"Medicine, Faculty of"@en . "Anesthesiology, Pharmacology and Therapeutics, Department of"@en . "DSpace"@en . "UBCV"@en . "Lum Min, Suyin Ann"@en . "2009-01-16T19:56:29Z"@en . "1995"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Presently, the literature regarding vascular smooth muscle contraction is\r\ncoloured with many contradictory observations and conclusions. However, like many\r\nphysiological systems, the biochemical pathways and functional events of vascular\r\nsmooth muscle contraction vary between individual species and/or tissues. Therefore,\r\ndetermination of a ubiquitous excitation-contraction coupling mechanism is unlikely;\r\nvariations between receptor classes, receptor density, excitation-contraction coupling\r\npathways and the efficiency of the receptor-pathway interaction contribute to the\r\nvarious observations and conclusions.\r\nThe inositol 1,4,5-trisphosphate (IP\u00E2\u0082\u0083) second messenger cascade regulates the\r\nmobilization of intracellular Ca\u00C2\u00B2\u00E2\u0081\u00BA, and subsequently contraction, in vascular smooth\r\nmuscle. However, phospholipase G-mediated production of IP\u00E2\u0082\u0083 appears to be\r\ncontrolled by tissue-specific regulatory factors. This study examines the effects of\r\nthree such factors, the presence of extracellular Ca\u00C2\u00B2\u00E2\u0081\u00BA, the sensitivity of the associated\r\nG-protein and inhibition by 8-bromoguanosine 3':5'-cyclic monophosphate (8-brombcGMP),\r\nin isolated rat caudal artery. Concentration-response curves were constructed\r\nfor phenylephrine and isometric contractions measured in isolated tissues. In addition,\r\nphosphatidylinositol turnover was assessed using anion exchange chromatography.\r\nThe effects of 8-bromo-cGMP on phenylephrine-induced contractions and\r\nphosphatidylinositol hydrolysis were compared to those of felodipine, a dihydropyridine\r\nCa\u00C2\u00B2\u00E2\u0081\u00BA-channel antagonist, and ryanodine, a putative depletor of intracellular Ca2* stores\r\nin rat caudal artery. Pertussis toxin was used to determine the identity of the G-protein\r\nregulating phenyiephrine-induced contraction. Further, the effects of felodipine and\r\nryanodine on contraction were determined in rat thoracic aorta to compare the\r\ncontribution of extracellular and intracellular Ca\u00C2\u00B2\u00E2\u0081\u00BA to contraction between a large\r\nconduit vessel and a small conduit vessel.\r\nThe results of this investigation suggest that phospholipase C-activated\r\nphosphatidylinositol hydrolysis in the rat caudal artery is dependent on extracellular\r\nCa\u00C2\u00B2\u00E2\u0081\u00BA, mediated, in part, through dihydropyridine sensitive Ca\u00C2\u00B2\u00E2\u0081\u00BA channels.\r\nPhospholipase C activity is not directly inhibited by 8-bromo-cGMP. However, the\r\nnucleotide may regulate vascular smooth muscle contraction by inhibition of Ca\u00C2\u00B2\u00E2\u0081\u00BA \r\nrelease from IP\u00E2\u0082\u0083-mediated intracellular stores, but it is unlikely that 8-bromo-cGMP\r\naffects ryanodine-sensitive stores. None of the G-proteins coupled to the ctiadrenoceptor\r\nmediated excitation-contraction pathway in rat caudal artery appear to be\r\nsensitive to pertussis toxin. Rat aortic tissue does not rely on intracellular Ca\u00C2\u00B2\u00E2\u0081\u00BA to the\r\nsame extent that rat caudal artery does, confirming the tissue specificity of ctiadrenoceptor\r\nagonist induced excitation-contraction in vascular smooth muscle."@en . "https://circle.library.ubc.ca/rest/handle/2429/3710?expand=metadata"@en . "3944003 bytes"@en . "application/pdf"@en . "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 THE 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 \u00C2\u00A9 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 ^ ^ ^ - T n t a c - o l o q y o<-~>ot ~TVic>-capc._rBc:-s . The University of British Columbia Vancouver, Canada Date A y y - i ' l 2&. ^ 5 , DE-6 (2/88) 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 (IP3) second messenger cascade regulates the mobilization of intracellular Ca 2 + , and subsequently contraction, in vascular smooth muscle. However, phospholipase G-mediated production of IP3 appears to be controlled by tissue-specific regulatory factors. This study examines the effects of three such factors, the presence of extracellular Ca 2 + , the sensitivity of the associated G-protein and inhibition by 8-bromoguanosine 3':5'-cyclic monophosphate (8-bromb-cGMP), 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 Ca2+-channel antagonist, and ryanodine, a putative depletor of intracellular Ca 2* stores 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 C a 2 + channels. Phospholipase C activity is not directly inhibited by 8-bromo-cGMP. However, the nucleotide may regulate vascular smooth muscle contraction by inhibition of C a 2 + release from IP3-mediated intracellular stores, but it is unlikely that 8-bromo-cGMP 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 cti-adrenoceptor agonist induced excitation-contraction in vascular smooth muscle. - i v -TABLE OF CONTENTS ABSTRACT.....: , ii TABLE OF CONTENTS iv LIST OF TABLES v ..'.,..!\u00E2\u0080\u00A2 v LIST OF FIGURES...., ..vi LIST OF ABBREVIATIONS..........; vii ACKNOWLEDGMENTS ...viii DEDICATION ix 1. INTRODUCTION .1 1.1. Vascular Smooth Muscle Contraction 1 1.2. Sources of Ca 2 + Utilized During Contraction-.'..,v.-...':.W: ....2 1.3. IPs-Mediated Ca 2 + Release.;!..,..........- 3 1.4. Phospholipase C ; ...5 1.5. cGMP Regulated Vasodilation 8 1.6. Experimental Objectives..:... 10 2. MATERIALS AND METHODS 15 2.1. Phosphatidylinositol Hydrolysis in Caudal Artery. :...15 2.2. Contractile Studies in Caudal Artery 16 2.3. Contractile Studies with Pertussis Toxin in Caudal Artery 17 2.4. Contractile Studies Thoracic Aorta .........................18 2.5. Data and Statistical Analysis... .........18 2.6. Chemicals ...... ......19 3. RESULTS.... 21 3.1. Phosphatidylinositol Hydrolysis in Caudal Artery... 21 3.2. Contractile Studies in Caudal Artery 24 3.3. Contractile Studies with Pertussis Toxin in Caudal Artery 28 3.4. Contractile Studies in Thoracic Aorta 28 4. DISCUSSION... !.........,... .39 4.1. Intracellular Versus Extracellular Ca 2 + ........39 4.2. cti-Agonist-lnduced Contraction in Rat Caudal Artery ...41 4.3. Ryanodine Sensitive Ca 2 + Store in Rat Caudal Artery......... 46 4.4. ai-Agonist-lnduced Contraction in Rat Thoracic Aorta... 47 4.5. Ryanodine Sensitive Ca 2 + Store in Rat Thoracic Aorta...., .......49 4.6. cGMP and Inositol Phosphate Accumulation 50 4.7. Experimental Design 51 4.8. Conclusion.. ; ......52 5. REFERENCES.... >:;,........... ...!........,.... ......54 - V -LIST OF TABLES T A B L E I. Pheny iephr ine - induced concentrat ion - response curve results obtained from rat cauda l artery preparations in the a b s e n c e and p resence of var ious antagonists.. . . . . : . : . : ; . : ; . . . . . .\u00E2\u0080\u009E . . : . . . . : . . : ; . . ..............26 T A B L E II. Pheny iephr ine - induced concentrat ion - response curve results obtained from rat cauda l artery preparations in the a b s e n c e and p resence of .. *> pertussis toxin. v..r..........36 T A B L E III. Pheny iephr ine - induced concentrat ion - response curve results obtained from rat aortic t issue in the a b s e n c e and p resence of var ious . antagonists.. . . . . : . . :.....;.....:... .........7...... :v.....v.........^...:.,38 LIST OF FIGURES FIGURE 1. Phosphatidylinositol accumulation in rat caudal artery in the presence and absence of 8-bromo-cGMP, felodipine or Ca2+-free buffer...22 FIGURE 2. Contractions in rat caudal artery in the absence and presence of 8-bromo-cGMP:..,; ..... 25 FIGURE 3. Contractions in rat caudal artery in the absence and presence of felodipine. v~28' FIGURE 4. Contractions in rat caudal artery in the absence and presence of ryanodine. \u00E2\u0080\u009E..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. ..!...:...,.........,!,..\u00E2\u0080\u009E...:.......;: 33 FIGURE 7. Contractions in rat caudal artery the presence and absence of. .* extracellular Ca 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'- EGTA tetraacetic acid Guanosine 3':5'-cyclic monophosphate cGMP Guanosine diphosphate GDP Guanosine triphosphate GTP Hill coefficient n Inositol 4,5-bisphosphate Ins (4,5)P2 Inositol 1,3,4,5-tetrakisphosphate Ins (1131415)P4 Inositol 1,3,4-trisphosphate lns(1,3,4)P3 Inositol 1,4,5-trisphosphate I P 3 Inositol 2,4,5-trisphosphate Ins (2,4,5)P3 Phosphatidylinositol PI Phosphatidylinositol 4,5-bisphosphate PIP2 Phospholipase C PLC Standard error of the mean S.E. - 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 (Ca2+) was first identified as a critical 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 Ca 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 increases from approximately 0.1 pM to 5 pM (Hartshorne, 1982). As the C a 2 + concentration rises, it binds the cytosolic Ca2*-bihding protein, calmodulin, to form a regulatory 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 (MLC2o); both the electric and steric effects of phosphorylation 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 Ca2*-dependent phosphorylation of MLC2o (Walker et al., 1994). 1.2. Sources of Ca 2 +Utilized During Contraction Cross-bridge cycling is regulated through the Ca2+-calmodulin-dependent phosphorylation of MLC20; therefore, increasing the free cytosolic Ca 2 * concentration is crucial for contraction. Ca 2 * can enter from the extracellular space through voltage-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 2 + pools have been identified within the sarcoplasmic reticulum of vascular smooth muscle cells; one controlled by inositol 1,4,5-trisphosphate (IP3) and the other sensitive to inhibition by ryanodine (Yamazawa 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 phospha-tidylinositol (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 IP3 was a second messenger capable of mobilizing stored Ca 2 + . Subsequently, results from rabbit pulmonary artery (Somlyo et al., 1985), 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 IP3 and the release of intracellular Ca 2 + . the effects of IP3 are not blocked by vanadate, an inhibitor of the sarcoplasmic reticulum Ca 2 +-ATPase re-uptake bump; therefore, IP3 acts by increasing the Ca 2* permeability of the sarcoplasmic reticulum, not by inhibiting the sequestering of free C a 2 + (Somlyo et al., 1985). However, IP3 may increase intracellular C a 2 + to a lesser extent by inhibiting extrusion from the cytosol by the sarcolemmal Ca2*-ATPase (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 IP3 (Seiler et al., 1987); however, 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 2 + from the ryanodine sensitive pools (White etal., 1993; Galione ef a/., 1993; Meszaros etai, 1993). . -Studies using various antagonists of IP3- and ryanodine-sensitive C a 2 + release , have provided evidence that the two intracellular Ca 2 * release pathways are functionally and spatially independent. IP3-induced release is blocked by cinnarizine, flunarizine, tetraethylammonium and the local anaesthetics benzocaine and lidocaine (Berridge & Irvine, .1989; Seiler et al., 1987). IP3-mediated Ca 2 * mobilization is not affected by nifedipine, diltiazem, verapamil, 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 IP3-induced Ca 2 * release, it does not affect ryanodine sensitive release (Berridge, 1993; White et al., 1993). The ryanodine sensitive store, can be mobilized by caffeine (Berridge, 1993); however, the IP3 sensitive store is blocked, not activated, by caffeine in Xenopus oocytes and rat cerebellar microsomes (Mignery ef al., 1990; Parker & Ivorra, 1991; Brown et al., 1992). Although caffeine-induced Ca 2 + release is insensitive to changes to the membrane potential, release, from the IP3 sensitive pool is regulated by potential dependent PI hydrolysis (Ganitkevich & Isenberg, 1993). The IP3 sensitive pool appears to sequester C a 2 + when the cytosolic concentration is high (~ 10\"6 M); however, the IP3 insensitive pool is the higher affinity C a 2 + buffer, capable of adjusting low intracellular C a 2 + concentrations (~ 10'7 M) (Thevenod et al., 1989). In brain microsomes, cADPR and IP3 released approximately 20 and 60 % of the stored Ca 2 + , respectively (White et al., 1993). Also, C a 2 + can induce C a 2 + release from the ryanodine sensitive stores (lino, 1989), but micromolar Ca 2 + inhibits C a 2 + release from IP3 sensitive stores by indirectly decreasing the affinity 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 vasopressin-induced IP3 synthesis in A7r5 cultured aortic smooth muscle cells were blocked in the presence of this antagonist (Berman etal., 1994): Therefore, IP3 and ryanodine appear - 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 IP3 can induce C a 2 + release from a ryanodine sensitive pool. Yamazawa and co-workers (1992) recorded an IPs-mediated rise in intracellular C a 2 + that was reduced by approximately 50 % after treatment with ryanodine. In skinned guinea pig taenia caeci, the amount of C a 2 + released by the application of IP3, or IP3 and caffeine, was approximately twice as large as that released by caffeine alone (lino et al., 1988). Finally, ryanodine blocked C a 2 + accumulation in both caffeine and IP3 sensitive stores (Kanmura et al., 1988). These results indicate that two C a 2 + pools may co-exist within some excitable cells; one sensitive to IP3l and the other sensitive to both IP3 and ryanodine (Yamazawa et al., 1992). Alternatively, an excitable tissue may contain only one C a 2 + pool which is sensitive to both second messengers. Undoubtedly, the functional and spatial characteristics of intracellular C a 2 + stores vary between tissues. 1.3. IPs-Mediated Ca 2 * Release Many of the physiological and biochemical characteristics of the IP3-mediated intracellular C a 2 + store have been investigated. The IP3 receptor consists of four 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 IP3 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 IP3 receptors on both central and peripheral sarcoplasmic - 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 IP3 receptor responds to ligand binding by undergoing a conformational change and increasing the opening frequency of the associated C a 2 + channel (Mignery & Sudhof, 1990; Berridge, 1993). Channel activation appears highly co-operative; at least four independently bound IP3 molecules may be required to open each channel (Meyer et al., 1990): Four hundred C a 2 + molecules are estimated to be released for every molecule of IP3 bound (Stauderman efa/.,1988). The IP3 receptor recognizes the D-isomer of IP3 more readily than the L-isorrier (Berridge & Irvine, 1989). However, release of C a 2 + can also be elicited by inositol 2,4,5-trisphosphate (lns(2,45)P3) and inositol 4,5-bisphosphate (lns(4,5)P2). Based on ECso values, the relative potencies are IP3 > lns(2,4,5)P3 > lns(4,5)P2 in rat cerebellum (Stauderman etai, 1988). In the absence of extracellular K\ or in the presence of tetraethylammonium, IP3-mediated C a 2 + release was reduced, although, other monovalent cations (Na + \u00C2\u00BBTris + >Li + ) could substitute effectively (Berridge & Irvine, 1989; Shah & Pant, 1988). C a 2 + alone could not stimulate K* influx; therefore, K* conductance was not a result of C 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 IP3, indicating that metabolism is the primary mechanism for terminating the effects of the second messenger (Stauderman et al., 1988; Berridge & Irvine, 1989). Repeated additions of IP3 to rat cerebellar microsomes did not - 8 -desensitize the receptor nor diminish the release of Ca 2 + . However, the receptor did desensitize in response to lns(2,4,5)P3 and lns(4,5)P2 (Stauderman et al., 1988). Stauderman and co-workers proposed two. explanations: (1) if lns(2,4,5)P3 and lns(4,5)P2 are not metabolized as quickly as IP3, the C a 2 + channel will remain open and refilling will be impossible, or (2) lns(2,4,5)P3 and lns(4,5)P2 cannot be metabolized to lns(1,3,4,5)P4 or Insd.S^Ps, metabolites of IP3 which may enhance the reaccumulation of stored Ca 2 + . 1.4. Phospholipase C The phosphoinositide-specific phospholipase C (PLC) isozymes are a series of catalytic proteins that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to water soluble IP3 and membrane bound diacylglycerol (DAG). IP3 induces C a 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 ct2-adrenoceptor antagonist (Cheung ef a/., 1990). 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 a2-adrenoceptor agonist UK 14304 stimulated PI 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 2 + stores as these antagonists did not affect acetylcholine- and caffeine-induced C a 2 + release (Lepretre et al., 1994). 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 IP3 and a phasic contraction of rabbit mesenteric artery in a Ca2+-free solution (Itoh et al., 1992); but, acetylcholine-induced stimulation of PI hydrolysis in rabbit iris smooth muscle was increased from 16 % above unstimulated preparations in the absence of extracellular C a 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 2 + release and C a 2 + 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., 1985; Stauderman et al., 1988; Chiu et al., 1986; Thevenod et al., 1989). 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 noradrenaline-induced 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 3 cells was maximized by the presence of Mg 2 + , ATP and extracellular C a 2 + (Martin et al., 1986). Finally, Ca 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 2 + through voltage sensitive channels for activation. However, C a 2 + 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 Ca2+-free medium. 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 2 + and the means by which C a 2 + is acquired. According to Rhee and Choi (1992), the activities of all PLC isozymes are dependent on Ca 2*. Therefore, differences between tissues may reflect the variation in the C a 2 + sensitivities of the isozymes. 1.5. 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 oxide-induced 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 endothelium-derived relaxing factor which stimulates production of cGMP by soluble guanylate cyclase (Furchgott et al., 1984; Rapoport & Murad, 1983; Furchgott, 1988; Ignarro et al, 1988). 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*/Ca2* exchanger (Furukawa et al., 1991), (4) inhibition of Ca 2 * translocation across the plasma membrane (Collins et al., 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 2 * release from intracellular storage sites (Collins et 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 2 * release, there are three mechanisms by which cGMP could Operate, (1) blocking production of IP3 through PLC, (2) blocking IP3 binding or activation of the sarcoplasmic reticulum receptor, or (3) hastening IP3 inactivation or metabolism. A cGMP dependent binding 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, dibutyryl-cGMP 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 IP3 second messenger 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 Ca 2 + , (2) to determine whether or not a pertussis toxin sensitive G-protein mediates cti-adrenoceptor contraction, (3) to establish whether or not cGMP. blocks IP3 production, (4) to - 14 -determine whether or not rat caudal artery contains two functionally distinct intracellular C a 2 + stores, one sensitive to ryanodine, the other to IP3, and (5) to compare the relative 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 Ca 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 Ca2+-channel antagonist, and ryanodine, a putative 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\"1) i.p.. The caudal artery was dissected free and cleaned of connective tissue in Krebs-bicarbonate buffer of the following composition (in mM): NaCI, 120; KCI, 4.6; glucose, 11; MgCI2, 1.2; CaCI2, 2.5; KH 2 P0 4 , 1.2; NaHC0 3, 25.3. The pH of the buffer following saturation with a 95 % O2: 5 % C 0 2 gas mixture was 7.4. The composition of the Ca2+-free buffer was the same, except that CaCI2 was omitted 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\u00C2\u00B0C 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\"1 [3H] myo-inositol to load for 90 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\u00C2\u00B0C. It has previously been demonstrated that Li + enhances the 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). Ca2+-free Krebs-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 -Ca 2 + . Individual assay tubes contained felodipine (10 nM), 8-bromo-cGMP (10 pM) or 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 NaHC0 3 (100 mM) 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 Scinti-Safe 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\u00C2\u00B0C under a force of 9.8 mN and gassed continuously with a mixture of 95 % 0 2 : 5 % C0 2 . Contractions 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 8-bromo-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 Ca 2 + , Ca2+-free buffer was in contact with the tissues for 15 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. 2.4. Contractile Studies Thoracic Aorta 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\u00C2\u00B0C under a force of 9.8 mN and gassed continuously with a mixture of 95 % 0 2 : 5 % C0 2 . Tissues were allowed to equilibrate for 60 minutes. Phenylephrine (10 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. Percent maximum, Hill coefficient and E C 5 0 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), L-phenylephrine 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-[2-3H(N)]-inositol (17.0 Ci mmol*1), ryanodine and salt free pertussis toxin were purchased from 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. Ail other chemicals were purchased from Fischer Scientific (B.C., Canada). . , 3. RESULTS 3.1. Phosphatidylinositol Hydrolysis in Caudal Artery 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'1 (Figure 1). In contrast, felodipine (10 nM) caused a noticeable decrease in PI hydrolysis at all concentrations of phenylephrine tested (Figure 1). This decrease was found to be statistically significant (n = 6; p < 0.05) at 1, 3 and 10 pM phenylephrine. Maximum accumulation was only 7-fold above basal dpm mg wet weight\"1. 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 + (Ca2+-free buffer containing 2 mM EGTA). It was noted that basal PI accumulation was not affected by 8-brbmo-cGMP, felodipine or Ca2+-free buffer. - 22 -Figure 1. Phosphatidylinositol accumulation in rat caudal artery in the presence and absence of 8-bromo-cGMP, felodipine or Ca2 +-free buffer. 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 Ca2+-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 \u00C2\u00B1 20 dpm mg wet weight'1 (mean \u00C2\u00B1 S.E.; n = 24). Each column represents the mean of six experiments \u00C2\u00B1 S . E . a Significantly different from control (p < 0.05). - 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 concentration-response 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 \u00C2\u00B1 8 % and 57 \u00C2\u00B1 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 \u00C2\u00B1 0.47 mN (mean \u00C2\u00B1 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). In contrast, concomitant application of 8- bromo-cGMP and ryanodine 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 8-bromo-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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 4.17 \u00C2\u00B1 0.68 mN (mean \u00C2\u00B1S.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 \u00C2\u00B1 S.E. Groups EC50 (uM) n % Maximum Control 1.25 \u00C2\u00B10 .18 1.10 \u00C2\u00B10 .06 102 + 1 8-Bromo-cGMP (10 M-M) 2.65 \u00C2\u00B1 0.35\" 1 .32\u00C2\u00B10.07 a 81 \u00C2\u00B1 3 a 8-Bromo-cGMP (10 uM) 3 .16\u00C2\u00B10 .49 a 1.25 \u00C2\u00B10 .06 80 + 5a Control 1.49 \u00C2\u00B10 .28 0.92 \u00C2\u00B1 0.04 104 \u00C2\u00B1 1 Felodipine (1 nM) 3.51 \u00C2\u00B10 .98 1.03 \u00C2\u00B10 .10 7 7 \u00C2\u00B1 8 a Felodipine (10 nM) 2.63 \u00C2\u00B1 0.45 1.11 \u00C2\u00B10 .09 5 7 \u00C2\u00B1 3 a b Control 1.29\u00C2\u00B10.19 1.10\u00C2\u00B10.10 102 \u00C2\u00B1 1 Ryanodine (3 pM) 1.96 \u00C2\u00B1 0.27a 1.03 \u00C2\u00B10 .06 81 \u00C2\u00B1 6 a Ryanodine (10 u.M) 2 .17\u00C2\u00B10 .32 a 1.03 + 0.08 76 \u00C2\u00B1 8 a Control 0.89 \u00C2\u00B10 .18 0.92 \u00C2\u00B10.04 104 \u00C2\u00B1 1 Fel (1 nM) + 8-br(10pM) 2 .60\u00C2\u00B10 .25 a 1.21 \u00C2\u00B10.05 7 4 \u00C2\u00B1 4 a Fel(10nM) + 8-br(10|iM) 3.83 \u00C2\u00B1 0.28 a b 1.25 \u00C2\u00B10.05 59 \u00C2\u00B1 5 a b Control 1.62 \u00C2\u00B10 .36 1.15 \u00C2\u00B10 .03 102 \u00C2\u00B1 1 Ry(3^M) + 8-br(10pM) 3.53 \u00C2\u00B10.81 120 \u00C2\u00B10 .09 52 \u00C2\u00B1 7 a c Ry(10pM) + 8-br(10pM) 3.52 \u00C2\u00B1 0.70 1.37 \u00C2\u00B10 .13 38 \u00C2\u00B1 9 a c Control 0.95 \u00C2\u00B10 .12 1.16\u00C2\u00B10.10 101 \u00C2\u00B1 1 Ca2+-free N N N + C a 2 + (2.5 mM Ca 2 +) 1.01 \u00C2\u00B10 .12 1.20 \u00C2\u00B10 .08 103 \u00C2\u00B1 3 Control 1.48 \u00C2\u00B10 .03 1.01 \u00C2\u00B10 .07 103 \u00C2\u00B1 1 Distilled H 2 0 (6 p.l) 1.56 \u00C2\u00B10.31 1.10 \u00C2\u00B10.05 102 \u00C2\u00B1 4 Distilled H 2 0 (20 u|) 1.52 \u00C2\u00B10 .25 1.05 \u00C2\u00B10.07 103 \u00C2\u00B1 6 Fel = felodipine; Ry = ryanodine; aSignificantly different from control, p < 0.05; \"Significantly different from the first concentration of drug, p < 0.05; cSignificantly different from the same concentration of ryanodine alone, p < 0.05; N = no measurable increase in contraction above resting tension. ; - 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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 3.04 \u00C2\u00B1 0.22 mN (mean \u00C2\u00B1 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 \u00C2\u00B1 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 Ca2 +-free buffer were unsuccessful (Figure 7). Addition of up to 300 pM phenylephrine did not produce contraction. 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 \u00C2\u00B1 0.29 mN (mean \u00C2\u00B1 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 \u00C2\u00B1 0.93 mN (mean \u00C2\u00B1 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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 4.70 \u00C2\u00B10.71 mN (mean \u00C2\u00B1 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-bromo-cGMP (squares) or 10 nM felodipine and 10 uM 8-bromo-cGMP (triangles). Each point represents the mean of six experiments \u00C2\u00B1 SE . Maximum tension in the absence of antagonists was 5.29 \u00C2\u00B1 0.97 mN (mean \u00C2\u00B1 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-bromo-cGMP (squares) or 10 pM ryanodine and 10 pM 8-bromo-cGMP (triangles). Each point represents the mean of six experiments \u00C2\u00B1 S.E. Maximum tension in the absence of antagonists was 3.79 \u00C2\u00B1 0.51 m'N (mean \u00C2\u00B1 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 2 + (circles), in the absence of C a 2 + (2 mM EGTA) (squares) and after Ca 2 * had been reintroduced (triangles). Each point represents the mean of six experiments \u00C2\u00B1 S.E. Maximum tension in the presence of extracellular Ca 2 * was 4.97 \u00C2\u00B1 0.63 mN (mean \u00C2\u00B1 S.E.; n = 6). - 34 T 120 c100 o 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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 3.76 \u00C2\u00B1 0.64 mN (mean \u00C2\u00B1 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. EC50, 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 \u00C2\u00B1 S.E. Groups ECwUiM) n % Maximum Control 1.51 \u00C2\u00B10.05 1.49 \u00C2\u00B10.06 100 \u00C2\u00B1 1 Pertussis toxin (100 ng/ml) 1.28 \u00C2\u00B10.10 1.37 \u00C2\u00B10.04 104 \u00C2\u00B1 4 Control 1.72 \u00C2\u00B10.21 1.49 \u00C2\u00B10.17 101 \u00C2\u00B1 1 Distilled H 20 (40 ul) 1.66 \u00C2\u00B10.22 1.48 \u00C2\u00B10.15 106 \u00C2\u00B1 6 - 36 -) 120 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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 11.64 \u00C2\u00B1 0.51 mN (mean \u00C2\u00B1 S.E.; n \u00E2\u0080\u00A2= 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 \u00C2\u00B1 S.E. Groups EC5o(p,M) n % Maximum Control 6.6 \u00C2\u00B12 .4 1.42 \u00C2\u00B10.24 93 \u00C2\u00B1 1 Felodipine (1 nM) 31.3 \u00C2\u00B17 .8 1.47 \u00C2\u00B10.17 86 \u00C2\u00B1 3 Felodipine (10 nM) 63 .0\u00C2\u00B117 .2 a 0.94 \u00C2\u00B10.12 7 4 \u00C2\u00B1 3 a Control 7.8 \u00C2\u00B12 .0 1.64 \u00C2\u00B10.12 94 \u00C2\u00B1 1 Ryanodine (3 pM) 14.0 \u00C2\u00B11 .0 \u00E2\u0080\u00A2 1.53 \u00C2\u00B10.06 97 \u00C2\u00B1 2 Ryanodine (10 pJvl) 13.0 \u00C2\u00B13 .0 1.67 \u00C2\u00B10.34 1 0 4 \u00C2\u00B1 2 a Control . 17.0 \u00C2\u00B18 .2 1.78 \u00C2\u00B10.61 103 \u00C2\u00B1 7 Distilled H 20 (20 pi) 16.0 \u00C2\u00B13 .4 1.79 \u00C2\u00B10 .04 106 \u00C2\u00B1 4 Distilled H 20 (20 ul) 22.0 \u00C2\u00B16 .0 1.44\u00C2\u00B10.12 112 \u00C2\u00B1 5 aSignificantly different from control, p < 0.05 - 38 -120 r 100 .2 80 ts 2 8 60 E ZD E x CO 40 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 \u00C2\u00B1 S.E. Maximum tension in the absence of antagonist was 10.84 \u00C2\u00B1 1.52 mN (mean \u00C2\u00B1 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, cti-adrenoceptors that induce an influx of extracellular Ca 2 * are designated au, while cti-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 1 a subtype mediates PLC activity that is induced by C a 2 + influx, while the a i b subtype activates a C a 2 + 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|/G0, G s , G12 or G q families (Simon et al., 1991). 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 0 subunit and Gp /G, complex. Gene transfection studies have demonstrated that aib-adrehoceptors can interact favourably with all four members of the Goq family, G a14i Gal6, Goq 3nd Ga11, coupling noradrenaline receptor-binding to inositol phosphate accumulation; however, aia-adrenoceptors can only interact favourably with G ^ and G a n (Wu et al., 1992). 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 a subunit or the Gp/Gy complex (Katz et al., 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 a n activated PLC-Bt, but not the\"-y1, -61 or -B2 isozymes (Wu etai., 1992; Smrcka etai., 1991; Aragayet al., 1992; Park etai., 1992; Lee etai 1992). In addition, Gaie, but neither Goq nor G a n , activated PLC-P2 (Schwinn 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/Gaii 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 ccia-adrenoceptors (Piascik etai, 1991). Therefore, PI hydrolysis in rat caudal artery is likely mediated through aia-adrenoceptors. 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 2 + through voltage gated C a 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 noradrenaline-induced PI turnover in rat caudal artery (Cheung etal., 1990). Therefore, the G protein coupling the 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 Ca 2 + . This is consistent with the previously reported observations. Phenylephrine induced a phasic contraction in rat thoracic aorta in the absence of extracellular Ca 2 * (Nishimura etai., 1991), and "Thesis/Dissertation"@en . "Fall 1995"@en . "10.14288/1.0086809"@en . "eng"@en . "Pharmacology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Guanosine 3\u00E2\u0080\u0099:5\u00E2\u0080\u0099-cyclic monophosphate and contraction in vascular smooth muscle"@en . "Text"@en . "http://hdl.handle.net/2429/3710"@en .