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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 T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Pharmacology & Therapeutics, Faculty of Medicine) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A April 2001 ©Yi H e , 2001 In p resen t ing this thesis in partial fu l f i lment of the r e q u i r e m e n t s for an a d v a n c e d d e g r e e at the Univers i ty of British C o l u m b i a , I agree that the Library shall m a k e it f reely available fo r re fe rence and study. I further agree that p e r m i s s i o n fo r ex tens ive c o p y i n g of this thesis fo r scholar ly p u r p o s e s may be g ran ted by the h e a d o f m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n of this thesis for f inancial ga in shall no t be a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t of The Un ivers i ty of Brit ish C o l u m b i a V a n c o u v e r , C a n a d a D E - 6 (2 /88) 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 of 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 of hypertension. The purpose of 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 of the study the contribution of 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 of CI" channels with niflumic acid (NFA) significantly inhibited cirazoline-induced vasoconstriction in isolated M A B from both groups of rats. Cirazoline-evoked vasoconstriction was also significantly inhibited following removal of CI" from the perfusion buffer. Removal o f CI" resulted in a significantly greater inhibition of cirazoline-mediated vasoconstriction in M A B from sham rats as compared with 2 K 1 C rats. In vivo, intravenous infusion of 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. U l To further investigate how the CI" channel blockade impaired ai-adrenoceptor-mediated vasoconstriction, the inhibitory effect of 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 (NFDP) . 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 of N F A and N F D P were not additive. In addition, in the absence of extracellular C a 2 + , the transient 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 of V O C s . The contribution of 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 of the dissertation, the role of CI" channels in endothelium-dependent 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 of CI" channels with N F A had no effect on the dilator responses to A C h in either mesenteric arteries or aorta. The B K C a antagonist, T E A , decreased the potency (pD 2 ) to A C h without affecting the maximum response (Rm a x) in mesenteric arteries, whereas it had no effect in aorta. In the presence of 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 a , CI" channels play a functional role in ACh-induced endothelium-dependent relaxation in aorta, possibly by preventing the depolarization-mediated inactivation of receptor-operated C a 2 + channels (ROC), thereby resulting in a sustained C a 2 + influx and N O synthesis. B y contrast, in mesenteric arteries, K + 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 of PGI2 is negligible, while the contribution of N O is small in mesenteric arteries. Furthermore, the L - N M M A / indomethacin-insensitive component of the relaxation response of mesenteric artery to A C h was greatly inhibited in the presence of S K c a and B K c a antagonists. H igh K + (30 mM) 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 of 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 of the dissertation, the contribution of endogenous E D R F (NO) 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 of 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 N E (90 nmol) tested. Inhibition of N O synthesis with L - N M M A enhanced the vasoconstriction to N E , while blockade of prostanoid production by indomethacin decreased the N E response. A pathophysiological concentration of insulin (200 mU/1) potentiated responses to the two lowest concentrations of 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 of N O synthesis, while it was prevented by inhibition of prostanoid production. These data suggested that NE-induced vasoconstrictor responses are normally modulated by concurrent release of N O and vasoconstrictor cyclooxygenase product(s) in M A B from both obese and lean Zucker rats. Insulin increases the release of contracting cyclooxygenase product(s) and enhances reactivity to N E in M A B from obese rats. This altered action of insulin may play a role in hypertension in this hyperinsulinemic/insulin resistant model. TABLE OF CONTENTS A B S T R A C T i i T A B L E OF C O N T E N T S v i L I S T OF T A B L E S x i i L I S T OF F I G U R E S x i i i L I S T OF A B B R E V I A T I O N S xv A C K N O W L E D G E M E N T S xv i i i D E D I C A T I O N x ix I N T R O D U C T I O N 1 I. OVERVIEW 1 II. 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 Bed 6 III. A L P H A I - A D R E N O C E P T O R S A N D VASOCONSTRICTION 7 1. The cti-Adrenoceptors 7 2. Alphai-Adrenoceptor Subtypes 8 3. Alphai-Adrenoceptor Signaling and C a 2 + M o b i l i z a t i o n 9 4. Calcium, and oci-Adrenoceptor-Induced Contraction 11 5. Calcium Influx Channels, Voltage Dependence and Activation by ai-Adrenoceptors 15 6 Possible Role of CI" Channels in C a 2 + influx and Smooth Muscle Contraction 18 IV. ENDOTHELIUM-MEDIATED REGULATION O F MESENTERIC ARTERIAL TONE 26 1. Endothelium-Derived Vasorelaxing Factors 26 1.1 Nitric Oxide (NO) 26 vii 1.2 Prostacyclin (PGI2) 37 1.3 EDHF 39 2. Endothelium-Derived Contracting Factors 45 2.1 Endothelin-1 45 2.2 Prostanoids: PGH2, TxA2 48 2.3 Superoxide Anion (02) 49 V. ABNORMALITIES IN HYPERTENSION 52 1. Goldblatt 2 K 1 C Renovascular Hypertension 52 2. Hypertension and Hyperinsulinemia/Insulin Resistance 58 V I . SUMMARY 66 P A R T I . T H E CONTRIBUTION O F C H L O R I D E C H A N N E L S T O a i - A D R E N O C E P T O R M E D I A T E D VASOCONSTRICTION IN R A T M E S E N T E R I C A R T E R Y 68 I. RATIONALE 68 II. WORKING HYPOTHESES AND SPECIFIC RESEARCH OBJECTIVES 73 III. METHODS AND MATERIALS 76 1. Surgical Preparation of Hypertensive Rats 76 2. Measurement of Plasma Renin Activity 77 3. Perfused Isolated Mesenteric Artery Preparation 77 4. Experimental Protocols in Perfused M A B 78 5. In vivo Measurement of Blood Flow and Vascular Conductance 81 6. Experimental Protocols for in vivo Experiments 81 7. Isolation of Small Mesenteric Arteries 82 8. Experimental Protocols for Measurement of 1 2 5 I Efflux in small mesenteric arteries 82 9. Chemicals 83 10. Data and Statistical Analysis 83 IV. RESULTS 85 1. Characteristics of 2 K 1 C Hypertensive Rats 85 2. Effect of N F A on Cirazoline-Induced Vasoconstriction in via Isolated Mesenteric Arteries Perfused with Normal Krebs 85 3. Effect of N F A on Cirazoline-Induced Vasoconstriction in Isolated Mesenteric Arteries Perfused with Cl"-Free Buffer 91 4. Influence of N F A on Cirazoline-Induced Change in Mesenteric Vascular Conductance in Anaesthetized 2 K 1 C Hypertensive and Sham Normotensive Rats 100 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 104 6. Effects of N F A on Cirazoline- and KCl-Induced Vasoconstriction in Isolated M A B Perfused with L o w C a 2 + Solution 107 7. Effect of N F A on Cirazoline-Induced Vasoconstriction in Isolated M A B Perfused with C a 2 + - F r e e - E G T A Solution 107 8. 1 2 5 I Efflux from Small Mesenteric Arteries 116 V . D I S C U S S I O N 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 V I S U M M A R Y 131 V I I C O N C L U S I O N S 133 P A R T 2. T H E M E C H A N I S M S O F A C E T Y L C H O L I N E - I N D U C E D R E L A X A T I O N IN R A T M E S E N T E R I C A R T E R Y : A C O M P A R I S O N W I T H A O R T A 134 I. R A T I O N A L E 134 II. W O R K I N G H Y P O T H E S E S 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 13 8 in. M E T H O D S A N D M A T E R I A L S 140 1. Isolated Artery Ring Preparation for Isometric Tension Measurement 140 2. Experimental Protocols 140 3. Chemicals 141 ix 4. Statistical Analysis 142 IV. RESULTS 143 1. ACh-induced Relaxation 143 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 151 2. A23187-Induced Relaxation 151 2.1 The Effect of NFA and TEA on A23187-Induced Relaxation in Rat Aorta and Mesenteric Arteries 151 2.2 Effect of L-NMMA and iC on A23187-Induced Relaxation of PE-Evoked Tension 152 3. Effect of KC1 and K(ca> Channel Blockade on ACh-induced NO-Independent Relaxation 152 V . DISCUSSION 161 Aorta 161 Effect of NFA and TEA 161 NO-Mediated and NO Independent Relaxation 164 Mesenteric Artery 167 Effect of NFA and TEA 167 NO-Mediated and NO Independent Relaxation 168 Aorta and Mesenteric Artery 171 Effect ofPGh in ACh- Induced Relaxation in Aorta and Mesenteric Arteries 171 EndotheHum-dependent relaxation to A23187 in aorta and mesenteric arteries 172 V i . SUMMARY 177 vn CONCLUSIONS 179 VIII PHYSIOLOGICAL SIGNIFICANCE 180 X PART. 3 NOREPINEPHRINE-INDUCED V A S O C O N S T R I C T I O N IN I S O L A T E D PERFUSED M A B F R O M O B E S E 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 8 6 1. General Methodology 186 Animals 186 Blood pressure measurement 186 Biochemical analysis of blood samples 186 Perfused isolated M A B preparation 187 2. Experimental Protocols 187 3. Chemicals 188 4. Statistical Analysis 189 IV. RESULTS 193 1. General Characteristics o f Zucker Rats 193 2. NE-Induced Vasoconstriction in Isolated Perfused M A B from Obese and Lean Zucker Rats 193 3. Effect of N O S and/or C O X Inhibition on NE-Induced Responses 193 4. Effect of Insulin on NE-Induced Vasoconstriction in Isolated Perfused M A B 197 5. Influence of N O S , C O X , PGH2/TXA2 Receptor and E T Receptor Inhibition on Insulin-Potentiation of N E Responses 202 V . DISCUSSION 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 206 2. Blockade of the NO synthesis enhanced vasoconstrictor responses to NE 207 3. 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 213 5. Lack of Influence of Endothelin on Responses to NE 213 Insulin Effect on Vasoconstrictor Responses to NE in MAB of Zucker Rats 214 1. Hyperinsulinemia elevated pressor responses to NE in MAB from obese rats 214 2. Blockade of NO synthesis enhanced vascular effect of insulin in obese rats 215 3. Inhibition of COX blocked insulin effect in MAB 216 4. ET-1 contributing to potentiating effect of insulin on responses to NE in obese rats 217 VI . SUMMARY 220 VII. CONCLUSIONS 222 C O N C L U D I N G R E M A R K S 223 BIBLIOGRAPHY 225 c Xll LIST OF TABLES Table Page 1.1 Physiological characteristics of 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 1.3 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 1.4 Effects o f N F A (3 mg/kg) on cirazoline-induced decreases in vascular conductance (% of control) in superior mesenteric artery in anesthetized 2 K 1 C and sham rats 103 1.5 Effects of prazosin and N F A on cirazoline-induced 1 2 5 I efflux in isolated small mesenteric arteries 119 2.1 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 2.2 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 of T E A or N F A plus T E A in intact rat aortic and mesenteric artery rings 150 2.3 Effects of K(ca) channel blockers or KC1 (30 mM) on sensitivity and maximum relaxation to A C h in intact mesenteric artery rings 160 3.1 Physiological characteristics of lean and obese Zucker rats 194 x i i i LIST OF FIGURES Figure Page 0.1 Alphai-adrenoceptor signaling pathway in vascular smooth muscle cells 12 0.2 ACh-induced release of E D R F s in endothelial cells 28 1.1 Effect of vehicle on vasoconstrictor responses to cirazoline in isolated M A B from 2 K 1 C or sham rats perfused with normal Krebs 87 1.2 Effect of 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 1.3 Effect of N F A on vasoconstriction to KC1 in M A B from 2 K 1 C or sham rats perfused with normal Krebs 92 1.4 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 1.5 Effects of 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 1.6 Effects of 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 1.7 Effect of nifedipine (NFDP) 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 SD rats perfused with normal Krebs 105 1.8 Effect of low C a 2 + buffer and vehicle on pressor responses to cirazoline (A) and KC1 (B) in M A B from SD rats 108 1.9 Effect of N F A (3 u M in A , 10 u M in B ) on pressor responses to cirazoline in M A B from SD rats perfused with low Ca + buffer 110 1.10 Effect of N F A (3 u M in A , 10 u M in B ) on contraction to KC1 in M A B from SD rats perfused with low C a 2 + buffer 112 1.11 Effect of N F A (10 u M ) on pressor response to cirazoline (0.3 nmol) in M A B from SD rats perfused with C a 2 + free-EGTA (1 mM) solution 114 1.12 Effects of cirazoline on 1 2 5 I efflux in isolated small mesenteric arteries of SD rats 117 XIV 2.1 A 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 144 2.1 B 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 144 2.2 A Effect of L - N M M A on relaxation responses to A C h in intact rat aorta, in the absence and presence of T E A or N F A plus T E A 148 2.2 B Effect of L - N M M A on relaxation responses to A C h in intact mesenteric artery rings, in the absence and presence of T E A or N F A plus T E A 148 2.3 A Effects of L - N M M A and KC1 on A23187-induced relaxation in intact rat aorta 153 2.3 B Effect of T E A , L - N M M A and KC1 on A23187-induced relaxation in intact rat mesenteric arteries 153 2.4 Representative traces showing the relaxation responses to A23187 in intact rings from aorta (A) and mesenteric artery (B) 155 2.5 Effect of KC1 and K ^ ) channel blockers on L-NMMA/indOmethacin-resistant responses to A C h in intact rat mesenteric artery rings 158 3.1 Control experiments for responses to N E in isolated M A B obtained from lean or obese Zucker rats 191 3.2 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 3.3 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 3.4 Concentration-response curves to N E in the absence and presence of insulin (200 mU/1) in isolated M A B from lean or obese Zucker rats 200 3.5 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 2 K 1 C A C h A n g l l A N O V A B K c a B P C a 2 + cr c o x C T X Eci E C s E D 5 0 E D H F E R E r ET-1 E T O H IbTX Id(Ca) 1 2 5 iodine Two-kidney, one-clip Acetylcholine Angiotensin II Analysis o f variance large conductance calcium-activated potassium channels Blood pressure Calcium ion Chloride ion Cyclooxygenase Charybdotoxin Chloride equilibrium potential Endothelial cells The molar concentration o f agonist which produces 50% of the maximum effect Endothelium-derived hyperpolarizing factor Endoplasmic reticulum Reverse membrane potential Endothelin-1 Ethanol Iberiotoxin 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(Ca) Calcium-activated potassium channels K + Potassium ion KC1 Potassium chloride L - N M M A N^-monomethyl-L-arginine M A B Mesenteric arterial bed M A P Mean blood pressure N a + Sodium ion N E Norepinephrine N F A Niflumic acid N F D P Nifedipine N O Nitric Oxide N O S Nitric oxide synthase 0 2 " Superoxide anion p D 2 -log E D 5 0 P G H 2 Prostaglandin endoperoxides H 2 P G I 2 Prostacyclin Popen Open probability Rmax The maximum response o f an agonist R O C s Receptor-operated calcium channels SKca Small conductance calcium-activated potassium channels S M A C Superior mesenteric arterial conductance S M A F Superior mesenteric arterial blood flow S O D Superoxide dismutase SR Sarcoplasmic reticulum T E A Tetraethylammonium T x A 2 Thromboxane A2 vm Membrane potential V O C s voltage -operated calcium channels V S M C Vascular smooth muscle cell XV111 ACKNOWLEDGEMENTS I would like to give my profound gratitude to my supervisor Dr. Kathleen M . MacLeod 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 Ph.D. student in the Department of Pharmacology. Hi 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 of my research committee: Dr. John H . M c N e i l l and Dr. Ismail Laher for their support and advice. M y special thanks to my former supervisor Dr. Reza Tabrizchi. Wi th his instruction I started my doctoral program and completed most of my initial research. I would like to thank Dr. David V . Godin, the head o f the Department of 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 Divis ion of 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 Chow and Violet Yuen for their help with glucose and insulin assay; to Dr. Linfu Yao 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 of 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 of the resistance of these vessels through changes in their lumen diameter permits regulation o f tissue blood flow and aids in control of blood pressure, thus allowing appropriate distribution of cardiac output. This hemodynamic characteristic of the resistance vessels is mainly determined by the smooth muscle tone of 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 of mechanisms involved in the regulation of smooth muscle tone in resistance arteries is thus of major importance for our understanding of 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% of total cardiac output in resting man (Folkow and N e i l 1971). It may possibly receive up to 30% of cardiac output in rats under resting conditions (Folkow and Nei l 1971; Nilsson 1985). This makes it an important region 2 for maintaining cardiovascular homeostasis (Lundgren 1983). The mesenteric vascular bed, one of the major vasculature beds within the splanchnic circulation, possesses great potential for demand related up- or down-regulation of the blood flow to the intestines (Mitchell and Blomqvist 1971). Resistance to blood flow in the mesenteric vascular bed is therefore of 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 of relationships between blood pressure, blood flow and vascular resistance, various preparations of 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 of 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 of the superior mesentery) are widely used to investigate the structure and function of 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 of 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 of the first part was to investigate the contribution of CI" channels to ai-adrenoceptor-mediated vasoconstriction in mesenteric arteries from normal and hypertensive rats. The second part of the study was designed to examine the role of CI" as well as K + channels in endothelium-dependent relaxation in superior mesenteric artery, and the factors that mediate the endothelium-dependent relaxation. And finally, the purpose of the third part o f the study was to evaluate the regulation of NE-induced vasoconstriction in the perfused mesenteric arterial bed from obese rats with hypertension. The effect of insulin was examined in the latter investigation. The following review focuses mainly on oti-adrenoceptor-mediated excitation-contraction coupling mechanisms and on mechanisms by which the endothelium modulates vascular tone, and how these are altered by hypertension. The general characteristics of 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 S O F R A T M E S E N T E R I C V A S C U L A T U R E 1. Structure and Constituents 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 . From 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 of an outer tunica adventitia, a central tunica media, and an inner tunica intima. The main cellular constituents of 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 of 60% of the total volume of tunica media. There are approximately 6-8 layers of smooth muscle cells ( S M C ) arranged between 4-6 layers of elastic laminae around the vessel wall . (Lee et al. 1983a; Lee et al. 1983b). The walls of 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 of S M C layers decreases with the decrease in diameter. The small arterioles have only a single layer of smooth muscle cells (Lee et al. 1983a; Mi l l e r et al. 1987); however the volume fraction of smooth muscle cells within the media increases with the decrease in the diameter of 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 of 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 of peripheral resistance is important in understanding how the mesenteric arterial bed regulates peripheral resistance. Therefore, measurements of pressure in individual vessels have been made. The percentage drop in pressure is indicative o f the portion of resistance formed in the specific vessels. Based on the results from direct measurements of the microcirculatory pressure in intestinal vasculature of anaesthetized rats, early work indicated that small arteries and the largest arterioles in the mesentery contribute approximately 23% to 57% and the small arterioles in the intestinal wall 18% to 47%, of 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 31% of the systemic blood pressure drop occurs in the arcade small arteries, about a 51% drop occurs in the intestinal microcirculation including arterioles, capillaries, venules and small veins, while 5% of 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-ct-adrenergic 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 post-junctional a-adrenoceptors (Bevan et al. 1980; Bowman 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 of 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 of cti-adrenoceptors by N E is vital to control mesenteric vascular resistance, thereby regulating blood flow and blood pressure. H I . 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. Many 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 of 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 of 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 c , an,, oiia/d, and were subsequently renamed oti a, an,, aw, respectively. They now have been classified on the basis of pharmacological evidence as aiA ( a i c ) , a i e (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 low affinity for prazosin (Oshita et al. 1991). Recently it has been suggested that the aiL subtype may represent a particular conformational state of 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 of 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 of aiA-adrenoceptors in the perfused mesenteric arterial bed o f rats (Chen et al. 1996; Kong et al. 1994; Will iams and Clarke 1995; Zhu et al. 1999). A small 9 number of 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 of N E or peripheral nerve stimulation in other resistance vascular beds (Blue et al. 1992; Eltze et al. 1991; Zhu 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 1997; Yous i f et al. 1998). Recently, using subtype-selective antibodies as tools, O I I A - , am-, 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 ( G C I A ) and femoral ( O C I D ) arteries (Hrometz et al. 1999). Based on our current knowledge, the role of 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 of the G protein-coupled receptor (GPCR) super family. Stimulation of ai-adrenoceptors results in activation of various effector enzymes including P L C , P L A 2 and P L D via different G proteins, (Insel et al. 1998; Llahi 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 of the 10 G q / i i family (Boyer et al. 1992; Smrcka et al. 1991; Taylor et al. 1991; W u et al. 1992). Activated P L C catalyzes the hydrolysis o f the membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), to yield the second messengers, diacylglycerol ( D A G ) and inositol-1,4,5-triphosphate (JP 3) (Abdel-Latif 1986). D A G activates protein kinase C (PKC) , which may play a central role in phosphorylation of variety of cellular proteins that involved in the transduction of 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 Ca 2 + release from the intracellular stores (Berridge 1993; l ino 1990; Lepretre et al. 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 SR pool (Baro and Eisner 1995; Karaki et al. 1997), and can activate several other classes of Ca 2 +-sensitive ion channels on the cell membrane, such as calcium-activated K + ( K (ca)) and CI" (Ici(Ca)) channels which modulate the cell membrane properties (Amedee et al. 1990a; Amedee et al. 1990b; Byrne and Large 1988b; Pacaud et al. 1989a). Activation of ai-adrenoceptors also leads to influx of 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 C a 2 + channels including voltage-operated C a 2 + channels (VOCs) (Bolton 1979; Bulbring and Tomita 1987; Nelson et al. 1988; Van Breemen et al. 1978), receptor-operated Ca 2 + channels (ROCs) (Bolton 1979; Bulbring and Tomita 1987; Ruegg et al. 1989; Van 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 ai-adrenoceptor activation to C a 2 + influx is still not very clear. Another C a 2 + influx pathway is the C a 2 + 11 release-activated Ca channels ( C R A C ) , which are activated by SR depletion after receptor stimulation, and have a variable sensitivity to dihydropyridine C a 2 + channel blockers (Low 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 of the depleted SR 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 2 + level ([Ca 2 +]i) by cti-adrenoceptor activation plays a predominant 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 of vascular smooth muscle by activation of ai-adrenoceptors mainly depends upon the increased [Ca 2 + ] i that results from both C a 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 2 + -calmodulin dependent activation of myosin light chain kinase, which then phosphorylates myosin light chain ( M L C ) . Phosphorylated myosin can interact with actin and so induce contraction. In addition, cti-adrenoceptor 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 [Ca2 +]i (Somlyo and Somlyo 1994). The enhancement of contractile filament Ca 2 + sensit ivity, and the fact that this enhancement may be mediated through P K C during oti- adrenoceptor 12 C a 2 + Figure 0.1 Major a [-adrenoceptor signaling pathways that mediate vascular smooth muscle contraction. (1) IP 3 receptor, (2) voltage-gated C a 2 + channels, (3) receptor-operated C a 2 + channels (4) I C i ( C a ) i (Please see text for details). 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). C a 2 + release from the SR by IP3 produced on receptor activation is mainly responsible for the initial peak of 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 of external C a 2 + , and that agonist-induced IP3 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 SR (Julou-Schaeffer and Freslon 1988; Sutko et al. 1985), inhibited only the initial portion, but not the sustained portion of agonist -induced contraction (lino et al. 1988; Julou-Schaeffer and Freslon 1988; Kanmura et al. 1988). On the other hand, it has been found that the tonic responses are largely inhibited in Ca 2 +-free solution or in the presence of C a 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 of contraction, whereas intracellular release could also contribute to the tonic response depending on the concentration of the agonist and the arteries (Weber et al. 1995). Several studies indicate that the contribution of 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 45% 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 C a efflux decreased in Ca 2 +-free 2 m M E G T A solution, while the sensitivity o f NE-induced 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 4 5 C a influx induced by N E in the resistance vessels was approximately 10,000 fold more sensitive to the action of diltiazem than that in rabbit aorta (Cauvin et al. 1984). These data indicate a decreasing release of 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 of 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 of the SR diminishes as the arteries become smaller, while C a 2 + fluxes across the plasma membrane predominate during oti- adrenoceptor activation (Low et al. 1996). In the perfused rat hindlimb, Zhu et al (Zhu et al. 1998) demonstrated that the NE-induced maximum response was decreased by 92% following perfusion with Ca 2 +-free medium. Nifedipine concentration-dependently inhibited NE-induced contractions with a maximum inhibition o f 65% and the residual nifedipine-insensitive response was further inhibited by C d 2 + , suggesting the N E response in this preparation is mediated largely via an influx of extracellular C a 2 + , mainly via nifedipine-sensitive C a 2 + channels. Furthermore, in rat mesenteric small arteries, the main effect of N E on [Ca 2 + ] i was suggested to be mediated through voltage-dependent, dihydropyridine-sensitive C a 2 + channels, because when force, 15 membrane potential and [Ca^j i were measured simultaneously during stimulation with N E or potassium, a similar relationship between [Ca 2 + ] , and membrane potential was seen for both forms of activation (Nilsson et al. 1994). The inhibition of 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 of resistance vessels are heavily dependent on the presence of 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. With patch-clamp and molecular biology techniques, six subtypes of 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 of 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). At present, the major C a 2 + influx pathway is considered to be voltage-dependent L-type C a 2 + channels (hereafter simply called C a 2 + channels), since both the maintained arterial tone and the increase in [Ca 2 + ] i upon 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 of time that a C a 2 + channel is open (P o p e n) increase exponentially with membrane depolarization from relatively hyperpolarized 16 potentials. That the voltage dependence of 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 of 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 m ) can be very steep, with 3 m V depolarization or hyperpolarization increasing or 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 m and arterial tone as the relationship between the V m and C a 2 + (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 KC1 is approximately -46mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. 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 of-40 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 of 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 oti-adrenoceptor 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 of nonselective cation channels (Amedee et al. 1990a), inhibition of K + channels and/or activation of 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 of single C a 2 + channels in patches on single cells 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 of the two mechanisms, parallel and synergistic. Nevertheless, in rat mesenteric small arteries, challenge with N E caused membrane depolarization, elevated [Ca 2 +]; , and induced contraction. There was a strong correlation between membrane potential and [Ca 2 + ] ; when membrane potential, [Ca 2 + ] i and force were simultaneously measured, suggesting that cti-adrenoceptor activation 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 2 + Influx and Smooth Muscle Contraction 6.1. Increase in CI Conductance Resulting in Membrane Depolarization in VSMC A possible role of 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 (Aickin and Brading 1982; Casteels 1981; Koncz and Daugirdas 1994). Vascular smooth muscle cells accumulate CI" intracellularly through several processes (Chipperfield et al. 1993; Davis 1992; Davis et al. 1993), including Na + -K + -2C1" cotransport, C17HC03" exchange, and a third component, possibly an ATP-dependent transporter (Davis 1996). Estimates of the CI" equilibrium potential { E C i = -60 log (extracellular CI" concentration /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 mV. In any given vascular tissue, Ec 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 mV) (Aickin and Brading 1990). Therefore, any neurotransmitter or local mediator which increases CI" conductance w i l l produce efflux of C f , drive the membrane potential toward E C i and hence evoke depolarization in V S M . If the 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 of 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 of 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; Van Helden 1988). In short segments of 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" in the external solution was replaced with an impermeant anion (Van Helden 1988). The alteration 20 of E r is in the same direction as the change in Eci on substitution with a low-Cl" solution (Aickin 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 of the CI" equilibrium potential produced similar changes in the reversal potential of 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 2 +-dependent vasoconstriction in response to a i -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 of blood vessels, and can be activated by a number of agonists that depolarize and contract arteries. NE-activated CI" currents, as mentioned above, were also found in cells of rabbit ear artery (Amedee e ta 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 ia 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; Van 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). With simultaneous patch-clamp recording and intracellular C a 2 + measurements, it was demonstrated in rat portal vein cells that N E did not open C a 2 + channels, but increased [Ca 2 + ] i and evoked a Ca 2 +-activated CI" current at a holding potential of -50 rriV, which is about the 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 of the agonists in the presence of caffeine failed to evoke a significant rise in [Ca 2 + ] i and did not induce Ici(Ca) (Pacaud et al. 1992). Furthermore, in Ca 2 +-free external solution, N E induced a transient rise in [Ca 2 + ] i and was still able to activate a CI" current (Pacaud et al. 1992; Pacaud et al. 1989b). NE-induced Ici(Ca) was blocked by caffeine, but could be recorded in a Ca 2 +-free (+EGTA) 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 oti-adrenoceptor activation results from an increase in the intracellular concentration of calcium released from internal stores. ICi(Ca) activated by the release of calcium from intracellular 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; Wang and Large 1993). Although it has been demonstrated that prolonged exposure to Ca 2 +-free solution gradually reduced and eventually abolished Ici(Ca) (Amedee et al. 1990b; Droogmans et al. 1991; Wang and Large 1993; Hirakawa et al. 1999) and that depolarizing pulses which produced C a 2 + entry through V O C s also activated ICi (ca) (Lamb et al. 1994; Pacaud et al. 1989a), the extent to which C a 2 + entry from extracellular sources can sustain activation of these channels is unknown. It has been suggested that in 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 of C a 2 + from intracellular stores could activate Ici(Ca)- On the other hand, Pacaud (Pacaud et al. 1991) suggested that the activation of CI" channels in rat portal vein is a prerequisite for enhanced opening of voltage-dependent C a 2 + channels in response to N E . 6.4. Physiological Role of ICi(Ca) 6.4.1. Ici(Ca) blockers The evaluation o f the physiological role of 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 4-isothocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2, 2'disulfonic acid (DJDS) and the fenamate, niflumic acid (NFA) , have been characterized as Ici(ca) inhibitors in V S M (Large and Wang 1996 and references therein); (Kirkup et al. 1996a). In addition, 5-nitro-2-(3-phenylpropylamino)-bezoic acid (NPPB) (Kirkup et al. 1996b), ethacrynic acid, indanyloxyacetic acid ( IAA) (Greenwood et al. 1995), diphenylamine-2, 2'-dicarboxylic acid (DPC) (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 of these compounds are specific for Ici(Ca)- For example, fenamates are also found to activate large conductance, Ca 2 +-sensitive K + 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 o f approximately 2 x 10"6 M but evoked potassium current only at concentrations greater than 1 x 10"4 M (Greenwood and 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 of Ici(Ca) and activation of BK(c a ) , indicating these antagonists exhibit a lower degree of selectivity for Ici(ca)- (Greenwood et al. 1995; Hogg 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 STIC (Ici(Ca)) amplitude, these compounds also reduced both the amplitude and frequency o f spontaneous transient outward currents (STOCs, Ca 2 +-activated K + currents, IK(C3)) (Greenwood et al. 1995). The potency of other channel blockers, such as D I D S and SITS, against Ici(Ca) (IC50 greater than TO" 4 M ) is less than that of their well-established effects on 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 in 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 of 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 of activity for some of these compounds in different tissues. A s mentioned above, it seems that N F A is the most potent and selective inhibitor of 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"6 M and 1-100 u M respectively. At concentrations up to 5 x 10' 5 M it did not inhibit the influx of divalent 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"6 and 5 xlO" 5 M ) has been shown 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 in response to stimulation with N E in rabbit portal veins (Hogg et al. 1994a). However, this characteristic of N F A may be tissue-specific, since in rat portal vein at concentrations less than 3 xlO" 5 M , N F A had no effect on the magnitude o f the caffeine- or NE-stimulated B K ( C a ) (Kirkup et al. 1996a). 6.4.2. Ici(Ca) blockers inhibit agonist-induced vasoconstriction. 25 Given the general lack of 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 of Ici(Ca) in contractile mechanisms under physiological condition. There have been several experiments with CI" channel antagonists in whole tissue preparations, suggesting the involvement of a CI" conductance in agonist-induced contraction of smooth muscle. For instance, it was demonstrated that IAA-94 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; He and Tabrizchi 1997; Hyvel in et al. 1998; Lamb and Barna 1998). N F A and DDDS were also reporte to inhibit oti-adrenoceptor-mediated vasoconstriction in mesenteric vascular bed in anesthetized rats (He and Tabrizchi 1997; Lamb et al. 2000). Recently, it has been reported that N P P B (3 uM) inhibited the contractile response and increase in 4 5 C a 2 + influx produced by P E , a selective cti-adrenoceptor agonist, in rat caudal arteries (Min et al. 1999). Removal o f chloride ions also impaired PE-induced contractions and 4 5 C a 2 + influx, while N P P B had no effect on PE-induced contraction in Cl"-free buffer. These results thus provide some evidence of the role of CI" channels in cti-adrenoceptor-mediated Ca influx and contraction. It is of interest to further assess the contribution o f CI" conductance to contractile responses to cti-adrenoceptor in mesenteric arteries since, as mentioned previously, in 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 2 + ] i has been found to be mediated though voltage-dependent, dihydropyridine-sensitive C a 2 + channels (Nelson et al. 1988). IV. 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 F M E S E N T E R I C A R T E R I A L T O N E The single layer of endothelial cells (ECs) that lines the luminal side of mesenteric arteries and all other blood vessels plays an important role in regulating blood vessel function. The E C layer serves in part as a protective covering and permeability barrier to the movement of substances through the blood vessel wall . In addition, E C s also have an active role in 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 of the smooth muscle cells to respond to them. 1. Endothelium-Derived Vasorelaxing Factors A large body of evidence shows that E C s synthesize and release nitric oxide (NO), prostacyclin (PGI2) and an unidentified endothelial-derived hyperpolarizing factor(s) (EDHF) 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. NO 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 (NOS) (Ignarro 1990a; Palmer et al. 1988). There are three isoforms of 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 of the constitutive N O S is Ca 2 +-calmodulin-dependent and requires reduced nicotinamide adenine dinucleotide phosphate ( N A D P H ) , 5,6,7,8-terahydrobiopterin (BH4) and flavin mononucleotide for optimal activity (Busse et al. 1993; Knowles and Moncada 1994; Mayer and Werner 1995; Moncada et al. 1991). The production of 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 of 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 of 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 etal . 1989; Moncada et al. 1991; Rees et al. 1989; Rees et al. 1990). 1.1.2. Mechanism of NO 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 [Ca 2 + ] ; (Busse et al. 1989; Busse et al. 1993; Freay et al. 1989; Furchgott and Vanhoutte 1989; Griffith et al. 1986; Long and Stone 1985; Lopez-Jaramillo et al. 1990; Luckhoff and Busse 1986; Luscher 1990; Singer and Peach 1982) (Fig. 0.2). The agonist-induced increase in [Ca 2 + ] i involves both a transient nVmediated release of Ca 2 + f rom 28 EC VSMC Figure 0.2 Schematic diagram illustrating pathways of Ach-induced release of E D R F s in endothelial cells and relaxation of smooth muscle by the E D R F s . Modified from Vanhoutte (1997) . A C h binding to muscarinic receptor increase in IP 3 which contrabutes to the increase in cytoplasmic C a 2 + by releasing it from endoplasmic reticulum (ER). Muscarinic receptor activation induces an influx of C a 2 + into the cytoplasma. The resulting increase in [Ca 2 +]j activates N O S to produce N O and leads to the release of E D H F . The increased [Ca 2 + ] i also accellerates the formation of P G I 2 from arrachidonic acid ( A A ) by C O X . Stimulation with A C h also produces hyperpolarization of membrane potential (Vm) due to activation of K ( C a ) and CI" channels by the rise in C a 2 + - The resuling hyperpolarization accentuates C a 2 + influx due to the increased electrochemical gradient for C a 2 + and thereby by a positive feed back loop potentiaes the release of E D R F s . N O causes relaxation by activating the formation of c G M P from GTP . P G I 2 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. 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 of C a 2 + from the extracellular space (Wang et al. 1995a). Several lines of evidence have shown that release of N O absolutely requires C a 2 + influx from the extracellular space (Luckhoff and Busse 1990; Luckhoff et al. 1988; White and Mart in 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 2 + channels 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 2 + entry. Another suggestion is that agonist-induced C a 2 + entry is a consequence o f depletion o f an endoplasmic reticulum C a 2 + (via store -operated C a 2 + channels) (Putney 1991), which can be achieved in the absence of 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 of Ca 2 +-permeable channels that are responsible for receptor-mediated C a 2 + entry, it is generally accepted that agonist-induced C a 2 + influx is controlled by membrane potential (Demirel et al. 1994; Ni l ius et al. 1997b and references therein). A membrane hyperpolarization caused by agonist opening K + channels provides an electrochemical gradient for maintained C a 2 + entry during agonist stimulation. A similar mechanism for modulation of 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 + concentration or preincubation of endothelial cells or intact arteries with K + channel blockers decreased the duration and the magnitude o f agonist-induced C a 2 + influx. This in turn reduced the production of 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 of 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 of 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 eNOS may be differentially activated by receptor-dependent agonists and mechanical stimuli (Fleming et al. 1997). It has been observed that a rapid N O release in response to an onset of flow or an increase in flow above preexisting levels, like in response to receptor-dependent agonists, was Ca 2 +/camodulin-dependent (Busse et al. 1993; Kuchan and Frangos 1994). On the other hand, the constitutive sustained release of 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 2 +]i redistribution within the cytoskeleton/caveolae and the activation of one or more 31 regulatory eNOS-associated proteins without any apparent rise of [Ca 2 + ] i (Busse and Fleming 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 of soluble guanylate cyclase in the smooth muscle cells o f vascular wall , leading to increase in guanosine 3',5'-cyclic monophosphate (cGMP) and the subsequent activation o f cGMP-dependent protein kinases, such as protein kinase G (PKG) , which may modulate C a 2 + metabolism resulting in smooth muscle relaxation (Cornwell et al. 1991; Ignarro 1990b; Lincoln and Cornwell 1991; Salomone et al. 1996; Tewari and Simard 1997). The reliance of endothelium-dependent vasodilation on this mechanism is based on the parallel drawn between the increase in c G M P content of arterial tissue caused by endothelium-dependent vasodilators and those of 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 of the enzyme (Edwards et al. 1986; Gruetter et al. 1981; Ignarro et al. 1987; Ignarro et al. 1986; Kruszyna et al. 1987; Mart in et al. 1986; Wol in 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 of isolated arteries (Archer et al. 1994; Bolotina et al. 1994; Bychkov 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 of the resting membrane potential have been observed, and the hyperpolarization was sensitive to glibenclamide, implicating K A T P channels (Garland and Plane 1996; Garland and McPherson 1992). Electrophysiological experiments have revealed that N O and N O donors produced a cGMP-independent activation of large conductance Ca 2 +-activated K + 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 (ca), but this effect 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 2 +-dependent K + current (Weidelt et al. 1997). The 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 of 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 of large and intermediate Ca 2 +-activated K+channels. 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; Wol in et al. 1998) (and references above). 1.1.4. NO regulates mesenteric vascular tone a). Effect of basal N O release: Several lines of in vivo evidence suggest that constitutive levels of expression o f N O S in endothelium are sufficient to influence tone in mesenteric blood vessels under basal conditions. Inhibition of N O S has been shown to produce constriction of 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 of the N O S inhibitor, N^-nitro-L-arginine (L-N N A ) or the guanylate cyclase inhibitor, methylene blue (Wu 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 of vasoconstrictor (Simonsen et al. 1999). These results suggest the presence o f continuous basal release of 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 of N O requires active tone (Adeagbo et al. 1994; Amerini et al. 1995; Baisch et al. 1994; Dohi 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 (400-500 pm) and small (150-250 pm) arteries (Takamura et al. 1999). The contribution of N O , 34 which was evaluated by the use of 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 of arteries and was inhibited by K + channel blockers (Takamura et al. 1999). b). Effects of agonist-induced N O release In the isolated perfused mesenteric arterial bed, as in other arteries, A C h , histamine, A V P and the C a 2 + ionophore A23187 all induced endothelium-dependent relaxation (Adeagbo and M a l i k 1990; Bhardwaj and Moore 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 in the mesenteric arterial bed as compared to large blood vessels such as aorta (Furchgott et al. 1987; Khan 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 + concentration [ K + ] 0 decreased L-NAME-resistant vasodilation, indicating a role of the putative E D H F . These observations were supported by other investigators (Kamata et al. 1996a; McCul loch 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 in the presence o f L - N A M E , a greater contribution of 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; McCul loch 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 of the relaxant, or with a small reduction in maximum relaxation in the presence of N O S inhibitors among these studies. In addition, Kamata et al showed that the effects of the endothelium-dependent relaxation induced by platelet-activating factor (PAF) (Kamata et al. 1996b), or by the Ca 2 + -ATPase inhibitor cyclopiazonic acid (Kamata et al. 1996a) were different as 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 of 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 of 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 Kyoto 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 of 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. On 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 of the A C h - induced relaxation (Hwa et al. 1994; Plane and Garland 1996; Waldron and Garland 1994; W u et al. 1993). In isolated superior mesenteric artery, the relative contribution of N O also varied among different studies. It has been reported that N O solely contributed to ACh-induce relaxation in SD rats (Hwa 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 C a 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 Hi ley 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 of 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 agonist-induced 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 of rats, in the way o f handling the tissues, such as the initial stretch of vessels (Parkington et al. 1993; Zygmunt et al. 1994a), the nature of the relaxing agonists, and that of 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 via both N O and N O synthase-independent smooth muscle repolarization, whereas the reversal o f contraction to the thromboxane-mimetic U46619 by A C h was entirely mediated by the action of 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 of 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 2 was discovered in 1976 (Moncada et al. 1976a). P G I 2 production is initiated by the enzyme P L A 2 , which liberates arachidonic acid from membrane phospholipids. The 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 ip 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 of PGI2 and other prostanoids, thereby preventing their actions. L ike 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 of PGI2 is also believed to be triggered by an increase in [Ca 2 + ] i (Hallam et al. 1988; Long and Stone 1985). However, in bovine cultured aortic endothelial cells, N O release correlates most closely with transmembrane C a 2 + influx rather than C a 2 + 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 2 + ] i are required for P G I 2 release than for N O release. Furthermore, inhibition o f intracellular Ca 2 + mobilization by T M B - 8 attenuated bradykinin-induced PGI2 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 2 (Fig. 0.2). 1.2.2. Actions Physiologically, PGI2 is a local autacoid (Blair et al. 1982). In the lumen of blood vessels PGI2 prevents platelet aggregation, and thus the release of 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 of 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 of specific receptors and activation of 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 of 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 of the synthesis o f N O and P G I 2 does not result in complete loss of endothelium-dependent relaxation in response to variety of agonists, such as A C h , histamine, bradykinin, or substance P. A putative non-NO/PGI 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 o f E D H F is not yet established. It has been proposed that E D H F could be K + in small resistance arteries of rats (Edwards et al. 40 1998) , or may be a metabolite of 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 of 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 of 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 of 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 of 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 of N O and PGI2, elevation of [Ca 2 + ] i in endothelial cells has also been proposed to be essential for the release of E D H F (Chen and Suzuki 1990). This hypothesis has been supported by the finding that the C a 2 + ionophore A23187 induces endothelium-dependent membrane hyperpolarization (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 in 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 of phospholipid hydrolysis due to phospholipase C activation, and is maintained by C a 2 + influx via a N i 2 + - and Mn 2 + -sensitive pathway. They also indicated that the C a 2 + influx mechanism seems to be activated following HVinduced depletion of the C a 2 + pool. Thus, the E D H F release from mesenteric arteries seems to rely on both C a 2 + release from intracellular stores and C a 2 + entry from the extracellular space (Fig. 0.2). 1.3.3. E D H F - mediated vasodilation in mesenteric arteries. a). Mechanisms of EDHF-mediated relaxation The action of E D H F is believed to occur via the activation of K + channels, leading to hyperpolarization of the vascular smooth muscle membrane and vasorelaxation. This is based on evidence that variations in the extracellular K + concentration ( [K + ] 0 ) control the amplitude of 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 + ] D or K + channel antagonists (Adeagbo and Triggle 1993; Fukao et al. 1995; Garland and McPherson 1992; Hansen and Olesen 1997; McCul loch 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 (SKc a ) antagonist, significantly inhibited ACh-induced smooth muscle hyperpolarization and relaxation that were resistant to the action of N O S inhibitors. C T X , a large- and intermediate- Ca 2 +-activated K + channel antagonist, marginally inhibited both responses. However, the combination of 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 . Most of 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 2 +-activated K + channel ( B K c a ) blocker, T E A (5mM), 4-aminopyridine (4-AP), a blocker of delayed rectifier, voltage-dependent, K + 43 channels (Kv), and BaCi2 (100 uM) , 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 ( ImM) and 4 -AP each significantly reduced N O - and PGMndependent 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 of arterial segments to the combination of apamin and C T X abolished EDHF-mediated hyperpolarization (Chataigneau et al. 1998b) and N O - and PGI 2-independent relaxation (Plane et al. 1997; White and Hiley 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 Hi ley 1997). Furthermore, in pressurized small mesenteric arteries (diameter: 70-120 uM), Lagaud et al (Lagaud et al. 1999) showed that apamin alone completely blocked, while IbTX 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 of apamin plus IbTX, 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 of either PE-stimulated or myogenic tone in the presence of 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 of the shear stress-induced relaxation was almost abolished by T E A and was significantly inhibited by the combination of C T X and apamin (Takamura et al. 1999). Most 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; McCul loch et al. 1997). Collectively, the data suggest that in the mesenteric vascular bed, several K + channels are involved in EDHF-mediated relaxation, particularly Ca 2 +-activated K + channels. The heterogeneity of 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 of 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 of 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; McCul loch 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 (Hwa 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) (EDCF) (Luscher and Vanhoutte 1990; Vanhoutte 1989). The nature of E D C F varies with the species and anatomical site of its production. Prostaglandin endoperoxides H2 (PGH 2 ) , thromboxane 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; Wi lcox etal. 1996). 2.1. Endothelin-1 2.1.1. Synthesis and release The endothelins are a family of contractile peptides made up of 21-amino acids (Yanagisawa et al. 1988). They are synthesized from larger precursors and expressed in different tissues. ET-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. ET-1 release from perfused rat mesenteric arterial bed and its enhancement by hypoxia has been observed (Rakugi et al. 1990). The circulating levels of ET-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, ET-1 mainly acts in a paracrine and autocrine manner through two subtypes of receptors: E T A and E T B , which have been cloned (Arai et al. 1990; Sakurai et al. 1990). 2.1.2. ET receptors and their function in rat mesenteric vascular bed E T 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 of N O and P G I 2 (Luscher et al. 1993a; Matsuda et al. 1993; Warner et al. 1989; Wright and Fozard 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), ET-1 causes vasodilation by activation of endothelial E T B receptors, while at higher concentrations it provokes sustained contractions by activation of 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, ET-1 may interact with other vasoactive substance to affect smooth muscle function. It was shown that threshold doses o f ET-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; Yang et al. 1990). Recently Ki t a etal (Kita et al. 1998) confirmed that ET-1 at sub-pressor 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 of C O X pathway-derived contracting factors to mediate endothelium-dependent vasoconstriction. It has been shown that E T stimulated T x A 2 release and evoked an endothelium-dependent contraction in aorta from spontaneously hypertensive rats (SHR) but not Wistar-Kyoto ( W K Y ) rats (Taddei and Vanhoutte 1993). ET-1 stimulated release of 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 A receptors were thought to predominate and seemed to be the critical ones involved in vasoconstriction (Deng et al. 1995). However, a clear role for E T B receptors in mediating constrictor responses to ET-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 of both E T A and E T B receptors may be required for effective inhibition of E T - 1 -induced vasoconstriction. (Fukuroda et al. 1996; Mickley et al. 1997). 2.2. Prostanoids: PGH2, TxA2 2.2.1. Synthesis /release and receptor blockade The prostaglandin endoperoxide P G H 2 , as mentioned above in section III. 1.2, is an intermediate in the C O X pathway o f arachidonic acid metabolism (Moncada and Vane 1978a). Like PGI2, thromboxane A 2 ( T x A 2 ) is transformed enzymatically from P G H 2 , but via an alternative metabolic pathway. T x A 2 synthase, which catalyzes the transformation of P G endoperoxides into T x A 2 , has been found mainly in platelets, as well as in blood vessels (Moncada and Vane 1978a). In blood vessels, prostaglandins are mainly synthesized in and released from endothelial cells (Smith 1986). T x A 2 and P G H 2 stimulate contraction o f vascular smooth muscle via 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 P G H 2 / T X A 2 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 of C O X , occur in veins (De M e y and Vanhoutte 1982; Mi l le 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; Kung and Luscher 1995; L i n et al. 1994; Luscher and Vanhoutte 1986) of 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 2 and T x A 2 have been reported to be involved in endothelium-dependent 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 of P G H 2 and T x A 2 may be different in different models of hypertension. For example, in mesenteric resistance arteries of adult SFfR and DOCA-sa l t hypertensive rats, P G H 2 seems to mediate the endothelium-dependent 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 of T x A 2 / P G H 2 receptors with ridogrel and inhibition o f T x A 2 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 of smooth muscle to A C h resulted from an increase in T x A 2 formation rather 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. Superoxide Anion (Oi) 2.3.1 Formation 50 0 2" is generated though one-electron reduction of 0 2 by N A D ( P ) H oxidases, and other enzymes, such as xanthine oxidase (Land and Swallow 1971). It is known that 0 2" is produced via the side-chain reaction of P G H synthase in the presence of N A D H or N A D P H (Kukreja et al. 1986). The rate of 0 2" generation was markedly inhibited by C O X inhibitors 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 (Ang II) (Katusic 1996 and references therein; Zhang et al. 1999). Recently accumulated evidence has shown that Ang II stimulates production of O2" in blood vessels throughout the vascular wall , especially in the endothelium and adventitia (Di Wang et al. 1999; Nakane et al. 2000). Ang 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 of 0 2" in the vascular endothelium (Cosentino et al. 1994; Holland et al. 1990; 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 (SOD) or may react with other free radicals, such as N O (Pryor 1994). Interaction of 0 2" with N O is very rapid and leads to production of an oxidant, peroxynitrite (Beckman et al. 1990). Thus, O2" may decrease the concentration of N O , favoring an increase in arterial tone, and increase formation of a potentially toxic free radical that may cause oxidative injury. In addition, increased 51 production of O2" in the blood vessel wall inhibits synthesis of PGI2, but not that o f TxA2 (Katusic and Vanhoutte 1989; Moncada et al. 1976b). This effect may also contribute to impairment of endothelium-dependent relaxation and favor an increase in arterial tone. 2.3.3. Effects of 0 2 ~ on endothelial function in mesenteric vasculature 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 of 0 2" in vascular smooth muscle cells is missing (Katusic 1996). However, it has been repeatedly reported that vasoconstriction in response to agonists, such as A23187, U46619, N E and Ang 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 of S O D or abolish the potentiation produced by inhibitors of S O D (Laight et al. 1998; Schnackenberg et al. 2000). The results suggested that the major mechanism responsible for participation of O2" in endothelium-dependent contractions is inactivation of N O . O2" was also reported to suppress the modulatory influence o f endogenous N O on Ang 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 (Wu et al. 1998). O2" effects on endothelium function by increasing the breakdown of 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 of NE-induced tone and potentiated 52 endothelium-dependent relaxation to A C h (Sunman et al. 1993). This effect of 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 of rats, application of A n g II induced an immediate production of 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 in adult (15 week-old) stroke-prone spontaneously hypertensive rats (SHRSP) as compared with age-matched normotensive Wistar-Kyoto rats, although N O release remained unaffected (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 of 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 in 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; Folkow 1982; Mulvany 1994). This section wi 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 of the models of Goldblatt hypertension that also includes the one-kidney, one-clip (1K1C) model of 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 of a dog with adjustable silver clamps. Very quickly, this technique was adapted for use in other small mammals. In 1939 Wilson and Byrom (Wilson and Byrom 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 of human renovascular hypertension, and has been widely used for exploring the mechanisms of the genesis and sustenance of 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 (Averi l l 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 of 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 of 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 of 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-to-lumen ratio in small mesenteric arteries from 2 K 1 C rats. The alteration occurred within 6 weeks of development of hypertension and was accompanied by significant increases in active wall pressure produced in 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 of the elevated blood pressure. However, structural changes in the resistance vessels cannot be the sole determinant o f the raised peripheral resistance, because removal o f the constricting clip in 2 K 1 C hypertensive rats resulted in a rapid fall in 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 (Ang U) concentrations occur in the period immediately following constriction of 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 of the renin-angiotensin system with antibody to Ang II, a specific competitive antagonist o f Ang II or an angiotensin-converting enzyme inhibitors, lowers the blood pressure of 2 K 1 C hypertensive rats in the early phase (Bennett and Thurston 1996; B ing et al. 1981; Brunner et al. 1971; Pals et al. 1971; Tokioka et al. 2000). In addition, an increase in vascular responsiveness to Ang 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 of 2 K 1 C hypertension (Yoshida et al. 1987). Moreover, studies in whole animals showed an increase in the pressor response to low levels of 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 Ang II was reported to be decreased in rat mesenteric arteries in the early phase of 2 K 1 C hypertension (Benedetti and Linas 1987). Another study also 56 showed that the pressor response to Ang 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 Ang 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 of 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; McQueen 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 McQueen (McQueen 1961) in hindquarters. Furthermore, it was found that intravenous injection of nanonmolar concentrations of 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 of 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) of 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 of 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; Van 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 of 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 of 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 of 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 2 receptor antagonists inhibited the 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 A n independent association of 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; Modan 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 of type 2 diabetes mellitus and obesity, that also share an association with hypertension (Manicardi et al. 1986; Modan et al. 1985; Reaven 1988). Compensatory hyperinsulinemia is a signal o f the presence of 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 of the effect of age, obesity, fasting glycaemia and antihypertensive medications (Denker and Pollock 1992; Modan et al. 1985; Salonen et al. 1998; Tsuruta et al. 1996). In addition, hyperinsulinemia occurs in the young normotensive offspring of patients with essential hypertension (Ferrari et al. 1991; Grunfeld et al. 1994), and is associated with an increased incidence of 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 S H R and fructose fed hypertensive rats, also demonstrate insulin resistance and hyperinsulinemia (Hwang et al. 1987; Mondon 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 of 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 of 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 of 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 of 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; Rowe 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 of 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 (Wu et al. 1994). The insulin-induced potentiation of the response o f M A B to N E has been reported to be further augmented in 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 of vascular smooth muscle cells (Ridray 1995; Stout et al. 1975) and to increase ET-1 gene expression (Oliver et al. 1991) and ET-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 of ET-1 receptor antagonists, suggesting a role for ET-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 of ET-1 than the control rats (Verma et al. 1995). They also reported that chronic ET-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 ET-1 synthesis, leading to increased peripheral resistance and raised B P . Furthermore, insulin may also exert a vascular action through regulating production of C O X pathway metabolites (Axelrod and Levine 1983; Axelrod et al. 1986; Keen et al. 1997; Rebolledo et al. 1998; van Veen and Chang 1997). It has been reported that inhibition of T x A 2 synthesis attenuated hypertension induced by chronic insulin infusion in SD 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 of skeletal muscle vascular beds in humans (Laakso et al. 1990). This vasodilatory action has been shown to be impaired in states of insulin resistance, such as obesity and type 2 diabetes (Laakso et al. 1990; Laakso et al. 1992). It has been suggested that impairment of 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 of NE-induced vasoconstriction by stimulation of N O release was also observed in isolated rat mesenteric resistance arteries 62 (Walker et al. 1997b). Recently, insulin was reported to directly increase NO production in cultured human umbilical vein endothelial cells (Zeng and Quon 1996). Insulin may also regulate NO production by increasing availability of the cofactor BH4 for activation of NO synthas (Verma et al. 1998). Insulin may also exert a modulatory effect on local vasodilator responses by increasing Na + -K + ATPase and Ca 2 +-ATPase activity (Sowers et al. 1991; 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 of 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 of 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 (TG). 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 Kik ta 1992; Kurtz et al. 1989). Cox and Kik ta (Cox and Kik ta 1992) measured systolic B P of Zucker rats indirectly on a weekly basis from the age of 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 of 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 of 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 of 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 Ang 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 of endothelium function and structural changes (Cox and Kik ta 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 of 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 (Wu 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 of 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 of 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 (Wu et al. 1996) and A23187 (Walker et al. 1997a) were not significantly different from those in lean Zucker rats. Endothelium-independent relaxation of 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 of insulin on the reactivity o f mesenteric vasculature from Zucker obese rats have not been well characterized. Two studies examined the influence of 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 of 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 of NE-mediated vasoconstriction is impaired in the obese Zucker rat, and that this defect precedes, and therefore could contribute to, the development of hypertension in this insulin-resistant animal model (Walker et al. 1997a). V I . SUMMARY The mesenteric arterial bed plays an important role in the maintenance and control o f peripheral resistance. The sympathetic neuronal control of 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 of the excitation-coupling properties of the mesenteric artery smooth muscle, especially the smooth muscle o f small arteries, appears to be the dependence of 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 of 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 of the three parts of 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 of 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 of 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 of 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. R A T I O N A L E cti-adrenergic receptors play an important role in the control of vascular smooth muscle contraction and thereby, in regulation of 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 of inositol 1,4,5,-triphosphate which promotes C a 2 + release from intracellular stores. The mechanism by which the receptor activation opens cell surface C a 2 + 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 (Aick in 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 of 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 of 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 Eci . 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 of 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 of vasoconstrictor agonists (Amedee et al. 1990b; Byrne and Large 1988b; Droogmans et al. 1991; Klockner 1993; Pacaud et al. 1989a; Van Renterghem and Lazdunski 1993; Wang and Large 1993). The properties of 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 mV. At these values, 7 0 the open-state probability of Caz+ 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; Hogg et al. 1993; Pacaud et al. 1991; Pacaud et al. 1992; Pacaud et al. 1989b). Thus, the most likely role of 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 of a potent selective antagonist has been an obstacle in evaluating the physiological role of 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 of 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" 5 M , N F A did not (1) reduce the NE-evoked non-specific cation current (Hogg et al. 1994a), (2) inhibit voltage-dependent C a 2 + channels (Hogg et al. 1994a; Lamb et al. 1994); or (3) evoke a K + 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 of C a 2 + from the intracellular stores, since N F A did not inhibit NE-evoked Ca 2 +-activated K + current (Greenwood and Large 1995; Hogg et al. 71 1994a). Therefore, N F A seems to be a potentially useful tool for evaluation of the role o f Ici(Ca) in ai-adrenoceptor-induced contraction. At the time this project was started, no studies on the functional role of CI" channels in ai-adrenoceptor-mediated vasoconstriction in mesenteric arteries were available. However, during the course of the investigation, Criddle et al (Criddle et al. 1996) demonstrated that at a concentration of l O u M , N F A produced a comparable attenuation o f a component of 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 of NE-induced pressor responses in rat mesenteric arteries (Criddle et al. 1997). However, they did not examine in either study whether the reduction of 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 of 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 of the oci-adrenoceptor that are responsible for membrane depolarization, and thereby C a 2 + influx in this type of hypertension (see Introduction for more details). 73 n. WORKING HYPOTHESES AND SPECIFIC RESEARCH OBJECTIVES The major aim o f this part of the study was to obtain further information about the possible physiological role of the Ici(Ca) in the process of ai-adrenoceptor-mediated vasoconstriction, and therefore in regulation of blood flow and blood pressure. In these experiments, we have used N F A as a tool to analyze the functional role of the Ici(ca) in cti-adrenoceptor-mediated vasoconstriction both in vitro and in vivo in mesenteric resistance arteries. The change in the function of 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 of calcium-activated chloride channels with niflumic acid ( N F A ) inhibits oti-adrenoceptor-induced vasoconstriction in rat mesenteric artery both in vitro and in vivo. The inhibitory effect of 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 , in rat mesenteric artery, results from an indirect inhibition o f voltage-gated nifedipine-sensitive C a 2 + channels. In other words: A . Niflumic acid (NFA) , 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 of 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 of voltage-gated nifedipine-sensitive C a 2 + channels. Specific Objectives: Functional studies. 1) To 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) To investigate the effects of 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 N F A on oti-adrenoceptor-stimulated vasoconstriction in two kidney one-clip ( 2 K 1 C ) 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 2 + release or C a 2 + entry and to confirm that N F A indirectly blocks cirazoline-induced C a 2 + influx in smooth muscle of M A B , the following specific objectives were proposed: 4) To examine the effects of N F A on cirazoline-induced vasoconstriction in low C a 2 + and Ca 2 +-free, EGTA-containing solution in rat isolated perfused M A B . 5) To investigate the influence of nifedipine, an L-type calcium channel blocker, alone and in combination with N F A on cirazoline-induced vasoconstriction in rat isolated M A B . 75 Ion efflux study: 1) To assess the action of cirazoline on C f ion efflux ( 1 2 5 I efflux was measured as an index of membrane CI" conductance) in absence or presence of prazosin in rat isolated small mesenteric arteries (2 n d or 3 r d order branches of the superior mesenteric artery) Only preliminary experiments were done. 2) To examine the effect of 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. METHODS 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 of 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 of arterial blood pressure and removal of blood samples, and the left femoral vein for administration of drugs. The catheters were filled with heparinized saline (25 IU/ml in 0.9% NaCl) and tunneled subcutaneously to the back of the neck, exteriorized and secured. Bupivacaine (1%) was applied topically to the site of incision and animals were allowed to recover for 24 hr. On the following day, blood pressure was recorded using a pressure transducer (PD23ID Gould Statham, C A , USA) and Grass polygraph (Model 79D Grass Instruments, MA, USA) and the heart rate was measured using a tachograph (Model 7P4G Grass Instruments, MA, USA) continuously for 30-45 minutes in free-moving conscious rats. After 30-45 minutes, a blood sample was taken for 77 measurement of renin activity. 2 K 1 C rats with diastolic blood pressure of > 100 mmHg were used, and animals with malignant phase hypertension, as evidenced by the onset of weight loss, were excluded from the study. 2. Measurement of Plasma Renin Activity Renin-dependent hypertension was verified by determination of plasma renin activity. Blood (1 ml) was collected into a pre-chilled syringe containing E D T A to yield a final concentration of 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 of E D T A , dimercaprol and 8-hydroxyquinoline. The amount of 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 of the following composition (in m M ) : N a C l 120, KC1 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 of the buffer following saturation with a 95%0 2 : 5 % C 0 2 gas mixture was 7.4. The other perfusion buffers used in the experiments were: 1) Cl'-free buffer of the following composition (in m M ) : C 2 H 5 C O O N a 120, C 2 H 5 C O O K 3.5, glucose 11, M g S 0 4 1.2, Ca ( C 6 H n 0 7 ) 2 2.5, K H 2 P 0 4 1.2, N a H C 0 3 25; 2) L o w C a 2 + buffer: C a 2 + was decreased to 0.5 m M in normal Krebs; 3) Ca 2 +-free E G T A containing buffer: C a 2 + 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 of 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 of cirazoline (9 nmol) to check the viability and responsiveness of the preparations, and then were allowed to further equilibrate for 1 hr. A control dose-response curve for cirazoline was constructed by injection of 6 separate bolus doses of cirazoline (0.09 - 30 nmol). Perfusion pressure was allowed to return to baseline between each injection of agonist. The second and the third dose-response curve to cirazoline were determined in the presence of vehicle (0.03 or 0.1% alcohol), or N F A (3 or 10 p M ) in the perfusion media. Blood vessels were perfused with buffer containing either vehicle or N F A for 20 min and thereafter dose-response curves for 7 9 the agonist were determined. After the completion of each dose-response curve for cirazoline, a single bolus injection o f KC1 (60 jumol) was also made. Series 2. Effects of 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 curve to cirazoline was determined. After the completion of 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 uM), with which blood vessels were perfused for 20 min and thereafter the determination of the final dose-response curve to cirazoline. The perfusion time is long enough to greatly decrease [Cl"]i in smooth muscle cells (Aickin and Brading 1982). Separate tissues were used for each concentration o f N F A . 4.2. Effects of NFA on Cirazoline-Induced Vasoconstriction in MAB Perfused with Low Ca2* and C^-free Solution, and Compared with Effect of Nifedipine. In the presence of nifedipine The M A B from 2 K 1 C and sham rats were initially exposed to a submaximal dose of 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 of cirazoline (0.09 - 30 nmol). Perfusion pressure was allowed to return to baseline between each injection of agonist. The first dose-response curve served as control. The second dose-response curve to cirazoline was performed with nifedipine (3uM) in the perfusion media, while the third dose response curve was determined in the presence o f 80 nifedipine (3pM) 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 of each dose-response curve for cirazoline, a single bolus injection of KC1 (60 pmol) was also made. Low Ca 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 2 + , while the third dose response curve was determined in the presence of 0.5 m M C a 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 Ca -free, EGTA-containing solution (preliminary experiments, data not shown), only a single bolus dose of cirazoline was applied each time. In addition, a single bolus injection of KC1 (30 pmol) was made before each cirazoline dose was given. A total of 5 sets of injections of KC1 and cirazoline were given in each M A B under different perfusion buffer conditions. The perfusing sequence of the different perfusion buffers was as follows: normal Krebs, Ca 2 +-free solution, Ca 2 +-free solution in the presence 2+ of N F A , Ca -free solution again, and Ca-free solution in the presence of nifedipine. After each injection of cirazoline, tissue was allowed to equilibrate for 40 min by perfusing with normal Krebs to refill the internal C a 2 + stores. Antagonists were added in the perfusion buffer 20 min before injection of KC1 and were present until the pressor response to cirazoline returned to baseline. The Ca 2 + - f ree -EGTA containing buffer was perfused for 10 min before 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 of 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. Blood 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 . ) . Blood pressure and heart rate were continuously monitored. Body temperature in 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 of 60 min.. 6. Experimental Protocols for in vivo Experiments Effects of N F A on blood pressure, blood flow and mesenteric vascular conductance were examined in four groups of rats. Each animal initially received a cumulative continuous infusion of 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. 8 2 Ion Efflux Studies: 7 . Isolation of Small Mesenteric Arteries Male 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 1 2 5 I Efflux in Small Mesenteric Arteries 1 2 5 I was chosen as a marker of CI" channel activity because: 1) it has higher specific activity than 3 6 C1 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 of I" through calcium-activated CI" channels is greater than that of CI" (Amedee et al. 1990b); 3) measurement of 1 2 5 I efflux from cells and tissues has been used by other investigators as an index of membrane CI" conductance (White et al. 1995).  L"l efflux was measured using a washout method (McMahon and Jones 1988; Smith 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. For 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 of vigorously gassed (with 95% 02: 5% C 0 2 ) tubes containing 1 ml non-radioactive Krebs in the absence or presence of 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 of the antagonists. The total washout time was 32 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. At the end of the washout, tissues were blotted and then the radioactivity in 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 Co (St. Louis, M O , U S A ) or Research Biochemical International (Natick, M A , U S A ) . Angiotensin I [ 1 2 5I] radioimmunoassay kits and carrier- free 1 2 5 I were purchased from D u Pont Company (Mississauga, Ont., Canada). A stock solution of N F A (10"1 M ) was prepared in 100% ethanol (ETOH) 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 in N a H C 0 3 with 5% glucose (4 x 10"1 M , p H 8.5) and prepared as a stock solution (10 mg/ml) for in vivo studies. Cirazoline and prazosin were dissolved in normal saline (0.9% NaCl) 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 in perfusion pressure following bolus injection of each dose of cirazoline were plotted. Vascular conductance in vivo was calculated as flow divided by mean blood pressure ( M A P ) . Conductance was calculated in order to assess active changes in vascular tone (Lautt 1999; Tabrizchi and Pang 1993). M A P 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 of 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 of the efflux curve were averaged and compared with the averaged k of 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 of drugs on 1 2 5 I efflux were plotted as percentage of 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 of rats (i.e. 2 K 1 C and sham). One-way A N O V A was used for multiple comparisons in one group of rats (normal rats). Duncan's multiple range test was used to compare between multiple means. P < 0.05 was considered as significant in the analysis. 85 I V . RESULTS 1. Characteristics of 2K1C Hypertensive Rats Systolic and diastolic blood pressure and heart rate of conscious 2 K 1 C rats were significantly (n= 42; P < 0.05) higher than those of 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 of 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 in 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 of 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, cirazoline-mediated vasoconstriction was significantly (n = 6; P < 0.05) inhibited at all doses (0.09-30 nmol) in presence of the higher concentration of 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. On the other hand, vasoconstriction evoked by bolus injection of 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 of 2 K 1 C and Sham rats: Blood pressure (mmHg), heart rate (beats/min), plasma renin activity (mg ml" 1 h"1) and body weight (g) of 2 K T C hypertensive and sham normotensive rats. 2 K 1 C Sham Arterial pressure Systolic 2 4 4 ± 5 a 134±2 Diastolic 166±4 a 94±2 Heart rate 4 1 7 ± 8 a 370±5 Plasma renin activity 18.37±2.10 a 3.03±0.28 Body weight 367±6 392±6 Values are pooled and shown as mean ± S E M n = 42 for each group of rats. aSignificantly different from sham, P < 0.05 (unpaired t-test). 87 FIGURE 1.1 Effect of vehicle ( E T O H , 0.03% and 0.1%) on vasoconstrictor responses to bolus injection of 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. a P < 0.05 vs. sham (two-way A N O V A followed by Duncan's test). 2K1C o5 2 5 0 x E E 2 0 0 CD i_ ~j co co CD Q_ CD co 03 CD i_ o c 150 h c o CO => 100 50 h 0 • C o n t r o l 0 E T O H ( O . O 3 % ) • E T O H ( 0 . 1 % ) 0 .03 0 .09 0.3 0.9 C i r a z o l i n e (nmol) Sham _ 2 5 0 X E f 2 0 0 rj co CO CD c g CO tr. Q_ 0) CO 03 CD o c 150 100 50 0 • C o n t r o l • E T O H ( 0 . 0 3 % ) • E T O H ( 0 . 1 % ) 0 .03 0 .09 0.9 C i r a z o l i n e (nmol) 89 FIGURE 1.2 Effect of N F A (3 u M and 10 uM) 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 . a P < 0.05 vs. sham, b P < 0.05 vs. control , 0 P < 0.05 vs. N F A (3 p M ) (two-way A N O V A followed by Duncan's test). 2 K 1 C 200 r X E 180 -E 0 160 -CO 140 -CO CD u. 120 -D_ c o 100 -CO 3 80 -t CD Q. 60 -40 -ase ase 20 -CD b 0 -0.09 0.30 0.90 3.00 Cirazoline (nmol) 9.00 30.00 Sham ' 3 200 r X E 180 -E, o 160 -CO 140 -CO 0 1_ 120 -0_ c o 100 -CO 80 -t: 0 Q. 60 -c 40 -0 CO CD 20 -0 b c 0 -Control WZtt NFA (3uM) H i NFA(10uM) be 0.09 0.30 0.90 3.00 9.00 30.00 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 mmHg 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 mmHg 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 of 3, 9 and 30 nmol. Perfusion of 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). We did find that in Cf-free buffer, cirazoline-mediated vasoconstriction was further inhibited by the presence of N F A (Fig. 1.5, 1.6), but not vehicle (Fig. 1.4) in the perfusion media. N F A (3 uM) significantly (P < 0.05) inhibited cirazoline-mediated vasoconstriction at doses of 0.9, 3, 92 FIGURE 1.3 Effect of N F A (3 u M and 10 uM) 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, (two-way A N O V A ) 93 2 K 1 C 100 r C o n t r o l 3 M M N F A 10 u - M N F A KCI ( 6 0 u m o l ) Sham 100 r C o n t r o l 3 u M N F A 10 u M N F A KCI (60 nmol ) 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 . a P < 0.05 vs. sham, b P < 0.05 vs. control, (two-way A N O V A followed by Duncan's test). 2 K 1 C 250 r CD CO CD CD i_ o c 200 h CD X E E , CD i— Zi CO CO 2? 150 Q. o CO t CD Q_ 100 h 50 h • C o n t r o l 0 C l - f r e e • E T O H / C I - f r e e 0 J X 0.03 0.09 0.3 0.9 3 C i r a z o l i n e (nmol) Sham oS 180 x E E c g 'co t CD CL CD CO CD CD i_ o c 160 h 2 140 CO _ _ s 1 2 0 100 80 60 40 20 0 • C o n t r o l 0 C l - f r e e • E T O H / C I - f r e e C i r a z o l i n e (nmol) 96 FIGURE 1.5 Effect of N F A (3 p M ) on pressor responses to cirazoline in 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 (2K1C) 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, d P < 0.05 vs. 3 u M N F A + Cl"-free buffer in 2 K 1 C rats (unpaired student's t-test). 97 2 K 1 C 0.09 0.30 0.90 3.00 9.00 30 .00 Cirazoline(nmol) Sham 0.09 0.30 0.90 3 .00 9 .00 30 .00 Cirazoline (nmol) 98 FIGURE 1.6 Effect of 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 (2K1C) 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, d P < 0.05 vs. 10 u M N F A + CI" free in 2 K 1 C rats (unpaired student's t-test). 99 0.09 0.30 0.90 3.00 9.00 30.00 Ci razo l ine (nmol) 2! Sham = 100 0.09 0.30 0.90 3.00 9.00 30.00 Ci razo l ine (nmol) 100 9 and 30 nmol in 2K1C hypertensive rats and sham normotensive rats. The magnitude of blockade produced by N F A of 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 of a higher concentration of N F A significantly (n = 6; P < 0.05) inhibited cirazoline-mediated vasoconstriction at doses of 0.9, 3, 9 and 30 nmol in 2K1C hypertensive rats and in normotensive rats. The magnitude of 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 of N F A was tested in vivo in anaesthetized rats. There was no significant difference in the baseline values of 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 of either N F A or vehicle did not alter the baseline values of MAP, superior mesenteric flow or conductance in anaesthetized rats (Table 1.2). Intravenous infusion of cumulative doses of cirazoline caused dose-dependent increases in MAP (Table 1.3), and decreases in superior mesenteric vascular conductance in 2K1C hypertensive and sham normotensive rats (Table 1.4). However, the degree of 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"1) values before and after injection of vehicle (NaHC03, 0.3 ml/kg) or N F A (3 mg/kg) in 2 K 1 C hypertensive and sham normotensive rats. M A P S M A F S M A C 2 K 1 C Sham 2 K 1 C Sham 2 K 1 C Sham Pre-vehicle 164±14 a 98±3 13.9±0.9 14.1±2.5 0 .088±0.012 a 0.140±0.028 Post-vehicle 148±8 a 92±3 14.2±0.9 14.5±1.9 0.097±0.009 a 0.158±0.021 P r e - N F A 162±13 a 97±5 11.9±1.3 15.5±1.8 0 .076±0.012 a 0.165±0.027 Pos t -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 aSignificantly different from sham rats, p < 0.05 (two-way A N O V A followed by Duncan's test). 102 TABLE 1. 3. Effects of 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 3 , 0.3 ml/kg) or N F A (3 mg/kg). Cirazoline (u.g kg" 1 min"1) 0.13 0.34 1.00 2.77 2 K 1 C Control 171±16 a 181±17 a 200±14 a 250±19 Vehicle-treated 160±9 168±10 188±13 243±15 Control 170±13 a 183±15 a 215±14 a 2 5 3 ± 5 a NFA-treated 154±15 163±14 188±14 237±5 Sham 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 Each value represents the mean ± S E M , n = 5 aSignificantly 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 (% of control) in superior mesenteric artery ( S M A C ; ml mmHg" 1 min" 1, control conductances were shown in Table 1.2) in anesthetized 2 K 1 C hypertensive and sham normotensive rats before (control) and after treatment with either vehicle ( N a H C 0 3 , 0.3 ml/kg) or N F A (3 mg/kg), Cirazoline (pg kg" 1 min"1) 0.13 0.34 1.00 2.77 2 K 1 C 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 a 2 . 0 ± 6 . 0 a 2 4 . 3 ± 8 . 0 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 a 13 .6±4.1 a 3 4 . 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 of vehicle (Table 1.3 and 1.4). In addition, in 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 of 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 of 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 of 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 SD rats perfused with normal Krebs. The presence of nifedipine (3 u M ) in the perfusion media significantly inhibited the vasoconstrictor action of 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 of nifedipine plus N F A (10 uM) , 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 of nifedipine (NFDP, 3 p M ) and N F D P (3pM) plus N F A (10 p M ) on vasoconstriction induced by bolus injection of cirazoline (A) or KC1 (B) in M A B from normal SD 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 of 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 • C o n t r o l • N F D P (3pM) • N F D P + N F A ( 1 0 u J V 1 ) b b b b mm. 0.03 0,09 0.3 0.9 C i r a z o l i n e ( n m o l ) 9 N o r m a l K r e b s NFDP (3uM) N F D P + NFA(10uM) KCI (60 p m o l ) 107 6. Effect of N F A on Cirazoline- and KCl-Induced Vasoconstriction in Isolated M A B Perfused with Low C a 2 + Solution Lowering C a 2 + 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 SD rats (Fig 1.8A & Fig . 1.9 A B ) , whereas the pressor response to K C I was significantly reduced (Fig, 1.8B & Fig . 1.10A B) . Addition of 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 l ow C a 2 + buffer alone (Fig 1.10A.B). In contrast, the presence of 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 of K C I , in the presence of vehicle, was not significantly different from the first in isolated M A B perfused with low Ca 2 + so lu t ion alone (Fig. 1.8B). 7. Effect of N F A on Cirazoline-Induced Vasoconstriction in Isolated M A B Perfused with Ca 2 + -free-EGTA Solution 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 m M C a 2 + (Fig l . H A : a ) . Perfusion with Ca 2 +-ffee solution containing 1 m M E G T A abolished the sustained plateau of pressor response, while leaving the initial transient peak intact (Fig. l . H A : b ) . The amplitude of the initial transient peak response to cirazoline with Ca 2 +-free solution did not differ from that obtained with normal Krebs (Fig. 1.1 IB) . In contrast, perfusing with Ca 2 +-free 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 of low Caz+ (0.5 m M ) buffer and vehicle (0.1% E T O H ) on the pressor response to bolus injection of cirazoline (A) and KC1 (B) in M A B from normal SD rats. Data are shown as mean ± S E M , n = 6. b P < 0.05 vs. normal Krebs. (one-way A N O V A followed by Duncan's test) 109 A x 160 r c 0.03 0 .09 0.3 0.9 3 9 C i r a z o l i n e (nmol) B D5 120 r X E £ 100 -N o r m a l K r e b s C a ( 0 . 5 m M ) C a ( 0 . 5 m M ) + E T O H KCI (60 Li mol) 110 FIGURE 1.9 Effect of N F A (3 u M in A , 10 u M in B) on pressor response to bolus injection of cirazoline in M A B from SD rats perfused with low C a 2 + buffer (0.5 m M ) . Data are shown as mean ± S E M , n = 6. b P < 0.05 vs. normal Krebs , 0 P < 0.05 vs. low C a 2 + (0.5 m M ) buffer (one-way A N O V A followed by Duncan's test) I l l -j? 180 r 0.03 0.09 0.3 0.9 3 9 C i r a z o l i n e (nmol) 0.03 0.09 0.3 0.9 3 9 C i r a z o l i n e (nmol) 112 FIGURE 1.10 Effect of N F A (3 u M in A , 10 u M in B) on KC1 evoked contraction in M A B from normal SD rats perfused with low Ca 2 + (0 .5 m M ) buffer. Data are shown as mean ± S E M , n = 6. b P < 0.05 vs. normal Krebs. (one-way A N O V A followed by Duncan's test) 120 r 100 h C o n t r o l C a ( 0 . 5 m M ) N F A ( 3 u M ) / C a ( 0 . 5 m M ) KCI (60 umol ) B 120 r C o n t r o l C a ( 0 . 5 m M ) NFA(10uiVI) / C a ( 0 . 5 m M ) K C I ( 6 0 u m o l ) 114 FIGURE 1.11 Effect of N F A (10 p M ) on the pressor response to bolus injection of cirazoline (0.3 nmol) in M A B from SD rats perfused with C a 2 + free-EGTA (1 mM) solution (B). Upper panel (A) is a representative trace obtained from one of 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 + ( 30umo l ) C i r a z o l i n e ( 0 . 3 n m o l ) B oS30 x E E • Control (Krebs) • C a f r e e + E G T A ( 1 m M ) • C a f ree+EGTA (1mM)+NFA ( 1 0 u M ) • C a f r e e + E G T A ( 1 m M ) mCa f r ee+EGTA (1mM)+NFDP ( 3 u M ) 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 of N F A on the cirazoline-induced vasoconstrictor response that mediated by C a 2 + released from intracellular store independently o f the influence of C a 2 + 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 Ca 2 + - f r ee -EGTA containing solution (Fig 1.11 A:c .d & B ) 8. 1 2 5 1 Efflux from Small Mesenteric Arteries The effect of cirazoline on 1 2 5 I efflux was evaluated, and preliminary experiments on the effects of prazosin and N F A were carried out. A s illustrated in Fig . 1.12, cirazoline caused an increase in 1 2 5 I efflux. F ig . 1.12A shows the 1 2 5 I efflux curve from an individual vessel; the effect of 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 of 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 1 2 5 I efflux, but inhibited cirazoline-induced increase in 1 2 5 I efflux (Table 1.5). 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 1 2 5 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 T i m e (min) B 250 , C o n t r o l 1 M-V1 3|iv1 10 p M C i r a z o l i n e 119 T A B L E 1.5 A . Effect of Prazosin on cirazoline-induced 1 2 5 I efflux (n=2) Control Cirazoline Prazosin Cirazoline+Prazosin (3uM) (0.3uM) (3uM) (0.3uM) kfmin" 1 ) 0.098±0.0016 0.129±0.0095 0.095±0.00115 0.072±0.0010 %Control 100 145.1±12.1 101.2±4,1 75.6±1.8 B . Effect of N F A on cirazoline induced I efflux (n=2) Control Cirazoline N F A Cirazoline+NFA (3uM) ( lOuM) (3uM) ( lOuM) kCmin"1) 0.096±0.00076 0.154±0.0020 0.076±0.0037 0.085±0.0082 %Control 100 160.1±16.4 88.3±2.3 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. To 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 DIDS, A - 9 - C I A A (Hogg et al. 1994a; Large and Wang 1996). We found that N F A was capable of inhibiting cirazoline-induced vasoconstriction both in vitro as well as in vivo. It failed to produce additive inhibition in the presence of the C a 2 + channel inhibitor nifedipine, which also attenuated the cirazoline-induced contraction significantly. We also found that removal of 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 of 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 via the stimulation of cti-adrenoceptor in rat mesenteric arteries. Based on our results, it also seems that the role of 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 of 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 of 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 of 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; Hogg et al. 1994b; Kirkup 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 of CI" inhibited c i i -adrenoceptor-mediated vasoconstriction in perfused mesenteric blood vessels. In addition, as expected, nifedipine, a specific blocker of 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 of mesenteric arteries by the combination of 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 via the cti-adrenoceptors have been shown to be sensitive to the actions of 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 of N F A in rat 122 mesenteric arteries is an inhibition of C f current evoked by cirazoline, thereby indirectly preventing C a 2 + from entering via voltage-gated C a 2 + channels. The role of 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 of extracellular CI" concentration decreases [Cl"]i in smooth muscle cells (Aickin and Brading 1982). In addition, the probability o f opening of certain CI" channels is dependent on the intracellular and extracellular concentrations o f CI", and the CI" current is much more affected by changes of 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 of smooth muscles including V S M (Bulbring and Tomita 1987; Large 1984; Van Helden 1988; Van Renterghem and Lazdunski 1993). Van Helden has studied the kinetics of the effect of low-Cl" on changes in CI" conductance induced by N E in smooth muscle of guinea pig mesenteric veins (Van Helden 1988). He observed that the currents increased in amplitude during about the first 40s exposure to low-C l " solution and decreased afterward. However, complete suppression was not always observed even after a 15min exposure to low-Cl" solution. He also found that the initial increase in amplitude of the CI" current was consistent with an increase in the driving force for CI". He 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 in 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 of 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 in 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 of explanations for this observation, which could account for the further inhibitory actions of N F A in Cf-free buffer. First, it is possible that N F A inhibited the efflux of residual CI" that remained inside the vascular smooth muscle cells in Cf-free buffer. This speculation is supported by observations in a number of articles. McMahon and Jones demonstrated that in Cf-free propionate substituted solution, the reduction of [Cl"]i was slower than the loss of CI" from the extracellular site in rat aorta strips (McMahon 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 of [Cl"]i in Cf-free solution one hour later still showed a positive value. The value of [Cf ]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 of agonist-induced depolarization via efflux through the CI" channels, while in the presence of 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 of 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" via 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 1 2 5 I efflux in vascular smooth muscle cells (White et al. 1995). The results of 1 2 5 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 of 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 of cti-adrenoceptor in rat mesenteric arterial bed are mediated by the activation of ctiA-adrenoceptor sub-types (Chen et al. 1996; Kong et al. 1994; Will iams 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 aid 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 2 + containing buffer. Second, N F A remained ineffective in low C a 2 + 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 cirazoline-induced vasoconstriction is not due to a direct blockade of voltage-gated Ca2+channels, or to non-specific effects on the contractile apparatus. Additionally, N F A did not affect cirazoline-induced 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 SR (Hogg et al. 1994a). In our experiments, in which rat mesenteric arteries were employed, this seems not to be the case. We 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 of K + channels. Altered Function of Ct Channels in mediating cci-Adrenoceptor-Induced Vasoconstriction in MAB from 2K1C Hypertensive Rats. In a previous study, McGregor 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 of 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 of 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 of blood vessels in 2 K 1 C hypertensive versus normotensive rats, since no difference in vasoconstrictor responses evoked by KC1 was observed in the present study. 127 The results of 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 in 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 of these results may not be straightforward. Differences in membrane potential between blood vessels from 2 K 1 C hypertensive and those of sham normotensive rats in Cf-free buffer may have been responsible for the increased ability o f N F A to inhibit cirazoline-induced vasoconstriction in 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 of 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 of [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 of 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. We also found that the magnitude o f the spontaneous contraction was greater in 128 2 K 1 C hypertensive rats than that in sham rats, implying that the depolarization produced upon removal of 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 m ) in M A B from 2 K 1 C rats. It has been shown that in one-kidney, one-clip (1K1C) hypertension, the resting tail artery was depolarized by about 7 mV. This depolarization may be caused by a humoral substance since plasma supernatants from hypertensive rats also depolarized the muscle cells in control animals (Pamnani et al. 1985). However, V m o f mesenteric smooth muscle in 2 K 1 C hypertensive rats may be not changed under control conditions as compared with normotensive control rats because the vasoconstrictor responses to KC1 in M A B were similar between the two groups of rats in our experiments. Thus, the greater transient contraction induced by initial removal of 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 m . It has been demonstrated 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. Not 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 of N a + / K + pump would hyperpolarize the membrane 129 (Cheung 1989). On the other hand, the N a + / K + / C l " cotransporter accumulates intracellular CI" above equilibrium. Although it is electroneutral (Aickin and Brading 1990; Chipperfield 1986), by raising [Cl"]i, it has a depolarizing influence on V m (Davis 1992; Davis et al. 1991). Thus, the V m 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 of [Cl"]i may be higher in smooth muscle of the hypertensive M A B perfused with physiological salt solution than that of 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 of 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 of ion currents flowing through open channels is determined by the number of ion channels, the single channel current and the channel activity (or Popen) (Nelson and Quayle 1995). The single channel current depends 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 of 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 of CI" channels could account for the smaller reduction in contractile responses and the lesser inhibitory effect of 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 of membrane potential, [Cl"]i and channel activity. In addition, a future study involving blockade of N a + / K + / C l " cotransport may help to resolve the involvement of 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 cti-adrenoceptor stimulation in 2 K 1 C hypertension. Yoshida et al (Yoshida et al. 1989) had previously reported that an increase in intracellular calcium content of 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 of altered function of CI" channels in blood vessels in experimental hypertension. 131 V I . S U M M A R Y 1. 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 Cf-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 KCI was already attenuated, N F A reduced cirazoline-induced contraction with no effect on KCl-evoked contraction. 132 6. Cirazoline elicited contraction in Ca 2 +-free, EGTA-containing solution. The contraction was transient in response to 3 nmol of cirazoline, and was not inhibited by either 10 p M N F A or 3 p M nifedipine. However, in the Ca 2 +-free, EGTA-containing solution, responses to KC1 were abolished. 7. In small mesenteric arteries, cirazoline caused a dose-dependent increase in 1 2 5 I efflux. The increased 1 2 5 I efflux was inhibited by prazosin and blocked by N F A . 133 V I I CONCLUSIONS Overall, our results demonstrated that N F A is capable of inhibiting cti-adrenoceptor-mediated vasoconstriction o f rat mesenteric artery both in vitro and in vivo. The mechanism of this action of 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 of voltage-gated C a 2 + channels, and consequently, a sustained contraction wi l l be maintained though C a 2 + influx. This contribution of CI" in blood vessels from hypertensive rats appears to be reduced. 134 PART 2. THE MECHANISMS OF THAT ACETYLCHOLINE-INDUCED 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/PGI2-independent hyperpolarization by activation of 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 of K + channels activated by E D H F have not yet been definitely identified. In addition, the role of 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 Hiley 1997; W u et al. 1997) (and please see Introduction). The release of N O , E D H F and P G I 2 by A C h acting at muscarinic receptors has been studied (Fukao et al. 1997c; Luckhoff and Busse 1990). There is evidence that an agonist-induced increase C a 2 + influx from the extracellular space is essential for the maintained production of 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 Mart in 1989). In endothelial cells, opening of C a 2 + entry channels is believed to require agonist binding to membrane receptor and / or depletion of intracellular C a 2 + stores (Putney 1991; Wang and 135 van Breemen 1997). Unlike in smooth muscle cells, opening Ca 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 of ion channels. Membrane hyperpolarization augments the electrochemical driving force promoting C a 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 of muscarinic receptors with A C h activates Ca 2 +-dependent K + channels leading to transient membrane hyperpolarization, which is sensitive to tetraethylammonium (TEA) 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 of 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 [Ca 2 + ] ; and greatly inhibited N O release in response to A C h (Luckhoff and Busse 1990), while in intact rabbit aorta, T E A was capable o f inhibiting ACh-induced C a 2 + elevation and E D R F synthesis/release as well as vasorelaxation based on results of 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; Nil ius et al. 1996; Ni l ius 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 2 + signaling stimulated by agonists such as A T P and histamine and triggered by C a 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 + equilibrium potential, C f removal had no effect on A C h - induced C a 2 + entry, indicating that C f current modulates C a 2 + influx by maintaining a polarized membrane potential after A C h activation. Thus, C f channels may act in conjunction with K + channels on regulating ACh-induced E D R F synthesis, thereby causing vasorelaxation. However, there is no evidence yet for a functional role of 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 of 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. We 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 of these two drugs on ACh-induced relaxation in isolated rings of 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 of ACh-mediated relaxation in the rat mesenteric vascular bed, we compared the characteristics of the muscarinic receptor-induced and the receptor-independent calcium ionophore A23187-induced relaxation in mesenteric arteries to that in rat aorta. We 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 H Y P O T H E S E S 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 : Working Hypotheses: A . The relative importance of endothelial CI" channels and K + channels in modulating ACh-induced relaxation is different in muscular mesenteric arteries vs. elastic aorta. B . The relative contributions of N O , E D H F , and PGI2 to ACh-induced relaxation are different in rat mesenteric artery and aorta. Specific Objectives: 1) . To examine the effects of niflumic acid (NFA) , tetraethylammonium ( T E A ) , a relatively selective large conductance Ca 2 +-activated K + channel antagonist and the combination of these two drugs on A C h - induced relaxation in intact rat aortic and mesenteric rings. 2) . To test the influence of L - N M M A , a N O synthase inhibitor, on ACh-induced relaxation in the absence or presence of T E A or N F A plus T E A in isolated rat aorta and mesenteric arteries. 3) To examine the effects of N F A , T E A , and the combination of these two drugs on C a 2 + ionophore A23187-induced relaxation in intact rat aortic and mesenteric rings. 4) To compare the influence of 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) To examine the effects of 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 of isotonic high K + buffer, K + channel blockers and their combinations {including charybdotoxin ( C T X ) , a intermediate and large conductance Ca 2 +-activated K + channel blocker, apamin ( A M P ) , a small conductance C a 2 + -activated K + channel blocker, the combination of C T X and A M P , the combination of T E A and A M P } on ACh-induced relaxation resistant to L-NMMA/indomethac in in isolated rat mesenteric arteries. 140 ELL M E T H O D S A N D M A T E R I A L S 1. Isolated Artery Ring Preparation for Isometric Tension Measurement Male Wistar rats (250-350g) were anaesthetized by intraperitoneal injections of sodium pentobarbitone (65mg/kg). The thoracic aorta and superior mesenteric arteries were removed and placed in Krebs solution o f composition (in mM): N a C l 113, K C I 4.7, CaCl2 2.5, KH2PO4 1.2, M g S 0 4 1.2 NaHCOs 35 and dextrose 11.5, at room temperature. The arteries were carefully cleaned of 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 of 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 ml Krebs solution maintained at 37° C and gassed with 95% O2 - 5 % C 0 2 resulting in p H 7.4. Rings of aorta and mesenteric arteries were placed under resting tension of 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 7E 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 of each experiment. 2. Experimental Protocols The rings were initially stimulated with a submaximal concentration (EDgo) o f 7 6 phenylephrine (PE, 10" M and 3x 10 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"7 M and 3 x 10"6 M for aorta and mesenteric arteries, respectively) again. When a stable contraction was obtained, A C h (10"8 to 10"4 M ) or A23187 (3 xlO" 9 to 3 x l O ^ M ) was 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 of 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 of 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 of N F A (0.1 M ) and indomethacin (0.1 M ) were prepared in ethanol. A23187 (10~2 M ) was dissolved in dimethyl sulphoxide ( D M S O ) and diluted with ethanol. 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 2 values {defined as -log (ED50)} and percentage o f maximum relaxation (Rmax) as determined from individual curves were fitted according to the following logistic equation: ED5Q"H +An" where R is the relaxation (%), A is the concentration of 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 2 and Rmax- Two-way 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 I V . R E S U L T S 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 of intact rings of aorta and mesenteric arteries precontracted with P E (Fig. 2.1). This relaxation effect was endothelium-dependent since removal o f endothelial cells abolished the relaxation in both aorta and mesenteric arteries (data not shown). Addition of 30 p M N F A did not affect baseline or P E -induced tension in either arteries (legend to Fig. 2.1). Incubation of 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 of 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 of N F A (30 p M ) plus T E A (3mM), the A C h concentration-relaxation curve was shifted to the right (Fig. 2.1 A , Table 2.1). Combined use of N F A and T E A significantly decreased the sensitivity (pD 2 ) to A C h without changing the maximum 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 of concentration-response curve to A C h in mesenteric arteries (Fig. 2. IB) . T E A significantly decreased the A C h p D 2 value without affecting the maximum 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 of N F A (30 uM) and T E A (3 mM) or N F A (30 u M ) plus T E A (3 mM) 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 (NFA) , 1.62 ± 0.11g a b (TEA) , 1.51 ± 0.10g a b ( N F A +TEA) ( a P < 0.05 vs. control, b P < 0.05 vs. N F A ) . n= 6. B: Effect of N F A (30 uM) , T E A (3 mM) or N F A (30 u M ) plus T E A (3 mM) 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 (NFA) , 0.88 ± 0.15g a b (TEA) , 0.85 ± 0.10g a b ( N F A +TEA) ( a P < 0.05 vs. control, b P < 0.05 vs. N F A ) . n = 5. 145 146 TABLE 2.1 Potency (pD 2 ) and maximum relaxation (Rm a x , % loss of P E tone) to A C h or A23187 (aorta only) in the absence (Control) and in the presence of N F A (30 p M ) , T E A (3 mM) or N F A (30 p M ) plus T E A (3 mM) in isolated aortic and mesenteric artery rings with intact endothelium. Arteries were precontracted with P E (10"? M and 3 x 10" 6 M for aorta and mesenteries, respectively). Aorta Mesentery A C h A23187 A C h p D 2 Rmax (%) p D 2 Rmax (%) p D 2 Rmax(%) Control 7.41±0.13 102±1 7.88±0.06 86±4 7.94 ± 0.09 102±2 N F A 7.11±0.14 105±1 7.55±0.13 80±4 7.84 ± 0 . 5 9 107±5 T E A 7.06±0.41 102±1 7.63±0.10 72±12 7 . 0 7 ± 0 . 1 6 a b 111±6 N F A + T E A 6 . 1 1 ± 0 . 2 9 a b c lOOtfclO 7.73±0.15 83±5 6 . 9 4 ± 0 . 1 7 a b 101±1 Each value represents the mean of six (aorta) and five (mesentery) experiments ± S E M . ( a p < 0.05 vs. control, b p < 0.05 vs. N F A , c p < 0.05 vs. T E A ) . 147 1.2. , Effect of L-NMMA on ACh-induced Relaxation of PE-Evoked Tension In order to elucidate the nature of the inhibition by N F A in combination with T E A of 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 of 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 of 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). Pre-contractions produced by EDgo of 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 mM) alone nor N F A (30 u M ) plus T E A (3 m M ) further increased the P E tone in the presence of 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 of 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 2 value and maximum relaxation in comparison with L - N M M A alone (n=6, P < 0.05) (Fig 2.2A, Table 2.2). However, there was no difference between the inhibitory effect of T E A alone and N F A plus T E A in the presence of 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 of 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 mM) further reduced the A C h p D 2 value (n = 5, P 148 FIGURE 2.2 A : Effect of L - N M M A (300 p M ) on the relaxation response to A C h in intact rat aorta, in the absence and presence of T E A (3 mM) or N F A (30 p M ) plus T E A (3 mM). The initial tensions induced by P E were 0.90 ± 0.19g (Control); 1.66 ± 0.38g a ( L - N M M A ) ; 1.74 ± 0.29g a ( T E A + L - N M M A ) ; 1.75± 0.21g a ( N F A + T E A + L - N M M A ) ( aP < 0.05 vs. control), n = 6 B: Effect of 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 of T E A (3 mM) or N F A (30 p M ) plus T E A (3 mM) . The initial tensions induced by P E were 0.62 ± 0.14g (Control); 1.11 ± 0.12g a ( L - N M M A ) ; 1.15 ± 0.07g a ( T E A + L - N M M A ) ; 1.12 ± 0.1 l g a ( N F A + T E A + L - N M M A ) ( aP < 0.05 vs. control), n = 5. 149 150 TABLE 2.2 Effects of L - N M M A (300 uM) (A) and L - N M M A plus indomethacin (Indo, 20uM) (B) on potency (pD 2 ) and maximum relaxation (Rmax) to A C h in the absence and in the presence of 3 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"7 M and 3 x l 0 - 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) A C h (Mesentery) p D 2 Rmax (%) p D 2 Rmax (%) A . Control 7.44 + 0.12 98 + 3 7.57 + 0.04 111 + 5 L - N M M A 6.40 ± 0.25 a 40 + 9 a 7.09 ± 0 . 1 4 a 103 + 6 T E A + L - N M M A 5.67 ± 0.28 a b 21 ± 13 a b 5.97 + 0.23 a b 101 + 2 N F A + T E A + L - N M M A 5.64 ± 0.30 a b 26+ 9 a b 6.20 ± 0.06 a b 90+14 B. Control 7.47 ± 0.22 106 + 7 7.75 + 0.12 1 0 1 + 2 L - N M M A ± Indo 6.83 ± 0.24 a 34 +7 a 6.99 +0.15 a 91 + 5 T E A + L - N M M A ± Indo 6.06 ± 0.26 a b 23 ± 4 a 5.71 + 0.19 a b 102 + 2 N F A + T E A + L - N M M A ± Indo / / 5.85 + 0.21 " b 85+14 Values represent the mean ± S E M . from six (aorta) and five (mesenteric arteries) experiments. ( a P<0.05 vs. control, b P<0.05 vs. L - N M M A in A or vs. L - N M M A + I n d o in 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 of 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 2 values or the maximum relaxations to A C h (n = 5 for each artery, P > 0.05) between the curves obtained in the absence or in the presence of indomethacin under each of the conditions (Table 2.2). Indomethacin alone had also no effect on the response o f control to A C h obtained in absence of any inhibitor (pD 2 : control, 7.47 ± 0.22, indomethacin, 7.40 ± 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 of P G I 2 to ACh-induced 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 of endothelium-intact rings of 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 mM), or a combination of N F A (30 u M ) and T E A (3 mM) had no effect on the A23187-induced relaxation (Table 2.1). In mesenteric arteries, pretreatment with T E A (3mM) (n = 5, Fig. 2.3B) reduced the maximum relaxation of 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 of 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 of A C h (30uM) did not further relax the aorta in the presence of 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 uM) 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 of K + (Fig. 2.4 B(c)) in the mesenteric arteries. 3. Effect of KCI and K+ Channel Blockade with Apamin, C T X , T E A and Their Combinations on ACh-induced NO- Independent Relaxation The ACh-induced relaxation of 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 of L - N M M A (300 u M ) and KC1 (30 mM) 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 .14g a (L-N M M A ) and 1.70 ± 0.18g a ( K + ) ( a P < 0.05 vs. control), n = 6 B : Effect of T E A (3 mM) , L - N M M A (300 u M ) and KC1 (30 mM) 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 a (L-N M M A ) , 0.89 ± 0.09 a ( K + ) , and 0.90 ± 0.12 a (TEA) (B) (a P < 0.05 vs. control), n = 5 154 B - l o o 1 1 1 1 1 1 1 — • • — ' 1 9 8 7 6 5 - L o g [ A 2 3 1 8 7 ] M 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 uM) , A P M (0.3 uM), a small conductance Ca -dependent K channel blocker, and C T X (0.1 uM), a large- and intermediate- conductance Ca 2 +-dependent K channel blocker had no effect on baseline tension or P E (3xl0" 6 M ) tone (Fig. 2.5, legend). 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 of P E tension (Fig. 2.5). The A C h p D 2 value was significantly decreased by C T X as compared to control, but the magnitude of 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 of reduction in p D 2 value and maximum relaxation was similar (P > 0.05) between the two combinations (Table 2.3). Changing Krebs buffer to high K + (30 mM) solution caused a small contraction o f artery rings (0.40 ± 0.06g, n = 5). However, the initial maximum tension, after addition of P E ( 3 x l 0 6 M ) , was not significantly (P < 0.05) different from L - N M M A alone (Fig. 2.5 legend). 30 m M K + further reduced the maximum relaxation to 38 ± 4 %. The blockade was greater than that of co-application of 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 of 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 (TEA) , 1.08 ± 0.14g ( C T X + A P M ) , 1.38 ± 0 .09g a ( T E A + A P M ) , .1.1.1 ± 0.15g (K+) (P > 0.05, one-way A N O V A ) . n = 5-7. 159 -Log [ACh]M 160 TABLE 2.3 Potency (pD 2 ) and maximum relaxation (R^x, % loss of P E tone) to A C h in the absence (Control) and in the presence of A P M (0.3 uM) , C T X (0.1 uM) , T E A (3 mM), A P M (0.3 u M ) plus C T X (0.1 uM) , A P M (0.3 u M ) plus T E A (3 mM) or K C I ( K + , 30 m M ) in isolated mesenteric artery rings with intact endothelium and precontracted with P E (3x10 - 6 M ) . The experiments were performed in the presence of L - N M M A (300 u M ) plus indomethacin (10 uM) . A C h P D 2 Rmax (%) Control 7.27 ± 0 . 1 1 100 ± 1 A P M 6.98 ± 0.07 96 ± 5 C T X 6.37 ± 0.07 a 91 ± 9 T E A 5.85 ± 0.24 a b 95 ± 4 C T X + A P M 5.88 ± 0 . 3 3 a b 6 4 ± 8 a c T E A + A P M 5.48 ± 0.25 a b 69 ± 5 a c K + 5 . 6 0 ± 0 . 1 5 a b 3 8 ± 4 a Each value represents the mean of five to seven experiments ± S E M . ( a P<0.05 vs. Control; b P<0.05 vs. C T X ; CP<0.05 vs. K + ) . 161 V. DISCUSSION The aim of the second part of 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 in endothelium-dependent smooth muscle relaxation. We also examined the component of ACh-induced relaxation that is resistant to inhibition of 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 of 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 of the two compounds decreased the potency o f A C h approximately 10 fold, although it had no effect on the magnitude of the maximal relaxation. N F A is a potent calcium-activated CI- channel blocker in both endothelium and smooth muscle (Hogg et al. 1994a; Nil ius 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 of 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 endothelium-dependent relaxation induced by A23187. L ike A C h , A23187 evokes relaxation by releasing 162 N O (White and Martin 1989) and also induces endothelium-dependent hyperpolarization (Chen and Suzuki 1990). A23187 is a receptor-independent C a 2 + ionophore, which increases [Ca 2 + ] i by causing C a 2 + entry from the extracellular space via electroneutral exchange of one C a 2 + for two Ff" ions (Reed and Lardy 1972). On the other hand, A C h activates the muscarinic receptor, and elevates [Ca 2 + ] i by releasing C a 2 + from D?3-sensitive intracellular stores (Wang et al. 1995b) and stimulating transmembrane C a 2 + influx through receptor-and/or store-operated C a 2 + channels (Putney 1991; Wang and van Breemen 1997). The elevation of [Ca 2 + ] i in endothelial cells is believed to trigger the release of N O , PGI2 and E D H F that evoke endothelium-dependent smooth muscle relaxation (Busse et al. 1989; Fukao et al. 1997c; Whorton et al. 1984). Activation of CI" or K + channels by A C h in aortic endothelial cells has been shown to facilitate C a 2 + influx by providing a constant driving force for C a 2 + entry, as well as by preventing the depolarization-mediated inactivation of 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; Hosoki 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 of the combination of N F A and T E A on A C h -induced relaxation in aorta is due to an action of 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 of either of 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 of K + channels and vise versa in aorta. 163 Activation of 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. We have reported that N F A inhibited oti-adrenoceptor-induced 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 of 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 of cirazoline, a specific ctiA-adrenoceptor agonist, is apparently less than that of P E , a nonselective cti-adrenoceptor agonist. Therefore, we chose P E to precontract the arterial segments in these experiments. Among 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 Mart in 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 of N O synthesis due to the competitive inhibition of 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 of endothelium-dependent relaxation to A C h in 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 of a higher concentration of 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. We 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 of 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 of L - N M M A , the maximum relaxation to A C h was decreased nearly 60% and the p D 2 was reduced by approximately 10 fold, and T E A caused a further significant reduction in 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 of 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 a channels, but not C f channels mediate the NO-independent relaxation response to A C h in rat aorta. NO-Mediated and NO-Independent Relaxation In the presence of 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 of which is mediated via N O , while the other is probably mediated via E D H F because it is sensitive to K c a blocker T E A . Although the chemical nature of 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 via opening of K + channels. That A C h could induce an endothelium dependent hyperpolarization of 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; Komor i and Vanhoutte 1990). The existence of 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 of 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 of 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 of the first transient peak although it diminished the second component of 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 of those functional studies, by suggesting that K c a is, at least 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 % of 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 of N O S inhibitor (Chen and Suzuki 1989; Hatake et al. 1995). The different effects of 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 in 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 in the presence o f N O S inhibitor, such as the strains o f rats and the anatomical location of 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 of 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-NMMA-resis tant , 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 of ACh-induced relaxation (in the presence of 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 Ca2+-activated K + channels which 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 of C f channels has been further confirmed by the observation that there was no difference between the inhibitory effects of 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 of an inhibitory effect of 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 of 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 of a large N O S inhibitor-resistant 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 (Hwa et al. 1994), and the other using young female Wistar (Van de Voorde and Vanheel 1997) rats showed a greater inhibition of 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 of L - N M M A , T E A further decreased potency of 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 of 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 via different mechanisms. Although T E A significantly decreased the sensitivity of 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. Using 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 of 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 of 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 of 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 of C T X was smaller. However, in their study, the combination of 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 of these two toxins. Nevertheless, these results suggest that in superior mesenteric arteries A C h simultaneously activates both S K c a and B K c a , and that combined inhibition of both channels is necessary to inhibit E D H F . Elevation of the extracellular K + concentration [K +]o to above 25 m M (25, 30 or 60 mM) 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 wi l l decrease the K + equilibrium potential to the extent low enough to prevent hyperpolarization to K + 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% of the control, indicating that besides K c a channels other K + channels may be also involved. However, ACh-induced vasorelaxation was not completely inhibited by the combination of 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 + mobilizing compound like A C h , as well as A23187, induced an endothelium-dependent relaxation that was affected by neither 60 m M K + nor the combination of K + and N O pathway inhibitors in the rat mesenteric arterial bed (Kamata et al. 1996b). However, the actual characteristics of 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 of arachidonic acid, is produced by C O X in endothelium of most blood vessels including aorta and mesenteric arteries (Moncada et al. 1977; Peredo et al. 1997). It mediates endothelium-dependent relaxation, probably via the c A M P pathway and evokes membrane hyperpolarization of 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 of C O X with indomethacin either alone or in the presence of a N O S inhibitor does not interfere with the relaxing effect of 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 COX-dependent, indomethacin-sensitive relaxation and hyperpolarization to A C h were revealed after inhibition of both N O and E D H F pathway in rat hepatic and rabbit mesenteric arteries, and raised concerns that the role of P G I 2 may have been overlooked (Murphy and Brayden 1995b; Zygmunt et al. 1998). In this study, we systemically compared the effects of 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 of 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. We 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 of the arteries, since responses to E D R F release by calcium ionophore wi 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-NMMA-sens i t ive and insensitive relaxations although the latter was less prominent in aorta. The lack of L-NMMA-insens i t ive response to A23187 could be due to the influence of the degree of 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 o f 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 of A C h in the presence o f A23187 fully reversed the inhibitory effect of L - N M M A , but had only small relaxant effect on the P E tone in the arterial rings challenged with K + . Addition of A C h did not stimulate further relaxation in the presence of 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 of A23187 in mesenteric arteries. The lack of further relaxation to A C h in the presence of L - N M M A in aorta is perhaps due to the minimal contribution of E D H F in this vessel. In this situation, the presence of A23187, which could interfere with the initial C a 2 + profile,- may have an effect on the synthesis/release of 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 of K + conductance is involved in regulating both N O -dependent and NO-independent relaxation. That N O mediated vasorelaxation may involve hyperpolarization of vascular smooth muscle by activation of K + channels has been proposed. The contribution of N O to A C h - or carbachol-induced hyperpolarization of 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 a channels in rabbit 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 of a CTX-sensitive pathway with little or no contribution from a pathway activated by increased levels of cyclic G M P in rat mesenteric arteries (Plane et al. 1996). In this study we did not attempt to characterize the specific type of K channels implicated in N O action on smooth muscle cells. We 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 2 +-activated K + channels contribute to the process in mesenteric arteries but not in aorta. In addition, the differential effects of 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 a channels. Different abilities of A23187 and A C h to release N O and/or E D H F in the same preparations have been suggested in other studies. It has been suggested that A23187 only evokes the release of N O from the endothelium in rabbit carotid artery preconstricted with P E , whereas A C h can induce release of both N O and E D H F (Dong et al. 1997). In rabbit femoral artery, the relaxation to A23187 of NE-induced tension seems to be mediated predominantly via E D H F , while ACh-induced relaxation has been explained solely in terms of 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 2 + ] i into different regions of endothelial cells, leading to activation of different enzymes that are responsible for synthesis of N O and EDFfF. The question of cellular compartmentalization with respect to the synthesis o f N O , PGI2 and E D H F , and also colocalization of 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 in response to these factors in 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 of the contractile agonist can determine the release and/or effects of 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 a was different in porcine endothelial cells stimulated chemically with A23187 compared with cell stimulated mechanically (Erdbugger et al. 1997). Our study did not explore the chemical nature of 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 of 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 Weid 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 in 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 of 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 A23187-evoked hyperpolarization of the rabbit mesenteric artery, and it was concluded that A23187-mediated 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 V I . S U M M A R Y 1. A C h induced a concentration- and endothelium-dependent relaxation of 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 (CRC) to A C h was shifted to the right without a change in maximum relaxation (Rmax). In intact mesenteric arteries, the presence of T E A alone resulted in a rightward displacement of 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 2 value and RmaX. In the presence of L - N M M A , T E A further shifted the C R C for A C h to the right without change in RmaX as compared to L -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 of 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 T E A 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 of L - N M M A or K C I (30 mM) the response to A23187 was abolished in both arteries. The P E response resistant to A23187 in the presence of L - N M M A was completely reversed by subsequent addition of A C h in mesenteric arteries but not in aorta. A C h had no further effect on the tension remaining in the presence of K C I (30 mM) 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 m a x, while high K + (30mM) buffer had a greater inhibitory effect on the Rmax than the combinations in mesenteric arteries. 179 vn. C O N C L U S I O N S 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. C a 2 + ionophore A23187-induced relaxation is solely N O dependent, which is mediated by K + conductance in both aorta and mesenteric artery. Blockade of 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 2 + influx, thus ensuring a sustained N O synthesis and release. 180 v m P H Y S I O L O G I C A L S I G N I F I C A N C E The physiological importance of the relative contribution by N O and E D H F released by a variety of 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 of 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 of 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 of 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 of E D H F and other EDRF(s) , the cellular target of 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 R A T I O N A L E The hemodynamic hallmark of most forms of 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 of 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 of hypertension (Luscher et al. 1993b; Mistry and Nasjletti 1988; Purkerson et al. 1986; Wi lcox et al. 1996). In states of 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 Ca 2 + -ATPase gene expression and activity (Sowers et al. 1991; Tirupattur et al. 1993), and on the other, promoting ET-1 gene expression (Oliver et al. 1991) and increasing ET-1 release (Hu et al. 1993; Nava 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 of 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 of 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 insulin-resistant states. The genetically obese Zucker rat (fa/fa) is an insulin-resistant animal model with early onset severe hyperinsulinemia, hyperlipidemia and normal plasma glucose (York 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 of arteries from obese Zucker rats has been investigated in a number of studies (e.g. Bohlen and Lash 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 of insulin on vascular reactivity in Zucker rats has not yet been defined (Turner et al. 1995; Walker et al. 1997a). Thus, the purpose of the present study was to investigate whether altered vascular reactivity to N E could be detected, in the absence and/or presence of a pathophysiologically relevant concentration of insulin, in obese Zucker rats with established hypertension. In 183 addition, the contribution of endogenous vasoactive substances including N O , prostanoids and ET-1 to N E responses and to the vascular actions of 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 H Y P O T H E S I S 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 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 ET-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 in 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) . To compare the vasoconstrictor responses to N E in isolated perfused M A B from obese Zucker rats and their lean littermates. 2) . To 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) . To investigate the effects of N ° - monomethyl-L-arginine ( L - N M M A ) , a nitric oxide synthase inhibitor, alone and in the presence of insulin on the vasoconstriction to N E in M A B from Zucker rats. 4) . To examine the effects o f indomethacin, a C O X inhibitor, alone and in the presence of insulin, on NE-induced contraction in M A B from Zucker rats. 185 To test the effect of SQ 29,548, a TXA2/PGH2 receptor antagonist, alone and in the presence of insulin, on pressor response to N E in M A B from Zucker rats. To evaluate the effects of 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 of bosentan, a non-selective endothelin receptor (both E T B , and E T A ) antagonist, alone and in the presence of insulin on vasoconstriction to N E in M A B from Zucker rats. To examine the effect of B Q 788, a selective E T B receptor antagonist, or B Q 123, a selective E T A receptor antagonist, alone and B Q 788 or B Q 123 in the presence o f insulin on pressor responses to N E in M A B from Zucker rats 186 III METHODS AND MATERIALS 1. General Methodology Animals Male obese (fa/fa) Zucker rats and their lean (Fa/?) littermate controls were obtained at age 8 to 10 weeks from the Department of Physiology, University o f Brit ish 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 (SBP) 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. SBP 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 Hi l l s , C A , U . S . A ) . The inflated cuff pressure was 250 mmHg and pressure was released by 500 mmHg min" 1. To accustom them to the setting, rats were placed in the apparatus once each day for 3 days prior to the actual day o f measurement. B lood pressure was recorded and calculated as the mean o f five to six measurements. Biochemical analysis of blood samples B lood 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 Linco Research, Inc. (St. Charles, Missouri, U . S . A . ) Perfused isolated M A B preparation On each day o f experiments, one obese rat and a lean littermate were anaesthetized with sodium pentobarbital (120 mg kg" 1, subcutaneously over 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 McGregor (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 2 : 5% C 0 2 . The Krebs-bicarbonate buffer was o f the following composition (in mM): N a C l 113, K C I 4.7, glucose 11.5, M g S 0 4 1.2, CaCl22 .5 , K H 2 P 0 4 1.2, N a H C 0 3 25.0. The p H of the buffer following saturation with a 95% 0 2 : 5% C 0 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 ID 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 of the experiment. 2. Experiment Protocols The tissues were initially treated with a maximal concentration of K C I (120 urnol) by bolus injection 4 times. Perfusion pressure was allowed to return to baseline between each 188 injection of 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 (DRCs) 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 of 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 of a combination of 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 of KC1 (60 pmol) was made. The three D R C s were constructed at fixed time intervals. A time control experiment that consisted of three D R C s for N E without the addition of insulin or any inhibitors was also done. To confirm the effect of insulin on the N E response, another set of 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 r d D R C was constructed. 3. Chemicals (-)-Norepinephrine hydrochloride and indomethacin were obtained from Sigma Chemical Co. (St. Louis M O , U .S .A . ) . L - N M M A and B Q 788 were purchased from Calbiochem Corporation (La Jolla, C A , U . S . A . ) . Bosentan (R047-0203) was a gift from 189 Hoffmann-La Roche Ltd. (Bazel Switzerland). BQ 123 and SQ 29,548 were purchased from Research Biochemical International (Natick, MA, 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"1 ascorbic acid. The volume of N E for each injection 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 .02%, 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 of 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 of N E (control-1, control-2 and control-3, respectively) were obtained 2.5 h apart. Data represent the mean ± S E M of seven (lean) and six (obese) experiments. a P < 0.05 vs. control-1; b P<0.05 vs. control-2. L e a n 192 _ 2 5 0 n cn X E £ 2 0 0 C D i— CO 2 150 c g CO t C D CL CD co ro CD o c: 100 H • Control -1 0 Contro l -2 • Contro l -3 0.9 3.0 9.0 N E ( n m o l ) 30.0 90.0 O b e s e o> 250 -, x E f 200 H L— CO CO 0) c g ' co t CD Q_ CD CO ro CD i_ o c 150 A 100 H 0.9 3.0 9.0 N E (nmol) 30.0 90.0 193 I V . RESULTS 1. General Characteristics of Zucker Rats A t 25 weeks of 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 of obese and lean rats were 7.1 ± 0.9 and 6.6 ± 0.7 mmHg (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 of 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 of lean and obese Zucker rats Lean Obese Body 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) a Plasma triglyceride (mmol/1) 1.48 ± 0 . 1 4 (25) 36.93 ± 0.20 (27) a Plasma glucose (mmol/1) 7.34 ± 0 . 2 7 ( 5 ) 6.92 ± 0:31 (7) Values are shown as mean ± S E M (number of rats in parentheses). a P < 0.05, vs. lean (Student unpaired t-test). 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. a P < 0.05 vs. 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). On the other hand, the effects of 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 of indomethacin plus L - N M M A , responses of 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 of obese M A B to 0.9 nmol N E was significantly potentiated in 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 of indomethacin alone on responses of lean and obese rats to N E is due to reduction in the release of the C O X pathway contracting factor P G H 2 / T x A 2 , the effect of SQ 29,548, a P G H 2 / T x A 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 of hyperinsulinemia on reactivity of 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 of M A B from lean rats to any concentration of 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 of 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. a P < 0.05 vs. control (one-way ANOVA followed by Bonferroni post test: compare all column vs. control column). L e a n 250 -| 0 C O c 200 -o Q . C O 0 E 150 -E X ro 100 -ro '_c 50 -0 -a • Control -2 B L - N M M A (300uM) • Indomethacin (20nM) • L - N M M A + l n d o 0.9 JUL NE (nmol) O b e s e 250 n C O § 200 Q . C O 0 % 150 H E J 100 ro 50 H 0 • Control -2 B L - N M M A (300nM) • Indomethacin (20nM) • L - N M M A + l n d o a ==J a A 0.9 NE (nmol) 2 0 0 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. a P < 0.05 vs. lean; b P < 0.05 vs. control (two-way A N O V A followed by Newman Keuls post test) Lean 180 -. C D C O c o C L C O C D or E E x TO C O • Control • Insulin (200mU/i) 0.9 3 9 NE (nmol) Obese 180 -| 160 -C D C O c 140 H • Control • Insulin (200mU/i) NE (nmol) 202 from obese rats. However, responses of 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 , P G H 2 / T X A 2 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 of insulin were determined. None of the inhibitors tested ( L - N M M A , indomethacin, SQ 29,548, the selective E T B antagonist, B Q 788, the selective ETA 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. As shown in F ig 3.5, insulin further enhanced the L - N M M A -induced potentiation of 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 of indomethacin. In addition, indomethacin prevented insulin from further increasing the responses to N E in the presence of 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 of bosentan were similar to those of SQ 29,548, in that it blocked the potentiating effect of insulin on the responses of 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 of 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 of various inhibitors on potentiating effect of 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 in the absence ( • ) and presence of 200 mU/1 insulin ( • ) under control condition without any inhibitors (n=8) or in the presence of 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 9.0 nmol NE CD CO § 180-, Q . • 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 of N O and the potentiating effect of 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 of N E in obese M A B , which was mediated by C O X metabolites and was enhanced after inhibition of 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 SBP of these animals was on average 29 mmHg greater than that of 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) of 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 of 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 PE 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 age-matched 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 KCI were attenuated in obese M A B . In these experiments, we did not further explore the 207 mechanisms of the decreased responsiveness to KC1 in M A B from obese rats. However, it is known that challenge with KC1 stimulates endogenous N E release from peripheral sympathetic nerve endings (Vanhoutte et al. 1981) and it has been shown that inhibition of the endogenous N E with phentolamine decreases KCl-evoked tension. Therefore, it is possible that the reduced response to KC1 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 of 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 low to attenuate perfusion pressure under the basal perfusion conditions (perfusion rate of 3ml/min). Thus, the enhancement of 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 ct-adrenoceptor 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 of L - N M M A induced a 50% reduction of mesenteric vascular conductance, indicating a physiological role of N O in the control of the tone in the mesenteric vascular bed and even in the absence o f functional 208 sympathetic activity. Recently, Hor i et al (Hori et al. 1998) have directly measured changes in N O metabolite (NOx) 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 of 4ml/min) in control rats was very low 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 of N O x released only decreased significantly at a flow rate of 48 ml/min. We 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 of effect of L - N M M A on basal perfusion pressure observed in our study is probably due to the low perfusion rate plus the very low viscosity o f the Krebs solution so that the shear stress is too low to evoke a significant N O release. The mechanisms that mediate the release o f N O in the presence of 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 2 + / camodulin-dependent (Busse et al. 1993; Kuchan and Frangos 1994). This concept was supported by a study in Wistar rat perfused mesenteric arterial bed, in which the inhibition of N O synthesis or endothelium denudation was able to comparably potentiate the response to either receptor-mediated (NE) 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 of rats, data not shown). Both cti and ©^-adrenoceptors have been implicated in the endothelium-dependent NO-mediated depression of N E contraction in rat aorta (Kaneko and Sunano 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 Mi l l e r 1989). The partial ct2-adrenoceptor agonist clonidine induced relaxation via 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 2-adrenergic, endothelium-dependent responses are much less pronounced in mesenteric arteries compared to femoral, carotid, and coronary arteries of dog (Angus et al. 1986). This may reflect a lower density o f endothelial 0C2-adrenoceptors in this vascular bed. In addition, there still is an uncertainty with regard to 012-adrenoceptor 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 of cti-adrenoceptors and making any possible direct effect of ©^-adrenoceptor unlikely. It is known that in rat mesenteric vasculature, N E causes contraction predominantly, i f not exclusively, via ai-adrenoceptors (Chen et al. 1996; Colucci et al. 1980; McPherson et al. 1984; Nielsen et al. 1991; P ip i l i 1986). However, there 210 is no evidence so far to demonstrate the presence of ai-adrenoceptors on vascular endothelium of rat mesenteric arteries. On 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 of 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 of vascular smooth muscle may result in activation of eNOS 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 of L - N M M A , the application of the C O X inhibitor indomethacin to mesenteric arterial beds induced a significant suppression of pressor responses to N E in both lean and obese rats. Because indomethacin did not affect the responses to KC1 in preparations from either group of 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 of 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 of 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; P ip i l i et al. 1988), and to modulate the N E response (Coupar 1980; Mal ik 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 of adrenergic receptors (Pipili et al. 1988). In the present study, we found that indomethacin had no effect on basal perfusion pressure in Zucker mesenteric arterial bed. This may be due to the removal o f a balanced basal release of vasodilator and vasoconstrictor prostaglandins (PGs). Indeed, unbalanced release of vasodilator and vasoconstrictor PGs in rat mesenteric resistance vessels in S H R rats has been reported (Matrougui et al. 1997; Soma et al. 1985). Prostanoid modulation of 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; Manku and Horrobin 1976), aspirin, mefenamic acid (Manku and Horrobin 1976) and 5, 8, 11, 14-eicosatetraynoic acid (Coupar 1980) caused a significant depression of pressor responses to N E in rat isolated perfused mesenteric blood vessels. In addition, indomethacin reduced an increase in release of a P G E 2 - l i k e activity stimulated by N E to below resting values (Coupar 1980), and P G E 2 , as well as other prostaglandins restored the indomethacin-depressed response to N E (Coupar 1980; Coupar and McLennan 1978; Ma l ik et al. 1976; Manku and Horrobin 1976). Furthermore, arachidonic acid potentiated NE-induced vasoconstriction, which was abolished by simultaneous infusion of 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 45% 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 2 via the C O X pathway in Zucker mesenteric vessels. In addition, since indomethacin blocks all pathways of 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 of 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; Yokota 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 of 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 of the C O X pathway known to produce vascular contractions include P G H 2 , T x A 2 , PGF2«, and in rat, P G E 2 . O f these, PGH2 and T x A 2 , which interact with a common receptor, have been proposed to be endothelium-derived contracting factors (Luscher et al. 1992; Vanhoutte 1996) and therefore, may be responsible for indomethacin-induced 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 of 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 of the C O X pathway by N E , that was not inhibited by SQ 29,548, may reduce the biological activity of N O and indirectly enhance the NE-induced tone, resulting in a smaller effect of SQ 29,548 than of 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 of responses to N E by L - N M M A alone is the result of the concomitant activation of C O X leading to release of predominantly vasoconstrictor prostanoids, while the attenuation of responses to N E with indomethacin alone is due to the concomitant release of N O . In the presence of 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 N O S and C O X , we found that the pressor responses tended to be greater at most concentrations of N E , but it was only statistically significant at 0.9 nmol N E . Thus, a slight imbalance in the release of these factors, favoring vasodilation, may account for the decreased maximum response of M A B from obese rats to N E in the absence of insulin. 5. Lack of influence of endothelin on responses to NE. Pretreatment with bosentan, a non-selective ET-1 receptor antagonist, or blocking of 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, ET-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 Two studies have previously addressed the effects of 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 of insulin was impaired in tissues from pre-hypertensive obese rats, suggesting obese mesenteric arteries are resistant to a vasodilator action of insulin. The significance of 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. On 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 of age. However, this concentration of insulin is lower than that which obese rats are exposed to in vivo. In contrast with these reports, we found potentiation of pressor responses to 0.9 and 3.0 nmol N E on M A B from obese rats by a concentration of 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 of N E that are within the physiological range for circulating N E in rats (Dargie et al. 1977; Katholi et al. 1982). In contrast, perfusion with the same concentration of 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 of hypertension in 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 of 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 of vasodilators and vasoconstrictors it releases. For instance, Baron and Brechtel (Baron and Brechtel 1993) reported that in lean human subjects, a physiological concentrations of 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 in 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 of insulin appeared to be mediated by the release of N O (Chen and Messina 1996; Steinberg et al. 1994; Walker et al. 1997b), while the vasoconstrictor effect of insulin may be linked to changes in production o f C O X pathway metabolites (Wu et al. 1994) or increased release of endothelin (Nava et al. 1997). Recently, Schroeder et al. (Schroeder et al. 1999) demonstrated that insulin induced a concentration-dependent increase in diameter of endothelium-intact arterioles isolated from male Wistar rat gastrocnemius muscle. However, inhibition o f N O synthesis or removal of the endothelium inhibited the insulin-induced arteriolar dilation and revealed an insulin-induced vasoconstriction. Consistent with this, in the present investigation L - N M M A further enhanced the insulin-mediated potentiation of vasoconstriction to N E in M A B from obese rats. The amplification by L - N M M A of the potentiating effect of 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 of prostaglandins in the mechanisms o f insulin action has been reported. It was demonstrated that indomethacin treatment caused a metabolic state of 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 of 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 of 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 of adrenergic agonists on the production of these vasodilative eicosanoids in adipose tissue (and perhaps other tissues). Insulin has also been shown to specifically enhance TxA 2 - induced vasoconstriction in porcine coronary vascular beds (Yanagisawa-Miwa et al. 1990). Moreover, Keen et al. (Keen et al. 1997) recently reported that inhibition of TxA2 synthase markedly attenuated mean blood pressure increased by chronic insulin infusion in rats, suggesting that TXA2 is a key component of the chronic hypertensive effect of insulin. In this study, we demonstrated that application of indomethacin and SQ 29, 548 completely inhibited the potentiating effect o f insulin on pressor responses to N E in M A B from obese Zucker rats, suggesting that the potentiating effect of insulin is mediated by release of vasoconstrictor C O X metabolites, possibly PGH2 and/or T x A 2 . In addition, the lack of further effect of insulin in the presence o f indomethacin plus L - N M M A on responses to N E in 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 of endothelin in the effect of insulin on vasoconstrictor responses to N E in obese Zucker rats. We 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 of 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 of previous studies, which have implicated the vasoconstrictor E T in the vascular actions of insulin. For instance, insulin was reported to increase ET-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 of both indomethacin and bosentan, B y contrast indomethacin completely prevent the insulin response in M A B from control rats, suggesting a role of 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 A receptor expression in V S M cells (Hopfner et al. 1998) and both E TA and E TB receptor expression have been found to be increased in mesenteric arteries and aorta from obese Zucker rats with hypertension although local ET-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 of 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 of endothelin enhanced pressor responses to N E in human coronary arteries, rabbit aorta and perfused rat mesenteric arterial beds (Henrion and Laher 1993; Ki t a et al. 1998; Tabuchi et al. 1989b; Yang et al. 1990). The failure of either of the selective E T receptor antagonists to mimic the effects of bosentan means that the effect of endothelin cannot be attributed to its actions on either of the 219 receptor subtypes alone. Although the effects of bosentan might have resulted from an action unrelated to antagonism of E T receptors, blockade of both receptor subtypes has been previously shown to be required for antagonism o f some actions of E T (Fukuroda et al. 1996; Mick ley et al. 1997). 220 V I . SUMMARY 1. Zucker obese rats were moderately hypertensive and severely hyperinsulinemic at 25 weeks of age. 2. In perfused M A B from both obese and lean Zucker rats, N E induced a concentration-dependent 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 of 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 of 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 of indomethacin, insulin no longer had any effect on the N E responses in the obese M A B . 6. The presence of indomethacin inhibited the potentiating effect of 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 in the presence of L -N M M A in the obese M A B . 7. B Q 123, a selective E T A receptor antagonist, and B Q 788, a selective antagonist for ETB , alone had no effect on pressor responses to concentrations of N E in both lean and obese M A B . However, in the presence of 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 ET-1 receptor inhibitor, alone had no effect on pressor responses to concentrations of N E in both lean and obese M A B . The presence o f bosentan abolished the potentiating effect of insulin on vasoconstrictor responses to the lower concentration of N E . 222 VII. CONCLUSIONS We present evidence that NE-induced vasoconstriction is normally regulated by release of 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 of contracting C O X product(s) and enhanced contractile responses to physiological concentrations of N E in M A B from obese, but not from the lean rats. The effects of insulin in obese rats may be partially mediated by ET-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 of hypertension in obese Zucker rats. Whether the coexistent hyperlipidemia wi l l interfere with the bioavailability of 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 of insulin leading to the release o f contracting factor (s) in insulin-resistant animals has been demonstrated. 223 CONCLUDING REMARKS Mesenteric arteries and arterioles play an important role in the maintenance and control of peripheral resistance, thereby regulating blood flow and blood pressure. A particular feature of ai-adrenoceptor-mediated excitation-contraction coupling o f mesenteric arterial smooth muscle appears to be the dependence of the contractile response on V O C s , and thus on the membrane potential. Information on the role of 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 via opening of nifedipine-sensitive C a 2 + channels. This contribution of CI" channels in mesenteric arteries from 2K1C hypertensive rats appears to be reduced. The diminished role of 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 of the muscular mesenteric artery compared to the elastic aorta. Endothelial-dependent smooth muscle relaxation in the mesenteric arteries appears mainly to reflect the action of 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 of 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 of 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 2 + and membrane potential and in turn the regulation of contractile mechanisms by C a 2 + 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 of mechanisms that mediate relaxation of smooth muscle in the mesenteric arteries. 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