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Guanosine 3’:5’-cyclic monophosphate and contraction in vascular smooth muscle Lum Min, Suyin Ann 1995

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GUANOSINE 3':5*-CYCLIC MONOPHOSPHATE AND CONTRACTION IN VASCULAR SMOOTH MUSCLE  by SUYIN ANN LUMMIN B.Sc., Simon Fraser University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics Faculty of Medicine We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 12, 1995 © Suyin Ann Lum Min, 1995  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study! I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  Of  F^^^-Tntac-oloqy  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  Ayy-i'l  2&.  ^5  ,  o<-~>ot ~ T V i c > - c a p c . _ r B c : - s .  ABSTRACT  Presently, the literature regarding vascular smooth muscle contraction is coloured with many contradictory observations and conclusions. However, like many physiological systems, the biochemical pathways and functional events of vascular smooth muscle contraction vary between individual species and/or tissues. Therefore, determination of a ubiquitous excitation-contraction coupling mechanism is unlikely; variations between receptor classes, receptor density, excitation-contraction coupling pathways and the efficiency of the receptor-pathway interaction contribute to the various observations and conclusions. The inositol 1,4,5-trisphosphate (IP ) second messenger cascade regulates the 3  mobilization of intracellular Ca , and subsequently contraction, in vascular smooth 2+  muscle.  However, phospholipase G-mediated production of IP appears to be 3  controlled by tissue-specific regulatory factors. This study examines the effects of three such factors, the presence of extracellular Ca , the sensitivity of the associated 2+  G-protein and inhibition by 8-bromoguanosine 3':5'-cyclic monophosphate (8-brombcGMP), in isolated rat caudal artery. Concentration-response curves were constructed for phenylephrine and isometric contractions measured in isolated tissues. In addition, phosphatidylinositol turnover was assessed using anion exchange chromatography. The effects of 8-bromo-cGMP on phenylephrine-induced contractions and phosphatidylinositol hydrolysis were compared to those of felodipine, a dihydropyridine Ca -channel antagonist, and ryanodine, a putative depletor of intracellular Ca * stores 2+  2  in rat caudal artery. Pertussis toxin was used to determine the identity of the G-protein  - iii regulating phenyiephrine-induced contraction.  Further, the effects of felodipine and  ryanodine on contraction were determined in rat thoracic aorta to compare the contribution of extracellular and intracellular C a  2 +  to contraction between a large  conduit vessel and a small conduit vessel. The results of this investigation suggest that phospholipase C-activated phosphatidylinositol hydrolysis in the rat caudal artery is dependent on extracellular Ca , 2+  mediated,  in  part,  through  dihydropyridine  sensitive  Ca  Phospholipase C activity is not directly inhibited by 8-bromo-cGMP.  2+  channels.  However, the  nucleotide may regulate vascular smooth muscle contraction by inhibition of C a  2+  release from IP -mediated intracellular stores, but it is unlikely that 8-bromo-cGMP 3  affects ryanodine-sensitiVe stores.  None of the G-proteins coupled to the cti-  adrenoceptor mediated excitation-contraction pathway in rat caudal artery appear to be sensitive to pertussis toxin. Rat aortic tissue does not rely on intracellular C a  2+  to the  same extent that rat caudal artery does, confirming the tissue specificity of ctiadrenoceptor agonist induced excitation-contraction in vascular smooth muscle.  -iv-  TABLE OF CONTENTS ABSTRACT.....: TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES...., LIST OF ABBREVIATIONS..........; ACKNOWLEDGMENTS DEDICATION  , v  ..'.,..!•  1. INTRODUCTION 1.1. 1.2. 1.3. 1.4. 1.5. 1.6.  .1  Vascular Smooth Muscle Contraction Sources of Ca Utilized During Contraction-.'.., .-...':.W: IPs-Mediated C a Release.;!..,..........Phospholipase C cGMP Regulated Vasodilation Experimental Objectives..:... 2+  1 ....2 3 ...5 8 10  v  2+  ;  2. MATERIALS AND METHODS 2.1. 2.2. 2.3. 2.4. 2.5. 2.6.  15  Phosphatidylinositol Hydrolysis in Caudal Artery. :...15 Contractile Studies in Caudal Artery 16 Contractile Studies with Pertussis Toxin in Caudal Artery 17 Contractile Studies Thoracic Aorta .........................18 Data and Statistical Analysis... .........18 Chemicals ...... ......19  3. RESULTS.... 3.1. 3.2. 3.3. 3.4.  21  Phosphatidylinositol Hydrolysis in Caudal Artery... Contractile Studies in Caudal Artery Contractile Studies with Pertussis Toxin in Caudal Artery Contractile Studies in Thoracic Aorta  4. DISCUSSION... 4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 4.8.  ii iv v ..vi vii ...viii ix  !.........,...  .39  Intracellular Versus Extracellular Ca cti-Agonist-lnduced Contraction in Rat Caudal Artery Ryanodine Sensitive Ca Store in Rat Caudal Artery......... ai-Agonist-lnduced Contraction in Rat Thoracic Aorta... Ryanodine Sensitive Ca Store in Rat Thoracic Aorta...., cGMP and Inositol Phosphate Accumulation Experimental Design Conclusion.. ;  5. REFERENCES....  2+  2+  2+  >:;,...........  21 24 28 28 ........39 ...41 46 47 .......49 50 51 ......52  ...!........,.... ......54  - V  -  LIST OF TABLES  T A B L E I. P h e n y i e p h r i n e - i n d u c e d c o n c e n t r a t i o n - r e s p o n s e curve results o b t a i n e d from rat c a u d a l artery preparations in the a b s e n c e a n d p r e s e n c e of v a r i o u s antagonists......:.:.:  ;.:;......„..:....:..:;..  ..............26  T A B L E II. P h e n y i e p h r i n e - i n d u c e d c o n c e n t r a t i o n - r e s p o n s e c u r v e results o b t a i n e d from rat c a u d a l artery preparations in t h e a b s e n c e a n d p r e s e n c e of  v.. ..........36  .. *> p e r t u s s i s toxin.  r  T A B L E III. P h e n y i e p h r i n e - i n d u c e d c o n c e n t r a t i o n - r e s p o n s e c u r v e results o b t a i n e d from rat aortic t i s s u e in the a b s e n c e a n d p r e s e n c e of v a r i o u s . antagonists......:..  :.....;.....:...  .........7......  : .....v.........^...:.,38 v  LIST OF FIGURES  FIGURE 1. Phosphatidylinositol accumulation in rat caudal artery in the presence and absence of 8-bromo-cGMP, felodipine or Ca -free buffer...22 2+  FIGURE 2. Contractions in rat caudal artery in the absence and presence of 8bromo-cGMP:..,;  .....  25  FIGURE 3. Contractions in rat caudal artery in the absence and presence of felodipine.  ~28'  v  FIGURE 4. Contractions in rat caudal artery in the absence and presence of ryanodine.  „..31  FIGURE 5.; Contractions in rat caudal artery in the absence and presence of felodipine plus 8-bromo-cGMP.  ......................32  FIGURE 6. Contractions in rat caudal artery in the absence and presence of ryanodine plus 8-bromb-cGMP. ..!...:...,.........,!,..„...:.......;:  33  FIGURE 7. Contractions in rat caudal artery the presence and absence of. .* extracellular C a . 2+  ;........:..........,...........34'  FIGURE 8. Contractions ih rat caudal artery in the absence of pertussis toxin and following pretreatment with pertussisToxin:  35  FIGURE 9. Contractions in rat aortic tissue in the absence and presence of felodipine.  .37  FIGURE 10. Contractions in rat aortic tissue in the absence and presence of ryanodine.  ;39  LIST OF ABBREVIATIONS  WORD  ABBREVIATION  Adenosine 3':5' cyclic monophosphate  cAMP  Adenosine trisphosphate  ATP  8-Bromoguanosine 3':5'-cyclic monophosphate  8-bromo-cGMP  Diacylglycerol  DAG  Effective concentration for 50 % of maximum response  EC50  Ethylene glycol-bis (B-amino-ethyl ether) N, N, N\ N'tetraacetic acid  EGTA  Guanosine 3':5'-cyclic monophosphate  cGMP  Guanosine diphosphate  GDP  Guanosine triphosphate  GTP  Hill coefficient  n  Inositol 4,5-bisphosphate  Ins (4,5)P  Inositol 1,3,4,5-tetrakisphosphate  Ins (1 3 4 5)P  Inositol 1,3,4-trisphosphate  lns(1,3,4)P  Inositol 1,4,5-trisphosphate  2  1  1  1  3  IP3  Inositol 2,4,5-trisphosphate  Ins (2,4,5)P  Phosphatidylinositol  PI  Phosphatidylinositol 4,5-bisphosphate  PIP  Phospholipase C  PLC  Standard error of the mean  S.E.  3  2  4  - viii-  ACKNOWLEDGMENTS Many thanks to Dr. Reza Tabrizchi for his patience, guidance and humor; Dr, Morley Sutter, Dr. Richard Wall, Dr. Catherine Pang and Dr. Michael Walker for their advice; and, Eugene Lam, Yi He and Ali Nekooeian. Special thanks to,my Mum, Dad, Francis, Michael, Meiko, Nathaniel, Kira, Meiyen, Trevor and Clay. -  - ix -  DEDICATION  This work is dedicated to a dear friend and mentor, Dr. Fred Einstein.  1.  INTRODUCTION 1.1.  Vascular Smooth Muscle Contraction  The vasculature is a closed system of vessels responsible for transporting nutrients and oxygen to cells while simultaneously removing metabolic waste products. The circulatory system is a dynamic organ, capable of adapting to changes in individual tissue needs and blood volume. The etiology of several cardiovascular disease states, including hypertension, has been associated with a failure of the vasculature to adequately adapt to changes.  More specifically, rigorous regulation of the total  peripheral resistance of the vascular circulation is critical to homeostasis. The most important determinant of total peripheral resistance is blood vessel diameter. Vessel diameter is regulated by local chemical and extrinsic neural and hormonal mechanisms that can induce both dilation and constriction. Constriction of conduit blood vessels is a function of the smooth muscle surrounding the vessels. Extracellular calcium (Ca ) was first identified as a critical 2+  factor in smooth muscle contraction in 1961, when Bohr arid Goulet recorded a decrease in epinephrine-induced tension in rabbit and dog aorta and rabbit mesoappendix resistance vessels when C a  2+  was removed from the bathing medium.  Even at this time, Bohr and Goulet (1961) were able to appreciate the complexity and variability of the intracellular signal transduction mechanisms responsible for smooth muscle contraction. Since 1961, many of the details of excitation-contraction coupling have been established; however, the mechanisms mediating vascular smooth muscle tone remain poorly understood.  Vascular smooth muscle tone  is regulated. by pharmacomechanical and  electromechanical coupling mechanisms under the control of the autonomic nervous system (Somlyo & Somlyo, 1994). Each smooth muscle fibre is a spindle shaped cell containing two contractile proteins: actin, a thin filament anchored to the plasma membrane through cytoplasmic dense bodies, and myosin, a thick filament whose mobile head forms transient cross-bridges with actin (Somlyo et al., 1985). The extent of overlap between the two contractile proteins, and degree of muscle tension, is determined by cross-bridge cycling (Bagby & Corey-Kreyling, 1985; Mulvany, 1985): During  contraction, . the  intracellular  Ca * 2  concentration  approximately 0.1 pM to 5 pM (Hartshorne, 1982). As the C a  2 +  increases  from  concentration rises, it  binds the cytosolic Ca *-bihding protein, calmodulin, to form a regulatory 2  Ca *2  calmodulin complex that activates the catalytic subunit of myosin light-chain kinase (MLCK) (Ruegg et al., 1985). MLCK then phosphorylates serine-19 on the regulatory light chain of myosin (MLC o); both the electric and steric effects of phosphorylation 2  activate myosin's ATPase. activity and motility (Sweeny et al., 1994).  A four-state  cross-bridge model described by Hai and Murphy (1988) successfully predicts the mechanical properties and energetics of smooth muscle contraction. Cycling between the four states requires adenosine triphosphate (ATP), but the primary regulatory mechanism is the Ca *-dependent phosphorylation of MLC o (Walker et al., 1994). 2  2  1.2.  Sources of Ca Utilized During Contraction 2+  Cross-bridge cycling is regulated phosphorylation of  MLC20;  crucial for contraction.  through the  Ca -calmodulin-dependent 2+  therefore, increasing the free cytosolic Ca * concentration is 2  Ca * can enter from the extracellular space through voltage2  gated and ligand-gated channels (Karaki et al., 1984) or C a  2+  can be released from  intracellular storage sites within the sarcoplasmic reticulum, mobilized through the activity of intracellular second messengers (Brown etal., 1984; Streb et al., 1984). Two functionally and spatially distinct C a  pools have been identified within the  2+  sarcoplasmic reticulum of vascular smooth muscle cells; one controlled by inositol 1,4,5-trisphosphate (IP ) and the other sensitive to inhibition by ryanodine (Yamazawa 3  et al., 1992; Tribe et al., 1994). The relative contribution of each C a  2+  source is tissue-  specific, dependent on variations in receptor classes and density, excitation-contraction coupling mechanisms and the efficiency of the receptor-signal transduction interaction (Vila etal., 1993).  ^  .  :V  '  Berridge (1983) observed a rapidly increased concentration of phosphatidylinositol (PI)  metabolites in insect salivary glands upon stimulation with 5-  hydroxytryptamine.  Since activation of plasmalemma receptors had previously been  associated with the release of internal C a  2+  (Schulz & Stolze, 1980; Exton, 1981),  Berridge (1983) suggested that IP was a second messenger capable of mobilizing 3  stored C a . Subsequently, results from rabbit pulmonary artery (Somlyo et al., 1985), 2+  rat thoracic aorta (Chiu et al., 1987), rat cerebellar microsomes (Stauderman et al., 1988) and rat pancreatic acinar cells (Thevenod et al., 1989) have demonstrated a close correlation between the generation of IP and the release of intracellular C a . 2+  3  the effects of IP are not blocked by vanadate, an inhibitor of the sarcoplasmic 3  reticulum Ca -ATPase re-uptake bump; therefore, IP acts by increasing the Ca * 2+  2  3  permeability of the sarcoplasmic reticulum, not by inhibiting the sequestering of free Ca  2+  (Somlyo et al., 1985). However, IP may increase intracellular C a 3  2+  to a lesser  extent by inhibiting extrusion from the cytosol by the sarcolemmal Ca *-ATPase 2  (Popescu era/., 1986). In 1958, duodenal smooth muscle was reported to be irreversibly contracted by the plant alkaloid ryanodine (Hillyard & Procita, 1958). Subsequent studies, reported, however, that ryanodine inhibited agonist-induced contractions in smooth muscle (Kanmura et al., 1988).  It now appears that low concentrations of ryanodine are  contractile, while higher concentrations inhibit contraction (Berridge, 1993). Recently, the'putative mechanism of action of ryanodine in smooth muscle was proposed. Ryanodine inhibits smooth muscle contraction by stimulating a slow C a  2+  efflux from an  intracellular store; as a result, the store is depleted and subsequent agonist stimulation futile (Kanmura et al., 1988; Hwang & van Breemen, 1987; Julou-Schaeffer & Freslon, 1988). The intracellular C a  2 +  store that is sensitive to blockade by ryanodine, does not  appear to be mobilized by the second messenger IP (Seiler et al., 1987); however, 3  cyclic adenosine 5'-diphosphoribose (cADPR), a metabolite of, B-nicotinamide adenine dinuclebtide (P-NAD), has been tentatively identified as the endogenous second messenger capable of mobilizing intracellular C a  from the ryanodine sensitive pools  2+  (White etal., 1993; Galione ef a/., 1993; Meszaros etai, 1993).  .  -  Studies using various antagonists of IP - and ryanodine-sensitive C a 3  , have provided evidence that the two intracellular functionally and spatially independent.  Ca * 2  2+  release  release pathways are  IP -induced release is blocked by cinnarizine, 3  flunarizine, tetraethylammonium and the local anaesthetics benzocaine and lidocaine (Berridge & Irvine, .1989; Seiler et al., 1987). affected  by nifedipine,  diltiazem,  IP -mediated Ca * mobilization is not  verapamil,  2  3  dantoline,  methylenedioxyihdene,  Tu-conotoxin or ryanodine (Seiler ef a/., 1987; Shah & Pant, 1988).  However, pre-  treatment with ryanodine does inhibit cADPR-indueed release (Seiler et al., 1987; lino  et al., 1988; Yamazawa et al, 1992;  White et al.,  1993); and, although heparin  completely blocks IP -induced Ca * release, it does not 2  3  affect  ryanodine sensitive  release (Berridge, 1993; White et al., 1993). The ryanodine sensitive store, can be mobilized by caffeine (Berridge, 1993); however, the IP sensitive store is blocked, not 3  activated, by caffeine in Xenopus oocytes and rat cerebellar microsomes (Mignery ef  al., 1990; Parker & Ivorra, 1991; Brown et al., 1992). Although caffeine-induced C a  2+  release is insensitive to changes to the membrane potential, release, from the IP  3  sensitive pool is regulated by potential dependent PI hydrolysis (Ganitkevich & Isenberg, 1993). The  IP  3  sensitive pool appears to sequester C a  when  2+  the cytosolic  concentration is high (~ 10" M); however, the IP insensitive pool is the higher affinity 6  3  Ca  2+  buffer, capable of adjusting low intracellular C a  2+  concentrations (~ 10'  7  M)  (Thevenod et al., 1989). In brain microsomes, cADPR and IP released approximately 3  20 and 60 % of the stored C a , respectively (White et al., 1993). 2+  induce C a Ca  2+  2+  Also, C a  2+  can  release from the ryanodine sensitive stores (lino, 1989), but micromolar  inhibits C a  2+  release from IP sensitive stores by indirectly decreasing the affinity 3  of the agonist for its low affinity receptor (Benevolensky ef al., 1994).  And finally,  ryanodine does not affect PI hydrolysis as neither basal nor 8-arginine vasopressininduced IP synthesis in A7r5 cultured aortic smooth muscle cells were blocked in the 3  presence of this antagonist (Berman etal., 1994): Therefore, IP and ryanodine appear 3  -  6 -  to regulate functionally and spatially distinct C a  2+  pools within the same cell  (Yamazawa et al., 1992). There is some evidence, however, to suggest that IP can induce C a 3  2+  release  from a ryanodine sensitive pool. Yamazawa and co-workers (1992) recorded an IPsmediated rise in intracellular C a treatment with ryanodine.  2+  that was reduced by approximately 50 % after  In skinned guinea pig taenia caeci, the amount of C a  2+  released by the application of IP , or IP and caffeine, was approximately twice as large 3  3  as that released by caffeine alone (lino et al., 1988). Finally, ryanodine blocked C a  2+  accumulation in both caffeine and IP sensitive stores (Kanmura et al., 1988). These 3  results indicate that two C a  2+  pools may co-exist within some excitable cells; one  sensitive to IP and the other sensitive to both IP and ryanodine (Yamazawa et al., 3l  1992).  3  Alternatively, an excitable tissue may contain only one C a  sensitive to both second messengers. characteristics of intracellular C a 1.3.  2+  2+  pool which is  Undoubtedly, the functional and spatial  stores vary between tissues.  IPs-Mediated Ca * Release 2  Many of the physiological and biochemical characteristics of the IP -mediated 3  intracellular C a  2+  store have been investigated.  The IP receptor consists of four 3  identical 313 kDa subunits (Furuichi et al., 1989; Mignery et al., 1990, Supattapone et  al., 1988). Each subunit contains an N-terminal cytosolic extension responsible for IP  3  binding and regulation by ATP and phosphorylation, and eight transmembrane spanning regions which form the C a  2+  channel through which stored C a  2 +  is released  (Mignery & Sudhof, 1990; Ehrlich & Watras, 1988; Mignery et al., 1990). Immunogold labelling has identified IP receptors on both central and peripheral sarcoplasmic 3  -  7 -  reticulum (Nixon et al., 1994); however, only part of the organelle, the calciosome, may actually be involved in C a  2+  release (Meldolesi etai., 1990).  The IP receptor responds to ligand binding by undergoing a conformational 3  change and increasing the opening frequency of the associated C a  channel (Mignery  2 +  & Sudhof, 1990; Berridge, 1993). Channel activation appears highly co-operative; at least four independently bound IP molecules may be required to open each channel 3  (Meyer et al., 1990): Four hundred C a  molecules are estimated to be released for  2+  every molecule of IP bound (Stauderman efa/.,1988). 3  The IP receptor recognizes the D-isomer of IP more readily than the L-isorrier 3  3  (Berridge & Irvine, 1989).  However, release of C a  2 +  can also be elicited by inositol  2,4,5-trisphosphate (lns(2,45)P ) and inositol 4,5-bisphosphate (lns(4,5)P ). Based on 3  2  ECso values, the relative potencies are IP > lns(2,4,5)P > lns(4,5)P in rat cerebellum 3  (Stauderman etai,  3  2  1988).  In the absence of extracellular K\ or in the presence of tetraethylammonium, IP -mediated  Ca  3  2+  release was  reduced, although,  other  monovalent  cations  ( N a » T r i s > L i ) could substitute effectively (Berridge & Irvine, 1989; Shah & Pant, +  +  1988). C a  +  2+  result of C a  alone could not stimulate K* influx; therefore, K* conductance was not a 2+  efflux, but was required for release and may neutralize the charge  displacement resulting from the release of bound C a  2 +  (Shah & Pant, 1988).  Following inositol phosphate production, the reaccumulation of C a  2+  coincided  wjth the degradation of IP , indicating that metabolism is the primary mechanism for 3  terminating the effects of the second messenger (Stauderman et al., 1988; Berridge & Irvine, 1989).  Repeated additions of IP  3  to rat cerebellar microsomes did not  -  8 -  desensitize the receptor nor diminish the release of C a .  However, the receptor did  2+  desensitize in response to lns(2,4,5)P and lns(4,5)P (Stauderman et al., 1988). 3  2  Stauderman and co-workers proposed two. explanations: (1) lns(4,5)P are not metabolized as quickly as IP , the C a 2  2 +  3  if lns(2,4,5)P  3  and  channel will remain open and  refilling will be impossible, or (2) lns(2,4,5)P and lns(4,5)P cannot be metabolized to 3  lns(1,3,4,5)P  or  4  Insd.S^Ps,  metabolites  2  of  IP  which  3  may  enhance  the  reaccumulation of stored C a . 2+  1.4.  Phospholipase C  The phosphoinositide-specific phospholipase C (PLC) isozymes are a series of catalytic proteins that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP ) to water 2  soluble IP and membrane bound diacylglycerol (DAG). IP induces C a 3  3  2 +  release from  intracellular stores, while DAG activates protein kinase C. PLC can be activated by receptor occupation, increased intracellular C a  2+  or Na , or increased extracellular K* +  (Akhtar & Abdel-Latif, 1978; Eberhard & Holz, 1988; Martin et al., 1986; Kendall & Nahorski, 1985). PLC activation in vascular smooth muscle appears to be coupled to the a r adrenoceptor.  Prazosin, an cn -adrenoceptor antagonist, inhibited  noradrenaline-  induced PI hydrolysis in rat aorta (Rapoport, 1987; Manolopoulos et al., 1991), guinea pig cerebral cortical synaptoneurosomes (Gusovsky et al., 1986) and rat caudal artery (Cheung et al., 1990). Noradrenaline-induced PI hydrolysis in rat caudal artery was insensitive to rauwolscine, an ct -adrenoceptor antagonist (Cheung ef a/., 1990). 2  In  addition, in rat portal vein, noradrenaline-induced PI hydrolysis was shown to be insensitive to chlorethylchlonidine, an irreversible cti^adrenoceptor alkylating agent  -  9 -  (Lepretre et al., 1994). Although the a -adrenoceptor agonist UK 14304 stimulated PI 2  accumulation in rat caudal artery, its effects failed to plateau with increasing concentrations and were sensitive to prazosin rather than rauwolscine (Cheung et al., 1990). The cti-adrenoceptor antagonists did not directly affect intracellular C a as these antagonists did not affect acetylcholine- and caffeine-induced C a (Lepretre et al., 1994).  2 +  2 +  stores  release  PLC activation, in both vascular and nonvascular tissues,  appears to be coupled to adrenoceptor occupation through an unidentified pertussis toxin insensitive G-protein (Martin etal., 1986; Cheung etal., 1990; Eberhard & Holz, 1988; Vallar et al., 1987; Lepretre etal., 1994; Muntzef al., 1993; Nichols etal., 1989). While some tissues appear to require extracellular C a  2 +  as a PLC activator or  activation-dependent co-factor, PI hydrolysis in other tissues seems to be C a  2+  independent. For example, noradrenaline induced a transient rise in IP and a phasic 3  contraction of rabbit mesenteric artery in a Ca -free solution (Itoh et al., 1992); but, 2+  acetylcholine-induced stimulation of PI hydrolysis in rabbit iris smooth muscle was increased from 16 % above unstimulated preparations in the absence of extracellular Ca  2+  to 32 % when C a  2 +  was included in the extracellular medium (Akhtar & Abdel-Latif,  1978), PLC activity has also been investigated by measuring intracellular C a and C a  2+  2 +  release  efflux; although less direct than measuring inositol phosphate accumulation,  these techniques are validated by the close correlation between PLC hydrolysis and intracellular C a  2+  release (Somlyo etal.,  1986; Thevenod et al., 1989).  1985; Stauderman et al., 1988; Chiu et al.,  Results from these studies also demonstrate the  variable dependence of PLC on extracellular C a  2 +  amongst excitable tissues. Thrombin  caused a rapid elevation of intracellular C a  2+  in the absence of extracellular C a  2+  in  human platelets (Nakashima et al., 1986). In addition, noradrenaline-stimulated C a  2+  efflux and contraction were transiently increased in rabbit aorta in the absence of extracellular C a  2+  (Meisheri et al., 1986).  However, in guinea pig cerebral cortical  synaptoneurosomes (Gusovsky et al., 1986), permeablized RINm5F cells (Vallar et al., 1987)  and rat cerebellar cortical slices (Kendall & Nahorski, 1985), removal of  extracellular  Ga , 2+  or addition of a potent dihydropyridine inhibitor, completely  abolished agonist-, Bay-K-8644- (a dihydropyridine C a  2 +  channel blocker) and K*-  induced inositol, phosphate accumulation. Furthermore, stimulation of noradrenalineinduced PI hydrolysis in rat caudal artery Was enhanced by exogenous C a  2 +  in a  concentration-dependent manner (Cheung et al., 1990), and thyrotropin-releasing hormone-mediated activation of PLC in permeablized G H cells was maximized by the 3  presence of Mg , ATP and extracellular C a 2+  2+  (Martin et al., 1986).  Finally, C a 2+  induced contractions in permeabilized rabbit mesenteric artery were not impaired by felodipine (Hagiwara et al., 1993), noradrenaline-stimulated PI accumulation in canine femoral artery was not affected by diltiazem (Eskinder et al., 1989), and in rat portal vein myocytes, oxodipine did not affect noradrenaline-induced inositol phosphate accumulation (Lepretre et al., 1994).  These latter results suggest that in rabbit  mesenteric artery, canine femoral artery and rat portal vein myocytes, PLC does not require an influx of C a Ca  2+  2+  through voltage sensitive channels for activation.  However,  influx may have occurred through dihydropyridine insensitive channels, as  observed in rabbit urethral smooth muscle.  Garcia-Pascual and co-workers (1993)  found that endothelin-1-induced inositol phosphate accumulation and contraction in  - 11 rabbit urethra were abolished in a Ca -free medium. 2+  However, pre-treatment with  nifedipine did not reduce PI hydrolysis, and contraction was sensitive to Ni . +2  Therefore, PLC isozymes may differ with respect to their sensitivity to extracellular C a and the means by which C a  2+  2+  is acquired. According to Rhee and Choi (1992), the  activities of all PLC isozymes are dependent on Ca *. Therefore, differences between 2  tissues may reflect the variation in the C a 1.5.  2+  sensitivities of the isozymes.  cGMP Regulated Vasodilation  Researchers who first recorded the association between increased guanosine 3':5'-cyclic monophosphate (cGMP) levels and increased muscle tone suggested that the nucleotide functioned as mediator of contraction (Dunham et al., 1974). Soon after, however, Gruetter and co-workers (1975) observed that inhibitors of nitric oxideinduced relaxation also blocked cytosolic guanylate cyclase, the enzyme responsible for producing cGMP from guanosine triphosphate (GTP). This led Gruetter to suggest that cGMP was involved in vasodilation, not contraction.  During contraction, cGMP  apparently acted as a negative feedback inhibitor of induced C a  2 +  influx to prevent  over-stimulation (Schultz etal., 1977). In 1980, Furchgott and Zawadzki demonstrated that relaxation of vascular smooth muscle required the presence of functional endothelial cells, and that acetylcholine, acting at muscarinic receptors on the endothelial cells stimulated the release of a substance that caused relaxation. Since then, it has been shown that endothelium-dependent relaxation in vascular smooth muscle is mediated through cGMP, and nitric oxide is the diffusible endotheliumderived relaxing factor which stimulates production of cGMP by soluble guanylate cyclase (Furchgott et al., 1984; Rapoport & Murad, 1983; Furchgott, 1988; Ignarro et  1988).  al,  However, to date, the mechanism of action of cGMP in vasodilation in  vascular smooth muscle has not been identified. Some of the mechanisms presently considered to explain the effects of cGMP in vascular smooth muscle relaxation include: (1) stimulation of a sarcolemmal Ca *2  ATPase (Popescu et al., 1985), (2)  activation of the Na /K7CI" +  co-transporter  (O'Dorinell & Owen, 1986), (3) activation of the Na*/Ca * exchanger (Furukawa et al., 2  1991), (4) inhibition of Ca * translocation across the plasma membrane (Collins et al., 2  1985; Ishikawa et al, 1993; Collins et al., 1986; Mery et al, 1991; Tohse & Sperelakis, 1991), (5) acceleration of C a  2+  uptake into the sarcoplasmic reticulum (Twort & van  Breemen, 1988), (6) inhibition of Ca * release from intracellular storage sites (Collins et 2  al, 1986), (7) phosphorylation of myosin light chain kinase (Nishikawa et al, 1984), (8) phosphorylation of cGMP-dependent cAMP phosphodiesterase (Mery et a/., 1991), or (9)  phosphorylation of the contractile  machinery  (Baltenspergeref  al,  1990).  Considering the significance of its role in the regulation of vascular muscle tone, it is unlikely a single mechanism is responsible for the effects of cGMP; rather, several mechanisms;probably contribute to cGMP-dependent vasodilation. If, as Collins and co-workers suggested, cGMP inhibits intracellular Ca * release, 2  there are three mechanisms by which cGMP could Operate, (1) blocking production of IP through PLC, (2) blocking IP binding or activation of the sarcoplasmic reticulum 3  3  receptor, or (3) hastening IP inactivation or metabolism. A cGMP dependent binding 3  protein,  cGMP-regulated  ion  channel  or  cGMP-binding  cyclic  nucleotide  phosphodiesterase, may mediate the effects of the nucleotide (Lincoln & Cornwell, 1993).  -  13-  Conflicting results have been reported for the effects of cGMP on PI hydrolysis. 8-Bromoguanosine S'iS'-cyclic monophosphate (8-bromo-cGMP), a stable, membrane permeable analogue of endogenous cGMP, inhibited PI accumulation induced by noradrenaline and thrombin in rat aorta and human platelets, respectively (Rapoport, 1986; Nakashima et ai, 1986).  Furthermore, PI hydrolysis in human platelets was  inhibited by sodium nitroprusside, which elevates cGMP (Takai et ai., 1981). However, the vasorelaxant osthole increased cGMP levels in rat aorta without affecting inositol phosphate formation (Ko et ai, 1992).  8-Bromo-cGMP did not block noradrenaline-  induced PI hydrolysis in canine femoral artery (Eskinder et ai, 1989). Finally, dibutyrylcGMP did not modify the carbachol-induced formation of inositol phosphates in rat gastric mucosal cells (Puurunen ef ai, 1987). 1.6.  Experimental Objectives  The regulatory effects of cGMP op the biochemical IP  second messenger  3  cascade of vascular smooth muscle contraction were investigated.  The rat caudal  artery was the preparation used in this study. It has been previously demonstrated that agonist-mediated PI hydrolysis is 10-100 times greater in the rat caudal artery than the larger, more frequently employed rat thoracic aorta (Labelle & Murray, 1990). Therefore, according to these researchers, the rat caudal artery appears to be the better preparation to investigate the relationship between PI hydrolysis and contraction. Specifically, the objectives of this study were, (1) to determine whether or not PLC activity in rat caudal artery is dependent on extracellular C a , (2) to determine 2+  whether or not a pertussis toxin sensitive G-protein mediates cti-adrenoceptor contraction, (3) to establish whether or not cGMP. blocks IP  3  production, (4) to  - 14 determine whether or not rat caudal artery contains two functionally distinct intracellular Ca  2+  stores, one sensitive to ryanodine, the other to IP , and (5) to compare the relative 3  contributions of intracellular and extracellular C a  2+  to contraction in rat caudal artery  and rat thoracic aorta. Phenyiephrine-induced PI accumulation and contraction were measured in denuded rat caudal artery in the presence and absence of extracellular C a . 2+  the  effects of 8-bromo-cGMP on phenyiephrine-induced contraction and PI hydrolysis were investigated in rat caudal artery. The effects of the nucleotide were compared to those of felodipine, a dihydropyridine Ca -channel antagonist, and ryanodine, a putative 2+  depletor of intracellular C a  2+  stores. The effect of pertussis toxin on phenyiephrine-  induced contractions was investigated in rat caudal artery.  Finally, comparative  contractile studies in rat aortic rings were carried out in the presence and absence of felodipine and ryanodine.  - 15 2.  MATERIALS AND METHODS 2.1.  Phosphatidylinositol Hydrolysis in Caudal Artery  Male Sprague-Dawley rats (300-400 grams) were anaesthetized with sodium pentobarbital (65 mg kg" ) i.p.. The caudal artery was dissected free and cleaned of 1  connective tissue in Krebs-bicarbonate buffer of the following composition (in mM): NaCI, 120; KCI, 4.6; glucose, 11; MgCI , 1.2; CaCI , 2.5; K H P 0 , 1.2; NaHC0 , 25.3. 2  2  2  4  3  The pH of the buffer following saturation with a 95 % O2: 5 % C 0 gas mixture was 7.4. 2  The composition of the Ca -free buffer was the same, except that CaCI was omitted 2+  2  and replaced with ethylene glycol-bis(B-amino-ethyl ether) N, N, N', N'-tetraacetic acid (EGTA) (2 mM).  The endothelial cell layer was removed from cleaned arteries by  inserting a wire through the lumen and rubbing gently. Phosphatidylinositol hydrolysis was assayed as described previously by Cheung ef al. (1990). Arteries were cut into 1 cm segments and incubated in buffer at 37°C for 60 minutes; the incubating buffer was changed every 20 minutes. The tissues were transferred into 2 of mi fresh buffer containing 6 pCi ml" [ H] myo-inositol to load for 90 1  3  minutes. Loaded tissues were washed five times with ice-cold Krebs before being put into individual assay tubes containing 10 mM LiCI in 300 pi buffer at 37°C. previously been demonstrated  that Li  +  enhances the  It has  accumulation of inositol  phosphates by inhibiting metabolism by monophosphatase; maximum enhancement resulted from 10 mM Li with an EC50 of 0.2 mM (Cheung ef al., 1990). Ca -free Krebs+  2+  bicarbonate buffer containing EGTA (2 mM) was used to wash, and later incubate those tissues that would be stimulated by phenylephrine in the absence of extracellular  - 16 C a . Individual assay tubes contained felodipine (10 nM), 8-bromo-cGMP (10 pM) or 2+  double distilled water (9 pi), and tissues were allowed to incubate for 20 minutes before phenylephrine (0.3-30.0 pM) from serial dilutions or double distilled water (9 pi) was added.  Individual tubes were gassed continuously with a mixture of 95 % 0  2  : 5 %  C0 . 2  Stimulation with phenylephrine was stopped after 45 minutes by adding .300 pi of ice-cold trichloroacetic acid (1M) to each sample. Tubes were left on ice for 30 minutes and then vortexed. Aliquots (500 pi) were transferred to clean assay tubes and washed with 2 volumes of water-saturated diethyl-ether five times. After the final wash, residual ether was rapidly evaporated by blowing air across, the surface of the sample. Part of each sample (400 pi) were then transferred to a clean tube and N a H C 0 (100 mM) 3  added to adjust the pH to 7-8.  Aliquots (400 pi) were then applied to Dowex-1 (x8)  anion-exchange columns (formate form, 100-200 mesh, 1 ml). Columns were washed with 12 ml of unlabeled myo-inositol (5 mM). Tritiated inositol phosphates were then eluted with 12 ml of 0.1 M formic acid/1 M ammonium formate. Two volumes of ScintiSafe 30 % scintillant were added to the two 6 ml aliquots collected and the radioactivity was counted in a Packard 1600TR liquid scintillation counter.  The efficiency of the  counter was 67 %. At the completion of each experiment, each tissue was blotted and weighed to normalize the radioactive counts per mg wet weight. 2.2.  Contractile Studies in Caudal Artery  Caudal arteries  were isolated as described for the  phosphatidylinositol  hydrolysis studies, except that the tissues were cleaned in ice-cold buffer and cut into  - 17 0.6 cm lengths. Tissues-were mounted in 20 ml organ baths at 37°C under a force of 9.8 mN and gassed continuously with a mixture of 95 % 0  2  : 5 % C 0 . Contractions 2  were measured through a force transducer and recorded on a Grass Model 7 Polygraph. The tissues were equilibrated for 60 minutes. Phenylephrine (1 u.M) was then used to contract the tissues and acetylcholine (10 u,M) was applied to ensure the functional endothelial response had been removed. Tissues were left for 30 minutes before a control concentration-response curve to phenylephrine (0.01 pM-100 pM) was constructed.  Following construction of control concentration-response curves,  felodipine (1 & 10 nM), ryanodine (3 & 10 uM), 8-bromo-cGMP (10 uM), distilled water (6 or 20 u,l) or a combination of felodipine and 8-bromo-cGMP or ryanodine and 8bromo-cGMP were left in contact with the tissues for 30 minutes to be followed by construction of another concentration-response curve to phenylephrine in the presence of the antagonists or water.  Tissues were allowed to equilibrate for 60 minutes  between concentration-response curves.  When responses were generated in the  absence of extracellular C a , Ca -free buffer was in contact with the tissues for 15 2+  2+  minutes before a concentration-response curve to phenylephrine was constructed. 2.3.  Contractile Studies with Pertussis Toxin in Caudal Artery  Caudal arteries were isolated and mounted as described for the contractile studies in caudal artery detailed above. Following the control concentration-response curve to phenylephrine, tissues were allowed to rest for 30 minutes and then pertussis , toxin (100 ng/ml) or distilled water (40 pi) was added to the bath. The tissues were incubated for 2 hours and then washed 5 times.  A second concentration-response  -  18-  curve was then generated for phenylephrine following the pretreatment with pertussis toxin. Contractile Studies Thoracic Aorta  2.4.  Rats were anaesthetized as described for the phosphatidylinositol hydrolysis studies in caudal artery detailed above, and the thoracic aortae rapidly removed and placed in Krebs-bicarbonate buffer.  Aortae were cleaned of extraneous connective  tissue and the endothelial layer removed by inserting a 20 gauge needle into the lumen and gently rubbing. Aortas were cut into 0.5 cm segments, and mounted in 20 ml organ baths at 37°C under a force of 9.8 mN and gassed continuously with a mixture of 95 % 0 : 5 % C 0 . Tissues were allowed to equilibrate for 60 minutes. Phenylephrine (10 2  2  pM) was then used to contract the tissues and acetylcholine (10pJv1) was applied to ensure the functional endothelial response had been removed. Tissues were left for 30 minutes before a concentration-response curve to phenylephrine (0.1 nM-10 pM) was constructed.  Following construction of control concentration-response curves,  felodipine (1 nM & 10 nM), ryanodine (3 uM and 10 uM) or distilled water (80 pi) were added and left in contact with,the tissues for 30 minutes to be followed by construction of another concentration-response curve to phenylephrine in the presence of felodipine or ryanodine.  Tissues were allowed  to equilibrate  for 60  minutes  between  concentration-response curves. 2.5.  Data and Statistical Analysis  In the absence of an antagonist, PI hydrolysis was maximum in the presence of 10 uM phenylephrine. Therefore, in experiments involving the use of an antagonist,  - 19 results are expressed as a percentage of the maximum PI turnover induced by 10 pM phenylephrine in untreated control tissues. Each experiment was run parallel to two such controls, of which the average radioactivity per mg wet weight was calculated. Results from contractile studies were calculated as a percentage of maximum contraction induced by phenylephrine in the absence of antagonists. maximum, Hill coefficient and  EC50  Percent  values were calculated for individual curves using a  program executed on an IBM compatible microcomputer (Wang & Pang, 1993). These parameters were determined by fitting the percent contractile response at increasing concentrations of phenylephrine, [PE], by non-linear least squares to the relation Y= a + bX, where Y = response and X = [PEf / ([PE]" +  [EG50]"  ) with n fixed at "floating"  integral values to obtain the best fit. Comparison of PI hydrolysis between control and corresponding experiments in the presence of an antagonist were made using an unpaired Student's t-test. For the results of the contractile studies, an analysis of variance block design was used for comparisons between control and treated tissue values for % maximum, Hill coefficient and  EC50.  For multiple comparisons, Duncan's multiple range test was used to  compare between means. For all cases, a probability of error of less than 0.05 was selected as the criterion for statistical significance. 2.6.  Chemicals  8-Bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-bromo-cGMP), Lphenylephrine HCI and ethylene glycol-bis(B-amino-ethyl ether) N, N, N', N'-tetraacetic acid (EGTA) were purchased from Sigma Chemical Co. (Ca., USA).  %o-[2- H(N)]3  inositol (17.0 Ci mmol* ), ryanodine and salt free pertussis toxin were purchased from 1  Amersham (Ont., Canada), Calbiochem (Ca., USA) and List Biological Laboratories (Ca, USA), respectively.  Felodipine was a gift from Hassle (Sweden).  With the  exception of felodipine, all drug solutions were prepared in double distilled water. A 10 mM felodipine stock solution was made in 80 % ethanol; dilutions were made with double distilled water. (B.C., Canada).  .  Ail other chemicals were purchased from Fischer Scientific ,  RESULTS  3.  Phosphatidylinositol Hydrolysis in Caudal Artery  3.1.  Phenylephrine (0.3-30 u.M) increased PI hydrolysis in a concentration dependent manner.  A maximum accumulation, approximately  10-times greater than- basal  accumulation, was achieved at a concentration of 10 pM phenylephrine (Figure 1). The addition of phenylephrine greater than 30 pM significantly decreased PI hydrolysis when compared to maximal PI accumulation (results not shown). Similar effects have been observed in rat caudal artery with supermaximal concentrations of noradrenaline (Labelle & Murray, 1990). Phenylephrine-induced PI hydrolysis was not affected by 8-bromo-cGMP (10 pM) and maximal accumulation remained approximately 10-fold above basal dpm mg wet weight' (Figure 1). In contrast, felodipine (10 nM) caused a noticeable decrease in 1  PI hydrolysis at all concentrations of phenylephrine tested (Figure 1). This decrease was found to be statistically significant (n = 6; p < 0.05) phenylephrine. weight" . 1  at 1, 3 and 10 pM  Maximum accumulation was only 7-fold above basal dpm mg wet  Maximum hydrolysis was restored by 30 pM phenylephrine, although  accumulation remained noticeably below the control value.  We also found that PI  accumulation induced by phenylephrine could be completely abolished in the absence of C a  2 +  (Ca -free buffer containing 2 mM EGTA). 2+  It was noted that basal PI  accumulation was not affected by 8-brbmo-cGMP, felodipine or Ca -free buffer. 2+  - 22 Figure 1. Phosphatidylinositol accumulation in rat caudal artery in the presence and absence of 8-bromo-cGMP, felodipine or Ca -free buffer. 2+  Phosphatidylinositol accumulation in rat caudal artery induced by phenylephrine in the absence (open) and presence of 8-bromo-cGMP (left-to-right hatched) or 10 nM felodipine (right-to-left hatched) or Ca -free buffer (2 mM EGTA) (cross hatched). Percent accumulation calculated relative to the maximum accumulation induced by 10 pM phenylephrine in the absence of an antagonist. Basal accumulation for control and treated tissues was 215 ± 20 dpm mg wet weight' (mean ± S.E.; n = 24). Each column represents the mean of six experiments ± S . E . Significantly different from control (p < 0.05). 2+  1  a  - 23 -  (|04uoo %) uoueinwnoov |o;isou!|Ap!;eL|clsoL|d  - 24 3.2.  Contractile Studies in Caudal Artery  Concentration-response curves to phenylephrine were displaced to the right in the presence of 8-bromo-cGMP (10 pM) (Figure 2). 8-Bromo-cGMP significantly (n = 6; p < 0.05) decreased the maximum tension and increased the EC50 of the concentrationresponse curve; the Hill coefficient was not affected. These effects were unchanged by 8-bromo-cGMP  (10  pM)  in  a  subsequent  concentration-response  curve  to  phenylephrine (Table I). In the presence of felodipine (1 & 10 nM) contractions induced by phenylephrine were attenuated (Figure 3).  Felodipine at 1 nM and 10 nM significantly (n = 6; p <  0.05) reduced the maximum tension to 77 ± 8 % and 57 ± 3 % of control, respectively. However, the EC50 and Hill coefficient values were unchanged in the presence of felodipine (Table I).  Ryanodine (3 & 10 pM) also inhibited phenylephrine-induced  contractions (Figure 4), and it significantly (n = 6; p < 0.05) reduced the maximum response and increased the EC50 value without affecting the Hill coefficient (Table I). Addition of ryanodine did not affect baseline tension.  Maximum response, Hill  coefficient and EC50 were not affected following addition of distilled water over time (Table I). The maximum tension of the control curve in the time study was 2.69 ± 0.47 mN (mean ± S . E ; n = 6). Contractions  induced  by  phenylephrine  were  not  further  inhibited  by  simultaneous application of 8-bromo-cGMP and felodipine as compared to felodipine alone (Table I; Figure 5). ryanodine  In contrast, concomitant application of 8- bromo-cGMP and  significantly  (n = 6;  p < 0.05)  inhibited  contractions  - 25-  120  r  -2  -1  0  1  2  3  Log [Phenylephrine] (jiM) Figure 2. Contractions in rat caudal artery in the absence and presence of 8bromo-cGMP.  Cumulative concentration-response curves in rat caudal artery to phenylephrine in the absence (circles) or in the presence of 8-bromo-cGMP (10 u,M) for the first time (squares) and for the second time (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the absence of antagonist was 4.17 ± 0.68 mN (mean ±S.E.; n = 6).  - 26 Table I. Phenyiephrine-induced concentration-response curve results obtained from rat caudal artery preparations in the absence and presence of various antagonists. EC50, Hill coefficient (n) and % maximum response values obtained from individual concentration-response curves in rat caudal artery preparations. Each value ^ represents the mean of six experiments ± S.E.  Groups  EC50  n  (uM)  % Maximum  Control 8-Bromo-cGMP (10 M-M) 8-Bromo-cGMP (10 uM)  1.25 ± 0 . 1 8 2.65 ± 0.35" 3.16±0.49  1.10 ± 0 . 0 6 1.32±0.07 1.25 ± 0 . 0 6  Control Felodipine (1 nM) Felodipine (10 nM)  1.49 ± 0 . 2 8 3.51 ± 0 . 9 8 2.63 ± 0.45  0.92 ± 0.04 1.03 ± 0 . 1 0 1.11 ± 0 . 0 9  104 ± 1 77±8 57±3  Control Ryanodine (3 pM) Ryanodine (10 u.M)  1.29±0.19 1.96 ± 0.27 2.17±0.32  1.10±0.10 1.03 ± 0 . 0 6 1.03 + 0.08  102 ± 1 81 ± 6 76 ± 8  Control Fel (1 nM) + 8-br(10pM) Fel(10nM) + 8-br(10|iM)  0.89 ± 0 . 1 8 2.60±0.25 3.83 ± 0.28  0.92 ± 0 . 0 4 1.21 ± 0 . 0 5 1.25 ± 0 . 0 5  104 ± 1 74±4 59 ± 5  Control Ry(3^M) + 8-br(10pM) Ry(10pM) + 8-br(10pM)  1.62 ± 0 . 3 6 3.53 ± 0 . 8 1 3.52 ± 0.70  1.15 ± 0 . 0 3 120 ± 0 . 0 9 1.37 ± 0 . 1 3  102 ± 1 52 ± 7 38 ± 9  Control Ca -free + C a (2.5 mM Ca )  0.95 ± 0 . 1 2 N 1.01 ± 0 . 1 2  1.16±0.10 N 1.20 ± 0 . 0 8  101 ± 1 N 103 ± 3  Control Distilled H 0 (6 p.l) Distilled H 0 (20 u|)  1.48 ± 0 . 0 3 1.56 ± 0 . 3 1 1.52 ± 0 . 2 5  1.01 ± 0 . 0 7 1.10 ± 0 . 0 5 1.05 ± 0 . 0 7  103 ± 1 102 ± 4 103 ± 6  2+  2  2  a  a  2+  2+  a  a  a b  a  102 + 1 81 ± 3 80 + 5  a  a  a  a b  a  a  a  ab  ac  ac  Fel = felodipine; Ry = ryanodine; Significantly different from control, p < 0.05; "Significantly different from the first concentration of drug, p < 0.05; Significantly different from the same concentration of ryanodine alone, p < 0.05; N = no measurable increase in contraction above resting tension. ; a  c  - 27 -  120  r  -2  -1  0  1  2  3  Log [Phenylephrine] (uM) Figure 3. Contractions in rat caudal artery in the absence and presence of felodipine.  Cumulative concentration-response curves in rat caudal artery to.phenylephrine in the absence (circles) or in the presence of felodipine, 1 nM (squares) and 10 nM (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the absence of antagonist was 3.04 ± 0.22 mN (mean ± S.E.; n = 6).  - 28 induced by phenylephrine in comparison to ryanodine alone (Figure 6).  Ryanodine (3  pM & 10 pM) alone lowered the maximum contraction to 81 ± 6 % and 76 + 8 % of control, respectively, and when combined with 8-bromo-cGMP these values were further reduced by 29 % and 38 % of control, respectively (Table I). Attempts to produce contractions with phenylephrine in Ca -free buffer were 2+  unsuccessful (Figure 7). contraction.  Addition of up to 300 pM phenylephrine did not produce  However, re-introduction of C a  2 +  into the bath restored contraction to  phenylephrine without affecting the maximum tension, Hill coefficient or EC50 in comparison to the control (Table I). 3.3.  Contractile Studies with Pertussis Toxin in Caudal Artery  Concentration-response curves to phenylephrine were not affected by pertussis toxin (100 ng/ml) (Figure 8).  Maximum tension, EC50 and Hill coefficient were  unchanged by pertussis toxin (Table II).  Maximum tension, EC50 and Hill coefficient  were not affected by the addition of distilled water over time (Table II).  The maximum  tension of the control curve in the time study was 2.19 ± 0.29 mN (mean ± S.E.; n = 6). 3.4.  Contractile Studies in Thoracic Aorta  Concentration-response curves to phenylephrine were displaced to the right in the presence of felodipine (10 nM) (Figure 9). Felodipine (10 nM) significantly (n = 4; p < 0.05) lowered the maximum tension and increased the EC50 (Table III).  The Hill  coefficient was not affected by pretreatment with felodipine. Felodipine (1 nM) did not affect the concentration-response curves to phenylephrine.  - 29 Ryanodine (3 \M)  did not significantly (n = 4; p < 0.05)  affected the  concentration-response curve to phenylephrine, maximum tension, EC50 and Hill coefficient remained unchanged (Figure 10; Table III).  Ryanodine (10 pJVI) increased  the maximum tension without affecting EC50 and Hill coefficient; however, the time control showed a similar, but insignificant trend (Figure 10; Table III).  Maximum  tension, EC50 and Hill coefficient were not affected by the addition of distilled water over time (Table III).  The maximum tension of the control curve in the time study was  10.47 ± 0.93 mN (mean ± S.E.; n = 4).  - 30 -  120  r  -2  -1  0  1  2  3  Log [Phenylephrine] (pM) Figure 4. Contractions in rat caudal artery in the absence and presence of ryanodine. Cumulative concentration-response curves in rat caudal artery to phenylephrine in the absence (circles) or in the presence of ryanodine, 3 pM (squares) and 10 pM (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the absence of antagonist was 4.70 ± 0 . 7 1 mN (mean ± S.E.; n = 6).  - 31 -  120  r-  -3  - 2 - 1  0  1  2  3  Log [Phenylephrine] (uM) Figure 5. Contractions in rat caudal artery in the absence and presence of felodipine plus 8-bromo-cGMP.  Cumulative concentration-response curves in rat caudal artery to phenylephrine in the absence (circles) and in the presence of 1 nM felodipine and 10 \xM 8-bromocGMP (squares) or 10 nM felodipine and 10 uM 8-bromo-cGMP (triangles). Each point represents the mean of six experiments ± S E . Maximum tension in the absence of antagonists was 5.29 ± 0.97 mN (mean ± S.E.; n = 6).  Log [Phenylephrine] (uM) Figure 6. Contractions in rat caudal artery in the absence and presence of ryanodine plus 8-bromo-cGMP.  Cumulative concentration-response curves in rat caudal artery to phenylephrine in the absence (circles) and in the presence of 3 pM ryanodine and 10 pM 8-bromocGMP (squares) or 10 pM ryanodine and 10 pM 8-bromo-cGMP (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the absence of antagonists was 3.79 ± 0.51 m'N (mean ± S.E.; n = 6).  - 33 -  -3  -2  -1  0  1  2  3  Log [Phenylephrine] (uM) Figure 7. Contractions in rat caudal artery the presence and absence of extracellular Ca *. 2  Cumulative concentration-response curves in rat caudal artery to phenylephrine in the presence of C a (circles), in the absence of C a (2 mM EGTA) (squares) and after Ca * had been reintroduced (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the presence of extracellular C a * was 4.97 ± 0.63 mN (mean ± S.E.; n = 6). 2 +  2 +  2  2  - 34  T  120  100 o  c  t> 2  "Ef 80  o  O E | 60 x ^ 40  20  J_  -2  -1  0  1  Log [Phenylephrine] (uM)  Figure 8. Contractions in rat caudal artery in the absence of pertussis toxin and following pretreatment with pertussis toxin. Cumulative concentration-response curves in rat thoracic aorta to phenylephrine in the absence (circles) or in the presence of pertussis toxin (100 ng/ml) (triangles). Each point represents the mean of six experiments ± S.E. Maximum tension in the absence of antagonist was 3.76 ± 0.64 mN (mean ± S.E.; n = 6).  35 Table II. Phenyiephrine-induced concentration-response curve results obtained from rat caudal artery preparations in the absence and presence of pertussis toxin. Hill coefficient [n) and % maximum response values obtained from individual concentration-response curves in rat caudal artery preparations in the presence and absence of pertussis toxin. Each value represents the mean of six experiments ± S.E. EC50,  Groups  ECwUiM)  n  % Maximum  Control Pertussis toxin (100 ng/ml)  1.51 ± 0 . 0 5 1.28 ± 0 . 1 0  1.49 ± 0 . 0 6 1.37 ± 0 . 0 4  100 ± 1 104 ± 4  Control Distilled H 0 (40 ul)  1.72 ± 0 . 2 1 1.66 ± 0 . 2 2  1.49 ± 0 . 1 7 1.48 ± 0 . 1 5  101 ± 1 106 ± 6  2  )  120  - 36 -  r  -2  -1  0  1  2  3  4  5  Log [Phenylephrine] (u.M) Figure 9. Contractions in rat aortic tissue in the absence and presence of felodipine. Cumulative concentration-response curves in rat thoracic aorta to phenylephrine in the absence (circles) or in the presence of felodipine, 1 nM (squares) and 10 nM (triangles). Each point represents the mean of four experiments ± S.E. Maximum tension in the absence of antagonist was 11.64 ± 0.51 mN (mean ± S.E.; n •= 4).  - 37 Table III.  Phenyiephrine-induced concentration-response curve results obtained  from rat aortic tissue in the absence and presence of various antagonists. ECso, Hill coefficient (n) and % maximum response values obtained from individual concentration-response curves in rat thoracic aorta preparations. Each value represents the mean of four experiments ± S.E.  EC5o(p,M)  n  % Maximum  Control Felodipine (1 nM) Felodipine (10 nM)  6.6 ± 2 . 4 31.3 ± 7 . 8 63.0±17.2  1.42 ± 0 . 2 4 1.47 ± 0 . 1 7 0.94 ± 0 . 1 2  93 ± 1 86 ± 3 74±3  Control Ryanodine (3 pM) Ryanodine (10 pJvl)  7.8 ± 2 . 0 14.0 ± 1 . 0 13.0 ± 3 . 0  1.64 ± 0 . 1 2 1.53 ± 0 . 0 6 1.67 ± 0 . 3 4  94 ± 1 97 ± 2 104±2  Control . Distilled H 0 (20 pi) Distilled H 0 (20 ul)  17.0 ± 8 . 2 16.0 ± 3 . 4 22.0 ± 6 . 0  1.78 ± 0 . 6 1 1.79 ± 0 . 0 4 1.44±0.12  103 ± 7 106 ± 4 112 ± 5  Groups  2  2  Significantly different from control, p < 0.05  a  a  •  a  a  - 38 -  120 r 100 .2 80  ts 2  8 60 E ZD E x 40 CO  20  -2  0  1  2  3  Log [Phenylephrine] (u\M)  Figure 10. Contractions in rat aortic tissue in the absence and presence o f ryanodine. Cumulative concentration-response curves in rat thoracic aorta to phenylephrine in the absence (circles) or in the presence of ryanodine, 3 uM (squares) and 10 uM (triangles). Each point represents the mean of four experiments ± S.E. Maximum tension in the absence of antagonist was 10.84 ± 1.52 mN (mean ± S.E.; n = 4).  - 39 4.  DISCUSSION This  investigation  found  that  phenylephrine-induced  inositol  phosphate  accumulation and contraction in rat caudal artery were critically dependent on the presence of extracellular C a . 2 +  The influx of C a  2 +  required for phenylephrine-induced  inositol phosphate accumulation and contraction was regulated, in a small part, by felodipine sensitive C a  2 +  channels. Pre-treatment with pertussis toxin did not impair the  phenylephrine-mediated excitation-contraction coupling mechanism in rat caudal artery. 8-Bromo-cGMP lowered the maximum phenylephrine-induced tension when applied alone, but did not additively inhibit contraction when applied concomitantly with felodipine.  8-Bromo-cGMP did not block phenylephrine-induced inositol phosphate  accumulation.  Ryanodine inhibited phenylephrine-induced contractions, and these  inhibitory effects were significantly potentiated in the presence of 8-bromo-cGMP. In rat thoracic aorta, pre-treatment with felodipine weakly, but significantly, reduced the maximum phenylephrine-induced contraction; however, pre-treatment with ryanodine did not affect phenylephrine-induced tension under normal pharmacological conditions. 4.1.  Intracellular Versus Extracellular C a  2 +  According to a classification scheme derived by Han and co-workers in 1987, ctiadrenoceptors that induce an influx of extracellular Ca * are designated au, while cti2  adrenoceptors that mediate PI hydrolysis are ecu,. More recently, however, Wilson and Minneman  (1990) suggested that both ai  subtypes induce inositol phosphate  accumulation, but differ with respect to their mechanisms of activation. According to  Wilson and Minneman (1990), the a Ca  2+  1 a  subtype mediates PLC activity that is induced by  influx, while the a i subtype activates a C a  2+  b  insensitive PLC.  The heterotrimeric guanine nucleotide^-binding proteins (G proteins) that couple surface receptors to secondary effectors have been classified as members of the G|/G , 0  G , G12 or G families (Simon et al., 1991). s  q  Upon stimulation of the receptor, the  guanosine diphosphate ( G D P ) bound to the a subunit of a G protein is exchanged for GTP and the G protein trimer dissociates to the functional G complex.  0  subunit and G p / G ,  Gene transfection studies have demonstrated that ai -adrehoceptors can b  interact favourably with all four members of the  Goq  family,  G 14i a  G l6, a  Goq  3nd G 11, a  coupling noradrenaline receptor-binding to inositol phosphate accumulation; however, ai -adrenoceptors can only interact favourably with G ^ and G n (Wu et al., 1992). a  a  Bordetela pertussis toxin catalyzes ADP-ribosylation at the a subunit of sensitive G proteins, uncoupling the protein from its associated receptor and inhibiting activities mediated by the dissociated G  a  subunit and Gp/G^ complex.  The GJG\ family is  sensitive to pertussis toxin, although most tissues are insensitive to bacterial toxins (Wu et al., 1992; Smrckaef al., 1991). PLC is the enzyme responsible for hydrolyzing membrane phospholipids to intracellular second messengers. Three families of phosphoinositide selective PLC enzymes, B, 8 and y, have been identified and subclassified on the basis of their molecular structure and mechanism of regulation (Rhee & Choi, 1992). Although the activities of all three families are C a  2+  dependent, their mechanisms of activation  appear to differ (Rhee & Choi, 1992). The p isozymes appear to be activated through G proteins coupling surface receptors (Wu et al., 1992), the y isozymes may be  activated when phosphorylated by a tyrosine kinase/receptor (Rhee & Choi, 1992), and the 5 isozymes may be activated by C a  2+  (Hamet ef ai, 1995).  PLC can be activated by both the G subunit or the Gp/Gy complex (Katz et al., a  1992). However, gene transfection studies have demonstrated that certain G protein subunits couple significantly more effectively with specific PLC isozymes. For example, it has been reported that Goq and G n activated PLC-Bt, but not the"-y1, -61 or -B2 a  isozymes (Wu etai., 1992; Smrcka etai., 1991; Aragay et al., 1992; Park etai., Lee etai  1992;  1992). In addition, G ie, but neither Goq nor G n , activated PLC-P2 (Schwinn a  a  etai, 1991; Lee etai, 1992). 4.2.  cti-Agonist-Induced Contraction in Rat Caudal Artery  Unfortunately, the PLC isozymes in rat caudal artery have not yet been identified. However, the results of this study and results previously reported, strongly suggest that in rat caudal artery, ai adrenoceptors are coupled through a Gaq/G ii a  protein to PLC-pi and PLC-51. Agonist-induced contractile studies in the presence of the ccia-adrenoceptor antagonist SZL-49 demonstrated that nerve terminals in rat caudal artery are only associated with cci -adrenoceptors (Piascik etai, a  1991). Therefore, PI hydrolysis in rat  caudal artery is likely mediated through ai -adrenoceptors. a  Rosenthal  and  colleagues (1988)  noted  that  a  number  of systems,  adrenocortical cells and rabbit pulmonary artery included, were sensitive to both pertussis toxin and dihydropyridine C a  2+  channel blockers. Nicholas and co-workers  (1989) reported a similarity between the inhibition patterns of the dihydropyridine C a  2+  ".  - 42 -  channel blocker nifedipine and pertussis toxin on agonist-induced increased diastolic pressure in the pithed rat and suggested that tissues dependent on an influx of C a through voltage gated C a  2+  2+  channels would also be sensitive to pertussis toxin. It was  demonstrated in this study that pre-treatment with felodipine, but not pertussis toxin, affected phenyiephrine-induced contractions in rat caudal artery. Furthermore, it has been reported that pre-treatment with pertussis toxin did not affect noradrenalineinduced PI turnover in rat caudal artery (Cheung etal., 1990). Therefore, the G protein coupling the <xi -adrenoceptor to PLC in rat caudal artery is pertussis toxin insensitive, a  and a member of one of the G„ G or G12 families. According to Wu and co-workers q  (1992), the aia-adrenoceptor is effectively coupled to the Goq family members Goq and  G ii, and tissues expressing ai-adrenoceptors almost always simultaneously express a  Gaq/G n proteins. a  Therefore, it is likely that PI hydrolysis in rat caudal artery is  mediated through an oti -adrenoceptor coupled to a Gaq/G n protein: a  a  The present study demonstrated (Figure 1) that inositol phosphate accumulation in rat caudal artery was critically dependent on the presence of extracellular C a . This 2+  is consistent with previously reported results; in rat caudal artery, exogenous C a  2+  enhanced noradrenaline-induced PI hydrolysis in a concentration dependent manner (Cheung et al., 1990). Wilson et al. (1990) and Suzuki et al. (1990) suggested that activation of PLC by ai -adrenoceptor agonists is mediated through a G protein that a  activates sarcolemmal Ca * channels and the resulting C a 2  2+  influx activates PLC. The  PLC isozyme most likely activated in this manner is PLC-51. Gene transfection studies may fail to identify the indirect coupling between Gaq/G ii and PLC-51 if a compatible a  <xi -adrenoceptor-operated C a a  2+  channel is not included in the transfection system.  Therefore, PI hydrolysis in rat caudal artery is probably mediated through an a i a  adrenoceptor coupled to a receptor-operated C a Ca  2+  2+  channel through a Goq/G ii protein; a  influx through the receptor-operated channel then activates PLC-51. However,  the present  study also demonstrated  accumulation was partially dependent on C a channels.  2+  that  inositol phosphate  influx through felodipine sensitive C a  2+  Inhibition by felodipine is unlikely to have resulted from non-specific  intracellular effects as Ca -induced contractions in permeabilized smooth muscle cells 2+  were not affected by pre-treatment with felodipine (1 nM) (Hagiwara et ai, 1993). Therefore, two mechanisms of PI hydrolysis appear to operate simultaneously in rat caudal artery, but differ with respect to the manner of C a activation.  influx required for PLC  2+  It is possible that a single tissue may contain more than one  phosphoinositide selective PLC system; PI turnover in human umbilical vein endothelial cells reportedly contains two different G proteins coupled to histamine and bradykinin receptors (Voyno-Yasenetskaya ef al., 1989): In rat caudal artery, the second inositol phosphate pathway must share the cti -: a  adrenoceptor with the PLC-51 pathway as the cti -subtype is reportedly the only ai-a  adrenoceptor present (Piascik ef al., 1991). Similarly, a Gaq/G n protein likely mediates a  both the PLC-51 pathway and the second pathway. However, the second PLC isozyme is probably PLC-B1, as this is the PLC isozyme most effectively activated by Goq/Gan (Wu etai, 1992; Smrcka etai, 1991; Aragay ef ai, 1992; Park ef ai, 1992; Lee ef ai, 1992). In the absence of extracellular C a , the second pathway is inactive, as is the 2+  PLC-51 pathway. Below 0.1 pM C a , PLC-01 activity is reportedly negligible (Park ef 2+  ai, 1992). Therefore, if the free intracellular Ca * concentration of a resting myocyte is 2  - 44 approximately 0.1 pM (Hartshorne, 1982), C a operate effectively.  2 +  must enter the cell before PLC-01 can  These observations can be explained if the ai -adrenoceptor is a  coupled to an ion channel whose opening initiates a current that depolarizes the membrane  and activates  Subsequently, C a intracellular G a  2+  2+  Ca  influx  2+  through  felodipine  sensitive  channels.  influx through the L-type channels sufficiently increases the  concentration to allow activation of PLC-B1 upon ai-agonist binding.  These observations are effectively summarized by an excitation-contraction mechanism loosely based on models proposed by Van Renterghem et al. (1988) and Nichols et al. (1989). Van Renterghem et al. (1988) described a mechanism of action for endothelin-l in A7r5 cells that addressed the observed dihydropyridine sensitivity of the agonist-induced contractions.  Van Renterghem and co-workers suggested that  agonist interaction activated Ca -sensitive K* channels to induce a transient 2+  hyperpolarization followed by a sustained depolarization that opened non-specific cation channels, allowing an influx of C a L-type C a  2+  channels.  2+  and Mg ; this depolarization then activated 2+  Nichols et al. (1989) proposed a model for ai-adrenoceptor  mediated vasoconstriction in which the receptor was linked to two distinct G proteins, one sensitive to pertussis toxin and coupled the receptor to C a  2+  channels, and the  other insensitive to pertussis toxin and involved in the mobilization of intracellular Ga . 2+  Excitation-contraction coupling in the rat caudal artery appears to involve three <xi,receptor-coupled G proteins; one coupled to a receptor-operated C a  2+  channel, another  coupled to an ion channel whose opening depolarizes the cell and the last coupled directly to PLC-pi:  Under normal pharmacological conditions, ai-agonist binding  simultaneously opens the two receptor-operated channels and readies PLC-pi for  - 45 activation.  The membrane is depolarized and C a  gated channels and receptor-operated channels.  enters the cell through voltage-  2 +  The elevated intracellular C a  2+  concentration activates PLC-51 and completes the activation requirements for PLC-B1. However, in the presence of an L-type channel blocker, C a  2 +  influx is reduced and  subsequent activation of the Ca *-activated PLC-51 and Ca -dependent PLC-B1 2  2+  attenuated. In the absence of extracellular C a , neither excitation-contraction coupling 2+  system operates as both PLC isozymes rely to some extent on extracellular C a . 2+  Membrane potential and intracellular C a  2 +  concentrations have been strongly  correlated in rat mesenteric artery (Nilsson et al., 1994). Changes to the membrane potential of smooth muscle have been found to affect PLC activity; depolarization is positively associated with inositol phosphate accumulation, while hyperpolarization inhibits PI turnover (Itoh et al., 1992).  These observations are consistent with the  model of excitation-contraction coupling described above. Although depolarization can result from an influx of cations, or efflux of anions, recent evidence favours the latter mechanism  in  ai-adrenoceptor-mediated  activation  of  L-type  Ca  2 +  channels.  Noradrenaline increased CI" efflux, while depolarizing and contracting rat mesenteric arteries, but it did not alter the rates of K* efflux or Na influx (Videbaskef al., 1990). In +  guinea-pig mesenteric veins a rapid, noradrenaline-induced depolarization had a reversal potential of -22 mV and was suppressed in a low-chloride solution (Van Helden, 1988).  Furthermore, noradrenaline decreased the CI' concentration of rat  portal veins without effecting Na and K* concentration (Wahlstrom, 1973). It is unlikely +  that changes to the membrane potential are the result of contraction and relaxation, as drugs that cause relaxation (atrial natriuretic factor, substance P and sodium  nitroprusside) did not induce changes to the membrane potential (Videbaek ef al., 1990).  Therefore, it appears that the ion channel activated by ai -adrenoceptor a  agonists is an anion channel, and CI" js the anion whose efflux is responsible for myocyte depolarization. However, altering the membrane potential is unlikely to affect a simple electrostatic response in excitable cells. 4.3.  Ryanodine Sensitive Ca * Store in Rat Caudal Artery 2  In rat caudal artery, ryanodine inhibited phenylephrine-induced contractions, indicating that a ryanodine sensitive contractile mechanism is present in the tissue and required for maximal cti-agonist-induced contraction. Because phenylephrine-induced contractions were abolished in the absence of extracellular  Ca , 2+  all the cti-  adrenoceptor-mediated excitation-contraction coupling pathways in caudal artery , including that sensitive to ryanodine, are critically dependent on extracellular C a . The 2+  Ca -induced C a 2+  2+  release mechanism effectively explains these results within the  context of the cti-adrenoceptor excitation-contraction model described above.  Under  normal pharmacological conditions, ai-adrenoceptor agonists appear to activate Ca * 2  influx through receptor-operated and voltage-gated channels. In addition to regulating PLC activity, the increased intracellular C a  2+  may initiate C a  2+  release from the  ryanodine sensitive store, lino (1989) provided direct evidence for the existence of a Ca -induced C a 2+  2+  release mechanism in guinea-pig taenia caeci smooth muscle;  therefore, it is likely that similar Ca -regulated release mechanism exists in caudal 2+  artery smooth muscle.  - 47 The inhibitory effects of ryanodine in rat caudal artery were significantly increased with the simultaneous addition of 8-bromo-cGMP. This suggests that, (1) intracellular C a  2+  release from ryanodine-sensitive stores is not regulated by 8-bromo-  cGMP, (2) the ryanodine-sensitive C a  2+  store and IP -regulated C a 3  2+  store are  functionally distinct in the rat caudal artery, and (3) the ryanodine-sensitive and IP 3  regulated Ca * stores are functionally isolated. 2  intracellular C a  2+  Functionally and spatially distinct  stores have been identified in vascular smooth muscle cells cultured  from arterial myocytes (Tribe et al:, 1994). 4.4.  cci-Agonist-Induced Contraction in Rat Thoracic Aorta  Previously reported results and the results from this study suggest that in rat thoracic aorta cti-adrenoceptors are couple through a pertussis toxin insensitive G  a  protein to PLC-51. Little work has been done to indicate which G protein couples ctiadrendceptors and PLC activity in rat thoracic aorta.  Therefore, speculating with  respect to the identity of the G protein is impossible. However, while pertussis toxin reportedly did not impair noradrenaline-induced contractions in rat aorta, cholera toxin did (Tabrizchi, 1994). > In rat thoracic aorta, PLC-y1 and PLC-51 isozymes, but not PLC-B1, were identified, and only the PLC^/1 subtype was activated by angiotensin ll-induced tyrosine phosphorylation (Marrero et al., 1994). Previously, two PLC enzymes whose activities increased with increasing C a  2+  had been characterized in rat thoracic aorta  (Griendling et al., 1991). The results of the current study demonstrate that contraction in rat thoracic aorta is, in a small part, sensitive to inhibition by felodipine.  This  - 48 suggests that ai-adrenoceptor mediated contraction in rat thoracic aorta relies heavily on intracellular C a . 2+  This is consistent with the previously reported observations.  Phenylephrine induced a phasic contraction in rat thoracic aorta in the absence of extracellular Ca * (Nishimura etai., 1991), and <xi-agonist-induced PLC activation in rat 2  aorta does not depend on an influx of C a  2+  through voltage-operated channels as  nifedipine did not affect IP accumulation to noradrenaline, phenylephrine or cirazoline 3  (Chiu ef al. 1987; Legan ef al. 1985). Noradrenaline-stimulated C a  2+  efflux from rabbit  aorta was transiently increased in the presence and absence of extracellular C a  2+  (Collins ef al., 1986). Furthermore, a thromboxane A -induced increase in intracellular 2  Ca  2+  was not affected by removal of extracellular C a  2+  and was associated with an  increased accumulation of IP (Dorn II & Becker, 1992). 3  However, other studies have suggested that PLC activity in rat aorta is dependent on extracellular Ca * to some extent. A study by Rapoport (1987) using rat 2  aorta demonstrated that noradrenaline-induced contraction and PI hydrolysis in a C a 2+  free buffer were greatly reduced, but not completely abolished. Moreover, the addition of noradrenaline to rat aortic rings produced a notably reduced phasic contraction in the absence of extracellular C a noradrenaline-induced C a  2+  2+  (Manolopoulos ef al., 1991).  Only 38 % of  influx was insensitive to nisoldipine (Morel & Godfriand,  1991). Finally, noradrenaline induced a phasic contraction and an increase ip inositol phosphate accumulation in rat aorta in the absence of extracellular Ca *, although PI 2  turnover was less than that in a physiological solution containing C a  2+  (Heaslip &  Sickels, 1989). These results suggest that although PI hydrolysis in rat thoracic aorta  is not critically dependent on extracellular C a , contraction can be maximally induced 2+  when the ion is present. 4.5.  Ryanodine Sensitive C a * Store in Rat Thoracic Aorta 2  In rat thoracic aorta, ryanodine did not affect ai-agonist-induced contractions, which suggests that maximal contraction in aortic tissue is not affected through a ryanodine sensitive pathway under normal pharmacological conditions. These results do not rule out the possibility, however, that a ryanodine sensitive contractile pathway in aortic tissue is activated only after the dominant excitation-contraction mechanisms are impaired. This possibility is likely as Julbu-Schaeffer & Freslon (1988) found that ryanodine inhibited noradrenaline-induced contraction in rat aorta only after C a been removed from the extracellular flujd.  2+  had  Similarly, Low and co-workers (1993)  reported that phenyiephrine-induced contractions were inhibited by ryanodine in a Ca -free medium. Furthermore, ryanodine induced a slowly developing rise in aortic 2+  tension, yet subsequent addition of noradrenaline induced a contractile response that was not significantly different from that of the control (Julou-Schaeffer & Freslon, 1988). Tabrizchi (1994) reported that noradrenaline-induced contractions in aortic rings could not be impaired by ryanodine unless the animal had been pretreated with the ccadrenoceptor alkylating agent, phenyoxybenzamine. ryanodine inhibits C a  2+  It appears, therefore, that  release from an intracellular pool in aortic tissue, but maximum  contraction following cti-adrenoceptor activation in rat thoracic aorta is not dependent on release from this pool.  :/.'•..•' 4.6.  :  '. - so  cGMP and Inositol Phosphate Accumulation  The results of this study demonstrate that 8-bromo-cGMP does not block inositol phosphate accumulation in rat caudal artery.  This is consistent with previously  reported results (Eskinder et al., 1989; Ko et al., 1992; Puurunen et al., 1987), and indicates that the nucleotide neither interacts with PLC directly, nor interferes with the agonist-induced influx of extracellular C a  required for PLC activity.  2+  8-Bromo-cGMP and felodipine failed to induce additive inhibition of contraction when applied together. This suggests that the two inhibitors appear to operate along the same excitation-contraction coupling'pathway.  Since felodipine, but not 8-bromo-  cGMP, blocks inositol phosphate accumulation, the nucleotide's inhibition of the Plmediated contractile pathway appears to occur subsequent to IP production. Evidence 3  of an interaction between cGMP and the IP receptor has been reported. 3  A cGMP-  dependent protein kinase that closely resembles the IP receptor has been discovered 3  (Koga et al., 1994).  Furthermore,  purified rat cerebellum  IP  receptors  3  are  stoichiometrically phosphorylated at the serine-1755 residue by cGMP-dependent protein kinases (komalayilas & Lincoln, 1994).  Results from bovine trachea and rat  aortic smooth muscle cells suggest that the effects of cGMP are mediated through a cGMP dependent kinase (Felbel et al., 1988; Cornwell & Lincoln, 1989). The results of this study, however, do not rule out the possibility that cGMP hastens the rate of metabolism of IP by inositol 1,4,5-trisphosphate 3-kinase to inositol 3  1,3,4,5-tetrakisphosphate  (lns(1,3,4,5)P ) 4  (Irvine  et al., 1986)  or  by  inositol  trisphosphate 5-phosphatase to inositol 1,4-bisphosphate (IP ). It is unlikely, however, 2  that the rate IP metabolism by these two enzymes is increased significantly by cGMP 3  - 51 as they naturally operate quite rapidly (Irvine ef al., 1986; Storey ef al., 1984). However, if cGMP had accelerated IP hydrolysis, the assay used in this study to 3  measure inositol phosphate accumulation may not have recorded this effect. The Li  +  used in this study would have prevented recycling of most of the inositol phosphates back to inositol and the assay would have recorded them. However, IP is incapable of 2  releasing C a  2+  from the IP sensitive intracellular stores (Stauderman ef al., 1988) and 3  lns(1,3,4,5)P Is reportedly involved in C a  2+  4  re-uptake into the intracellular store (Irvine  and Moor, 1987) and activation of Ca *-dependent K* channels (Morris et al., 1987). 2  Therefore, we may have been recording the presence of ineffective IP metabolites 3  which are incapable of eliciting contraction. 4.7.  Experimental Design  Many studies have investigated the inhibitory effects of antagonists at a single agonist concentration. The importance of testing antagonist effects over an agonist concentration range is apparent from the felodipine experiments in the present study. Although a qualitative decrease in PI hydrolysis was apparent over the entire phenylephrine concentration range, significant changes could only be reported for three concentrations of the agonist. We also found that a supermaximal concentration of phenylephrine could restore maximum PI turnover in the presence of felodipine. This may be indicative of an ai-adrenoceptor reserve. Although PI response does not normally demonstrate a significant receptor reserve (Michell & Kirk, 1981), it must be sufficient in the caudal artery to overcome the effects of felodipine. Berta and co-worker (1986), investigating the influence of extracellular C a  2+  on  serotonin- and phenylephrine-induced contractions and phosphoinositide metabolism in  - 52 rat caudal artery, reported that although phenyiephrine-induced contractions were completely abolished by removal of extracellular C a , PI hydrolysis was unaffected by 2+  the omission. These results indicated that although extracellular C a  2+  was required for  contraction, it was not necessary for PLC activity. However, according to Cheung et al. (1990), agonist stimulated PI hydrolysis in rat caudal artery reaches a maximum at 5 mM extracellular C a  2 +  with an ECso of about 80 uJA/l. Therefore, the 0:5 mM EDTA used  by Berta to chelate C a contributed to the  2 +  may have been insufficient to completely immobilize the C a  bathing medium by the other  2+  ingredients; and, aggressive  concentrations of chelating agents are required to ensure a Ca -free environment. 2+  4.8.  Conclusion  The comparison Of phenyiephrine-induced contractions in rat caudal artery and rat thoracic aorta performed in this study has demonstrated that there is a significant difference between the relative contributions of intracellular and extracellular Ca * to 2  the excitation-contraction coupling mechanisms in these two tissues. Contraction in rat caudal artery is critically dependent on extracellular C a  2+  mediated, in part, through  dihydropyridine-sehsitive channels. The contractile mechanism in rat thoracic aorta, however, is not significantly dependent on C a channels nor on C a  2 +  2 +  influx through voltage dependent  release from ryanodine sensitive stores under normal  physiological conditions.  Contraction in rat caudal artery, however, is sensitive to  inhibition by ryanodine. cti-Agonist-induced contractions in rat caudal artery are not sensitive to pertussis toxin; therefore, the G protein coupling the ai-adrenoceptor and PLC activity is not a member of the G|/G, family.  - 53 This study also demonstrated that although 8-bromo-cGMP does not inhibit PLC activity in rat caudal artery, the nucleotide does impair ai-adrenoceptor-induced contraction. Inhibition of contraction by felodipine and 8-bromo-cGMP occurs along the same excitation-contraction  coupling pathway.  Since felodipine blocks inositol  phosphate accumulation, 8-bromo-cGMP likely blocks IP -induced C a 3  2 +  release from  intracellular stores. However, 8-bromo-cGMP does not appear to affect the ryanodinesensitive contractile mechanism which mediates C a  2+  release from an IP -insensitive 3  intracellular pool. 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