, M . , Waldron, G . J., Garland, C . J., Boyle, J. P.: Evidence that anandamide and E D H F act via different mechanisms in rat isolated mesenteric arteries. Br J Pharmacol 121: 1509-1511,1997 Plane, F., Hurrell, A . , Jeremy, J. Y . , Garland, C . J.: Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation o f rat isolated mesenteric artery. Br J Pharmacol 119: 1557'-1562, 1996 Plane, F . , Pearson, T., Garland, C . J.: Multiple pathways underlying endothelium-dependent relaxation in the rabbit isolated femoral artery. Br J Pharmacol 115: 31-38, 1995 Plane, F., Wiley, K . E . , Jeremy, J. Y . , Cohen, R. A . , and Garland, C . J.: Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery. Br J Pharmacol 123: 1351-1358, 1998 Popp, R . , Bauersachs, J., Hecker, M . , Fleming, I., and Busse, R . : A transferable, betanaphthoflavone-inducible, hyperpolarizing factor is synthesized by native and cultured porcine coronary endothelial cells. J Physiol 497: 699-709, 1996  268  Pryor, W . A . : Free radicals and lipid peroxidation: What they are and how they got that way. In B . Frei (ed.): Natural antioxidants in human health and disease, pp. 1-24, Academic Press, Boston, 1994 Purkerson, M . L . , Martin, K . J., Yates, J., Kissane, J. M . , and Klahr, S.: Thromboxane synthesis and blood pressure in spontaneously hypertensive rats. Hypertension 8: 1113-1120,1986 Putney, J. W . , JR.: Phosphoinositides and alpha-1 adrenergic receptors. In R. R. J. Ruffolo (ed.): The alpha-1 Adrenergic Receptors, pp. 189-208, Humana Press, Clifton, N J , 1987 Putney, J. W . , Jr.: Capacitative calcium entry revisited. Cell Calcium 11: 611-624, 1990 Putney, J. W . , Jr.: The capacitative model for receptor-activated Pharmacol 22: 251 -269, 1991  calcium entry.  Adv  Quignard, J. F., Feletou, M . , Edwards, G . , Duhault, J., Weston, A . H . , and Vanhoutte, P . M . : Role o f endothelial cell hyperpolarization in EDHF-mediated responses in the guineapig carotid artery. Br J Pharmacol 129: 1103-1112, 2000 Quignard, J. F., Feletou, M . , Thollon, C , Vilaine, J. P., Duhault, J., and Vanhoutte, P . M . : Potassium ions and endothelium-derived hyperpolarizing factor in guinea-pig carotid and porcine coronary arteries. Br J Pharmacol 127: 27-34, 1999 Quilley, J., Fulton, D . , and M c G i f f , J. C : Hyperpolarizing factors. [Review]. Biochem Pharmacol 54: 1059-1070, 1997 Quilley, J., M c G i f f , J. C , and Nasjletti, A : Role o f endoperoxides in arachidonic acidinduced vasoconstriction in the isolated perfused kidney o f the rat. Br J Pharmacol 96: 111-116, 1989 Raat, N . J., Wetzels, G . E . , and D e M e y , J. G . : Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects o f exogenous and neurogenic noradrenaline. Pflugers Arch 436: 262-269, 1998 Radomski, M . W . , Palmer, R . M . , and Moncada, S.: The anti-aggregating properties o f vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 92: 639-646, 1987 Rajagopalan, S., Kurz, S., Munzel, T., Tarpey, M . , Freeman, B . A . , Griendling, K . K., and Harrison, D . G : Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane N A D H / N A D P H oxidase activation. Contribution to alterations o f vasomotor tone. J Clin Invest 97: 1916-1923, 1996  269  Rakugi, H . , Tabuchi, Y . , Nakamaru, M . , Nagano, M . , Higashimori, K . , M i k a m i , H . , Ogihara, T., and Suzuki, N : Evidence for endothelin-1 release from resistance vessels o f rats in response to hypoxia. Biochem Biophys Res Commun 169: 973-977, 1990 Randall, M . D . , Alexander, S. P., Bennett, T., Boyd, E . A . , Fry, J. R., Gardiner, S. M . , Kemp, P. A . , M c C u l l o c h , A . I., and Kendall, D . A . : A n endogenous cannabinoid as an endothelium-derived vasorelaxant. Biochem Biophys Res Commun 229: 114-120, 1996 Randall, M . D . , K a y , A . P., and Fliley, C . R.: Endothelium-dependent modulation o f the pressor activity o f arginine vasopressin in the isolated superior mesenteric arterial bed o f the rat. Br J Pharmacol 95: 646-652, 1988 Randall, M . D . and Kendall, D . A . : Involvement o f a cannabinoid in endothelium-derived hyperpolarizing factor-mediated coronary vasorelaxation. Eur J Pharmacol 335: 205209, 1997 Randall, M . D . and Kendall, D . A . : Anandamide and endothelium-derived hyperpolarizing factor act via a common vasorelaxant mechanism in rat mesentery. Eur J Pharmacol 346: 51-53, 1998 Randall, M . D . , M c C u l l o c h , A . I., and Kendall, D . A . : Comparative pharmacology o f endothelium-derived hyperpolarizing factor and anandamide in rat isolated mesentery. Eur J Pharmacol 333: 191-197, 1997 Rapoport, R. M . , Schwartz, K . , and Murad, F.: Effects o f N a , K - p u m p inhibitors and membrane depolarizing agents on acetylcholine-induced endothelium-dependent relaxation and cyclic G M P accumulation in rat aorta. Eur J Pharmacol 110: 203-209, 1985 +  +  Reaven, G . M . : Banting lecture 1988. Role o f insulin resistance in human disease. Diabetes 37: 1595-1607, 1988 Rebolledo, A . , Milesi, V . , Alvis, A . G . , Rinaldi, G . J., and Grassi de Gende, A . O.: Role o f insulin preincubation i n the contractile reactivity o f rat aortic rings. Can J Physiol Pharmacol 76: 1066-1071, 1998 Reed, P. W . and Lardy, H . A . : A23187: a divalent cation ionophore. J Biol Chem 247: 69706977, 1972 Rees, D . D . , Palmer, R. M . , Hodson, H . F., and Moncada, S.: A specific inhibitor o f nitric oxide formation from L-arginine