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Adrenergic-cholinergic interactions in the heart Ray, Abhijit 1992

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inTHE FACULTY OF GRADUATE STUDIESPHARMACEUTICAL SCIENCESWe accept this thesis as conforming,ADRENERGIC-CHOLINERGIC INTERACTIONS IN THE HEARTbyABHIJIT RAY(M. Sc. PHARMACOLOGY)A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYOCTOBER, 1992.© ABHIJIT RAY^1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  C) I-1 A R M l3 CE C T 1 t_ R i— SCI e ;Qcial SThe University of British ColumbiaVancouver, CanadaDate 143 k2CC ,^i^92--DE-6 (2/88)ABSTRACTIn the mammalian atrial myocardium the muscarinic receptor agonist carbacholcan inhibit the positive inotropic responses to isoproterenol, a f3-adrenoceptor agonist,forskolin, a direct activator of adenylate cyclase, IBMX, a phosphodiesterase inhibitor,and phenylephrine, an a-adrenoceptor agonist. While the inhibitory effect of carbacholon the isoproterenol-stimulated cAMP generation is believed to contribute to the negativeinotropic effect of carbachol in the presence of isoproterenol, it is not known howcarbachol inhibits the positive inotropic responses to phenylephrine, forskolin and IBMXin the atrial myocardium. One of the objectives of the present study was to investigatewhether the reversal by carbachol of the positive inotropic responses of left atria tophenylephrine, forskolin and IBMX is related to the ability of carbachol to openpotassium channels.In rabbit left atria, carbachol exerted a direct negative inotropic response andinhibited the positive inotropic response to phenylephrine. Carbachol also promoted theefflux of 86 Rb in the presence and absence of phenylephrine from left atria. The abilityof carbachol to increase the rate-constant of 86 Rb-efflux was attenuated by atropine (100nM), 4-aminopyridine (50 and 500 gM), a potassium channel blocker, and pre-treatmentof rabbits with pertussis toxin (0.5 and 1 lig/kg), an uncoupler of muscarinic receptorsfrom potassium channels. Although both 4-aminopyridine and pertussis toxin attenuatedthe negative inotropic response to carbachol, both agents had a greater attenuating effecton the carbachol-stimulated 86 Rb-efflux than on the carbachol-induced negativeinotropy. The abilities of carbachol to promote the 86 Rb-efflux and to exert a negativeinotropic response in the presence of phenylephrine were attenuated by pertussis toxiniipre-treatment of rabbits. Although 4-aminopyridine was able to attenuate the inhibitoryeffect of carbachol on the phenylephrine-induced positive inotropy, only 500 1.1M 4-aminopyridine slightly reduced the carbachol-stimulated increase in the rate constant of86 Rb efflux in the presence of phenylephrine.4-Aminopyridine did not have any effect on the carbachol-induced inhibition ofthe isoproterenol-stimulated cAMP generation suggesting that 4-aminopyridine was notacting as a muscarinic receptor antagonist. 4-Aminopyridine inhibited only modestly theinhibitory effect of carbachol on the isoproterenol-induced positive inotropy, a cAMP-dependent response. The potassium channel openers, pinacidil and cromakalim, did nothave any inhibitory effect on the isoproterenol-induced positive inotropy but inhibited ina concentration-dependent manner the positive inotropic responses to phenylephrine.Uncoupling of muscarinic receptors from adenylate cyclase using pertussis toxin(2.2 µg/kg) attenuated only partially the negative inotropic responses of left atria tocarbachol in the presence of forskolin and IBMX suggesting that at least part of thereversal by carbachol of positive inotropic responses to forskolin and IBMX occurs by acAMP-independent mechanism. 4-Aminopyridine attenuated in a concentration-dependent manner the negative inotropic responses to carbachol in the presence offorskolin and IBMX in left atria from both saline and pertussis toxin pre-treated rabbits.These results suggest that the ability of carbachol to open potassium channelsmay not explain completely the direct negative inotropic response to carbachol but maycontribute to the negative inotropic responses to carbachol in the presence ofphenylephrine, forskolin and IBMX.It is established that muscarinic receptors are linked to potassium channels andadenylate cyclase in the atrium and only to adenylate cyclase in the ventricle by means ofiiipertussis toxin sensitive G-protein(s). The second objective of this study was toinvestigate if inhibition of the isoproterenol-stimulated adenylate cyclase by carbachol inthe rabbit atrium is more sensitive to pertussis toxin than either the same response tocarbachol in the ventricle or the ability of carbachol to open potassium channels in theatrium. Injection of rabbits with 0.5 µg/kg pertussis toxin, a dose which ADP-ribosylated60 % of atrial and ventricular G-proteins, resulted in complete uncoupling of muscarinicreceptors from the atrial adenylate cyclase. On the other hand, in rabbits injected with 1µg/kg pertussis toxin, the ability of carbachol to inhibit the ventricular adenylate cyclasewas not altered and to open the atrial potassium channels was only partially attenuated.These data suggest that the muscarinic receptor-mediated inhibition of adenylate cyclasein the atrium is more sensitive to pertussis toxin than either the muscarinic receptor-mediated activation of potassium efflux in the atrium or inhibition of adenylate cyclase inthe ventricle. This suggests that there are differences in the coupling of muscarinicreceptors to adenylate cyclase and potassium channels in the atrium and ventricle.ivTABLE OF CONTENTSPageABSTRACT^ iiLIST OF TABLESLIST OF FIGURES^ xiLIST OF ABBREVIATIONS^ xivACKNOWLEDGEMENTS xvi1. INTRODUCTION1.1. OVERVIEW^ 11.2. ADRENOCEPTORS 41.2.1. f3-adrenoceptors^ 41.2.2. a-adrenoceptors 51.3. CHOLINOCEPTORS^ 61.4. FUNCTIONAL CONSEQUENCES OF a- AND (3-^ 6ADRENO- AND MUSCARINIC CHOLINOCEPTORSTIMULATION IN THE HEART1.5. BIOCHEMICAL CONSEQUENCES OF ADRENOCEPTOR^7STIMULATION IN THE HEART1.5.1. 13-Adrenoceptor^ 71.5.1.1. Adenylate cyclase and cAMP^ 71.5.1.2. cAMP-independent effects 81.5.2. a-Adrenoceptor^ 91.5.2.1. a-Adrenoceptors and ion channels^ 91.5.2.2. a-Adrenoceptors and phosphoinositide^ 10turnover1.6. BIOCHEMICAL CONSEQUENCES OF MUSCARINIC^13CHOLINOCEPTOR STIMULATION1.6.1. Muscarinic receptors and adenylate cyclase^ 131.6.2. Adenylate cyclase-independent responses to muscarinic^13muscarinic receptor stimulation1.6.2.1. Contribution of cGMP^ 161.6.2.1.1. cGMP-dependent protein kinase^ 181.6.2.1.2. cGMP-stimulated phosphodiesterase 191.6.2.2. Activation of phosphatase^ 191.6.2.3. Alteration of ion channel activity 201.6.2.3.1. Potassium channel^ 201.6.2.3.2. Pacemaker current 221.6.2.3.2. Calcium current^ 221.6.2.4. Promotion of phosphoinositide turnover^ 231.7. G-PROTEINS^ 241.8. OBJECTIVES 312. MATERIALS AND METHODS2.1. MATERIALS^ 412.2. PREPARATION OF SOLUTIONS^ 452.3. GEL CASTING^ 512.4. ISOLATED TISSUE PREPARATION^ 51vi2.5. CONTRACTILITY STUDY^ 522.6. cAMP ASSAY^ 542.7. 86 Rb EFFLUX MEASUREMENT^ 542.8. HOMOGENIZATION OF TISSUE 572.9. ADP-RIBOSYLATION STUDY^ 572.10. ADENYLATE CYCLASE ASSAY 592.11. PROTEIN ASSAY^ 602.12. STATISTICS 613. RESULTS3.1. INTERACTION OF CARBACHOL WITH PHENYLEPHRINE^623.1.1. Effects of 4-aminopyridine on negative inotropic^ 62responses to carbachol alone and in the presenceof phenylephrine and isoproterenol3.1.2. Effects of pinacidil and cromakalim^ 643.1.3. 86 Rb efflux studies^ 653.1.3.1. Time-dependence of the 86Rb efflux rate constant^653.1.3.2. Effects of carbachol on the 86 Rb efflux rate constant 67and force of contraction3.1.3.3. Effects of 4-aminopyridine on carbachol-induced decrease^67in tension and increase in the rate constant of 86Rb efflux3.1.3.4. Effects of carbachol on the rate constant of 86Rb efflux^68and tension in the presence of phenylephrine3.1.3.5. Effects of 4-aminopyridine on responses to carbachol^68viiin the presence of phenylephrine3.1.3.6. Effects of pertussis toxin on carbachol-induced^ 70increase in the rate constant of 86Rb efflux anddecrease in tension3.1.3.7. Effects of pertussis toxin on responses to carbachol^71in the presence of phenylephrine3.2 EFFECTS OF 4-AMINOPYRIDINE AND PERTUSSIS^72TOXIN (2.2 pg/kg) ON THE INHIBITION BYCARBACHOL OF ISOPROTERENOL-STIMULATEDADENYLATE CYCLASE ACTIVITY AND cAMP GENERATION3.3. INTERACTION OF CARBACHOL WITH cAMP-^ 74ELEVATING AGENTS: EFFECTS OF4-AMINOPYRIDINE AND PERTUSSIS TOXIN3.3.1. Effects pertussis toxin and 4-aminopyridine on negative^74inotropic responses to carbachol in the absence andpresence of isoproterenol3.3.2. Effects of pertussis toxin and 4-aminopyridine on negative^76inotropic responses to carbachol in the presence offorskolin and IBMX3.4. ADP-RIBOSYLATION EXPERIMENTS^ 783.4.1. Selecting the dose of pertussis toxin for in vitro 79ADP-ribosylation3.4.2. Estimation of in vivo ADP-ribosylation by pertussis toxin^793.5. ADENYLATE CYCLASE ASSAY^ 82viii3.5.1. P-Adrenoceptor-stimulation of adenylate cyclase^ 823.5.2. Effect of muscarinic receptor stimulation on adenylate cyclase^85activity in atria and ventricle3.5.2.1. Basal adenylate cyclase^ 853.5.2.2. Isoproterenol-stimulated adenylate cyclase^ 853.5.2.3. Forskolin- and GTPyS- stimulated adenylate cyclase^863.5.3. Pertussis toxin treatment of rabbits^ 873.5.3.1. Muscarinic receptor-mediated response 873.5.3.1.1. Basal adenylate cyclase^ 873.5.3.1.2. Interaction of carbachol with isoproterenol^ 874. DISCUSSION^ 1474.1. DIRECT NEGATIVE INOTROPIC RESPONSE TO CARBACHOL^1484.2. INTERACTION OF CARBACHOL WITH PHENYLEPHRINE^151AND ISOPROTERENOL4.3. EFFECTS OF PINACIDIL AND CROMAKALIM^ 1534.4. INTERACTION OF CARBACHOL WITH FORSKOLIN AND^154IBMX4.5. DIFFERENTIAL EFFECT OF PERTUSSIS TOXIN^ 1585. SUMMARY AND CONCLUSIONS^ 1646. REFERENCES^ 168ixLIST OF TABLESTable^ Page1. Effects of 4-aminopyridine on maximum negative inotropic^63responses and pD2values of carbachol in the presence andabsence of phenyrephrine and isoproterenol.2. Effects of pinacidil and cromakalim on the maximum positive^66inotropic responses and pD2values to phenylephrine andisoproterenol.3. Effects of phenylephrine and 4-aminopyridine, aloe and in^69combination, on the contractile response and the Rb-efflux-rate-constant in electrically-stimulated rabbit leftatria.4. Effects of 4-aminopyridine and pertussis toxin, alone and in^75in combination, on maximum negative inotropic responsesand pD2values to carbachol in the presence and absenceof isoproterenol, forskolin, IBMX.5. In vitro ADP-ribosylation of G-proteins by pertussis toxin in^80rabbit atrial and ventricular homogenates.6. ADP-Ribosylation by exogenous pertussis toxin (30 pg/m1)^81of G-proteins in atrial and ventricular homogenates fromsaline and pertussis toxin (0.5, 1, 2, 3 µg/kg) pre-treatedrabbits.7. Adenylate cyclase activity in atrial and ventricular^83homogenates in the presence and absence of variousactivators.8. Effects of pre-treatment of rabbits with different doses^88of pertussis toxin on basal adenylate cyclase activityin the atrium and ventricle.9.^Effects of pertussis toxin pre-treatment of rabbits on^89isoproterenol (ISO) - and forskolin- stimulated adenylatecyclase activity in the atrium and ventricle.xLIST OF FIGURESFIGURE^ PAGE1. A schematic representation of the possible consequences^14of muscarinic receptor stimulation in the heart.2. A schematic representation of the role of G-proteins in the^2713-adrenoceptor-mediated activation and the muscariniccholinoceptor-mediated inhibition of adenylate cyclase inthe heart.3. Tracings showing the effect of pertussis toxin pre-treatment^37of rabbits on the direct negative inotropic response tocarbachol in the presence and absence of isoproterenol inin rabbit left atrium.4. Concentration-dependent positive inotropic effects of^914-aminopyridine in electrically-stimulated rabbit leftatria.5. Effects of 4-aminopyridine on negative inotropic responses^93to carbachol in the absence and presence of phenylephrine orisoproterenol in electrically-stimulated rabbit left atrialstrips.6. Effect of pinacidil on the phenylephrine-induced positive^95inotropic response in electrically-stimulated rabbit leftatrial strips.7. Effect of cromakalim on the phenylephrine-induced positive^97inotropic response in electrically-stimulated rabbit leftatrial strips.8. Effects of pinacidil and cromakalim on the isoproterenol-^99induced positive inotropic response in electrically-stimulatedrabbit left atrial strips.9. Time-dependence of the 86Rb-efflux-rate-constant in^101electrically-stimulated rabbit left atrial strips.10. Effects of carbachol on the rate-constant of 86Rb-efflux^103and the force of contraction in electrically-stimulatedrabbit left atria.11. Effect of atropine on the rate-constant of 86Rb-efflux^105in the presence of carbachol in electrically-stimulatedrabbit left atrial strips.12. Effects of 4-aminopyridigs on the negative inotropic response^107and the rate-constant of °uRb-efflux in the presence ofcarbachol in electrically-stimulated rabbit left atrial strips.xi13. Effects of 4-aminopyridine on the rate-constant of 86Rb-efflux and the negative inotropic response to carbachol in thepresence of phenylephrine in electrically-stimulated rabbitleft atria.14. Effects of pre-treatmot of rabbits with pertussis toxin onthe rate-constant of °°Rb-efflux and the negative inotropicresponse to carbachol in electrically-stimulated rabbit left atria.15. Effects of pre-treatment of rabbits with pertussis toxin oncarbachol-ingiced changes in the contractile force and rate-constant of °° Rb-efflux in the presence of phenylephrinein electrically-stimulated rabbit left atrial strips.16. Effects of 4-aminopyridine and pertussis toxin on cAMPlevels in electrically-stimulated left atrial strips fromsaline and pertussis toxin pre-treated rabbits in thepresence of isoproterenol alone and in combination withcarbachol.17. Effects of carbachol on the isoproterenol-stimulated adenylatecyclase activity in the atrial homogenates from saline andpertussis toxin pre-treated rabbits.18. Effects of 4-aminopyridine and pertussis toxin, alone and incombination on negative inotropic responses to carbacholin electrically-stimulated rabbit left atrial strips.19. Effects of 4-aminopyridine and pertussis toxin, alone and in^121combination on negative inotropic responses to carbacholin the presence of isoproterenol in electrically-stimulated rabbitleft atrial strips.20. Effects of 4-aminopyridine and pertussis toxin, alone and in^123combination, on negative inotropic responses to carbacholin the presence of forskolin in electrically-stimulated rabbitleft atrial strips.21. Effects of 4-aminopyridine and pertussis toxin, alone and in^125combination, on the negative inotropic responses to carbacholin the presence of IBMX in electrically-stimulated rabbit leftatrial strips.22. Autoradiogram showing the ADP-ribosylating effect of exogenous^127pertussis toxin in rabbit atrial and ventricular homogenates.23. Autoradiogram showing the effect of pre-treatment of rabbits with^129pertussis toxin on the ADP-ribosylation of G-proteins by exogenouspertussis toxin in atrial homogenates.24. Autoradiogram showing the effect of pre-treatment of rabbits with^131pertussis toxin on the ADP-ribosylation of G-proteins by exogenousxi109111113115117119pertussis toxin in ventricular homogenates.25^Concentration-response effects of isoproterenol on adenylate cyclase^133activity in atrial and ventricular homogenates of rabbit.26. Effects of timolol on the isoproterenol-stimulated adenylate cyclase^135activity in rabbit atrial and ventricular homogenates.27. Concentration-response effects to carbachol in the absence and^137and presence of isoproterenol in rabbit atrial and ventricularhomogenates.28. Effects of atropine on the carbachol-induced inhibition of^139adenylate cyclase activity in the absence and presence ofisoproterenol in rabbit atrial and ventricular homogenates.29. Effects of carbachol on the forskolin- and GTPyS- stimulated^141adenylate cyclase activity in rabbit atrial and ventricularhomogenates.30. Effects of pertussis toxin pre-treatment of rabbits on the^143inhibitory effect of carbachol on adenylate cyclase activityin the absence and presence of isoproterenol in rabbitventricular homogenates.31. Effects of pre-treatment of rabbits with different doses of^145pertussis toxin on the inhibitory effect of carbachol on theisoproterenol-stimulated adenylate cyclase activity in rabbitatrial homogenates.LIST OF ABBREVIATIONS4-AP^4-AminopyridineADP Adenosine diphosphateATP^Adenosine triphosphate[a-3213]ATP^ATP where the a-phosphate group is radioactiveC^ CentigradecAMP Adenonsine 3',5' cyclic monophosphatecpm^Counts per minute[31-1]-cAMP^Tritium labelled cAMPDMSO Dimethyl sulphoxidedpm^Disintegrations per minuteDTT DithiothreitolEDTA^Ethylenediamine tetra acetic acidEGTA Ethyleneglycol-bis-((3-amino ethyl ether)-N, N, N', N'-tetra acetic acidg^ Gram(s)G-protein^Guanine nucleotide binding proteinGp Beta and gamma subunits of G-proteinGi^ Inhibitory G-proteinGia^a—Subunit of the inhibitory G-proteinGp^G-protein mediating hormonal stimulation ofphospholipase CGs^Stimulatory G-proteinGsa a-Subunit of the stimulatory G-proteinGo^Other G-proteinGoa a-Subunit of the other G-proteinGz^A pertussis toxin-insensitive G-proteinxivGTP^Guanosine triphosphateGTPyS Guanosine 5'-O-(3-thiotriphosphate)cGMP^Guanosine 3',5' cyclic monophosphateHz HertzIBMX^Isobutylmethyl xanthineIf Pacemaker currentIP3^ Inositol trisphosphateIP4 Inositol tetrakisphosphateIP5^Inositol pentakisphosphateIP6 Inositol hexakisphosphatekDa^Kilodaltonkg Kilogram1^ LitreM MolarMCK^Modified Chenoweth-KoelleniAMP Milliamperemg^ Milligramml MillilitremM^Millimolar4Ci Microcurielig^ Microgram111^Microlitre1.1M MicromolarmRNA^messenger RNAmin MinuteN^ NormalNAD Nicotinamide adenine dinucleotideXV[3211-NAD^Nicotinamide adenine dinucleotide where onephosphorus is radioactiveng^ nanogramnm NanometernM^ NanomolarPMSF Phenylmethylsulphonyl Fluoridepmol^Picomole86 Rb Radioactive rubidiumRsp^ Specific radioactivity (dpm/pmol)SDS Sodium dodecylsulphateS.E.M.^Standard error of the meanTCA Trichloroacetic acidTEMED^N, N, N', N'-Tetra methyl ethylenediamineACKNOWLEDGEMENTSI gratefully acknowledge the constant support and guidance of my researchsupervisor Dr. K. M. MacLeod.Sincere thanks are also due to Drs. Stelvio Bandiera, Jack Diamond, DavidGodin, Sidney Katz and Keith McErlane for all their constructive criticisms.Last but not the least, I thank my colleagues in the laboratory Ms. SlavicaBosjnack, Ms. Lynn Weber , Ms. Yi Jia Bi, Mr. Ali Tabatabaie and Mr. Jack Zhang forall their cooperation and being so very patient with me.Financial support provided by the Heart and Stroke Foundation of Canada isgratefully acknowledged.xviiINTRODUCTION1.1. OVERVIEWThe force and rate of myocardial contraction is subject to modulation, dependingupon physiological needs, by the sympathetic and parasympathetic branches of theautonomic nervous system. The effects of sympathetic stimulation in the heart aremediated by the neurotransmitter norepinephrine which acts on the a- and J3-adrenoceptors. The parasympathetic neurotransmitter acetylcholine on the other hand actson muscarinic cholinoceptors. Stimulation of the a- and 13- adrenoceptors bynorepinephrine increases the rate and force of myocardial contraction. The consequencesof muscarinic receptor stimulation in the heart, however, are very complex and seem tovary from one part of the myocardium to the other. Depending upon the region of theheart and the animal species involved, muscarinic agonists can exert a direct negativeinotropic and chronotropic response, reverse positive inotropic and chronotropicresponses to both cAMP-independent a-adrenoceptor agonists and cAMP-generating [3 -adrenoceptor agonists, and can also exert a positive inotropic response. The biochemicaland/or electrophysiological changes accompanying muscarinic receptor stimulationinclude inhibition of adenylate cyclase activity and lowering of cAMP levels elevated byI3-adrenoceptor agonists, elevation of cGMP levels, activation of protein phosphatase,increase in an outward potassium current (seen only in atrium), inhibition of thepacemaker current (atrium only) and the slow inward calcium current and promotion ofphosphoinositide breakdown (see Loffelholz and Pappano, 1985; Hartzell, 1988;Pappano, 1990). It has been fairly well established that the inhibitory effect of muscarinicagonists on the 13-adrenoceptor agonist-mediated positive inotropy and chronotropy is1mediated largely through the adenylate cyclase and cAMP pathway, although theexistence of a cAMP-independent mechanism of action of muscarinic agonists has alsobeen suggested (Endoh et al., 1985; MacLeod, 1986). In addition, it has also been shownthat the negative inotropic responses to muscarinic agonists in the presence of cAMP-elevating agents like forskolin or isobutylmethyl xanthine (IBMX) are not alwaysassociated with a reduction in accompanying increases in cAMP levels (MacLeod andDiamond, 1986; Ray and MacLeod, 1992). On the other hand, muscarinic agonists havebeen reported to antagonize or reverse the cAMP-independent positive inotropicresponses to a-adrenoceptor stimulation without altering the basal cAMP levels(MacLeod, 1986; 1987). It has been suggested that the ability of muscarinic agonists toactivate a potassium current may contribute to the cAMP-independent negative inotropicresponse to muscarinic receptor stimulation (Ten Eick et al., 1976; Loffelholz andPappano, 1985). However, it remains to be seen if activation of a potassium current mayalso play a role in the cAMP-independent functional interaction of muscarinic agonistswith different positive inotropic agents.It is well known that the a- and [3- adrenoceptors and muscarinic cholinoceptorsin the heart are linked to their effectors by guanine nucleotide binding proteins(Robishaw and Foster, 1988; Fleming et al., 1982). The guanine nucleotide bindingproteins (G-proteins) consist of three subunits, a-, (3- and y. The a-subunit of astimulatory G-protein (Gs) mediates the stimulation of adenylate cyclase in response to(3-adrenoceptor stimulation. On the other hand, the message of muscarinic receptoroccupancy to adenylate cyclase and potassium channels is conveyed by a group ofpertussis toxin-sensitive G-proteins. These G-proteins can be further divided into twogroups, Gi which is a mixture of three different proteins Gil, Gi2 and Gi3 and G 0 . It is2not clear how and which of these G-proteins connect muscarinic receptors to adenylatecyclase and potassium channels. It is well known that pertussis toxin can uncouplemuscarinic receptors from adenylate cyclase and potassium channels in atria andventricles (Fleming et al., 1992). In a previous study from this laboratory (Ray andMacLeod, 1992) it was observed that in left atria from rabbits pre-treated with pertussistoxin the muscarinic receptor agonist carbachol lost its inhibitory effect on the 13-adrenoceptor agonist isoproterenol-induced increases in the force of contraction andcAMP levels but the direct negative inotropic response to carbachol was only partiallyattenuated. The same dose of pertussis toxin did not have any uncoupling effect on theability of carbachol to inhibit the isoproterenol-stimulated positive inotropic response inthe right ventricular papillary muscle of rabbits. This suggested that pertussis toxinuncoupled in a differential manner muscarinic receptors from potassium channels andadenylate cyclase in the atrium and muscarinic receptors from adenylate cyclase in theatrium and ventricle.The purpose of the present study was to investigate, using pharmacological andbiochemical techniques, (a) the role of muscarinic receptor-mediated potassium-efflux inthe functional interaction of a muscarinic agonist with various positive inotropic agents inatria, and (b) whether pertussis toxin uncouples in a differential manner (i) muscarinicreceptors from adenylate cyclase and potassium channels in atria, and (ii) muscarinicreceptors from adenylate cyclase in atria and ventricles.In the following pages an attempt will be made to describe in brief (i) thedifferent types of adrenoceptors and muscarinic cholinoceptors in the heart, (ii)functional, biochemical and electrophysiological consequences of adrenoceptor andmuscarinic cholinoceptor stimulation in the heart, with special reference to the3mechanism(s) of interaction of muscarinic cholinoceptor agonists with a- and 13-adrenoceptor agonists in the heart, and (iii) the G-proteins and their role in theadrenergic-cholinergic interactions in the heart.1.2. TYPES OF ADRENOCEPTORS IN THE HEART1.2.1 13-AdrenoceptorsIn the mammalian myocardium, stimulation of the sympathetic nervous systemresults in the release of norepinephrine. Although norepinephrine is capable ofstimulating both a- and f3- adrenoceptors, it is believed that under normal physiologicalconditions it is the 13-adrenoceptors which mediate the effects of sympathetic stimulationon myocardial function. The presence of f3-adrenoceptors in the heart can bedemonstrated using a variety of different techniques, including pharmacological, ligandbinding and autoradiographic localization (Stiles et al., 1984; Hartzell, 1988). Based onthese studies, we know that in the mammalian myocardium there exists at least twodifferent types of j3-adrenoceptors, namely the 131 and 132, while the presence of a thirdtype, 133, is being debated (Kaumann, 1988). It has been shown that in the mammalianmyocardium the ratio of 13032 receptors varies with the species and the region of theheart. Results of binding studies show that in rat, guinea pig, rabbit, cat, dog and humanhearts, it is the 131-adrenoceptors which predominate, usually constituting 80% or more ofthe total 13-adrenoceptor population (Stiles et al., 1984). The number of 132-adrenoceptorsis usually very low or absent in the ventricle but represents 20 - 30 % of the total 13-adrenergic receptors in the atrial myocardium (Stiles et al., 1984). Using selectiveagonists and antagonists it can be shown that the 131-adrenoceptors play an equally4important role in atria and ventricles, whereas the 132-adrenoceptors are more importantin the sinoatrial node, less so in the left atrium and even less so in the ventricularmyocardium (Kaumann, 1988).1.2.2. a-AdrenoceptorsIn contrast to the P-adrenoceptors, the role played by the a-adrenoceptors in theheart is not very clear. The presence of a-adrenoceptors can be demonstrated, usingligand binding techniques, in both atria and ventricles from different species of animals(Williams and Lefkowitz, 1978; Karliner et al., 1979; Yamada et al., 1980; Mukherjee etal., 1983). The number of a-adrenoceptors vary among different species of animals andbetween different regions of the heart. It has been shown that in rabbit and rat hearts, thenumber of a-adrenoceptor binding sites are almost equal to the number of 13-adrenoceptorbinding sites (Mukherjee et al. 1983). Much lower numbers of a-adrenoceptor bindingsites, approximately 3 - 60 times less, were reported in dog, guinea pig and hamsterhearts (Karliner et al., 1979; Mukherjee et al., 1983). The regional distribution of the a-adrenoceptors has been investigated by Yamada et al. (1980), who reported that in the ratheart the number of a-adrenoceptors are at least two times higher in ventricles comparedto atria. The fact that a-adrenoceptor stimulation in the heart, using selective agonists,results in dose-dependent positive inotropic and chronotropic responses suggests thatthese receptors are functionally coupled to some effector(s) (Benfey, 1980; 1982; Scholzet al., 1986). The a-aderenoceptors can be divided into two groups, al and a2, whichhave been further subdivided into am, alB , a lc and a 2A , a 213 , a 2C , a2Dcategories (Bylund, 1992). It has been established using selective agonists andantagonists that the a-adrenoceptors in the heart are of the al subtype (Bruckner et al.,51985; Minneman, 1988). More recently, in the rat heart, the messenger RNAs (mRNA)were reported for both the al A- and am- adrenoceptors (Bylund, 1992). The functionalsignificance of these different types of a-adrenoceptors and their distribution in differentanimal species, however, is not known at present.1.3. CHOLINOCEPTORS IN THE HEARTThe receptors which bind the neurotransmitter acetylcholine in the heart are of themuscarinic cholinergic subtype. With the help of pharmacological and radioligandbinding studies it has been established that the muscarinic receptors in the heart aremostly of the M2 subtype (Loffelholz and Pappano, 1985; Mei et al., 1989). When themyocardium from different species of animals was screened for the presence of mRNAfor different muscarinic receptor subtypes, the mRNA for only the M2 subtype wasdetected (Peralta et al., 1987; Maeda et al., 1988; Mei et al., 1989). Unlike theadrenoceptors, the number of binding sites for muscarinic cholinoceptors is much higherin the atrial myocardium compared to that in the ventricular myocardium from differentspecies of animals (Fields et al. 1978; Wei and Sulakhe, 1978; Loffelholz and Pappano,1985).1.4. FUCNTIONAL CONSEQUENCES OF a- AND (3- ADRENOCEPTOR ANDMUSCARINIC CHOLINOCEPTOR STIMULATION IN THE HEARTThe functional consequences of the adrenoceptor stimulation in the heart havebeen investigated widely and it is known that both a- and p- adrenoceptor stimulationcan increase the rate and force of myocardial contraction (Bruckner et al., 1985; Hartzell,61988). However, the functional effects of muscarinic receptor stimulation are not onlyvery diverse, but they seem to vary from one part of the myocardium to another(Loffelholz and Pappano, 1985). In the atrial myocardium, muscarinic receptor agonistshave been shown to exert a direct negative inotropic and chronotropic response and canalso antagonize the positive inotropic responses to both a- and 13- adrenoceptorstimulation. In contrast, in the ventricular myocardium the negative inotropic response tomuscarinic receptor stimulation is minimal and muscarinic receptor agonists can inhibitthe positive inotropic responses to (3- but not to a- adrenoceptor stimulation. However, itis important to note that the inhibitory effect of muscarinic receptor stimulation on boththe atrial and ventricular contractility becomes more pronounced in the presence of a (3-adrenoceptor agonist or a background of sympathetic activity. This phenomenon ofenhanced muscarinic response in the presence of a background sympathetic tone has beentermed "accentuated antagonism" or the "anti-adrenergic effect" of muscarinicstimulation (Loffelholz and Pappano, 1985). In addition to the inhibition of atrial andventricular contractility, it has been shown that in both atrial as well as in ventricularmyocardium muscarinic receptor agonists in high concentrations can exert a positiveinotropic response (Loffelholz and Pappano, 1985; Pappano, 1990).15. BIOCHEMICAL ANDIOR ELECTROPHYSIOLOGICAL CONSEQUENCESOF ADRENOCEPTOR STIMULATION IN THE HEART1.5.1. P-Adrenoceptor1.5.1.1. Adenylate cyclase and cAMPThe biochemical basis of the (3-adrenoceptor-mediated positive inotropy has beeninvestigated and reviewed very extensively (Drummond and Severson, 1979; Stiles et al.,71984; Hartzell, 1988; Lindemann and Watanabe, 1990). It is well established that 13-adrenoceptor agonists stimulate adenylate cyclase activity and increase intracellularcyclic AMP (cAMP) levels in a variety of different tissue and cell types includingmyocardial cells. Cyclic AMP in turn facilitates the entry of calcium through voltage-operated calcium channels (Hartzell, 1988; Brown, 1990), decreases the sensitivity ofcontractile proteins to calcium (Hartzell, 1988) and stimulates the uptake of calcium bythe sarcoplasmic reticulum (Lindemann and Watanabe, 1990). Cyclic AMP has also beenshown to stimulate the pacemaker current (If) in the sinoatrial node and to stimulate thedelayed rectifier potassium current. Most of the effects of cAMP are believed to berelated to its ability to activate a cAMP-dependent protein kinase which in turnphosphorylates various proteins. It has been shown that the cAMP-dependent proteinkinase can phosphorylate the calcium channel (Trautwein and Hescheler, 1990),phospholamban (Lindemann and Watanabe, 1985) and proteins associated with thecontractile machinery, namely troponin-I and tropomyosin (Hartzell, 1988). Althoughthere is no direct evidence that cAMP-dependent protein kinase phosphorylates thepotassium channel, it has been shown that at least part of the effects of 13-adrenoceptor-stimulation on the delayed rectifier current are phosphorylation-dependent (Szabo andOtero, 1990). However, in a recent study DiFrancesco and Tortora (1991) have shownthat cAMP can stimulate If channels without activating the cAMP-dependent proteinkinase.81.5.1.2. cAMP-independent effects of f3-adrenoceptor stimulationP-Adrenoceptor agonists have been shown to modulate potassium (Freeman et al.,1992; Szabo and Otero, 1990), calcium (Yatani et al., 1988; Yatani and Brown, 1990;Trautwein and Hescheler, 1990) and If channels (Yatani et al., 1990; Brown, 1990)without increasing cAMP levels or activating the cAMP-dependent protein kinase (seealso, Brown, 1990, Brown and Birnbaumer, 1990). However, it is worth mentioning thatHartzell and his colleagues were unable to detect the cAMP-independent effects of the p-adrenoceptor stimulation on calcium currents (Hartzell et al., 1991). It has been arguedthat activation of the delayed rectifier current by a f3-adrenoceptor agonist occurssecondary to activation of the calcium current (Hume, 1985; see also Hartzell, 1988).However, the fact that calcium and delayed rectifier currents are independent of eachother was later confirmed by the differential temperature sensitivity of the two currents(Walsh and Kass, 1988), and by the ability of P-adrenoceptor agonists and cAMP-dependent protein kinase to activate the potassium current in the presence of a calciumchannel blocker (Walsh and Kass, 1988; Walsh et al., 1991; Hartzell, 1988; Szabo andOtero, 1990).1.5.2. a-Adrenoceptor1.5.2.1. a-Adrenoceptors and ion channelsUnlike the 13-adrenoceptors, very little is known about the mechanism of the a-adrenoceptor-mediated positive inotropy in the myocardial tissue. The involvement ofcAMP or cGMP can be ruled out because the a-adrenoceptor agonists do not increase theintracellular cAMP and cGMP levels (Inui et al., 1982; Buxton and Brunton, 1986;MacLeod, 1986; see also Bruckner et al., 1985; Hartzell, 1988). a-Adrenoceptor agonists9were reported to stimulate the voltage-dependent slow inward calcium current (Miura etal., 1978; Bruckner and Scholz, 1984) and to increase the sensitivity of the myofilamentsto calcium (Endoh and Blinks, 1988; Puceat et al., 1992). However, it has been shownthat the increase in the slow inward calcium current in response to a-adrenoceptorstimulation is not only very small in magnitude (Handa et al., 1982), but it can beabolished in the presence of a 13-adrenoceptor antagonist (Sanchez-Chapula, 1981;Hartman et al. 1988). Many others have failed to observe any change in the slow inwardcalcium current in response to a-adrenoceptor stimulation (Hescheler et al., 1988a; Ertlet al., 1991). Handa et al. (1982) reported that a-adrenoceptor agonists prolong the actionpotential duration by blocking an outward potassium current. This was later confirmed bymany others (Apkon and Nerbonne, 1988; Fedida et al., 1989; Ravens et al., 1989; Braunet al., 1990; Fedida and Bouchard, 1992) and may contribute to the a-adrenoceptor-mediated positive inotropy by prolonging the action potential duration. It is, however, notknown how a-adrenoceptor agonists increase the sensitivity of myofilaments to calciumor block the outward potassium current in the heart.1.5.2.2. a-Adrenoceptors and phosphoinositide turnoverIn many tissues, including the myocardium, a-adrenoceptor agonists have beenshown to promote the turnover of a membrane phospholipid, phosphatidylinositol (Quistand Sanchez, 1983; Sekar and Roufogalis, 1984). The consequences ofphosphatidylinositol turnover have been reviewed extensively (see Brown and Jones,1986; Rana and Hokin, 1990). Briefly, phosphatidylinositol is sequentiallyphosphorylated to phosphatidylinositol mono- and bis- phosphates. Two different secondmessengers are generated as a result of phosphatidylinositol bisphosphate breakdown10under the influence of phospholipase C, namely inositol 1,4,5 trisphosphate (IP3), anddiacylglycerol. While IP3 is believed to release calcium from intracellular stores,diacylglycerol in turn activates protein kinase C, a calcium- and phospholipid- dependentprotein kinase, which phosphorylates a variety of intracellular proteins. In addition toIP 3, various products of phosphorylation of IP 3 , such as inositol tetrakis (IP4)-, pentakis(1P5)- and hexakis (IP6)- phosphates, are also believed to have second messengerfunctions.In the heart, a-adrenoceptor agonists have been shown to promote the breakdownof phosphatidylinositol bisphosphate (Otani et al., 1988) and accumulation of differentproducts of phosphatidylinositol breakdown namely, inositol mono-, bis- and tris-phosphates (Brown et al., 1985; Otani et al., 1988; Edes et al., 1991) and more recentlytetrakis-, pentakis- and hexakis- phosphates (Scholz et al., 1992). Scholz et al. (1992)showed that the a-adrenoceptor-mediated generation of IP3 precedes contraction and thatof IP4 coincides with increases in the force of contraction. They proposed that it is theIP3 which initiates, and IP4 which maintains, the a-adrenoceptor-mediated myocardialcontractility. However, exactly how IP3 and various phosphorylation products of IP3bring about changes in myocardial contractility is not known. Some workers have shownthat IP3 can release calcium from the sarcoplasmic reticulum (Nosek et al., 1986; Vitesand Pappano, 1990) while others either failed to see any effect of IP3 on calcium releasefrom the sarcoplasmic reticulum (Movsesian et al. 1985) or were unable to ascribe anyphysiological significance to the IP3 -induced calcium release in the heart (Fabiato,1986).While the presence of protein kinase C has been demonstrated in the heart byNishizuka and colleagues using selective antibodies (see Shearman et al., 1989), the1 1functional significance of the protein kinase C pathway in the a-adrenoceptor-mediatedpositive inotropy in the heart is not clear. Both a-adrenoceptor agonists and phorbolesters, direct activators of protein kinase C, have been demonstrated to causetranslocation of protein kinase C from the cytosol to sarcolemma (Yuan et al., 1987; Edesand Kranius, 1990; Edes et al., 1991; Talosi and Kranius, 1992). However, the similarityprobably ends there. While a-adrenoceptor agonists have been shown to exert a positiveinotropic response which is associated with an inhibition of the outward potassiumcurrent and an increase in intracellular calcium levels, activators of protein kinase C havevery diverse effects on the myocardial contractility. For example, depending on theanimal species and the type of activator used, phorbol esters can increase (Teutsch,1987), decrease (Leatherman et al., 1987; Yuan et al., 1987; Nakanishi et al., 1989;Capogrossi et al., 1990; Karmazyn et al., 1990) or have no effect (Otani et al., 1988;Kushida et al., 1988) on myocardial contractility. Similarly, some phorbol esters havebeen shown to stimulate (Lacerda et al., 1988) or inhibit (Leatherman et al., 1987)calcium influx and intracellular calcium levels, and activate an outward potassiumcurrent (Tohose et al., 1987; Walsh and Kass, 1988).Dissimilarity also exists at the level of protein substrates phosphorylated by a-adrenoceptor agonists and purified protein kinase C. In in vivo experiments a-adrenoceptor agonists and phorbol esters have been shown to promote phosphorylation ofa 15 kDa sarcolemmal and/or a 28 kDa cytosolic protein (Lindemann, 1986; Edes andKranius, 1990; Edes et al., 1991; Talosi and Kranius, 1992) in perfused guinea pig andrabbit hearts. On the other hand, purified protein kinase C has been shown tophosphorylate a variety of proteins in the sarcolemma and sarcoplasmic reticulum, mostnotably phospholamban (Movsesian et al., 1984; Yuan and Sen, 1986), and proteins12associated with contractile proteins, namely troponin C and T (Katoh et al., 1981; seealso Hartzell, 1988; Rana and Hokin, 1989; Shearman et al., 1989). However, thesignificance of phosphorylation of these proteins is not clearly understood.1.6 BIOCHEMICAL CONSEQUENCES OF MUSCARINIC RECEPTORSTIMULATIONFig. 1 is a schematic representation of various electrophysiological andbiochemical responses to muscarinic receptor stimulation in the heart. Muscarinicagonists exert their negative inotropic and chronotropic effects both in the presence andabsence of a- and (3- adrenoceptor agonists by a variety of mechanisms. These include:(a) inhibition of the (3-adrenoceptor agonist-stimulated increases in adenylate cyclaseactivity and intracellular cAMP levels, and (b) mechanisms independent of adenylatecyclase inhibition. Muscarinic inhibition of the 3-adrenoceptor agonist-stimulated cAMPgeneration is believed to contribute, at least in part, to the inhibitory effect of muscarinicagonists on the slow inward calcium current and the pacemaker current. The adenylatecyclase independent effects of muscarinic receptor stimulation include: (i)elevation of cGMP levels, which in turn may activate the cGMP-dependent proteinkinase and cGMP-stimulated phosphodiesterase, (ii) activation of a protein phosphatase,and (iii) altering various ion channels and currents such as an outward potassium current,the pacemaker current (If) and the calcium current. In addition, muscarinic agonists alsopromote phosphoinositide breakdown in the heart. While this does not play a role in theadrenergic-cholinergic interactions, it is believed to be responsible for the positiveinotropic responses to muscarinic stimulation.13Fig. 1: A schematic representation of the possible consequences of muscarinic receptorstimulation in the heart.(??) Indicates the mechanism of activation not known. (+) Represents stimulationand (-) represents inhibition of activity. G s, Gi and Go represent the stimulatory,inhibitory and the "other" guanine nucleotide binding proteins, respectively. G s, Gi andGo couple P-adrenoceptors and muscarinic cholinoceptors to adenylate cyclase andpotassium channels respectively. In addition to inhibiting the P-adrenoceptor agonist-stimulated adenylate cyclase, muscarinic agonists can open potassium channels (atriumonly), inhibit pacemaker (I f ) channels in the sinoatrial nodal cells, elevate cGMP levelsand increase phosphatase activity (for details see text). cAMP-dependent protein kinasecan stimulate the pacemaker channel (not shown in the figure).141.6.1. Muscarinic receptors and adenylate cyclaseSince the first report by Murad et al. (1962), numerous workers have shown thatmuscarinic agonists can inhibit the 0-adrenoceptor-stimulated adenylate cyclase activity,cAMP levels and positive inotropic responses in atria and ventricle from a variety ofdifferent animals (Watanabe et al., 1978; Jacobs et al., 1979; Sulakhe et al., 1985; Keelyand Lincoln, 1978; Endoh, 1980; Endoh et al., 1985; MacLeod, 1985; 1986; see alsoLoffelholz and Pappano, 1985; Hartzell, 1988). In addition, in the ventricularmyocardium a good correlation also exists between the muscarinic antagonism of the 13-adrenoceptor-mediated cAMP generation and inhibition of various processes activated byan increase in cAMP levels, such as the cAMP-dependent protein kinase activity (Keelyet al., 1978; Ingbretsen, 1980), slow inward calcium current (Biegon et al., 1980;Hescheler et al. , 1986; Rardon and Pappano, 1986), pacemaker current (DiFrancesco andTromba, 1987; 1988) and phospholamban phosphorylation (Lindemann and Watanabe,1985).1.6.2. Adenylate cyclase-independent responses to muscarinic receptorstimulationAlthough it has been shown that a good correlation sometimes exists between theability of muscarinic agonists to inhibit the 13-adrenoceptor-stimulated cAMP generationand force of contraction, many others have reported that the inhibition of cAMPgeneration does not always correlate with the inhibitory effect of muscarinic agonists onthe P-adrenoceptor agonist-mediated positive inotropy in atria and ventricles (Keely et al.1978; Endoh et al., 1985; MacLeod, 1986) and sometimes muscarinic agonists can inhibitthe positive inotropic responses to P-adrenoceptor stimulation without having any15inhibitory effect on the elevated levels of cAMP (Watanabe and Besch 1975; Schmiedand Korth, 1990). In addition, in atria and ventricles, muscarinic agonists have beenshown to inhibit the positive inotropic responses to various cAMP-elevating agentsincluding cholera toxin (Brown, 1980; Pappano et al, 1982), forskolin (Lindemann andWatanabe, 1985; MacLeod, 1985; MacLeod and Diamond, 1986) and isobutylmethylxanthine (Brown, 1979; Biegon et al., 1980; Schmied and Korth, 1990), withoutlowering the accompanying increases in cAMP levels. This suggested that somemechanism other than, or in addition to, inhibition of cAMP generation may contribute tothe functional interaction of muscarinic and cAMP-elevating agonists.It is not clear how muscarinic receptor agonists produce a direct negativeinotropic response or antagonize the cAMP-independent positive inotropic response to a-adrenoceptor stimulation in the atrial myocardium. Although it has been reported bysome that muscarinic agonists can inhibit the basal adenylate cyclase activity in the atrialmyocardium (Sulakhe et al., 1985), many others have shown that muscarinic agonists donot alter either the basal adenylate cyclase activity (Watanabe et al., 1978; Fleming et al.,1987) or the basal cAMP levels, in the absence (Keely and Lincoln, 1978; Linden andBrooker, 1979; Biegon et al., 1980; Pappano et al., 1982; Endoh et al., 1985; MacLeodand Diamond, 1986) or presence of an a-adrenoceptor agonist (Inui et al., 1982;MacLeod, 1986).1.6.2.1. Contribution of cGMPIn the search of an alternative mechanism, it was proposed that the ability ofmuscarinic agonists to elevate cGMP levels may explain the cAMP-independentresponses to muscarinic receptor stimulation in both atria and ventricles. In early studies16(George et al., 1970; 1973; 1975; Fink et al., 1976), a good correlation was reportedbetween the negative inotropic response to muscarinic agonists in atria and ventricles andthe ability of muscarinic agonists to elevate cGMP levels. Subsequently many othershave shown that the negative inotropic responses to muscarinic receptor stimulation inthe absence and presence of a 13-adrenoceptor stimulation were associated with anincrease in cGMP levels in both atria (Endoh and Yamshita, 1981) and ventricles(Watanabe and Besch, 1975; Endoh, 1980; Ingbretsen, 1980; Keely and Lincoln, 1978;Lincoln and Keely, 1980). It has also been shown that cGMP and various analogs ofcGMP are capable of mimicking the electrophysiological and contractile responses tomuscarinic receptor stimulation in the presence or absence of different cAMP-generatingagents in atria (Kohlhardt and Haap, 1978; Endoh and Yamashita, 1981) and ventricles(Watanabe and Besch, 1975; Wahler and Sperelakis, 1985; 1986).However, in the atrial myocardium several lines of evidence suggest thatelevation of cGMP levels is probably not causally related to the electrophysiological andcontractile responses to muscarinic receptor stimulation: (a) muscarinic agonists wereable to exert their functional and electrophysiological responses without elevating cGMPlevels (Brooker, 1977; Mirro et al., 1979; Brown, 1980), (b) interference with the abilityof muscarinic agonists to elevate cGMP levels, using LY 83583 and methylene blue, didnot affect the negative inotropic responses to muscarinic receptor stimulation in thepresence and absence of the cAMP-elevating agent forskolin (Diamond and Chu, 1985;MacLeod and Diamond, 1986; Groschner et al., 1986), (c) although the lipid solubleanalogs of cGMP and muscarinic receptor agonists were both capable of exerting a directnegative inotropic response in the mammalian atrial myocardium, Nawrath (1977) andLinden and Brooker (1979) have shown that the mechanism of the negative inotropic17response to a muscarinic receptor agonist differs from that of a cGMP-derivative, (d) andlastly, certain activators of the guanylate cyclase, such as sodium nitroprusside, in spiteof producing a massive increase in cGMP levels, did not decrease the atrial contractilityin the absence (Diamond et al., 1977) or presence of the various cAMP-elevating agents(Linden and Brooker, 1979).In spite of reports to the contrary (Linden and Brooker, 1979; Endoh andYamashita, 1981; Pappano et al., 1982), in the ventricular myocardium evidence exists tosuggest that cGMP elevation contributes to the negative inotropic responses tomuscarinic agonists in the presence of cAMP-elevating agents. MacLeod and Diamond(1986) have demonstrated that in the rabbit right ventricular papillary muscle interferencewith the ability of carbachol to elevate cGMP levels, using LY 83583, results in loss ofthe inhibitory effect of carbachol on the forskolin-induced increases in force ofcontraction. Several mechanisms have been suggested to account for the functionalresponses to cGMP elevation as described below.1.6.2.1.1. Activation of cGMP-dependent protein kinaseLincoln and Keely (1980) first demonstrated that the muscarinic receptor-stimulated elevation of cGMP levels in the rat heart was associated with activation of thecGMP-dependent protein kinase. It is believed that cGMP-dependent protein kinaseantagonizes the effects of cAMP-dependent protein kinase by phosphorylating differentproteins. In in vitro studies, the purified cGMP-dependent protein kinase has been shownto phosphorylate different sarcolemmal, sarcoplasmic reticular and cytosolic proteinsincluding phospholamban (see Lohmann et al., 1991). However, the physiologicalsignificance of this phosphorylation is not known. Evidence has been provided that18muscarinic agonists, cGMP, and cGMP analogs can inhibit the calcium current inventricles from different animal species, both in the presence and absence of cAMP-elevating agents and cAMP-analogs (Wahler and Sperelakis, 1985; 1986; Thakkar et al,1988; Levi et al., 1989; Wahler et al., 1989; Mery et al., 1991). The lowering ofintracellular calcium levels by cGMP, although so far not demonstrated experimentally,is proposed to be related to phosphorylation of the calcium channel protein in theventricle (Lohman et al., 1991; Mery et al., 1991).1.6.2.1.2. Activation of phosphodiesteraseCyclic GMP and cGMP analogs have been shown to promote the hydrolysis ofcAMP in the frog heart by activating a cGMP-stimulated phosphodiesterase (Hartzell andFischmeister, 1986; Fischmeister and Hartzell, 1987). Although this cGMP-stimulatedphosphodiesterase has been shown to be present in frog, rat and bovine hearts and hasbeen purified from the bovine heart (see Hartzell, 1988; Lohman et al., 1991), itscontribution to the inhibitory effects of muscarinic stimulation in the ventricle of speciesother than the frog is not clear at present (Lohman et al., 1991). In the hatched chickventricle (Biegon et al., 1980) and in rat ventricular myocytes (Mery et al., 1991)muscarinic agonists did not alter the phosphodiesterase activity. Similarly, muscarinic-inhibition of the pacemaker current in the rabbit sinoatrial nodal cells was not affected byphosphodiesterase or phosphodiesterase inhibitors (DiFrancesco and Tromba, 1987;1988b).1.6.2.2. Activation of phosphataseIt is well established that elevation of cAMP levels result in activation of cAMP-dependent protein kinase which phosphorylates various proteins such as the calcium19channel, phospholamban, troponin I and cardiac C protein (Hartzell, 1988; Trautwein andHescheler, 1990; Lindemann and Watanabe, 1990). Muscarinic agonists have beenshown to antagonize the protein-phosphorylating effects of several cAMP-elevatingagents (Loffelholz and Pappano, 1985). However, the mechanisms of these muscarinicresponses are not clear, because muscarinic agonists do not always reduce cAMP levelsincreased by different cAMP-elevating agents such as IBMX and forskolin, as discussedabove. On the other hand, the cGMP-dependent protein kinase has been shown tophosphorylate and not dephosphorylate proteins (discussed above). Thus, an alternativepossibility could be activation of protein phosphatases. Previously, Trautwein andcolleagues (Hescheler et al., 1987; 1988b; see also Trautwein and Hescheler, 1990) hadshown that a protein phosphatase could inhibit, and the inhibitor of the proteinphosphatase could enhance, the isoproterenol-induced increases in the calcium current.More recently, Watanabe and his group (Ahmad et al., 1989) have demonstrated thatcarbachol can also activate a type-I protein phosphatase in the guinea pig ventricle.However, the mechanism of muscarinic agonist-mediated activation of the proteinphosphatase is not known and it is also not known if a cause and effect relationship existsbetween the activation of protein phosphatase and the ability of muscarinic agonists toantagonize the cAMP-dependent phosphorylation of proteins and inhibit the positiveinotropic responses to cAMP-elevating agents.1.6.2.3. Alteration of ion channel activity1.6.2.3.1. Outward potassium currentIt has been known for a long time that the negative inotropic and chronotropicresponses to muscarinic receptor stimulation in the atrial myocardium are associated with20shortening of the action potential duration, hyperpolarization of the membrane, adecrease in the influx of calcium and an increase in the permeability to potassium(Burgen and Terroux, 1953; Raynor and Weatherall, 1959; Grossman and Furchgott,1964; Van Zwitten, 1968; Jakobs et al., 1989). Ten Eick et al. (1976) reported themuscarinic activation, in low concentrations, of an outward potassium current in the atrialmyocardium without inhibition of the slow inward calcium current. This was supportedby others (DiFrancesco et al., 1980; Inoue et al., 1983; Soejima and Noma, 1984, Iijimaet al., 1985; see also Hartzell, 1988; Pappano, 1990). It has also been shown thatactivation of the outward potassium current is independent of changes in intracellularcAMP, cGMP and calcium levels (Trautwein et al., 1982; Soejima and Noma, 1984; seealso Loffelholz and Pappano, 1985; and Hartzell, 1988 for a review). More recently it hasbeen shown that muscarinic receptors are linked to potassium channels by means of apertussis toxin-sensitive guanine nucleotide binding protein (Breitwieser and Szabo,1985; Pffafinger et al., 1985; Sorota et al., 1985; Hartzell, 1988; Brown and Birnbaumer,1990; Pappano, 1990; Szabo and Otero, 1990).Ten Eick et al (1976) proposed that the ability of muscarinic agonists to activatethe outward potassium current may contribute to their negative inotropic effect byshortening the duration of action potential and reducing the influx of calcium. It has alsobeen shown that the negative inotropic response to muscarinic agonists in the atrialmyocardium is associated with an efflux of potassium (42 K) (Nawrath, 1977) orrubidium (86 Rb), a tracer for potassium (Raynor and Weatherall, 1959; Van Zwitten,1968; Quast et al., 1988; Jakobs et al., 1989; Urquhart et al., 1991). Uncoupling ofmuscarinic receptors from potassium channels, using pertussis toxin, results inattenuation of the ability of muscarinic agonists to increase the outward potassium21current (Pffafinger et al., 1985; Sorota et al., 1985), promote the efflux of 86 Rb (Martinet al., 1985; Quast et al., 1988; Urquhart et al., 1991) and exert a direct negative inotropicresponse (Endoh et al., 1985; Sorota et al., 1985; Ray and MacLeod, 1992).1.6.2.3.2. Pacemaker current (If)This current, also known as the hyperpolarization-activated current, has beenstudied very extensively by DiFrancesco and colleagues (DiFrancesco and Tromba,1987; 1988a; 1988b; DiFrancesco et al., 1989; DiFrancesco and Tortora, 1991; see alsoHartzell, 1988; DiFrancesco, 1990 for a review). If is an inward current activated byhyperpolarization of the sinoatrial nodal cells and is believed to be the principal currentresponsible for the automaticity of the sinoatrial nodal cells. According to DiFrancesco etal. (1989), inhibition of the If is responsible for vagal slowing of the heart rate becauseinhibition of the If could be seen at a concentration at least 20 times less than that neededto promote the outward potassium current. However, it has been argued (Hirst et al.,1992) that whereas vagal stimulation produces cardiac arrest, inhibition of the If currentalone by a muscarinic agonist merely slows the heart rate, suggesting that thecontribution of some current in addition to inhibition of the If current cannot be ruled outin the process of vagal slowing of the heart rate. Muscarinic receptors are linked to the Ifchannel by means of a pertussis toxin-sensitive guanine nucleotide binding protein(DiFrancesco and Tromba, 1987; Yatani et al., 1990). Evidence exists to suggest thatmuscarinic agonists can inhibit the pacemaker current both directly, without involvingany second messenger (Yatani et al., 1990), as well as by inhibiting the basal and f3-adrenoceptor agonist-stimulated adenylate cyclase activity (DiFrancesco and Tromba,1987; 1988b; Yatani et al., 1990).221.6.2.3.2. Calcium currentMuscarinic agonists can inhibit the slow inward calcium current stimulated by aP-adrenoceptor agonist in atria and ventricles by decreasing the P-adrenoceptor agonist-stimulated cAMP levels. In addition, the ability of muscarinic agonists to elevate cGMPlevels may also contribute to the inhibitory effect of muscarinic stimulation on the basalcalcium current (Hino and Ochi, 1981; Wahler et al., 1989) or the calcium currentstimulated by different cAMP-elevating agents including isoproterenol, forskolin andIBMX (Wahler and Sperelakis, 1985; Rardon and Pappano, 1986; Thakkar et al., 1988).However, the mechanism of muscarinic inhibition of the basal calcium current in themammalian atrial myocardium is not clear (Ten Eick et al. 1976; Iijima et al., 1985;Cerbai et al. 1988; DiFrancesco and Tromba 1988b; see also Hartzell, 1988). Iijima et al.(1985) have suggested that the muscarinic-inhibition of the calcium current is mostlysecondary to activation of the outward potassium current. However, both Iijima et al.(1985) and Cerbai et al. (1988) have shown that a reduction of the calcium current can bemeasured even when the outward potassium current is blocked. An alternative possibilitysuggested by Cerbai et al. (1988) is that the ability of muscarinic agonists to inhibit thebasal calcium current is related to the muscarinic-inhibition of the basal adenylatecyclase activity in atria. However, as discussed earlier, muscarinic agonists do notconsistently inhibit either the basal adenylate cyclase activity or cAMP levels in theatrial myocardium.1.6.2.4 Promotion of phosphoinositide turnover23Like the a-adrenoceptor agonists, high concentrations of muscarinic receptoragonists, usually 10 i.iNI and higher, have been shown to promote phosphoinositidebreakdown in the heart (Brown and Jones, 1986; Tajima et al., 1987; Pappano, 1990).This effect is believed to contribute to the pertussis toxin-insensitive positive inotropicand chronotropic (Tsuji et al. 1987; Tajima et al. 1987a; 1987b; Agnarsson et al., 1988;see also Pappano, 1990) responses to muscarinic receptor stimulation in the heart and notto the adrenergic-cholinergic interactions, and will not be discussed any further.1.7. G-PROTEINSThe subject of the guanine nucleotide binding proteins has been reviewedextensively (see Gilman, 1987; Robishaw and Foster, 1988; Birnbaumer et al., 1990;Brown, 1990; Brown and Birnbaumer, 1990; Fleming et al., 1992; Lefkowitz, 1992)). Itis well established that a- and 0- adrenoceptors and muscarinic cholinoceptors arelinked to their effectors including phospholipase C, adenylate cyclase and ion channels(potassium, calcium and If), by means of G-proteins. In addition, evidence also existsthat G-proteins can activate phospholipase A2 , phospholipase D, ATP-dependentpotassium channels and sodium channels.G-proteins are heterotrimeric proteins consisting of three different subunits a, 13and y. G-proteins usually have common 13 and y subunits, whereas the a-subunits varyamong G-proteins. The a-subunit of G-proteins are hydrophilic in nature and have abinding site for GTP, GTPase activity, sites for ADP-ribosylation by different bacterialtoxins, namely cholera toxin and pertussis toxin, and myristoylation sites, although G-protein a-subunits which lack the ADP-ribosylation site for cholera toxin also lack themyristoylation site. However, in recent years G-proteins which lack the ADP-ribosylation24sites for either toxin have been identified . In contrast to the a subunit, the (3 and ysubunits are very hydrophobic in nature and are usually closely associated. In addition tohelping anchor the a-subunits to the membrane, 13y subunits have been shown to increasethe receptor-stimulated GTPase activity of a-subunits and may also have somephysiological effects of their own. For example 13y subunits have been reported to inhibitadenylate cyclase, open potassium channels, and activate phospholipase C andphospholipase A2.Bacterial toxins have been of great help to identify, classify and study thefunctional significance of different G-proteins (Wregget, 1986). More recently with thehelp of molecular biological techniques, the genes for many G-protein a-subunits havebeen cloned and expressed and their amino acid sequences determined. Using a-subunit-specific antibodies raised against peptide sequences typical of a particular a-subunit,many different G-protein a-subunits have been identified both in cardiac andextracardiac tissues. On the basis of the molecular weight of the a-subunits, the G-proteins present in the heart can be grouped into the following (Fleming et al., 1992):a. Gs  or the stimulatory G-protein Two different subtypes of the a-subunits, ofthe molecular weight 45 and 52 kDa, have been identified. G sa is known to mediate theagonist-stimulation of adenylate cyclase and voltage-dependent calcium channels. G sa isa substrate for ADP-ribosylation by cholera toxin.b. Gi  or the inhibitory G-protein, and G o the 'other' G protein a-Subunits of bothof these proteins are substrates for ADP-ribosylation by pertussis toxin. Gi a consists ofthree different proteins namely Gi a i and Gia3 each 40 kDa and the 41 kDa Gi a2. Atleast two different a-subunits of G o, each of molecular weight 39 kDa, have beenidentified in the canine heart and bovine brain and purified from the bovine brain25(Birnbaumer et al., 1990; Kobayashi et al., 1990). Functions of the different a-subunits ofGi in vivo are not entirely clear, but it is believed that the Gi a2 mediates the hormonal-inhibition of adenylate cyclase (McClue et al., 1992), while the Gi a3 opens potassiumchannels (see Birnbaumer et al., 1990). Recently, it has been shown that the G oa subunitis responsible for the hormonal-inhibition of the If channel in the heart (Brown, 1990;Yatani et al., 1990) and calcium channels in pituitary cells (Trautwein and Hescheler,1990; Kleuss et al., 1991).c. GLIGa This protein has recently been identified in the heart and has amolecular weight of 42 kDa. This protein lacks the site for ADP-ribosylation by bacterialtoxins. Its exact function is not known.d. Gz6_x has also been shown to be present in the heart (Spicher et al., 1988;Foster et al., 1990). However, according to Foster et al. (1990) this protein is present inthe membrane of non-myocardial cells. G z is not ADP-ribosylated by bacterial toxins anda 43 kDa subunit has been found in the cell membrane (Foster et al., 1990) and another40 kDa subunit in the cytosol (Spicher et al., 1988). The functional significance of G z inthe heart is not known. However, in transfection studies the wild type and mutationally-activated Gza inhibited the agonist-stimulated adenylate cyclase in the presence andabsence of various inhibitory receptor agonists (Wong et al., 1992).The remaining section on the G-proteins will focus mainly on the mechanism ofmuscarinic inhibition of adenylate cyclase and activation of potassium channels in theheart. The mechanism of hormonal, eg. P-adrenoceptor agonist, stimulation of adenylatecyclase has been elucidated very clearly (review Gilman, 1987; Birnbaumer, 1990).Briefly, as shown in fig. 2 (Fleming et al., 1987), occupation of the receptor by an agonistfacilitates exchange of GTP for GDP, and the GTP-bound G sa-subunit is dissociated26 I Inhibition^IStimulotion(GIP)GDP^ GOP[GardGB ,G.-GOP 6;9+( —) 1 IndirectinhibitionG. -GTPGTPose^ atv,,,3. 7 1(+)Fig. 2: A schematic representation of the role of G-proteins in the P-adrenoceptor-mediated activation and the muscarinic cholinoceptor-mediated inhibition of adenylatecyclase in the heart (Fleming et al., 1987). For details see text.27from (3y subunits. The GTP-bound G sa in turn activates adenylate cyclase. The effect isterminated when GTP is hydrolyzed by the GTPase of the a-subunit, and the GDP-boundGsa unites with the fry subunits. The purified, pre-activated, native and recombinant G sahas been shown to stimulate adenylate cyclase (see Birnbaumer et al., 1990). However,the mechanism of muscarinic inhibition of adenylate cyclase and activation of potassiumchannels in the heart is less well understood.In reconstitution studies it has been shown that purified muscarinic receptors caninteract with Go and all three Gi proteins (Ikegaya et al., 1990). However, in the rat heartthe agonist-stimulated muscarinic receptors have been shown to associate preferentiallywith Gi2 and Go in ventricles and only with G o in atria (Matesic et al., 1991). Thequestion then arises as to which G-protein(s) really connect muscarinic receptors toadenylate cyclase and potassium channels? According to Liang and Galper (1988), it isthe Gi-like protein(s) which mediate the muscarinic inhibition of adenylate cyclase in thechick ventricular myocytes. In reconstitution (Kobayashi et al., 1990) and transfection(Wong et al., 1992) studies, it was shown that the a-subunits of all three Gi proteins, butnot Go , were able to inhibit adenylate cyclase. However, the exact Gi protein whichconveys the message of muscarinic receptor occupancy to adenylate cyclase in the heartis not known. In extra-cardiac tissues, it has been shown that the Gia2 is the most likelycandidate that couples inhibitory receptors to adenylate cyclase (McClue et al., 1992). Asimilar problem exists regarding the identity of G-protein(s) that link muscarinicreceptors to potassium channels. According to Brown, Birnbaumer and group (Yatani etal., 1988; Birnbaumer et al., 1990), all three pure and recombinant Gi a subunits, but notthe Goa, can open potassium channels, and the authors believe that in intact tissue it isthe Gia3 which opens the agonist-stimulated potassium channels (see Birnbaumer et al.,281990). In contrast, Kobayashi et al. (1990) have shown that the G oa subunits, along withall three Gia subunits, are also capable of opening the atrial potassium channels.The second problem concerns which subunits of G-protein, a or fry, convey themessage of muscarinic receptor occupancy to adenylate cyclase and potassium channels.Watanabe and group (Fleming et al., 1987) in agreement with the observation of Katadaet al. (1984) have suggested that the agonist-stimulated inhibition of adenylate cyclase incardiac tissue is indirect and seen only when adenylate cyclase is stimulated previouslyby a stimulatory receptor agonist. According to this hypothesis, the 13y subunit ofinhibitory G-proteins, released as a result of inhibitory agonist stimulation, quenches theGTP-bound Gsa and prevents it from stimulating adenylate cyclase (fig. 2). In theirstudy, Fleming et al. (1987) failed to observe any direct inhibitory effect of methacholine,a muscarinic receptor agonist, and GPP(NH)P, a non-hydrolyzable GTP analog, on thebasal (in the absence of GTP) and forskolin-stimulated adenylate cyclase. Because theforskolin-induced activation of adenylate cyclase occurs independently of the stimulatoryG-protein, the lack of inhibitory effect of methacholine and GPP(NH)P on the forskolin-stimulated adenylate cyclase was considered to be evidence that the a-subunits of Gi andGo do not have any direct inhibitory effect on adenylate cyclase. However, several linesof evidence suggest that the a-subunits of inhibitory G-proteins may also contribute tothe hormonal-inhibition of adenylate cyclase in cardiac and extra- cardiac tissues:(a) In mutant S49 lymphoma cells which lack a stimulatory G protein,somatostatin, an inhibitory agonist for adenylate cyclase, was able to inhibit theforskolin-stimulated adenylate cyclase (see Birnbaumer, 1990 for a review). Thisobservation was used to support the hypothesis that the hormonal-inhibition of adenylatecyclase is mediated by the a-subunit of inhibitory G-proteins.29(b) Hildebrandt and Kohnken (1990) have shown that in normal S49 lymphomacells the mechanism of somatostatin-induced inhibition of the isoproterenol-stimulatedadenylate cyclase is different from that of the (3y subunit-mediated inhibition ofadenylate cyclase.c) More recently, McClue et al. (1992) have shown that inhibition of the GTPaseactivity of the Gia2 by a selective antibody resulted in loss of the ability of an a2-adrenergic agonist to inhibit adenylate cyclase activity. Bourne and his group have alsoshown that the mutationally-activated a-subunits of inhibitory G-proteins, whenexpressed in mouse fibroblast cells were capable of inhibiting the forskolin-stimulatedadenylate cyclase (Wong et al., 1991; 1992).d) Reithman et al. (1989) reported that prolonged exposure of the rat heart cells tonorepinephrine resulted in a loss of the ability of isoproterenol, forskolin and GTPyS tostimulate adenylate cyclase. This was associated with an increase in the immunoreactiveGia subunit but not the (3-subunit, suggesting that the a-subunit of inhibitory G-proteinsplay an important role in the inhibition of adenylate cyclase.e) Many workers have shown that muscarinic agonists can inhibit both basal andforskolin-stimulated adenylate cyclase in the heart (Jakobs et al., 1979; Martin et al.,1985; Sulakhe et al., 1985).Similar to adenylate cyclase, the mechanism of muscarinic receptor-mediatedactivation of potassium current is also not very clear. Birnbaumer, Brown and colleagueshave demonstrated that the purified and recombinant (Codina et al., 1987; Yatani et al.,1987; Kirsch et al., 1988; Yatani et al., 1988a; see also Birnbaumer et al., 1990; Brown,1990) a-subunits of inhibitory G-proteins open potassium channels and the (3y subunit ofinhibitory G-proteins was shown to inhibit potassium channels (Okabe et al., 1988). A30specific antibody to the purified a-subunit of the inhibitory G-protein, also called ak orcci3 by Brown, Birnbaumer and group, that opened potassium channels was shown toabolish the ability of carbachol and the pre-activated a-subunit to stimulate the potassiumcurrent (Yatani et al. 1988b).Neer, Clapham and colleagues (Logothetis et al., 1987), on the other hand, werethe first to report that the 13y subunits of inhibitory G-proteins are also capable ofopening potassium channels. However, the same group later proposed that themechanism of the fry subunit-mediated activation of potassium current is related tostimulation of phospholipase A2 and generation of lipoxygenase metabolites ofarachidonic acid (Kim et al., 1989). In a recent study, however, Ito et al. (1992) havereaffirmed the original observation that the f3y subunits of G-proteins open potassiumchannels. In their study, Ito et al. (1992) reported that the purified a-subunits of Gi 1' 2and 3 were very weak activators of potassium channels and failed to see any involvementof phospholipase A2 and lipoxygenase metabolites as mediators of the fry-inducedchannel opening.Thus, further work is required to settle the issue of which particular G-protein(s),and which subunit of the G-proteins, conveys the message of muscarinic receptoroccupancy to potassium channels and adenylate cyclase.1.8. OBJECTIVESIn previous sections, evidence has been discussed that in both atrium andventricle muscarinic agonists can antagonize the positive chronotropic and inotropicresponses to 13-adrenoceptor stimulation by inhibiting the (3-adrenoceptor agonist-induced31increases in cAMP generation. However, it is not clear how muscarinic receptor agonistsreverse the cAMP-independent positive inotropic responses to a-adrenceptor agonists inthe atrial myocardium. In addition, previous reports from this laboratory havedemonstrated that the negative inotropic responses to carbachol in the presence offorskolin and IBMX were not associated with any reduction in accompanying increasesin cAMP levels (MacLeod and Diamond, 1986; Ray and MacLeod, 1992). It is knownthat muscarinic agonists can open potassium channels in the atrial myocardium and thisis believed to contribute to the direct negative inotropic response to muscarinic agonistsby shortening the action potential duration and reducing the influx of calcium (Ten Eicket al., 1976; Cerbai et al., 1988). One of the objectives of the present study was to test thehypothesis that the ability of carbachol to open potssium channels contributes to thecAMP-independent negative inotropic responses to carbachol in the presence of differentcAMP-elevating and cAMP-independent positive inotropic agents in the rabbit left atrialmyocardium.In a previous study from this laboratory, it was shown that the cAMP-independentnegative inotropic responses of rabbit left atria to carbachol in the presence ofphenylephrine were attenuated by pre-treatment of rabbits with pertussis toxin (Ray andMacLeod, 1990). It is well established that pertussis toxin can uncouple muscarinicreceptors from potassium channels in the atrial myocardium (Wregget, 1986). It has beenshown that pre-treatment of animals with pertussis toxin results in the loss of the abilityof muscarinic agonists to open potassium channels (Pffafinger et al., 1985; Sorota et al.1985), to promote the efflux of 86 Rb (Martin et al., 1985; Quast et al., 1988; Urquhart etal., 1991) and to exert a direct negative inotropic effect (Endoh et al., 1985; Quast et al.,1988; Ray and MacLeod, 1990; 1992). Thus, attenuation by pertussis toxin of the cAMP-32independent negative inotropic response to carbachol in the presence of phenylephrinesuggested a role of the carbachol-stimulated potassium current in this process. In order toinvestigate the contribution of potassium channels, we studied the ability of 4-aminopyridine, a potassium channel blocker, to antagonize the inhibitory effect ofcarbachol on the phenylephrine-induced positive inotropy. Evidence fromelectrophysiological studies suggests that in cardiac tissue, 4-aminopyridine can prolongthe action potential duration by inhibiting the outward potassium current responsible forrepolarization of the membrane (Van Bogaert et al., 1982; Gilmour et al., 1986; see alsoRudy, 1988). 4-Aminopyridine has also been shown to antagonize the direct negativeinotropic (Freeman, 1979; De Biasi et al., 1989; Urquhart and Broadley, 1991) and actionpotential shortening effects of carbachol (Freeman, 1979). We reasoned that if carbacholexerts its inhibitory effect on the phenylephrine-induced positive inotropy by openingpotassium channels then 4-aminopyridine, by antagonizing the potassium channelopening effect of carbachol, should decrease the ability of carbachol to inhibit thephenylephrine-induced positive inotropy. At the same time, we also studied the effect of4-aminopyridine on the ability of carbachol to inhibit the isoproterenol-stimulatedpositive inotropy, a cAMP-dependent response.An attempt was also made to obtain more direct evidence for the muscarinicreceptor-mediated opening of potassium channels and its contribution to the negativeinotropic responses to carbachol in the presence and absence of phenylephrine. This wasachieved by measuring the ability of carbachol to promote the efflux of 86 Rb-labelledrubidium chloride and to exert a negative inotropic response in the absence and presenceof phenylephrine, in the same electrically-stimulated rabbit left atrium. Rubidium hasbeen known for a long time to pass through potassium channels and it has been shown33that the contractile responses of guinea pig atria in the presence and absence of positiveinotropic agents do not change if potassium is replaced by rubidium (Van Zwitten, 1968).In addition, radioactive rubidium (86 Rb) has a half life of 18.8 days compared toradioactive potassium (42 K) which has a half life of 12 hours. Thus, in spite of thecriticism that the passage of 86 Rb across the membrane is slower than that of potassium(Smith et al., 1986; Jahnel and Nawrath, 1989), 86 Rb has been used quite extensively asa tracer for potassium to study the effect of various drugs on potassium channel activityin the heart (Raynor and Weatherall, 1959; Van Zwitten, 1968; Hunter and Nathanson,1985; Quast et al., 1988; Kemmer et al., 1989; Urquhart et al., 1991) as well as in othertissues (Bolton and Clark, 1981; Smith et al.,1986). The effects of 4-aminopyridine andpertussis toxin were also tested on the contractile and 86 Rb-efflux promoting effects ofcarbachol in the presence and absence of phenylephrine.Using a different approach, the effects of the potassium channel openers,pinacidil and cromakalim, were tested on the ability of phenylephrine and isoproterenolto exert a positive inotropic response. A previous study from this laboratory has shownthat carbachol antagonized the development of a- and 13- adrenoceptor-mediated positiveinotropic responses in rabbit left atria (MacLeod, 1987). It was reasoned that if theseinhibitory effects of carbachol on the positive inotropic responses to a- and i3-adrenoceptor agonists were related to the ability of carbachol to open potassiumchannels, then potassium channel agonists should also be able to antagonize thedevelopment of the positive inotropic responses to a- and p- adrenoceptor agonists.Previous reports from this laboratory have shown that the negative inotropicresponses of left atria to carbachol in the presence of isoproterenol, but not in thepresence of forskolin or IBMX, are associated with a reduction in the accompanying34increases in cAMP levels (MacLeod, 1986; MacLeod and Diamond, 1986; Ray andMacLeod, 1992). The contribution of the carbachol-stimulated potassium current to thesecAMP-independent negative inotropic responses to carbachol in the presence of forskolinand IBMX was investigated using 4-aminopyridine. Again, very similar to the carbachol-phenylephrine interaction, the reasoning was if the ability of carbachol to open potassiumchannels contributes to the negative inotropic responses of left atria to carbachol in thepresence of forskolin and IBMX, then antagonism by 4-aminopyridine of the carbachol-stimulated potassium current should attenuate the negative inotropic responses tocarbachol in the presence of forskolin and IBMX.However, it has been suggested that carbachol inhibits the forskolin-stimulatedadenylate cyclase activity or cAMP generation in a compartment linked to the contractilemachinery and a small reduction in cAMP levels is usually not detectable when totaltissue cAMP levels are measured (Hartzell, 1988). In addition, methacholine, amuscarinic receptor agonist, was shown to inhibit the IBMX-stimulated cAMP levels inrat atria (Brown et al., 1980). In order to rule out the involvement of adenylate cyclase inthe functional interaction of carbachol with forskolin and IBMX, pertussis toxin was usedto uncouple muscarinic receptors from adenylate cyclase (Wregget, 1986; Fleming et al.,1988). The ability of carbachol to inhibit the isoproterenol-stimulated adenylate cyclaseactivity and cAMP levels was used as an index of uncoupling of muscarinic receptorsfrom adenylate cyclase. The effect of pertussis toxin pre-treatment of rabbits, alone andin combination with 4-aminopyridine, was investigated on the negative inotropicresponses to carbachol in the presence of forskolin and IBMX. The effects of 4-aminopyridine and pertussis toxin, alone and in combination, were also studied on thenegative inotropic responses to carbachol in the presence of isoproterenol. Since35inhibition of the isoproterenol-stimulated cAMP generation is believed to contribute tothe negative inotropic response to carbachol in the presence of isoproterenol (Ray andMacLeod, 1992), we reasoned that potassium channel blockade by 4-aminopyridineshould have very little effect on the isoproterenol-carbachol interaction. In contrast, weexpected that uncoupling of muscarinic receptors from adenylate cyclase by pertussistoxin would have a very pronounced inhibitory effect on the negative inotropic responseto carbachol in the presence of isoproterenol.It has been reported that 4-aminopyridine is capable of displacing muscarinicagonists from their receptor binding sites (Drukarch et al., 1988; Urquhart and Broadley,1991). Therefore, in some experiments the effect of 4-aminopyridine on the ability ofcarbachol to inhibit the isoproterenol-stimulated cAMP generation was tested.It has been discussed that muscarinic receptors are linked to potassium channelsand adenylate cyclase in atria and ventricles by means of pertussis toxin-sensitive G-protein(s). However, it is not known very clearly exactly which G-protein(s), and whichsubunit(s) of the G-protein(s), link muscarinic receptors to different effectors, namelypotassium channels and adenylate cyclase, in atria and ventricles. In a recent study fromthis laboratory (Ray and MacLeod, 1992), it was observed that a dose of pertussis toxin(1.75 1.1g/kg) completely attenuated the ability of carbachol to inhibit the isoproterenol-stimulated positive inotropic response (fig. 3 II B) and cAMP generation in rabbit leftatrial strips, attenuated only partially the direct negative inotropic response to carbachol(fig. 3 I B). The negative inotropic response to carbachol, as discussed earlier, is believedto be related to the ability of carbachol to open potassium channels. Thus, our resultssuggested that in the rabbit left atrium pertussis toxin uncouples in a differential mannermuscarinic receptors from potassium channels and adenylate cyclase. In addition,36Fig. 3 Tracings showing the effect of pertussis toxin pre-treatement of rabbits on thedirect negative inotropic response to carbachol in the presence and absence ofisoproterenol in rabbit left atrium_(1). Carbachol (3 pIVI) exerted a direct negative inotropic response (A) in rabbitleft atrium which was attenuated by pre-treatment of rabbits with pertussis toxin (1.75lig/kg) (B). (II). Carbachol (3 i_tM) attenuated the positive inotropic response to 100 nMisoproterenol (A). This inhibitory effect of carbachol on isoproterenol-induced positiveinotropy was completely attenuated by pre-treatment of rabbits with 1.75 µg/kg pertussistoxin (B).37II^AISO = Isoproterenol (100 nM)CCh = Carbachol (3 gM)B38the same dose of pertussis toxin that completely attenuated the inhibitory effect ofcarbachol on the isoproterenol-stimulated cAMP generation in the left atrium did nothave any uncoupling effect on the ability of carbachol to inhibit the positive inotropicresponse to isoproterenol in the right ventricular papillary muscle of rabbits. Thus, thesecond objective of the present study was to test the hypothesis that the muscarinic-inhibition of the isoproterenol-stimulated adenylate cyclase in the atrial myocardium ismore sensitive to uncoupling by pertussis toxin than either the muscarinic-inhibition ofthe isoproterenol-stimulated adenylate cyclase in the ventricle or the muscarinic-activation of potassium channels in the left atrium.The following were the specific goals of the present study:(a) Study the effects of 4-aminopyridine on contractile responses of rabbit leftatria to carbachol in the presence and absence of phenylephrine or isoproterenol.(b) Correlate the negative inotropic response to carbachol in the presence andabsence of phenylephrine with the ability of carbachol to promote the efflux of 86 Rb.Study the effects of 4-aminopyridine and pertussis toxin on these responses to carbachol.(c) Study the effects of cromakalim and pinacidil on the positive inotropicresponses to phenylephrine and isoproterenol in rabbit left atria.(d) Study the effects of 4-aminopyridine and pertussis toxin on the inhibitoryeffect of carbachol on the isoproterenol-stimulated cAMP generation and adenylatecyclase activity in rabbit left atria.(e) Study the effects of 4-aminopyridine and pertussis toxin, alone and incombination, on the negative inotropic responses to carbachol in the presence andabsence of the cAMP-elevating agents isoproterenol, forskolin and IBMX in rabbit leftatria.39(f) Measure adenylate cyclase activity in atria and ventricles.(g) Study the effect of pertussis toxin pre-treatment of rabbits on the ADP-ribosylation of G-proteins. Compare the degree of ADP-ribosylation of G-proteins andthe degree of uncoupling of muscarinic receptors from (i) adenylate cyclase in atria andventricles and (ii) potassium channels in left atria.40MATERIALS AND METHODS2.1. MATERIALSThe materials used in the study were obtained from the following sources:Aldrich Chemical Co, Wisconsin, U.S.A. Carbamylcholine chlorideAmersham Canada Ltd., Canada [a-32 P]-ATP,[86] Rb-labelled rubidium chlorideBDH Chemical Co., CanadaCalcium chloride dihydrate,Diethyl ether,Dimethyl sulphoxide,Glacial acetic acid,d-Glucose,Glycerol,Hydrochloric acid,Magnesium chloride hexahydrate,Methanol,Potassium chloride,41Sodium bicarbonate,Sodium chloride,Trichloroacetic acidBIO-RAD Laboratories, Ontario, CanadaAcrylamide,Ammonium persulphate,N'-N'-methylene-bisacrylamide,Bromophenol blue,Coomasie blue,Dithiothreitol,Dowex Resin (H± form)Glycine,Protein assay kit,Protein standards for electrophoresis,Sodium dodecylsulphate,TEMEDTris base,Tris hydrochloride,Calbiochem Corporation, California, U.S.A. Forskolin,Isobutylmethylxanthine,42Du Pont Canada Inc., Ontario, CanadaAquasol,[32P]-NAD,ProtosolICN Biomedicals Canada Ltd., Canada[3H]-cAMPList Biological Laboratories, California, U.S.A. Pertussis toxinSigma Chemical Co., St. Louis, U.S.A. Adenosine deaminase,Alamethicin,Alumina (neutral; activity grade I)4-Aminopyridine,cAMP sodium salt,Ascorbic acid,ATP disodium salt,Benzamidine hydrochloride,Benzethonium chloride,Creatine phosphate disodium salt,Creatine phosphokinase,43Dithiothreitol,EDTA,EGTA,Film, Kodak X-OMAT RPGTP sodium salt,GTPyS tetralithium salt,Imidazole hydrochloride,Isoproterenol hydrochloride,Myokinase,Nicotinamide adenine dinucleotide sodium salt,Phenylephrine hydrochloride,Phenylmethylsulphonylfluoride,Processing chemicals kit, Kodak,Sodium dodecylsulphateThymidine,Timolol maleate,Tris base,Tris hydrochloride,Trypsin inhibitor,Pinacidil and cromakalim were gifts from Eli-Lilly and Co., Indianapolis, U.S.A.and Beecham Pharmaceuticals, Surrey, U.K., respectively.442.2. PREPARATION OF SOLUTIONS2.2.1. Modified Chenoweth-Koelle solutionA ten times concentrated stock solution of the modified Chenoweth Koelle(MCK) solution was prepared by dissolving sodium chloride (140 g), potassium chloride(8.4 g), calcium chloride (6.4 g) and magnesium chloride (3.6 g) in 1000 ml of distilledwater. On the day of the experiment, 200 ml of the stock solution was diluted to 2000 mlwith water after adding sodium bicarbonate (3.2 g) and glucose (3.6 g) to obtain a bufferof the final composition in mM : NaCI 120, KC1 5.7, CaC1 2 2.2, MgC1 2 0.9,NaHCO3 25 and glucose 10.2.2.2. Pertussis toxin solutionPertussis toxin was dissolved in distilled water to obtain a stock solution of 5014/500 ill for injecting into rabbits. For electrophoresis experiments, a stock solution of50 µg/150 µl was prepared in distilled water.2.2.3. Drug solutionsSolutions of carbachol, phenylephrine, timolol, atropine and 4-aminopyridinewere prepared in distilled water. Isoproterenol was dissolved in distilled water in thepresence of ascorbic acid (5 mg/m1) to prevent oxidation of isoproterenol. Forskolin wasdissolved in 90 % ethanol to obtain a stock solution of 10 mM, from which subsequentdilutions were made using water. Cromakalim and pinacidil were dissolved in dimethylsulphoxide (DMSO).452.2.4. Solutions for the ADP-ribosylation experiments2.2.4.1. Pertussis toxin activation bufferA double strength pertussis toxin activation buffer was prepared by dissolving60.5 mg Tris HC1, 7.4 mg EDTA, 6 mg Dithiothreitol (D I 1), 12.4 mg ATP and 1 mllubrol (1.0%) in a final volume of 10 ml in water. The pH of this solution was adjusted to7.6 and stored in 500 ill aliquots at - 40 ° C. On the day of the experiment, 30 ill of theactivation buffer was incubated at 37 ° C for 15 min with an equal volume of pertussistoxin (50 lig/ 150 111) to obtain a final composition (in mM) : Tris HC1, 25; EDTA, 2;DTT, 20; ATP, 1 and Lubrol, 0.5%. A 9 ill aliquot of the mixture provided 30 14/m1pertussis toxin in a final reaction volume of 50 ill for in vitro ADP-ribosylation of G-proteins.2.2.4.2. ADP-ribosylation mixtureA 10 times concentrated ADP-ribosylation mixture was prepared by dissolving1.2 g Tris base, 37.2 mg EDTA, 62.2 mg ATP, 15.3 mg DTT, 242 mg thymidine, 6.2 mgGTP and 2 mg DNAase I in 10 ml of distilled water. The pH of this solution was adjustedto 8 and it was stored in 500 p1 aliquots at - 40 ° C. A 5 1.11 aliquot of this reactionmixture was added to a final volume of 50 ill to obtain the final composition (in mM) :Tris, 100; EDTA, 1; ATP, 1; MT, 1; Thymidine, 10, GTP, 0.1 and DNAase 20 µg/ml.462.2.4.3. Nicotinamide adenine dinucleotide (NAD)A stock solution of 10 mg/ml (14.5 mM) of the sodium salt of nicotinamideadenine dinucleotide was prepared in water and stored at - 40 ° C. On the day of theexperiment, a 4 ill aliquot of NAD was mixed with 2.5^of radioactive NAD in a finalvolume of 10^adjusted with distilled water. In a final reaction volume of 50 41, theamount of non-radioactive NAD present was 50 µM.2.2.4.4. Solubilization bufferThe double strength solubilization buffer was prepared by mixing 1 ml Tris (0.5M, pH 6.8), 1.6 ml sodium dodecylsulphate (10%), 0.8 ml glycerol, 0.4 ml 2-mercaptoethanol (5%) and bromophenol blue in sufficient quantity with 4 ml of water.This solution was stored at -40 ° C. On the day of the experiment, the solubilizationbuffer was diluted with an equal volume of water and 50 ill was added to each tube tosolubilize the precipitated G-proteins.2.2.5. Solutions for electrophoresis2.2.5.1. Electrode bufferA ten times concentrated electrode buffer was prepared by dissolving 30 g Trisbase, 144 g glycine and 10 g sodium dodecylsulphate in water to make a final volume of1000 ml. The stock solution was stored at room temperature. On the day of theexperiment, 400 ml of the stock was diluted to 4000 ml with water.472.2.5.2. Protein standardsBio-Rad low molecular weight protein standards (phosphorylase B, 97.4 kDa;serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybeantrypsin inhibitor, 21.5 kDa and lysozyme, 14.4 kDa) were used as the molecular weightmarkers. The stock solution of the standard proteins was diluted 20 times with thesolubilization buffer and heated in a boiling water bath for 3 min before loading on thegel.2.2.5.3. Staining solutionStaining solution was prepared by dissolving 2.5 g Coomassie blue in a mixtureof 100 ml acetic acid, 450 ml methanol and 450 ml water. The staining solution wasstored at room temperature.2.2.5.4. Destaining solutionDestaining solution consisted of a mixture of methanol 450 ml, acetic acid 100m1and water 450 ml and was stored at room temperature.2.2.5.5. Gel drying solutionOne litre of gel drying solution contained 20 ml glycerol and 980 ml of water andstored at room temperature.482.2.6. Solutions for the adenylate cyclase assay2.2.6.1. Reaction mixtureThe five times concentrated reaction mixture for the adenylate cyclase assay wasprepared by dissolving 155 mg magnesium sulphate, 41.3 mg cAMP, 18.8 mg DTT, 725mg sodium chloride, 27.5 mg isobutylmethyl xanthine and 988 mg Tris hydrochloride insufficient water to make up the volume to 25 ml. The pH was adjusted to 7.8, and thereaction mixture was stored at - 40 ° C prior to use. A 30 tl aliquot of this reactionmixture, when diluted with other components of the assay mixture to 150 containedin mM: magnesium sulphate, 5; cAMP, 1.0; DTT, 1.0; sodium chloride, 100;isobutylmethyl xanthine, 1 and Tris hydrochloride, 50.2.2.6.2. Regeneration mixtureA 100 mM solution of creatine phosphate was prepared by dissolving 255 mgcreatine phosphate in 10 ml of distilled water. A 15 pl aliquot of this solution was dilutedwith other components of the assay mixture to 150 so that the final concentration ofcreatine phosphate in each tube was 10 mM. Each tube also contained 1.6 U/150 p.1 ofcreatine phosphokinase (250 U/mg), 6 U/150 IA myokinase (200 U/mg) and 0.75 U/150.tl adenosine deaminase (200 U/mg).2.2.6.3. GTP solutionA 500 tM GTP solution was prepared by dissolving 2.62 mg GTP sodium salt insufficient water to make up the volume to 10 ml. A 15 pl aliquot of this solution, when49diluted with other components of the assay mixture to 150 tl gave a final concentrationof 50 1.tM in each tube.2.2.6.4. ATP solutionA 5 mM solution of disodium salt of ATP was prepared by dissolving 27.6 mg ofATP in 10 ml of water. Tris HCl was used to adjust the pH of this solution to 7.8. A 15 tlaliquot of this solution was added to each tube to obtain a final concentration of 0.5 mMwhen diluted with other components of the assay mixture. ATP solution was stored in500 pl aliquots at - 40 ° C prior to use. On the day of the experiment, 15 pl of non-radioactive ATP was mixed with 500,000 dpm of radioactive ATP and water to make upthe volume to 30 pl. Each 30 tl ATP solution was diluted with other components of theassay mixture to obtain a final volume of 150 pl.2.2.6.5. Stop solutionThe stop solution was prepared by dissolving 200 mg sodium dodecylsulphate,248.8 mg ATP and 4.61 mg cAMP in sufficient water to make up the volume to 10 ml.The pH was adjusted to 7.5 with Tris base. The solution was stored at -40 ° C prior touse. Each 100 1.11 aliquot of the stop solution when added to individual tubes to stop theadenylate cyclase activity contained 2 % sodium dodecylsulphate, 40 mM ATP and 1.4mM cAMP.2.2.6.6. 0.1 N Hydrochloric acidConcentrated hydrochloric acid (12 N) was diluted 120 times with distilled waterto obtain approximately 0.1 N hydrochloric acid.502.2.6.7. 0.1 M Imidazole hydrochloride bufferImidazole hydrochloride (10.04 g) was dissolved in 1000 nil water to obtain a 0.1M solution. The pH of this solution was adjusted to 7.5.2.3. GEL CASTINGThe separating gel was prepared by dissolving 4.25 g acrylamide and 56 mg BIS(N', N'-methylene-bis-acrylamide) in distilled water containing 8.75 ml Tris base (1.5M ;pH 8.8), 350 gl SDS (10% w/v) and 875 gl ammonium persulphate (20 mg/ml) to a finalvolume of 35 ml. The solution was poured in a BIO-RAD Protean II (16 cm) gel castingapparatus after adding 12 gl TEMED, a catalyst that facilitates polymerization ofacrylamide, and allowed to stand for 6 hours for the acrylamide solution to gel.The stacking gel was prepared by dissolving 350 mg acrylamide and 9.3 mg BISin a final volume of 10 ml distilled water containing 2.5 ml Tris base (0.5 M, pH 6.8),100 gl SDS (10 % w/v) and 250 gl ammonium persulphate (20 mg/m1). The reaction wasstarted by adding 10 gl TEMED and the solution was poured over the separating gel andallowed to stand for an hour to gel.2.4. ISOLATED TISSUE PREPARATIONRabbits of either sex were housed individually in cages and had free access tofood and water. For experiments involving pertussis toxin, animals were injected with asingle dose of pertussis toxin in a volume not exceeding 1 ml through the ear vein 4851hours before the experiments. It should be pointed out that in this study two differentbatches of pertussis toxin were used. For the contractility study, rabbits were injectedwith 2.2 gig/kg pertussis toxin (potency 0.1 ng/ml in CHO cell assay). On the other hand,rabbits were injected with 0.5, 1, 2 and 3 gig/kg pertussis toxin of a different batch(potency 0.03 ng/ml in CHO cell assay) for the ADP-ribosylation study, adenylatecyclase assay and rubidium efflux measurements. Control rabbits were injected withnormal saline. On the day of the experiment, animals were sacrificed by an injection ofpentobarbitone sodium (65 mg/kg) through the ear vein followed by exsanguination.Hearts were rapidly removed and placed in M. C. K. solution at room temperature,aerated with 95% 02 and 5% CO2. Left and right atria and the right ventricular free wallwere removed from the heart. In some experiments, left atria were used for thecontractility and 86 Rb efflux measurement studies. In other studies left and right atriaand the right ventricle were frozen in liquid nitrogen for adenylate cyclase assay andADP-ribosylation study.CONTRACTILITY STUDYLeft atria were cut into 4-5 strips, approximately 2 mm in width and 5 mm inlength, and one end of each strip was attached to a bipolar platinum electrode which wasplaced in a 20 ml tissue bath containing the M. C. K. solution maintained at 35° C andaerated with 95 % 02 and 5 % CO2. The other end of the muscle preparation wasattached by means of a cotton thread to a Grass FT .03 force displacement transducer.Tissues were stimulated to contract with pulses of 5 msec duration at a frequency of 1 Hzand a voltage two times threshold. Responses were recorded on a Grass polygraph52(model 7E). Atrial strips were placed under a resting tension of 0.5 g and the restingtension was adjusted throughout the 60 min equilibrium period to give the optimal basaldeveloped tension. This usually occurred at resting tensions between 1.0 and 1.5 g. Atrialstrips that developed contracture and/or arrhythmias during the equilibration period werediscarded.Cumulative concentration-response curves to carbachol alone or in the presenceof 100 nM isoproterenol, 100 1.1M phenylephrine, 3 gIVI forskolin and 50 WVI IBMX wereobtained under various conditions. All experiments involving phenylephrine wereconducted in tissues pre-treated with 1 j.tM timolol in order to block the P-adrenoceptor-mediated component of the phenylephrine-response. To determine the influence of 4-aminopyridine on responses to carbachol, tissues were randomly assigned to receiveeither 4-aminopyridine (50 or 500 [tM) or vehicle for 10 min, before the concentration-response curves to carbachol alone or in the presence of isoproterenol (100 nM) orphenylephrine (100 ptM) were obtained. For experiments involving forskolin and IBMX,tissues were exposed to 3 12M forskolin or 50 j.IM IBMX for 15 min each with 4-aminopyridine (50 and 500 1AM) being added for the final 10 min, before obtaining thecarbachol concentration-response curves.Cumulative concentration-response curves to isoproterenol and phenylephrinewere also obtained in the absence and presence of pinacidil and cromakalim. Tissueswere pre-treated with either vehicle (DMSO), pinacidil or cromakalim for 10 min beforethe concentration-response curves to isoproterenol or phenylephrine were obtained.In a separate set of experiments, atria were frozen for cAMP assay. For thispurpose, atrial strips were set up and equilibrated as described above, and then dividedinto seven groups. The first and second groups were treated with vehicle and 4-53aminopyridine (500 tiM), respectively and served as controls. The third and fourth groupswere treated with vehicle or 4-aminopyridine (500 1.1M) for 10 min, with isoproterenol(100 nM) being added for the final 6 min. The fifth and sixth groups were exposed toeither vehicle or 4-aminopyridine (500 1.1M) in combination with 100 nM isoproterenol(as described above) for 10 min with carbachol (3 iiM) being added for the final 3 min.The seventh group was exposed to carbachol (3 liM) for 3 min. At the specified timestissues were frozen with clamps cooled in liquid nitrogen and stored at -70°C prior toassay for cAMP levels.2.6. CYCLIC AMP ASSAYTissues were homogenized in 1 ml of 6% w/v trichloroacetic acid and centrifugedat 7000 rpm for a period of 40 min. Trichloroacetic acid was removed by extraction ofthe supernatant four times with 5 ml water-saturated ether. Cyclic AMP levels in thesupernatant were determined using a radioimmunoassay kit obtained from New EnglandNuclear (MacLeod, 1986).2.7. 86 Rb-EFFLUX MEASUREMENTAfter an hour of equilibration, an atrial strip was exposed to 3 - 5 pCi / ml of86Rb (in a total volume of 17 ml) for another 2 hour period, during which time the forceof contraction was monitored. At the end of the 2 hour loading period, the tissue waswashed with the non-radioactive M.C.K. buffer every 2 min for 20 min. The atrial stripwas then exposed to drugs dissolved in the M.C.K. buffer for various lengths of time54during which the tissue was washed every 2 min with the non-radioactive M.C.K. buffercontaining the drug, and the force of contraction was also monitored. In order for thecontractile response of the atrial strip to stabilize to the pre-drug treatment level, the atrialstrip was re-equilibrated for 40 min in 17 ml of the non-radioactive M.C.K. buffer whichwas changed and collected for counting every 20 min. After the 40 min washout period,the tissue was again washed every 2 min with the non-radioactive M.C.K. buffer foranother 10 min, followed by exposure to drugs as described above.The contents of the tissue bath were collected in polystyrene scintillation vialsand 86 Rb was counted by scintillation counting in the Cerenkov mode without theaddition of any scintillation fluid (Urquhart et al., 1991). The radioactivity remaining inthe atrial strip at the end of the experiment was obtained by dissolving the atrial strip in 2ml of Protosol for 24 hours followed by liquid scintillation counting after adding 11 ml ofAquasol. In order to permit comparison between the aqueous counts (each 2 min sample)and the counts obtained in the presence of Aquasol, a non-radioactive atrial strip wasspiked with a known amount of radioactivity and counted, after dissolution in Protosoland addition of scintillation fluid. At the same time, a 17 ml volume of M.C.K. solutionwas also spiked with same amount of radioactivity and was counted without adding anyscintillation fluid. The ratio of the aqueous to organic counts was obtained (Urquhart etal., 1991), which was 0.29 in our experiments. The counts present in the atrial strips weremultiplied by 0.29, and compared with other aqueous counts.The efflux rate constant for rubidium was calculated using the formulakt = Et/(CtX t) wherekt = efflux rate constant at time t,E t = efflux (measured as cpm) at time t,55C t = total tissue counts (cpm) of rubidium at time t,t = collection time in min.C t was calculated by back addition of radioactivity remaining in the tissue at the end ofthe experiment plus all the radioactivity that was released during the experiment fromtime t to the end.The efflux rate constant reflects the net movement of 86 Rb in inward andoutward directions across the membrane. However, because the M.C.K. solution used towash the tissues did not contain any rubidium, the efflux of 86 Rb predominated over theinflux in these experiments.Drugs were dissolved in 17 ml of warm M. C. K. solution containing 1 pMtimolol to block the 13-adrenoceptor-mediated component of the phenylephrine response.When the carbachol concentration-response curve was obtained, each concentration ofcarbachol was kept in contact with the tissue for an eight min period, with the solutionbeing changed every 2 min. When the carbachol-response was measured in the presenceof 4-aminopyridine or phenylephrine, tissues were exposed to them for a period of 16min, with carbachol being added for the final 8 min. When the carbachol-response wasmeasured in the presence of both 4-aminopyridine and phenylephrine, tissues wereexposed to 4-aminopyridine for 24 min with phenylephrine and carbachol being addedfor the last 16 and 8 min, respectively.In each experiment, the average of the final four values of the efflux rate constantor tension obtained immediately prior to addition of drug was considered the' basal orinitial response. In order to obtain the response to drug the four final values of the rateconstant and tension obtained in the presence of each concentration of drug wereaveraged. Average values from four or more such experiments were used to calculate the56mean ± S.E.M. In some experiments, data were expressed as a percent of the basalresponse using the formula% of basal response = (drug response/basal response)X1002.8. HOMOGENIZATION OF TISSUEFrozen atria and ventricles were ground under liquid nitrogen and homogenizedin 10 volumes of ice cold 10 mM Tris hydrochloride buffer (pH 7.5), in the presence ofprotease inhibitors (EGTA 1 mM, PMSF 174 µg/ml, trypsin inhibitor 100 pig/ml,benzamidine 20 pg/ml, and benzethonium chloride 20 j.tg/m1), using Polytron (setting 6)twice for 5 sec each. The homogenate was filtered through several layers of cheese cloth.A portion of the homogenate was used for the ADP-ribosylation study and anotherportion was incubated with alamethicin (100 pg/m1) for 10 min at 37°C and used for theadenylate cyclase assay. The third portion of the homogenate was used for the proteinassay.2.9. ADP-RIBOSYLATION STUDYIn vitro pertussis toxin-catalyzed incorporation of the [ 32P]-labelled ADP-ribosefrom [32 P]-NAD into G-proteins was studied by the method of Liang and Galper (1988)with certain modifications. Pertussis toxin was activated by incubation at 37°C for 15min with equal volumes of the activation buffer at pH 7.8. Atrial and ventricularhomogenates were incubated with the pre-activated pertussis toxin (30 tg/ml) and [ 3211-57NAD (2.5 .tCi) for 1 hour at 37 °C in a 50 ill reaction medium containing in mM:thymidine 10, EDTA 1, ATP 1, DTT 1, GTP 0.1, NAD 0.05, creatine phosphate 10 andcreatine phosphokinase 0.84 U. The reaction was started by adding the [ 32P]-labelledNAD and stopped by adding 1 ml ice-cold TCA (20 %) followed by centrifugation at3000 rpm for 30 min at 4 ° C. The supernatant was decanted and the precipitate waswashed with 1 ml of ice-cold ether and again centrifuged at 3000 rpm for 30 min. Theprecipitate was solubilized in the solubilization buffer at room temperature for 30 minand subjected to electrophoresis (35 mA and 500 Volts) using a 12 % polyacrylamidegel. The gel was stained with Coomasie blue for 30 min and destained to visualize themolecular weight standards using a destaining solution. The destained gel, after drying,was subjected to autoradiography by aligning against a Kodak X-OMAT-RP film for 6 -18 hours using an intensifying screen. The film was developed by immersing it in adeveloping solution for 2 min, followed by immersion in a fixing solution for 2 min inthe dark. The [32P]-labelled G-protein bands were cut from the gel, by aligning theautoradiogram against the gel, and the incorporation of [ 32 1]-labelled ADP-ribose wasmeasured by liquid scintillation counting in a Packard-Tricarb liquid scintillation counterin the presence of Aquasol.The incorporation of [32 P]-labelled ADP-ribose was calculated using theformula:pmol ADP-ribose per mg = [(S - B) / (R sp X E)] X 1000, whereS = counts in dpm from sample lanes,B = background dpm,Rsp = specific activity of NAD in dpm/pmol,E = protein concentration in jig,58The blank value was obtained by calculating the radioactivity incorporated in theG-proteins in the absence of pertussis toxin. The blank count was subtracted from thesample count to obtain the pmol ADP-ribose per mg.2.10. ADENYLATE CYCLASE ASSAYAdenylate cyclase was assayed by the method of Salomon (1979). Thealamethicin- treated atrial and ventricular homogenates were incubated in a final volumeof 150 gl for 10 min at 37 ° C with the [a-3211-ATP (500,000 dpm) in a reactionmedium (pH 7.8) of the composition in mM : MgSO4 5, cAMP 1, DTT 1, NaC1 100,IBMX 1, Tris HCl 50, GTP 0.05, ATP 0.5, creatine phosphate 10 mM, creatinephosphokinase 1.6 U, myokinase 6 U, and adenosine deaminase 0.75 U. The reaction wasstarted by adding the [ 32P]-labelled ATP and stopped by adding 100 gl stop solution.The [32I1-cAMP was separated from [32P]-ATP by sequential chromatographyon the Dowex and Alumina columns using [31-1]-cAMP (10000 cpm) as recoverymarker. Briefly, 50 gl of the [ 3H]-cAMP and 800 gl of water was added to each tubeafter the addition of the stop solution. The contents of each tube were added to theregenerated Dowex columns and the effluent was collected as waste. Individual reactiontubes were washed with 2 ml of water and the contents of each tube were transferred tothe Dowex columns and the effluent was again collected as waste. Dowex columns werethen placed on top of the regenerated Alumina columns and washed with 3 ml of water.Water was allowed to run through the Alumina columns and was collected as waste.Alumina columns were eluted with 4 ml of 0.1 M imidazole buffer and the effluent59collected in polystyrene scintillation vials. The [32P)- and [ 3E1]- labelled cAMP werecounted in a Packard-Tricarb liquid scintillation counter after adding 11 ml of Aquasol.Recovery of cAMP ranged from 50 - 80%. Effects of different drugs (isoproterenol,carbachol, atropine, timolol, forskolin and GTPyS) were tested by dissolving themdirectly in the reaction medium.Adenylate cyclase activity (pmol/min/mg) was calculated using the formulaproposed by Salomon (1979):cAMP (pmol/min/mg) = [H/S]C[(S'-B'-F)X1000]/R sp X EX tH and H'= 3 H-cAMP standard counts in 3 H and 32 P channelsrespectively,P and P'= 32 P ATP standard counts in 3 H and^32 P channelsrespectively,S and S'= Sample counts in 3 H and 32 P channels respectively,B and B'= Background counts in 3 H and 32 P channels respectively,F = (1-1 1-B)/H X SRsp = P'(dpm)/pmoles of ATP per tube,E = Protein concentration in pg,t = time of incubation in min.In one tube the [32-P]- ATP was added after the stop solution. This tube wasused as the blank. Blank value was calculated in the same way as the sample, and wassubtracted from the sample value to obtain net cAMP values in pmol/min/mg.602.11. PROTEIN ASSAYProtein was assayed by the method of Bradford (1976) using bovine serumalbumin as standard. Standard proteins, and proteins in the atrial and ventricularhomogenates were made to react with the dye solution and the absorbance was measuredat 594 - 596 nm using a spectrophotometer.2.12. STATISTICSThe results of the contractility studies were compared by one-way analysis ofvariance followed by Neuman Keul's multiple range test. Data from the saline- andpertussis toxin- treated groups were compared by two way analysis of variance followedby Neuman Keul's multiple range test. A P<0.05 was considered significantly different.Results obtained in the rubidium-efflux experiments and adenylate cyclase assayswere analyzed by Student's t-test. Mean values of tension, efflux rate constant andadenylate cyclase activity obtained for different treatments within the same experimentwere compared by paired t-test. Means obtained for the same treatments but fromdifferent experiments were analyzed by Student's unpaired t-test. A P<0.05 wasconsidered significantly different.61RESULTS3.1. INTERACTION OF CARBACHOL WITH PHENYLEPHRINE3.1.1. Effects of 4-aminopyridine on negative inotropic responses tocarbachol alone and in the presence of phenylephrine and isoproterenolIn preliminary experiments it was observed that 4-aminopyridine exerted aconcentration-dependent positive inotropic effect in the rabbit atrium over theconcentration range of 50 p.M to 5 mM (fig 4). The effects of 50 and 500 1.tM 4-aminopyridine were determined on the negative inotropic responses to carbachol in theabsence and presence of positive inotropic agents.The tension in left atrial strips in the presence of 50 p.M 4-aminopyridine (0.8 ±0.2 g) was not significantly different from that in its absence (0.6 ± 0.1 g). In comparison,500 i..tM 4-aminopyridine produced a much greater increase (1.2 ± 0.1 g) in thecontractile force (fig. 5A). Pre-treatment of left atria with 4-aminopyridine for 10 mincaused a small rightward shift of the carbachol concentration-response curve anddepressed the maximum response to carbachol (table 1). Increasing the concentration of4-aminopyridine to 500 gM produced a greater shift in the carbachol concentration-response curve although the overall percentage tension remaining in the presence of amaximum concentration of carbachol was not further increased (table 1).Phenylephrine (100 p.M) plus timolol produced a positive inotropic effect in atria,which was completely reversed by carbachol (fig. 5B). The left atrial tension in thepresence of 50 p.M 4-aminopyridine plus phenylephrine was not different from thatobtained with phenylephrine alone. However, in the presence of the combination of 5062TABLE 1. Effects of 4-aminopyridine on the maximum negative isotropic responsesand pD2 values of carbachol in the presence and absence of phenylephrine andIsoproterenol.Treatment Control50 1_11A 500 pIVICarbachol^Tension apD231.0±3.5^(6)7.26E103 (6)50.7±6.0 (8)6.94-1-0.05 (8)57.2±8.8 (9)6.36±0.2* (7)Carbachol^Tension 23.4±3.2 (10) 40.1±5.8 * (9) 79.5±5.0* (5)Phenylephrine pD2 6.80±0.17 (10) 6.48±0.07 (9) 5.86±0.24* (4)Carbachol^Tension 12.6±2.5 (6) 21.8±3.1^(8) 26.3±5.7* (8)Isoproterenol pD2 7.25±0:2 (5) 7.07±0.18 (8) 6.73±0.31 (6)(*) Represents significantly different from control within the same treatment group (one-way ANOVA).(a) Contractile response is expressed as percent of the initial tension.Each data point represents the mean ± S.E.M. of number of experiments shown inparentheses.63pM 4-aminopyridine and phenylephrine, the carbachol concentration-response curve wasshifted slightly to the right and the maximum negative inotropic response to carbacholwas significantly attenuated (fig. 5B, table 1). The positive inotropic effect of 500 tM 4-aminopyridine was additive with that of phenylephrine, and in the presence of thecombination of phenylephrine plus 500 pM 4-aminopyridine, the carbacholconcentration-response curve was shifted further to the right and the maximum inhibitoryeffect of carbachol was further reduced (fig. 5B, table 1).Carbachol also antagonized the positive inotropic response to 100 nMisoproterenol in a concentration-dependent manner (fig. 5C). The tension in the presenceof the combination of 100 nM isoproterenol and 50 1,LNI 4-aminopyridine was notsignificantly different from that in the presence of isoproterenol alone and thisconcentration of 4-aminopyridine had very little effect on either the carbachol pD2 value,or the maximum inhibitory effect of carbachol in the presence of isoproterenol (fig 5C;table 1). The combination of 500 4-aminopyridine and 100 nM isoproterenolproduced a further increase in tension (fig. 5C). Despite the high initial tension, there wasonly a small reduction in the magnitude of the maximum negative inotropic response tocarbachol in the presence of isoproterenol plus 500 j.tM 4-aminopyridine (fig 5C; table 1).No significant difference in the potency of carbachol in the presence of isoproterenol andeither concentration of 4-aminopyridine was detected (table 1).3.1.2. Effects of pinacidil and cromakalimBoth pinacidil and cromakalim were dissolved in DMSO. Tissues were treatedwith either DMSO alone (100 t1) or DMSO containing various concentrations ofpinacidil or cromakalim for 10 min prior to obtaining the concentration-response curves64^.to phenylephrine or isoproterenol. DMSO itself reduced the basal tension of left atrialstrips, and no significant difference in the initial tensions, prior to obtaining thephenylephrine- or isoproterenol- concentration-response curves, was observed whentissues were treated with either DMSO alone or with pinacidil or cromakalim (data notshown). Phenylephrine alone produced a concentration-dependent positive inotropiceffect which was not affected by DMSO. Pre-treatment of tissues with pinacidilantagonized the maximum positive inotropic response to phenylephrine in aconcentration-dependent manner, without affecting the phenylephrine pD2 value (fig. 6;table 2). The maximum inhibitory effect was observed in the presence of 1 mM pinacidil,which completely abolished the positive inotropic response to phenylephrine. Althoughcromakalim also attenuated the positive inotropic response to phenylephrine, in theconcentration range tested, cromakalim was found to be less effective than pinacidil (fig.7; table 2). Like pinacidil, cromakalim did not have any effect on the phenylephrine pD2value. The magnitude of the maximum positive inotropic response of left atria toisoproterenol was slightly less than double that to phenylephrine (fig. 8; table 2). Neitherpinacidil (1 mM) nor cromakalim (1 mM) antagonized the contractile response toisoproterenol (fig. 8). In fact, pinacidil enhanced the maximum positive inotropicresponse to isoproterenol (table 2).3.1.3. 86 Rb-Efflux studies3.1.3.1. Time-dependence of the 86 Rb efflux rate constantThe efflux of rubidium from left atrial strips in the absence of any drug over aperiod of 60 min is shown in fig. 9. The rate-constant for rubidium-efflux declined from a65TABLE 2. Effects of pinacidil and cromakalim on the maximum positive inotropicresponses and pD 2 values to phenylephrine and isoproterenol.TreatmentPhenylephrineTension (g) pD 2IsoproterenolTension (g) pD2Control 0.72±0.09 (9) 5.45±0.08 (9) 1.26±0.11 (7) 6.97±0.19 (7)Pinacidil100 pl■il 0.61±0.07 (11) 5.31±0.23 (11)30011M 0.42±0.05 (6) 5.21±0.23 (5)1 mM 0.18±0.05*(5) N.D. 1.72±0.1* (6) 7.08±0.07 (5)Cromakalim300 p.M 0.65±0.10 (6) 5.60±0.16 (6)1 mM 0.40±0.05 (6) 5.68±0.12 (4) 1.41±0.11 (6) 7.14±0.23 (6)(*) Represents significantly different from control within the same treament group (oneway ANOVA).(N.D.) Represents not determined.Each data point represents the mean ± S.E.M. of number of experiments shown inparentheses.66very high value to a plateau of approximately 0.01/min within the first 10 min afterloading and then remained stable for the remaining 60 min monitoring period.3.1.3.2. Effects of carbachol on the 86 Rb-efflux-rate-constant and force ofcontractionCarbachol produced an increase in the rate-constant of 86 Rb-efflux whichreached a maximum (138 ± 2 % of basal) in the presence of 10 JAM carbachol (fig. 10).Rubidium-efflux in response to 10 iiM carbachol was attenuated by 100 nM atropine to108 ± 2 % of basal (fig. 11). Atropine (100 nM) alone slightly reduced the basal efflux-rate-constant, to 88 ± 4% of basal. Carbachol also produced a negative inotropic responsein rabbit left atria which reached a maximum at 1 i_tM carbachol (fig 10). A furtherincrease in the carbachol concentration resulted in partial reversal of the negativeinotropic response. The lowest concentration of carbachol used (100 nM) produced onlya small increase in the efflux of 86Rb (to 105 ± 3 % of basal) but produced a largerdecrease (45 ± 7 %) in the basal developed force.3.1.3.3. Effects of 4-aminopyridine on carbachol-induced increase in the rate-constant of 86 Rb-efflux and decrease in tension4-Aminopyridine alone (50 and 500 1.1M) exerted a concentration-dependentpositive inotropic effect in the rabbit left atrium (fig. 12A), but had no significant effecton the 86Rb-efflux-rate-constant (fig. 12B). The same concentrations of 4-arninopyridine also attenuated the ability of carbachol to both reduce the tension (fig.12A) and to increase the rate-constant of 86Rb-efflux (fig. 12B). Carbachol alone (101.1M) produced a significant increase in the efflux-rate-constant to 128 ± 2% of the basal67and reduced the tension by 72 ± 4%. In the presence of 50 p.M 4-aminopyridine,carbachol still produced a significant increase in the efflux-rate-constant to 120 ± 2%,while reducing the tension by 59 ± 6%. In the presence of the higher concentration of 4-aminopyridine (500 11,M), carbachol had no significant effect on the efflux-rate-constant(fig. 12B), although it still produced a significant negative inotropic effect (fig. 12A),reducing the tension by 45 ± 7%.3.1.3.4. Effects of carbachol on the rate-constant of 86Rb-efflux and tension inthe presence of phenylephrinePhenylephrine alone (100 iiM) had a positive inotropic effect in left atria but hadno significant effect on the 86Rb-efflux-rate-constant (table 3). In the presence ofphenylephrine, 10 pM carbachol produced a significant decrease in the tension (fig. 13A)and increase in the 86Rb-efflux-rate-constant (fig. 13B). When expressed as percentageof the corresponding values obtained in the presence of phenylephrine alone, carbacholproduced a 65 ± 5% decrease in the tension elevated by phenylephrine, and increased the86 Rb-efflux-rate-constant to 119 ± 5%.3.1.3.5. Effects of 4-aminopyridine on responses to carbachol in the presence ofphenylephrineIn this series of experiments, 4-aminopyridine alone (50 and 500 ptM) producedonly a very small positive inotropic response and had little effect on the positive inotropicresponse of left atria to phenylephrine (table 3). No significant effect on the 86Rb-effluxwas detected when phenylephrine was administered in the presence of eitherconcentration of 4-aminopyridine (table 3).68Table 3. Effects of phenylephrine awl 4-aminopyridine, alone and in combination,on the contractile response and the °°Rb-efflux-rate-constant in electrically-stimulated rabbit left atriaTension(g)Efflux Rate Constant(X 10 -3 )BasalPhenylephrine(100 i.tM)0.7 ± 0.21.2 ± 0.2 *10.1 ± 0.610.4 ± 0.6Basal 0.5 ± 0.2 10.0 ± 0.54-Aminopyridine 0.6 ± 0.1 10.0 ± 0.4(5011M)4-Aminopyridine(50 p.M) + 1.2 ± 0.2* 10.3 ± 0.4Phenylephrine(100 p.M)Basal 0.6 ± 0.2 9.9 ± 0.54-Aminopyridine 0.8 ± 0.2 9.7 ± 0.5(500 gM)4-Aminopyridine(50012M) + 1.4 ± 0.1 * 9.7 ± 0.5Phenylephrine(100 iiM)(*) Represents significantly different from the corresponding basal response (paired t-test).Each data point represents the mean ± S.E.M. of 10 - 11 experiments.694-aminopyridine attenuated the reversal by carbachol of the positive inotropicresponse to phenylephrine (fig. 13A). In the presence of 50 p„M 4-aminopyridine,carbachol inhibited the positive inotropic response to phenylephrine by 51 ± 6%, while inthe presence of 500 1..tM 4-aminopyridine, carbachol reduced the positive inotropicresponse to phenylephrine by 25 ± 5%. 4-Aminopyridine appeared to have somewhatless effect on the increase in the 86Rb-efflux produced by carbachol in the presence ofphenylephrine (fig. 13B). The lower concentration of 4-aminopyridine (50 iiM) had nosignificant effect on the carbachol-induced increase in the efflux-rate-constant. Thehigher concentration of 4-aminopyridine (500 p.M) reduced the magnitude of thecarbachol-induced increase in the efflux-rate-constant to 113 ± 3%. However, theincrease in the 86Rb-efflux-rate constant produced by carbachol in the presence of thecombination of phenylephrine and 500 p„M 4-aminopyridine was still significant (fig.13B).3.1.3.6. Effects of pertussis toxin on carbachol-induced increase in the rate-constant of 86Rb-efflux and decrease in tensionPre-treatment of rabbits with 0.5 and 1 µg/kg pertussis toxin appeared to producea rightward shift in the carbachol-concentration-response curve, while having little effecton the maximum stimulatory effect of carbachol on the efflux-rate-constant of 86Rb (fig.14B). In contrast, pertussis toxin pre-treatment inhibited the maximum negativeinotropic response to carbachol, while having no effect on the reversal of the negativeinotropic effect by 100 iiM carbachol (fig. 14A). Responses to 1 p.M carbachol, whichproduced a near-maximal increase in the rate-constant of 86Rb-efflux and the maximumdecrease in tension in tissues from saline-treated rabbits, were both markedly inhibited by70pertussis toxin pre-treatment of rabbits (fig. 14A, B). No statistically significantdifference was observed in the abilities of 11.iM carbachol to increase the rate-constant of86 Rb-efflux in atria from rabbits pre-treated with 0.5 and 1 14/kg pertussis toxin (fig.14B). However, 1 i.tM carbachol reduced the tension by 36 ± 8% and 20 ± 5 % in atriafrom 0.5 and 1 tg/kg pertussis toxin pre-treated rabbits, respectively (fig. 14A). Nofurther reduction in tension of atria from pertussis toxin-treated rabbits was obtained byincreasing the carbachol concentration to 10 i_iM, despite the fact that this concentrationof carbachol increased the rate-constant of 86Rb-efflux to almost the same extent as inatria from saline-treated rabbits (fig. 14 A,B).3.1.3.7. Effects of pertussis toxin on responses to carbachol in the presence ofphenylephrinePre-treatment of rabbits with pertussis toxin had no significant effect on themagnitude of the positive inotropic response of left atria to 100 1..tM phenylephrine.However, the ability of 10 [tM carbachol both to reverse the positive inotropic responseto phenylephrine and to increase the efflux-rate-constant in the presence of phenylephrinewas essentially abolished by pertussis toxin pre-treatment (fig. 15 A,B). No carbachol-induced increase in the rate-constant of 86Rb-efflux could be detected in the presence of100 i_tM phenylephrine in atria from pertussis toxin pre-treated rabbits (fig. 15B). Whilecarbachol still produced a small reduction in the tension elevated by phenylephrine inatria from pertussis toxin-treated rabbits, this difference was not significant (fig. 15A).In the next series of experiments we studied the contribution of potassiumchannels to the cAMP-independent negative inotropic responses of left atria to carbachol71in the presence of forskolin and IBMX. In these studies pertussis toxin was used touncouple muscarinic receptors from adenylate cyclase. However, the batch of pertussistoxin used in this study was less potent than the one used in the rubidium efflux study. Inorder to ascertain that pertussis toxin was uncoupling muscarinic receptors fromadenylate cyclase, we measured the effect of pertussis toxin on the ability of carbachol toinhibit the isoproterenol-stimulated adenylate cyclase activity and cAMP generation. Inaddition, in order to ensure that 4-aminopyridine was not acting at the level of muscarinicreceptors, we also studied the effect of 4-aminopyridine on the inhibitory effect ofcarbachol on the isoproterenol-stimulated cAMP generation.3.2. EFFECTS OF 4-AMINOPYRIDINE AND PERTUSSIS TOXIN (2.2 Rglkg)ON THE INHIBITION BY CARBACHOL OF ISOPROTERENOL-STIMULATEDADENYLATE CYCLASE ACTIVITY AND cAMP GENERATIONIn atrial strips from saline-treated rabbits, isoproterenol (100 nM) elevated cAMPlevels from the basal value of 664 ± 17 pmol/g wet weight to a value of 1856 ± 102pmol/g wet weight. Carbachol (3 1.tM) alone did not alter cAMP levels (590.8 ± 64.4pmol/g wet weight) from the basal value, but when added in the presence ofisoproterenol, carbachol inhibited the isoproterenol-stimulated cAMP generation (fig.16A). Pre-treatment of rabbits with pertussis toxin (2.2 pg/kg) did not alter the basalcAMP levels (599 ± 70 pmol/g wet weight) or the ability of isoproterenol (100 nM) tostimulate cAMP generation (fig 16A). However, carbachol had no significant inhibitoryeffect on the isoproterenol-stimulated cAMP generation in atria from pertussis toxin pre-treated rabbits (fig 16A).72Basal adenylate cyclase activity in atrial homogenates from saline and pertussistoxin pre-treated rabbits was 115. 4 ± 18.5 (n=6) and 139.4 ± 7 (n=3) pmol/min/mg,respectively. Both isoproterenol and carbachol were able to stimulate and inhibitadenylate cyclase activity, respectively, although the potency of both agonists wasmarkedly reduced compared to their effects on cAMP levels in intact tissues.Isoproterenol (100 i.tM) increased adenylate cyclase activity in atria from saline-treatedrabbits and this increase was antagonized by 1 mM carbachol (fig. 17). Pertussis toxinpre-treatment of rabbits did not have any statistically significant effect on the ability ofisoproterenol to increase adenylate cyclase activity but attenuated the ability of carbacholto inhibit the isoproterenol-stimulated adenylate cyclase (fig. 17).We also investigated whether 500 pM 4-aminopyridine, the maximumconcentration used in the study, had any effect on the ability of carbachol to inhibit theisoproterenol-induced increases in cAMP levels. Basal cAMP levels in the presence andabsence of 4-aminopyridine were 825 ± 156 and 774 ± 60 pmol/g wet weight,respectively. 4-Aminopyridine did not have any effect on either the ability ofisoproterenol to promote cAMP generation or on the inhibition by carbachol ofisoproterenol-stimulated cAMP generation (fig. 16B).733.3. INTERACTION OF CARBACHOL WITH cAMP-ELEVATING AGENTS:EFFECTS OF 4-AMINOPYRIDINE AND PERTUSSIS TOXIN3.3.1. Effects of pertussis toxin and 4-aminopyridine on negative inotropicresponses to carbachol in the absence and presence of isoproterenolThe effects of pertussis toxin and 4-aminopyridine were compared on the cAMP-independent direct negative inotropic response to carbachol, and the cAMP-dependentresponse to carbachol in the presence of isoproterenol. Pre-treatment of rabbits withpertussis toxin had no significant effect on the basal developed tension (fig. 18) or on thepositive inotropic responses of rabbit left atrial strips to 100 nM isoproterenol (fig. 19). 4-Aminopyridine alone exerted a concentration-dependent positive inotropic response (fig.18), but had little effect on the positive inotropic responses to isoproterenol (fig. 19) inatria from both control and pertussis toxin pre-treated rabbits. No significant differencesin the magnitude of positive inotropic responses to 4-aminopyridine were observedbetween saline and pertussis toxin-treated rabbits (fig. 18).In left atrial strips from control rabbits, carbachol exerted a concentration-relatednegative inotropic response (fig. 18A). Pre-treatment of rabbits with the dose of pertussistoxin which completely uncoupled muscarinic receptors from adenylate cyclase (fig.16A, 17) had very little effect on the direct negative inotropic response to carbachol,producing only a slight reduction in the sensitivity, while having no effect on themaximum negative inotropic response, to this agonist (fig. 18, table 4). 4-Aminopyridineproduced a concentration-dependent attenuation of the sensitivity and maximum negativeinotropic response to carbachol (fig. 18; table 4). The antagonism by 4-aminopyridine ofthe direct negative inotropic response to carbachol was little affected by pertussis toxin74TABLE 4. ErrecIS of 4•;1111itiopyridine and pertussis toxin, alone and in combination, on the maximum negative Inotroplc responses and p1 2 values to carbachol in thepresence and absence of isoproterenol, rorskolin and 113MX.Tre:ItmenSaline-treatedControl^ 4•Aminopyridine50µM 500µM57.1±6.2t (15)6,29±0.12t(12)Pertussis toxinControl^34,0±8,6^(7)^6.68±0,15^(6)4-Aminopyridine50 p M^500 p M51.9±9,3^(7)^66.2±7, It (7)6.49±0.08^(6)^6,18±- 0.12t (6)Carbachol^Tension:IpD231.4±3,30 (9)7.08i-0.09 (9)45,3±4,90 (14)6.84±0.05 (14)Carbachol^Tension 1.7±3.1^(6) 18.9±3.5^(6) 35.1±4,91- (6) 68.7±10.6t(4) 75.4±11,6t(4) 81,1±3,6t(5)Isoproteienol^pD2 6.81±0,09 (6) 6.70±0.09 (7) 6,30±0.14(6) 6.02±0.23+(4) 6,15±0.09t(4) 6.02±0, 1 R t (5)Carbachol^Tension 13,0±4^(II) 25.1±2.61.(10) 52.3±3.7 t (9) 46,4±8,4+(8) 60.5±5.9' (7) t72.4±5,3^(5)Fors1;olin^pD2 6.62±0.08 (I I) 6.37±0.071-(10) 6.03±0.071(9) 6.51±.1^(7) 6,28±0,03^(7) 6.12±0.15^(4)Carbochol^Tension 7.70±3,7^(13) 16.9±4,4^(8) 3164±±0t9((99) 22.1±8,8^(7) 37.6±8.1^(7) 65.5±7.25)1131\1X^pD, 6,89±0,06(13) 6.801-0.07^(8)64.4..40t16.71±0.08^(4) 6.34±0.051(4) 6.1 I ±0.21t (5)(t) Represents significantly different from control within the same treatment group (one-way ANOVA).(+) Represents significantly different from the respective treatment in saline-treated group (two-way ANOVA).(a) Contractile response is expressed as percent of the initial tension.Each data point represents the mean ± S.E,M, of number of experiments shown in parentheses.pre-treatment of rabbits. There was no significant difference between atria from controland pertussis toxin pre-treated rabbits in the maximum response or sensitivity tocarbachol in the presence of either concentration of 4-aminopyridine (fig. 18, table 4).Carbachol also completely reversed the positive inotropic responses of left atrialstrips from saline-treated rabbits to isoproterenol (fig. 19A). The response to carbachol inthe presence of isoproterenol was affected to a much greater extent by pertussis toxin pre-treatment of rabbits than was the direct negative inotropic response to carbachol (fig.18B). Both the maximum response and the sensitivity to carbachol in the presence ofisoproterenol were significantly reduced in atria from pertussis toxin-treated rabbitscompared to atria from control rabbits (table 4). On the other hand, the response of atriafrom saline-treated rabbits to carbachol in the presence of isoproterenol was relativelyresistant to inhibition by 4-aminopyridine, being blocked to a small extent by 500 tM butnot by 50 11M 4-aminopyridine (fig. 19A, table 4). In atria from pertussis toxin pre-treated rabbits, 4-aminopyridine produced further concentration-dependent inhibition ofthe responses to carbachol in the presence of isoproterenol, although the carbacholresponse was not completely abolished by the combination of both pertussis toxin and 4-aminopyridine (fig.19B, table 4).3.3.2. Effects of pertussis toxin and 4-aminopyridine on the negativeinotropic responses to carbachol in the presence of forskolin and IBMX:In atria from saline-treated rabbits, both forskolin (3 i.tM) and IBMX (50 laM)produced a positive inotropic response which was slightly smaller in magnitude than thatproduced by 100 nM isoproterenol (fig. 20A, 21A). Pre-treatment of rabbits withpertussis toxin had no significant effect on positive inotropic responses of left atria to76either forskolin or IBMX (fig. 20, 21). Overall, the contractile force in the presence of thecombination of 4-aminopyridine plus forskolin or IBMX was greater than the positiveinotropic effect of either agent alone, in atria from both saline and pertussis toxin pre-treated rabbits (fig. 20, 21). However, this difference was not consistently significant.In left atrial strips from saline-treated rabbits, increasing concentrations ofcarbachol completely reversed the positive inotropic response to forskolin (fig. 20A).The ability of carbachol to overcome the response to forskolin was attenuated bypertussis toxin pre-treatment of rabbits (fig. 20B). While the carbachol pD2 value wasunaffected, the maximum decreases in tension produced by carbachol in the presence offorskolin was significantly reduced in atria from pertussis toxin pre-treated rabbits (fig.20, table 4). In atria from saline-treated rabbits, 4-aminopyridine produced aconcentration-dependent reduction of the response to carbachol in the presence offorskolin, reducing both the potency and maximum inhibitory effect of carbachol in thepresence of forskolin. 4-Aminopyridine produced a further inhibition of the response tocarbachol in the presence of forskolin in atria from pertussis toxin pre-treated rabbits,such that the maximum negative inotropic responses to carbachol in the presence offorskolin and 4-aminopyridine were less than those to carbachol in the presence offorskolin alone in atria from pertussis toxin-treated rabbits (fig. 20, table 4).Carbachol also produced a concentration-dependent reversal of the positiveinotropic response to 50 tM IBMX in left atrial strips from saline-treated rabbits. Thenegative inotropic response to carbachol in the presence of IBMX was very pronounced,and 7 out of 13 tissues stopped beating after the addition of 2 ptM carbachol. In atria fromrabbits pre-treated with pertussis toxin, the maximum negative inotropic response tocarbachol in the presence of IBMX was only slightly attenuated (fig. 21, table 4), but77only one tissue stopped beating in the presence of the maximum concentration ofcarbachol. The lower concentration of 4-aminopyridine (50 µM) also had relatively littleapparent effect on the response to carbachol in the presence of IBMX in atria fromsaline-treated rabbits, but only 2 out of 7 tissues stopped beating after 2 1.1M carbachol.The higher concentration of 4-aminopyridine (500 .tM) significantly reduced both thepotency and negative inotropic response to carbachol in the presence of IBMX in atriafrom saline-treated rabbits (fig. 21, table 4). In atria from pertussis toxin pre-treatedrabbits, 4-aminopyridine produced further attenuation of the response to carbachol in thepresence of IBMX (fig. 21, table 4). The maximum response to carbachol in the presenceof IBMX plus 500 .tM 4-aminopyridine in atria from pertussis toxin pre-treated rabbitswas significantly different from the corresponding value in atria from saline-treatedrabbits.3.4. ADP-RIBOSYLATION EXPERIMENTSBefore describing the next series of experiments, it is important to mention thatthe batch of pertussis toxin used in this series was the same as that used in the rubidium-efflux experiment. The reason for this was we wanted to make a direct comparison of theuncoupling effects of pertussis toxin on the carbachol-mediated inhibition of adenylatecyclase and the carbachol-stimulated rubidium-efflux in atria and ventricles.783.4.1. Selecting the concentration of pertussis toxin for in vitro ADP-ri bosylationAtrial and ventricular homogenates (50 - 100 [tg protein) were incubated withincreasing concentrations of pertussis toxin (10, 20, 30 and 100 µg/ml) in the presence of32 P-NAD. Two bands in the molecular weight range 45 - 39 kDa were seen uponelectrophoretic separation followed by autoradiography of the reaction mixture (fig. 22).The average molecular weight of the labelled proteins was 40.2 ± 0.9 and 41.5 ± 0.9 kDa(n=4) in the atrium and 41 ± 0.3 and 42.4 ± 0.2 kDa (n=4) in the ventricle. As shown intable 5, the maximum effect was observed in the presence of 30 µg/ml pertussis toxin.Increasing the concentration of pertussis toxin to 100 µg/ml did not result in anysignificant increase in the ADP-ribosylation of G-proteins.3.4.2. Estimation of in vivo ADP-ribosylation of G-proteins by pertussis toxinFigs. 23 and 24 are two representative autoradiograms showing the effect ofinjection of rabbits with pertussis toxin (0.5, 1, 2, 3 gg/kg) on the ability of exogenouspertussis toxin (30 1.1g/m1) to ADP-ribosylate the atrial and ventricular G-proteins. Thedegree of in vivo ADP-ribosylation was determined by comparing the ability of in vitropertussis toxin (30 µg/ml) to transfer the [ 32 P]-ADP-ribose from [32 P]-NAD to G-proteins in the atrium and ventricle from saline and pertussis toxin pre-treated rabbits. Ina typical experiment the amount of radioactivity incorporated by 30 p.g/m1pertussis toxinin atrial and ventricular homogenates of rabbits injected with normal saline was 1258dpm and 829 dpm respectively. In rabbits injected with 0.5 and 3.0 µg/kg pertussis toxin,the amount of radioactivity incorporated in the presence of 30 µg/ml pertussis toxin inatrial homogenates was 875 and 227 dpm respectively. In the presence of 30 µg/ml79Table 5. Invitro ADP-ribosylation of G-proteins by pertussis toxin in the rabbitatrial and ventricular homogenates.Concentration(iig/m1)Atrium^(n)(pmol ADP-Ribose/mg)Ventricle^(n)(pmol ADP-Ribose/mg)10 85.11-21.0 (6) 75.5+19.1 (4)20 73.2±10.3 (3)30 1212+41_4 (6) 95.2±15.7 (4)100 133.0±48.2 (6) 65.9±5.3 (4)Each data point represents the mean ± S.E.M. Values in parentheses represent number ofanimals.80Table 6. ADP-Ribosylation by exogenous pertussis toxin (30 1.1g/m1) of G-proteins in atrialand ventricular homogenates from saline and pertusssis toxin (0.5, 1, 2 and 3 gig/kg) pre-treated rabbits.(pmolIn vitro In vivo(n)ADP-Ribose/mg) (% of control) (% of control)Control Atrium 238.6±48.5 100.0 0.0 (9)Ventricle 156.9±34.3 100.0 0.0 (9Pertussis Atrium 77.4±14.1 32.4±5.9 67.6±5.9 (4)toxin Ventricle 77.3±20.4 49.3±12.8 50.7±12.8 (5)(0.5 µg/kg)Pertussis Atrium 71.8±28.1 30.1±10 69.9±10.8 (6)toxin Ventricle 63.4±9.3 40.4±5.9 59.6±5.9 (6)( 1 1-ig/kg)Pertussis Atrium 10.9±10.2 4.6±4.2 95.4±4.3 (5)toxin Ventricle 12.4±16.4 7.9±10.5 92.1±10.5 (6)(2 µg/kg)Pertussis Atrium 0 0 100.0 (5)toxin Ventricle 0 0 100.0 (5)(3 µg/kg)Each data point represents the mean ± S.E.M. of number of rabbits shown in parentheses.81pertussis toxin, incorporation of radioactivity in ventricular homogenates from rabbitsinjected with 0.5 and 3.0 lag/kg pertussis toxin was 711 and 194 dpm respectively. Blankcounts in atrial and ventricular homogenates typically were 105 and 196 dpmrespectively. In the atrium and ventricle from control rabbits, the amounts of ADP-riboseincorporated were 238.6 ± 48.5 pmol/mg (n=9) and 156.9 ± 34.3 pmol/mg (n=9)respectively. Pre-treatment of rabbits with pertussis toxin resulted in a loss of the abilityof exogenous pertussis toxin to ADP-ribosylate G-proteins (table 6). In the atrium, 0.5ag/kg pertussis toxin ADP-ribosylated 68 % of all pertussis toxin-sensitive G-proteins.In the ventricle, the same dose of pertussis toxin ADP-ribosylated 51 % of all G-proteins.Increasing the dose of pertussis toxin to 1 µg/kg resulted in very small increase in the invivo ADP-ribosylation of G-proteins in the atrium and ventricle. Pre-treatment of rabbitswith 2 .tg/kg pertussis toxin resulted in 95 % and 92 % ADP-ribosylation of G-proteinsin the atrium and ventricle respectively. In the atrium and ventricle from rabbits pre-treated with 3 µg/kg pertussis toxin, external pertussis toxin was unable to ADP-ribosylate any G-protein.3.5. ADENYLATE CYCLASE ASSAYThe alamethicin-treated atrial and ventricular homogenates were used to measureadenylate cyclase activity. The basal adenylate cyclase activity in the atrial andventricular homogenates were found to be 121.9 ± 12.9 and 156.2 ± 15.5 pmol/min/mg,respectively. The basal adenylate cyclase activity was responsive to stimulation byforskolin and GTPyS (table 7). This suggested that the crude homogenates of atrium and82Table 7. Adenylate cyclase activity in the rabbit atrial and ventricular homogenates in thepresence and absence of various activators.Adenylate cyclase activity(pinolimin/mg)^% of basal (n)Basal Atrium 121.9 ± 12.9 (15)Ventricle 156.2 ± 15.5 (10)Isoproterenol Atrium 163.3 ± 13.3 144.2 ± 11 (15)Ventricle 252.4 ± 27.8 198.2 ± 22 (7)Forskolin Atrium 907.0 ± 92.1 689.4 ± 38 (14)(I 1-IM) Ventricle 1115 ± 92 780 ± 50 (11)GTPyS Atrium 245.6 ±.23.9 241.7 ± 23 (7)(101.1M) Ventricle 311.0 ± 47 215.0 ± 19 (5)Each data point represents the mean ±-*S.E.M. of adenylate cyclase activity in the atrial andventricular homogenates from number of animals shown in parentheses.Concentrations of isoproterenol used were 100 p.M in atrium and 1 1.11■4 in ventricle.83ventricle had a functioning adenylate cyclase enzyme which was stimulatory G-proteincoupled.3.5.1. 13-Adrenoceptor stimulation of adenylate cyclaseIsoproterenol stimulated adenylate cyclase activity in a concentration-dependentmanner in atrial and ventricular homogenates (fig. 25). In the concentration range used inthe present study (100 nM - 1 mM) the ventricular adenylate cyclase was moreresponsive to isoproterenol, both in terms of potency and maximum stimulatory effect,than that of the atrium (fig. 25). Whereas 100 nM isoproterenol was able to stimulateadenylate cyclase activity in the ventricle, the same concentration of isoproterenol didnot stimulate the basal adenylate cyclase activity in the atrium (fig. 25). Similarly, 100tM isoproterenol increased adenylate cyclase activity 289.5 pmol/min/mg from the basalvalue in the ventricle. The same concentration of isoproterenol, however, produced onlya 36.3 pmol/min/mg increase in the enzyme activity in the atrial homogenate (fig. 26).In order to ascertain that the stimulatory effect of isoproterenol on adenylatecyclase was mediated through /3-adrenoceptors, timolol, a P-adrenoceptor blocker wasused to antagonize the isoproterenol response. Timolol (1 1.tM) alone did not have anyeffect on the basal adenylate cyclase activity in homogenates of atrium and ventricle.However, when combined with 100 1.1M isoproterenol, timolol (1 JIM) attenuated theability of isoproterenol to stimulate adenylate cyclase activity (fig. 26).843.5.2. Effect of muscarinic receptor stimulation on adenylate cyclase activityin the atrium and ventricle3.5.2.1. Basal adenylate cyclaseIn both atrium and ventricle the inhibitory effect of carbachol on adenylatecyclase activity was seen at concentrations of 10 tM and higher. In the ventricle,carbachol, 10 tM - 1 mM, produced a concentration-dependent inhibitory effect onadenylate cyclase activity (fig. 27A). The maximum concentration of carbachol used inthe study (1 mM) produced a 27.7 ± 2 % inhibition of the basal adenylate cyclaseactivity. The inhibitory effect of carbachol (1 mM) was sensitive to antagonism byatropine, a muscarinic receptor blocker. Atropine (10 pM), alone had a slight stimulatoryeffect on the basal adenylate cyclase activity (fig. 28A), but when used with carbachol,atropine antagonized the ability of carbachol to inhibit adenylate cyclase activity (fig.28A).In the atrium, on the other hand, a measurable inhibition of 11.8 ± 4.3 % (fig27A) of the basal adenylate cyclase activity was seen only in the presence of 1 mMcarbachol. In two atria, out of a total of eleven, carbachol did not inhibit the basaladenylate cyclase activity at all. In the remaining nine, inhibition of the basal adenylatecyclase activity ranged from 1.8 - 33 %. Atropine, 10 j.IM, did not have any statisticallysignificant attenuating effect on the ability of carbachol to inhibit the basal adenylatecyclase activity (fig 28A).3.5.2.2. Isoproterenol-stimulated adenylate cyclaseCarbachol (10 p.M - 1 mM) exerted a concentration-dependent inhibitory effecton adenylate cyclase activity stimulated by 1 j.tM isoproterenol in the ventricle (fig. 27B).85In the concentration range used in the present study a maximum inhibition of 54.7 ± 5.7% of the increase produced by isoproterenol was seen in the presence of 1 mM carbachol(fig 27B). Atropine (10 i.tM) completely blocked the inhibitory effect of carbachol on theisoproterenol-stimulated adenylate cyclase activity (fig 28B).Carbachol also inhibited in a concentration-dependent manner the ability ofisoproterenol to stimulate adenylate cyclase activity in the atrium (fig 27B). In theconcentration range used in the present study (10 j.tM - 1 mM), a maximum of 57.2 ±10.2 % inhibition of the isoproterenol-stimulated adenylate cyclase was observed in thepresence of 1 mM carbachol. The inhibitory effect carbachol on the isoproterenol-stimulated adenylate cyclase activity (fig 28B) was attenuated by atropine (10 41\4).3.5.2.3. Forskolin- and GTP7S- stimulated adenylate cyclaseSince the maximum inhibitory effect on the basal and isoproterenol-stimulatedadenylate cyclase activity was seen in the presence of 1 mM carbachol, this concentrationof carbachol was used to study the interaction with forskolin and GM'S.In the ventricle, carbachol inhibited the forskolin-stimulated adenylate cyclaseactivity (fig. 29A). The inhibitory effect of carbachol (18.4 ± 3.6 %) on the forskolin-stimulated adenylate cyclase activity was attenuated by atropine (data not shown).Carbachol also exerted a 17 ± 2.4% inhibition of the GTPyS-stimulated adenylate cyclaseactivity (fig. 29B).In contrast, in the atrium carbachol did not have any inhibitory effect on adenylatecyclase activity stimulated by either forskolin or GTRyS (fig. 29 A,B).863.5.3. Pertussis toxin treatment of rabbitsRabbits were treated with 4 different doses of pertussis toxin (0.5, 1, 2, 3 'Az/kg)48 hours prior to sacrifice. In the ventricle, the effect of 0.5 µg/kg pertussis toxin onadenylate cyclase activity was not tested. In both atrium and ventricle, pertussis toxinpre-treatment of rabbits did not have any effect on the basal adenylate cyclase activity(table 8). Similarly, the abilities of isoproterenol or forskolin to stimulate adenylatecyclase activity were not altered by pre-treatment of rabbits with pertussis toxin (table 9).3.5.3.1. Muscarinic receptor-mediated response3.5.3.1.1. Basal adenylate cyclaseIn the ventricle, pertussis toxin pre-treatment of rabbits resulted in a dose-dependent attenuation of the ability of carbachol to inhibit the basal adenylate cyclaseactivity (fig 30A). In rabbits pre-treated with 1 pertussis toxin, a dose that ADP-ribosylated 60 % of G-proteins, carbachol retained its ability to inhibit (28 ± 4%) thebasal adenylate cyclase activity. Increasing the dose of the toxin to 2 and 3 pg/kg, whichresulted in 92% and 100% ADP-ribosylation, respectively, attenuated the maximuminhibitory effect of carbachol. The inhibitory effects of carbachol (1 mM) in the ventriclefrom rabbits pre-treated with 2 and 314/kg pertussis toxin were 21 and 7 %, respectively.3.4.3.1.2. Interaction of carbachol with isoproterenol:In the ventricle, pertussis toxin pre-treatment of rabbits resulted in a dose-dependent attenuation of the inhibitory effect of carbachol on the isoproterenol-stimulated adenylate cyclase activity. As shown in fig. 30B, pre-treatment of rabbits with1 pg/kg pertussis toxin had no effect on the ability of carbachol to• inhibit the87Table 8. Effects of pre-treatment of rabbits with different doses of pertussis toxin on basaladenylate cyclase activity in the atrium and ventricle.Adenylate cyclase activity(pmol/min/mg,)Treatment Atrium (n) Ventricle (n)Saline 116.5±16 (12) 156.0±16 (10)Pertussistoxin 128.5+23 (6)(0.5 p.g/kg)Pertussistoxin 148.8+29 (6) 301.0±82 (6)(1 lig/kg)Pertussistoxin 140.8±19 (7) 172.7±26 (6)(2 lig/kg))Pertussistoxin 123.9±36 (6) 140.6±20 (5)(3 ttg/kg)Each data point represents the mean ± S.E.M. of adenylate cyclase activity in the atrial andventricular homogenates of number of animals shown in parentheses.88Table 9_ Effects of pertussis toxin pre-treatment of rabbits on the isoproterenol (ISO)- andforskolin- stimulated adenylate cyclase activity in the atrium and ventricle.Adenylate cyclase activity(°/0 of Basal) , Atnuni^ Ventricle eTreatment ISO Forskolin ISO ForskolinSaline 149±14 684±39 198+72 825+45Pertussistoxin 131+5 612±26(0 - 5 ligilcg)Pertussistoxin 131±9 542±60 198±32 810±115(1 p.g/kg)Pertussistoxin 148+11 563±62 181±7 794±67(2 µg/kg))Pertussistoxin 139±10 553±48 245±30 975±87(3 p.g/kg)Each data point represents the mean ± S.E.M. of adenylate cyclase activity in the atrial andventricular homogentes from 5 - 15 animals.Concentrations of isoproterenol used were 11.1M in ventricle and 100 p.M in atrium.89isoproterenol-stimulated adenylate cyclase. In rabbits pre-treated with 1 ig/kg pertussistoxin carbachol produced 59.8 ± 8.5 % inhibition of the isoproterenol-stimulatedadenylate cyclase which was not significantly different from the inhibitory effect of 1mM carbachol (54.9 ± 5.7%) on the isoproterenol-stimulated adenylate cyclase observedin atria from saline-treated rabbits. Carbachol inhibited the isoproterenol-stimulatedadenylate cyclase activity by 18 ± 11 % in rabbit pre-treated with 2 tg/kg pertussis toxin.In rabbits pre-treated with 3 tg/kg pertussis toxin the inhibitory effect of 10 .tMcarbachol (18.4±10%) on the isoproterenol-stimulated adenylate cyclase activity washigher than the inhibitory effect (-17.1 ± 9.9 %) observed in the presence of the sameconcentration of carbachol in saline-treated rabbits. This inhibitory effect of carbachol,however, was not concentration-dependent. Increasing the concentration of carbachol to1 mM did not result in any further increase in the inhibitory effect of carbachol (27.4 ±7.9%) on the enzyme activity in the presence of isoproterenol.In the atrium, the effect of pertussis toxin pre-treatment of rabbits on theisoproterenol-carbachol interaction was very pronounced and complex (fig 31). In theatrium from rabbits pre-treated with 0.5, 1 and 3 1.1g/kg pertussis toxin, which resulted in68, 70 and 100 % ADP-ribosylation of G-proteins respectively, the inhibitory effect ofcarbachol (1 mM) on the isoproterenol-stimulated adenylate cyclase activity was almostcompletely attenuated (fig 31). In contrast, in rabbits pre-treated with 2 .tg/kg pertussistoxin, which ADP-ribosylated 95% of G-proteins, the inhibitory effect of carbachol onthe isoproterenol-stimulated adenylate cyclase activity was very similar to that observedin saline-treated rabbits (fig. 31).90Fig. 4: Concentration-dependent positive inotropic effects of 4-aminopyridine inelectrically-stimulated rabbit left atria.Cumulative concentration-response curves to 4-aminopyridine were obtained inrabbit left atrial strips. Data are expressed as the developed tension in grams. Each datapoint represents the mean ± S.E.M. of 9 experiments.91- LOG 4-AMINOPYRIDINE (M)92Fig. 5: Effects of 4-aminopyridine on negative inotropic responses to carbachol in theabsence and presence of phenylephrine or isoproterenol in electrically-stimulated rabbitleft atrial strips.Left atrial strips were treated with normal saline (o), or 50 (A) or 500 1..tM (v) 4-aminopyridine either alone (A) or in combination with 100 pM phenylephrine plus 1 i.tMtimolol (B) or 100 nM isoproterenol (C) prior to obtaining cumulative concentration-response curves to carbachol. Tissues were treated with 4-aminopyridine for 10 min, withphenylephrine or isoproterenol being added for the final 3 min. Data are expressed as thethe mean ± S.E.M. of developed tension in grams. B represents the basal developedtension prior to addition of any drug. In panel A, the contractile force of atria treated withsaline, 50 and 500 11M 4-aminopyridine was 0.9 ± 0.2 (n=8), 0.8 ± 0.2 (n=8), and 1.2 ±0.1 g (n=8), respectively. In panels B and C, P and I represent the phenylephrine- andisoproterenol- induced increases in force of contraction in the presence of saline or 4-aminopyridine. In panel B, the tension in the presence of phenylephrine alone and incombination with 50 and 500 ?AM 4-aminopyridine was 0.7 ± 0.1 g (n=9), 0.7 ± 0.1 g(n=9) and 1.2 ± 0.1 g (n=9) respectively. In panel C, the positive inotropic response toisoproterenol alone and in combination with 50 and 500 1..tM 4-aminopyridine was 1.1 ±0.1 g (n=6) , 1 ± 0.1 (n=8) and 1.7 ± 0.2 g (n=6) respectively.932.0 (A)0.(B)^2.01 .51 .00,52.01,51.00,5^0.0 ... __1^L--1^0,0 ^^B 8^7^6^5^4^B P 8j 0 . 0 _7^6^5^4^B I 8^7- LOG CARBACHOL (M)Fig. 6: Effect of pinacidil on the phenylephrine-induced positive inotropic response inelectrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to phenylephrine were obtained aftertreating left atrial strips with DMSO (o) (n=9) or 100 JIM (o) (n=11), 300 11M (A) (n=6)or 1 mM (A) (n=5) pinacidil. Atrial strips were exposed to DMSO or pinacidil for 10 min.Data are expressed as the mean ± S.E.M. of the developed tension in grams (g).95- LOG PHENYLEPHRINE (M)Fig. 7: Effect of cromakalim on the phenylephrine-induced positive inotropic response inelectrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to phenylephrine were obtained aftertreating left atrial strips with DMSO (o) (n=9) or 300 1.1M (A) (n=6) and 1 mM (A) (n=6)cromakalim. Atrial strips were exposed to DMSO or cromakalim for 10 min. Data areexpressed as the mean ± S.E.M. of attained tension in grams.97- LOG PHENYLEPHRINE (M)98Fig. 8: Effects of pinacidil and cromakalim on the isoproterenol-induced positiveinotropic response in electrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to isoproterenol were obtained aftertreating left atrial strips with DMSO (o) (n=7), 1 mM pinacidil (0) (n=5) or 1 mMcromakalim (6) (n=6). Atrial strips were exposed to DMSO, pinacidil or cromakalim for10 min. Data are expressed as the mean ± S.E.M. of the developed tension in grams.99- LOG ISOPROTERENOL (M)1 00Fig. 9: Time-dependence of the 86 Rb efflux rate constant in electrically-stimulatedrabbit left atrial strips.Efflux of 86 Rb was monitored from pre-loaded and electrically-stimulated rabbitleft atrial strips every 2 min for 60 min. Data are expressed as the efflux rate constant.Each data point represents the mean of 3 experiments.1010.050.04E-■0.03zCf)0U▪ 0.02X 0.01 -rx,▪ 0.000 I 10 20 30 40 50 60TIME (min)102Fig. 10: Effects of carbachol on the rate constant of 86 Rb efflux and the force ofcontraction in electrically-stimulated rabbit left atria.Concentration-response effects of carbachol on the rate constant of 86 Rb efflux(o) and force of contraction (o) were determined in left atrial strips. Atrial strips wereexposed to each concentration of carbachol dissolved in 17 ml of M.C.K. buffer for 8 minduring which time the tissue was washed every 2 min with non-radioactive M.C.K.solution containing the drug and the force of contraction of the tissue was also monitored.In order for the contractile response of the atrial strip to stabilize to the pre-drugtreatment level, the atrial strip was equilibrated for 40 min in 17 ml of the non-radioactive M.C.K. solution which was changed and collected for counting every 20 min.After the 40 min washout period, the tissue was again washed every 2 min with the non-radioactive M.C.K. solution for another 10 min, followed by exposure to anotherconcentration of carbachol as described above. Data are expressed as percent of the initialefflux or force of contraction immediately prior to addition of each concentration ofcarbachol. Each curve represents the mean ± S.E.M. of 5 experiments.103140120•....-,Z 1000E----11U80HZ0 60Urz-I040UC40 20G--i0 ^I^f^1^I^18 7 6 5^4^3- LOG CARBACHOL (M)Fig 11: Effect of atropine on the rate constant of 86Rb efflux in the presence of carbacholin electrically-stimulated rabbit left atrial strips.The rate constant of 86Rb efflux was monitored in the presence of 10 i_tMcarbachol (A) alone, 10 JIM carbachol plus 100 nM atropine (B) or 100 nM atropinealone (C). Atrial strips were exposed to atropine or carbachol alone for 8 min each. Whenatropine was combined with carbachol, tissues were exposed to atropine for 16 min, withcarbachol being added for the final 8 min. Data are expressed as percent of the initialefflux-rate-constant prior to addition of any drug. The initial efflux-rate-constants prior toaddition of carbachol (10 1.1M), atropine (100 nM) and atropine plus carbachol were0.0078 ± 0.0006, 0.0094 ± 0.0006 and 0.0081 ± 0.0008 per min respectively. Data wereanalyzed by paired t-test. Each bar represents the mean ± S.E.M. of 4 experiments. (*)Represents significantly different from carbachol alone.105106Figure 12: Effects of 4-aminopyridine on the negative inotropic response and the rateconstant of 86 Rb efflux in the presence of carbachol in electrically-stimulated rabbit leftatrial strips.Effects of carbachol were monitored on the contractile force (A) and the rateconstant of 86Rb efflux (B) in electrically-stimulated rabbit left atrial strips in theabsence (CON) and presence of 4-aminopyridine (4-AP, 50 and 500 1.1M). Open barsrepresent the basal response prior to addition of drug, cross-hatched bars represent the 4-aminopyridine response, and hatched bars represent the response to carbachol (10 1.1M).Atrial strips were exposed to carbachol for 8 min. When carbachol was combined with 4-aminopyridine, atrial strips were exposed to 4-aminopyridine for 16 min, with carbacholbeing added for the final 8 min. Contractile response is expressed as the attained tensionin grams and efflux is expressed as the efflux rate constant. Each bar represents the mean± S.E.M. of 6-8 experiments. (*) Represents significantly different from thecorresponding response immediately prior to addition of 4-aminopyridine and carbachol(P<0.05, paired t-test). (+) Represents significantly different from carbachol response inthe control group (P<0.05, t-test).107(A) (B)1. 8X^3Ca-4W^0 ^CON 4-AP 4-AP^CON 4-AP 4-AP50 uM 500 uM 50 uM 500 uMFig 13: Effects of 4-aminopyridine on the rate constant of 86 Rb efflux and the negativeinotropic response to carbachol in the presence of phenylephrine in electrically-stimulated rabbit left atria.Effects of carbachol were monitored in the presence of phenylephrine on thecontractile force (A) and the rate constant of 86 Rb efflux (B) in electrically-stimulatedrabbit left atrial strips. Open bars represent the response to phenylephrine (10011.M) aloneand hatched bars represent the response to carbachol (10 p.M) in combination withphenylephrine, in the absence (CON) and presence of 50 and 500 AM 4-aminopyridine(4-AP 50 and 50011M). Tissues were treated with 4-aminopyridine for 24 min, andphenylephrine for 16 min, with carbachol being added for the final 8 min. Contractileresponse is expressed as the attained tension in grams and efflux is expressed as theefflux rate constant. Each bar represents the mean ± S.E.M. of 10-11 experiments. (*)Represents significantly different from the corresponding response immediately prior toaddition of carbachol (P<0.05, paired t-test). (+) Represents significantly different fromcarbachol response in the control group (P<0.05, t-test).109N0.0CO1.51.00 5(A)4-AP 4-AP50 uM 500 uM2.0 - (B)1 5 -*,0CON 4-AP 4-AP50 uM 500 uM\\N^Fig 14: Effects of pre-treatment of rabbits with pertussis toxin on the rate constant of 86Rb efflux and the negative inotropic response to carbachol in electrically-stimulatedrabbit left atria.Effects of carbachol were monitored on the contractile force (A) and the rateconstant of 86Rb efflux (B) in left atrial strips from saline (o), 0.5 (o) or 1 gg/kg (v)pertussis toxin pre-treated rabbits. Atrial strips were exposed to a concentration ofcarbachol dissolved in 17 ml of M.C.K. solution for 8 min during which the tissue waswashed every 2 min with non-radioactive M.C.K. buffer containing the drug and theforce of contraction of the tissue was also monitored. In order for the contractile responseof the atrial strip to stabilize to the pre-drug treatment level, the atrial strip wasequilibrated for 40 min in 17 ml of the non-radioactive M.C.K. buffer which waschanged and collected for counting every 20 min. After the 40 min washout period, thetissue was again washed every 2 min with the non-radioactive M.C.K. solution foranother 10 min, followed by exposure to another concentration of carbachol as describedabove. Data are expressed as percent of the initial efflux or force of contractionimmediately prior to addition of each concentration of carbachol. Each curve representsthe mean ± S.E.M. of 4 - 5 experiments.111__JOG CARBAC'OL12007) 10050604, 400200160( B)7 61003^8 57 6 45(A)Fig. 15: Effects of pre-treatment of rabbits with pertussis toxin on carbachol-inducedchanges in the contractile force and the rate constant of 86Rb efflux in the presence ofphenylephrine in electrically-stimulated rabbit left atrial strips.Force of contraction (A) and the rate constant of 86Rb efflux (B) were monitoredin left atrial strips from saline (S), and pertussis toxin 0.5 (P 0.5) and 1 lig/kg (P 1.0)pre-treated rabbits in the presence of 100 .tM phenylephrine alone (open bars) or with 10p.M carbachol in combination with phenylephrine (hatched bars). Atria were treated withphenylephrine for 16 min, with carbachol being added for the final 8 min. Contractileresponse is expressed as the attained tension in grams and rubidium efflux is expressedas the efflux rate constant. Each bar represents the mean ± S.E.M. of 4-6 experiments. (*)Represents significantly different from the response immediately prior to addition ofcarbachol (P<0.05, paired t-test).113-1.^-■N)^01TENSION (G)0^0O^(3) ^NJ-^CO1EFFLUX RATE CONSTANT (min 1)0^,,^(x JO 3) co1^1i^1^1^1cn//*    -ci 0  ^1  ^IAFig. 16: Effects of 4-aminopyridine and pertussis toxin on cAMP levels in electrically-stimulated left atrial strips from saline and pertussis toxin pre-treated rabbits in thepresence of isoproterenol alone and in combination with carbachol.(A). Atrial strips from saline (CON) and 2.2 pg/kg pertussis toxin pre-treated(PTX) rabbits were exposed to 100 nM isoproterenol alone for 6 min, or to 100 nMisoproterenol for 6 min with 3 gM carbachol being added for the final 3 min. (B) Atrialstrips were exposed to 500 1.1.M 4-aminopyridine (4-AP) or the M.C.K. solution (CON)for 10 min. Atria were then treated with isoproterenol and carbachol as described in partA. Data are expressed as pmol cAMP / g wet weight. Open bars represent theisoproterenol response and hatched bars represent responses in the presence ofisoproterenol in combination with carbachol. Each bar represents the mean ± S.E.M. of 4- 6 experiments. (*) Represents significantly different from isoproterenol alone.115CON PTX(2.2 uG/KG)CON 4-APFig. 17: Effects of carbachol on isoproterenol-stimulated adenylate cyclase activity inatrial homogenates from saline and pertussis toxin pre-treated rabbits.Adenylate cyclase activity was measured in atrial homogenates from saline(CON) and 2.2 lig/kg pertussis toxin (PTX) pre-treated rabbits in the presence ofisoproterenol (100 j.tM) alone and in combination with carbachol (1 mM). Open barsrepresent the isoproterenol response and hatched bars represent isoproterenol incombination with carbachol. Data are expressed as change from the basal adenylatecyclase activity which was 115.4 ± 18.5 (n=6) in saline and 139.4 ± 7 (n=3) in pertussistoxin pre-treated rabbits Each bar represents the mean ± S.E.M. (*) Representssignificantly different from isoproterenol alone.117Fig. 18: Effects of 4-aminopyridine and pertussis toxin, alone and in combination, onnegative inotropic responses to carbachol in electrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to carbachol were obtained in theabsence (a) and presence of 4-aminopyridine 50 (v) or 500 f.tM (A) in left atrial stripsfrom saline (A) and pertussis toxin (B) pre-treated rabbits. Atrial strips were exposed to4-aminopyridine for 10 min before carbachol concentration-response curves wereobtained. B represents the basal developed tension prior to addition of any drug. In panelA, the contractile force of atria treated with normal saline, 50 and 500 1.1,M 4-aminopyridine was 0.8 ± 0.2 g (n=9), 1.0 ± 0.1 g (n=14) and 1.3 ± 0.1 g (n=15). In panelB, the contractile response of atria treated with saline, 50 and 500 mM 4-aminopyridinewas 0.6 ± 0.1 g (n=7), 1.1 ± 0.2 (n=7) and 1.4 ± 0.3 (n=7) respectively. Data areexpressed as the developed tension in grams. Each data point represents the mean ±S .E.M.1192.5 (B) 2.01.51.00.50.0B 8^7^6^52.5 - (A)2.01.5z0'--C7z 1 0H0,50.0B^8^7^6^5^4)- LOG CA-RBACHOL (1v-Fig. 19: Effects of 4-aminopyridine and pertussis toxin, alone and in combination, onnegative inotropic responses to carbachol in the presence of isoproterenol in electrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to carbachol were obtained in thepresence of 100 nM isoproterenol alone (o) and in combination with 50 (v) or 500 1.IM( A) 4-aminopyridine in left atrial strips from saline (A) and pertussis toxin (B) pre-treated rabbits. Atrial strips were exposed to 4-aminopyridine for 10 min withisoproterenol being added for the final 3 min. Data are expressed as the developedtension in grams. Each curve represents the mean ± S.E.M. B represents the basaldeveloped tension prior to addition of any drug. I represents the isoproterenol-inducedincreases in contractile response prior to addition of carbachol. In panel A, the positiveinotropic response to isoproterenol in atria treated with normal saline, 50 and 500 tM 4-aminopyridine was 1.9 ± 0.4 (n=6), 1.8 ± 0.3 (n=7) and 2.1 ± 0.4 (n=6) respectively. Inpanel B, the contractile response to isoproterenol in atria treated with saline, 50 and 5001.1M 4-aminopyridine was 2.0 ± 0.3 g (n=4), 2.3 ± 0.4 g (n=4) and 2.5 ± 0.2 g (n=5)respectively.1212 01 . 50Cip1.0WH0.50 .03.02.5(A)T3,0 -2.52.01 .51,00.5(B)1 ^0B I 8^7^6^5^4 B I 8^7^6^5- LOG CARBACF_OL CFig. 20: Effects of 4-aminopyridine and pertussis toxin, alone and in combination, onnegative inotropic responses to carbachol in the presence of forskolin in electrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to carbachol were obtained in thepresence of 3 1.1M forskolin alone (o) and in combination with 50 (v) or 500 1AM (o,) 4-aminopyridine in atrial strips from saline (A) and pertussis toxin (B) pre-treated rabbits.Atrial strips were exposed to forskolin for 16 min with 4-aminopyridine being added forthe final 10 min. Data are expressed as the developed tension in grams. Each curverepresents the mean ± S.E.M. B represents the basal developed tension prior to additionof any drug. F represents the forskolin-induced increases in contractile response prior toaddition of carbachol. In panel A, forskolin-induced positive inotropic response in atriatreated with normal saline, 50 and 500 tiM 4-aminopyridine was 1.2 ± 0.2 g (n=11), 1.7 ±0.2 g (n=10) and 1.9 ± 0.2 g (n=9) respectively. In panel B, the contractile response ofleft atria to forskolin in the presence of normal saline, 50 and 500 gM 4-aminopyridinewas 1.3 ± 0.2 (n=7), 1.6 ± 0.2 (n=7) and 1.7 ± 0.3 (n=6) respectively.123B )2 5 (A) 2 01 .50.52.52.01.51.00.5- -0,0B F 8^I^0 , 07^6^5 4 B F 8^7^6^5- LOG CARBAC 0 L (Fig. 21: Effects of 4-aminopyridine and pertussis toxin, alone and in combination, onnegative inotropic responses to carbachol in the presence of IBMX in electrically-stimulated rabbit left atrial strips.Cumulative concentration-response curves to carbachol were obtained in thepresence of 50 p.M IBMX alone (o) and in combination with 50 (v) or 500 ii.M (o) 4-aminopyridine in atrial strips from saline (A) and pertussis toxin (B) pre-treated rabbits.Atrial strips were exposed to IBMX for 16 min with 4-aminopyridine being added for thefinal 10 min. Data are expressed as the developed tension in grams. Each curverepresents the mean ± S.E.M. B represents the basal developed tension prior to additionof any drug. X represents the IBMX-induced increases in force of contraction prior toaddition of carbachol. In panel A, the IBMX-induced positive inotropic response in atriatreated with normal saline, 50 and 500 µM 4-aminopyridine was 1.1 ± 0.2 g (n=12) 1.0 ±0.3 g (n=8) and 1.5 ± 0.1 g (n=10) respectively. In panel B, the positive inotropicresponse of atria to IBMX in the presence of saline, 50 and 500 i.tM 4-aminopyridine was1.2 ± 0.2 g (n=7), 1.7 ± 0.1 g (n=7) and 1.9 ± 0.2 g (n=5) respectively.1252 5 (A )2 0(B)hT T^T,21-T\ I-1. 1^.c9\T,zOC7)^1.00.51 52.5 -6^5^4^B X 8^7^6^5- LOG CARBACHOL (v2.01.51,00.5Fig. 22: Autoradiogram showing the ADP-ribosylating effect of exogenous pertussistoxin in rabbit atrial and ventricular homogenates.Atrial and ventricular homogenates (50 - 70 .tg protein) were incubated in theabsence (-) or presence of increasing concentrations (10 - 100 p.g/m1) of pertussis toxin .Reaction mixtures were subjected to gel electrophoresis followed by autoradiography.The numbers 97 - 31 indicate the position of proteins of known molecular weight (kDa)on the gel.12797►66►45►31►PTX^10 30 so 70 100(U9/M1)10 20 30 100ATRIUM^VENTRICLEFig. 23: Autoradiogram showing the effect of pre-treatment of rabbits with pertussistoxin on the ADP-ribosylation of G-proteins in atrial homogenates by exogenouspertussis toxin.Atrial homogenates (50 - 70 jig protein) from rabbits pre-treated with normalsaline (-) or 0.5, 1, 2 or 3 µg/kg pertussis toxin were incubated in duplicate in the absence(-) or presence of 30 µg/ml pertussis toxin. The reaction mixture was subjected toelectrophoresis followed by autoradiography. The numbers 97 - 31 indicate the positionof proteins of known molecular weight (kDa) on the gel.129PTXIn-vivo - - - 0.5 0-5(pgikg)In-vit roChtgimi)97►66145►31►1 1 2^2 3 330 30 30 30 30 30- 30 30 30 30130Fig. 24: Autoradiogram showing the effect of pre-treatment of rabbits with pertussistoxin on the ADP-ribosylation of G-proteins in ventricular homogenates by exogenouspertussis toxin.Ventricular homogenates (50 - 70 pg protein) from rabbits pre-treated withnormal saline (-) or 0.5, 1, 2 or 3 pg/kg pertussis toxin were incubated in duplicate in theabsence (-) or presence of 30 µg/ml pertussis toxin. The numbers 97 - 31 indicate theposition of proteins of known molecular weight (kDa) on the gel.131PTXIn-ViVO019/kg) - - 0.5 1 2 3^- 0.5 1^2 3In-vitro01,9111D - 30 30 30 30 30^30 30 30 30 3097,66045r31►132Fig. 25: Concentration-response effects of isoproterenol on adenylate cyclase activity inatrial and ventricular homogenates or rabbit.Atrial (v) and ventricular (•) homogenates were incubated in the presence ofincreasing concentrations of isoproterenol for 10 min. Adenylate cyclase activity isexpressed as percent of the basal activity which was 130 ± 26.8 in atria and 179.8 ± 29.2(pmol/min/mg) in ventricles. Each curve represents the mean ± S.E.M. of 4 experimentsdone in duplicate using atrial and ventricular homogenates from 4 different rabbits.133Fig. 26: Effects of timolol on the isoproterenol-stimulated adenylate cyclase activity inrabbit atrial and ventricular homogenates.Atrial and ventricular homogenates were incubated with 100 1.1M isoproterenol(solid bars), 1 p.M timolol (open bars) or isoproterenol plus timolol (hatched bars), for 10min. Data are expressed as change from the basal adenylate cyclase activity which was130 ± 26.8 in atria and 179.8 ± 29.2 (pmolimin/mg) in ventricles respectively. Each barrepresents the mean ± S.E.M. of 3 - 4 separate experiments done in duplicate in atrial andventricular homogenates obtained from separate rabbits. (*) Represents significantlydifferent from isoproterenol alone (paired t-test; P<0.05).135z:10400~0 300Cf)2000:1E'^100uLT-1-U)^0100500VENTRICLE^ATRIUMFig. 27: Concentration-response curves to carbachol in the absence and presence ofisoproterenol in rabbit atrial and ventricular homogenates.Atrial ( A ) and ventricular ( o ) homogenates were incubated with increasingconcentrations of carbachol in the absence (A) and in the presence (B) of 1 j.IM(ventricle) or 100 i_tM (atria) isoproterenol for 10 min. Data are expressed as percent ofthe basal adenylate cyclase activity, which is shown in Table 6. I represents the responseto isoproterenol alone. The effect of carbachol on basal adenylate cyclase activity wasdetermined in duplicate in atrial and ventricular homogenates from 13 and 10 differentrabbits, respectively. The effect of carbachol on isoproterenol-stimulated adenylatecyclase was determined in duplicate in atrial and ventricular homogenates from 15 and 7separate rabbits, respectively.13710080HUCf)Cr),60L-r-,40H204^3I^515016^5^ 00 4^3200it0120^(A)^250^(B)- LOG CARBACHOL (M)Fig. 28: Effects of atropine on the carbachol-induced inhibition of adenylate cyclaseactivities in the absence and presence of isoproterenol in rabbit atrial and ventricularhomogenates.(A). Atrial and ventricular homogenates were incubated with 1 mM carbachol(open bars) or 10 1.1M atropine (solid bars) alone or in combination (hatched bars) for 10min. The effect of atropine was tested in duplicate in atrial and ventricular homogenatesfrom 3 - 4 separate rabbits. The effect of atropine in combination with carbachol wastested in duplicate in 3 separate ventricles and 9 separate atria. (B). Atrial (n=11) andventricular (n=5) homogenates were incubated with isoproterenol alone (open bars) or incombination with either carbachol alone (hatched bars) or carbachol plus atropine (solidbars) for 10 min. Each assay was done in duplicate. Concentrations of isoproterenol usedwere 1 in ventricle and 100 1.1M in atrium. Data are expressed as change from thebasal adenylate cyclase activity (pmol/min/mg).139( B )VENTRICLE ATRIUM^ VENTRICLE^ATRIUMFig. 29. Effects of carbachol on forskolin- and GTPyS- stimulated adenylate cyclaseactivities in rabbit atrial and ventricular homogenates.Atrial and ventricular homogenates were incubated with 1 p.M forskolin or 10 ;AMGTPyS in the absence or presence of 1 mM carbachol. Data are expressed as change fromthe basal adenylate cyclase activity, which was 142.6 ± 15.3 (n=10) and 162.5 ± 22.2(n=8) pmol/min/mg, in atrial and ventricular homogenates exposed to forskolin. Thebasal adenylate cyclase activity in atrial and ventricular homogenates exposed to GTPySwas 106.9 ± 17.7 (n=7) and 156.9 ± 34.3 (n=5) pmol/min/mg, respectively. Each assaywas done in duplicate in atrial and ventricular homogenates prepared from separaterabbits.141(A) (B)r!TP ,■/VENTRICLE^ATRIUM^VENTRICLE^ATRIUMFig. 30: Effects of pertussis toxin pre-treatment of rabbits on the inhibitory effect ofcarbachol on adenylate cyclase activity in the absence and presence of isoproterenol inrabbit ventricular homogenates.Ventricular homogenates from rabbits pre-treated with normal saline (o), 1 (A), 2( v ) or 3 tg/kg (^) pertussis toxin were incubated with increasing concentrations ofcarbachol in the absence (A) and presence (B) of 1 tM isoproterenol. Data are expressedas percent of the basal activity, which is shown in table 8. Each curve represents themean ± S.E.M. of 6 - 7 separate experiments each done in duplicate in ventricularhomogenates from different rabbits.143V1I^5i 14 3(A) (B)›-4HII'-E:::^900W .----..Cf) ,.4 80i— cil00^70W 0H< Ni-4'- 60ZWQ50 — 1^1 i^1005 4^3300250200150- LOG CARBACHOL (M)Fig. 31: Effects of pre-treatment of rabbits with different doses of pertussis toxin on theinhibitory effect of carbachol on isoproterenol-induced increases in adenylate cyclaseactivity in rabbit atrial homogenates.Atrial homogenates from saline (A) (n=15), 0.5 (B) (n=6), 1 (C) (n=6), 2 (D)(n=7) and 3 pg/kg (E) (n=6) pertussis toxin pre-treated rabbits were incubated with 1001.1M isoproterenol alone (open bars) or in combination with 1 mM carbachol (hatchedbars) for 10 min. Each bar represents the mean ± S.E.M. of adenylate cyclase activity,expressed as percent of basal activity (table 8), done in duplicate in atrial homogenatesfrom separate rabbits.145A B CDDISCUSSIONThe results of the present study suggest that: (a) the ability of carbachol to openpotassium channels contributes to the negative inotropic responses to carbachol in thepresence of phenylephrine, forskolin and IBMX in the rabbit left atrium, and (b) pertussistoxin is more effective in uncoupling muscarinic receptors from adenylate cyclase in atriathan either muscarinic receptors from potassium channels in atria or muscarinic receptorsfrom adenylate cyclase in ventricles.It is well established that in the atrial myocardium muscarinic receptors are linkedto potassium channels by means of a pertussis toxin-sensitive G-protein (Fleming et al.,1992). The cyclic nucleotide-independent direct negative inotropic response tomuscarinic receptor stimulation in the atrial myocardium is believed to be related to theability of muscarinic agonists to open potassium channels (Ten Eick et al., 1976; Cerbaiet al., 1988). One of the objectives of the present study was to investigate the contributionof potassium channel opening in the functional interaction of carbachol with a variety ofcAMP-elevating and cAMP-independent positive inotropic agents in rabbit left atria.Phenylephrine, an fa-adrenoceptor agonist, was used as a cAMP-independent positiveinotropic agent. The cAMP-elevating agents used in the study were isoproterenol, a 0-adrenoceptor agonist, forskolin, a direct activator of adenylate cyclase and IBMX, aphosphodiesterase inhibitor.1474.1. DIRECT NEGATIVE INOTROPIC RESPONSE TO CARBACHOLIn this series of experiments, 4-aminopyridine and pertussis toxin were used tointerfere with the ability of carbachol to open potassium channels. In the present study, 4-aminopyridine, in the concentration range of 1 - 5 mM exerted a concentration-dependent positive inotropic response in rabbit left atria, in agreement with previousreports (Yanagisawa and Taira, 1979; Wollmer et al., 1981). Based on preliminaryexperiments, the effects of two different concentrations of 4-aminopyridine (50 and 500i.tM) were studied on the negative inotropic responses to carbacnol in the presence andabsence of various positive inotropic agents. It has been reported previously that 4-aminopyridine in high concentrations is capable of displacing muscarinic agonists fromtheir receptor sites (Drukarch et al. ,1989; Urquhart and Broadley, 1991). In order toinvestigate this possibility, the effect of 500 i.tM 4-aminopyridine on the ability ofcarbachol to inhibit the isoproterenol-stimulated cAMP generation was tested. 4-Aminopyridine did not interfere with the ability of carbachol to inhibit the isoproterenol-stimulated cAMP generation, suggesting that 4-aminopyridine was not acting at the levelof muscarinic receptors.In the present study, carbachol exerted a concentration-dependent negativeinotropic response which was attenuated by 4-aminopyridine in a concentration-dependent manner. This was in agreement with previous reports of De Biasi et al (1989)and Urquhart and Broadley (1991). However, 500 1.1M 4-aminopyridine did notcompletely attenuate the negative inotropic response to carbachol suggesting that either500 4-aminopyridine is not capable of completely blocking the carbachol-stimulated148potassium current or activation of the potassium current may not completely explain thedirect negative inotropic response to carbachol in left atria.In order to obtain more direct evidence for the carbachol-stimulated opening ofpotassium channels and its contribution to the negative inotropic response to carbachol,we monitored the effects of carbachol on the rate-constant of 86 Rb-efflux andcontractile response in the same electrically-stimulated left atria. Carbachol produced anincrease in the efflux-rate-constant of 86 Rb from rabbit left atrial strips which wasblocked by atropine and was attenuated by pre-treatment of rabbits with pertussis toxin.In agreement with previous reports in rat (Quast et al., 1988) and guinea pig (Urquhart etal., 1991) atria, this suggests that in the rabbit left atrium, muscarinic receptors arecoupled to potassium channels by means of a pertussis toxin-sensitive G-protein.Carbachol exerted a direct negative inotropic response over the sameconcentration-range that it increased the 86Rb-efflux-rate-constant. However, the lowestconcentration of carbachol used caused a very marked decrease in the left atrial tension,while producing only a very small increase in the rate-constant of 86Rb-efflux. Therelative effects of carbachol on the tension and 86Rb-efflux appear to be very similar tothose reported in rat left atria (Quast et al, 1988). It is possible that either a very smallincrease in the potassium-efflux can produce a very marked effect on the tension, or thatchanges in the inotropic response can be measured with greater sensitivity than changesin the potassium-efflux with the methods employed, as has been suggested to occur invascular smooth muscle (Smith et al, 1986; Quast and Baumlin, 1988). Thesepossibilities seem unlikely, since Urquhart et al (1991) have shown that a goodcorrelation exists between the negative inotropic response and increases in the rate-constant of 86Rb-efflux in response to adenosine receptor stimulation in guinea pig left149atria. A third possibility is that mechanisms other than increases in the potassium-effluxcontribute to the direct negative inotropic response to carbachol. To further investigatethis, we used 4-aminopyridine and pertussis toxin to interfere with the ability ofcarbachol to open potassium channels.4-Aminopyridine alone had no significant effect on the basal 86Rb-efflux-rateconstant, but it reduced the increases in the rate-constant of 86 Rb-efflux produced bycarbachol in rabbit left atria. However, a concentration of 4-aminopyridine (500 ptM)which completely blocked the carbachol-induced increase in the efflux-rate-constant,only partially attenuated the direct negative inotropic response to carbachol. This, inagreement with our observation in functional studies, suggests that some process inaddition to the increased potassium-conductance contributes to the direct negativeinotropic response of rabbit left atria to carbachol.Pre-treatment of rabbits with 0.5 and 1 tg/kg pertussis toxin resulted in a markedreduction of the ability of 1 1.1.M carbachol to exert a negative inotropic response and toincrease the rate-constant of 86 Rb-efflux. However, 0.5 jig/kg pertussis toxin appearedto cause a relatively greater depression of the increase in the 86 Rb-efflux-rate-constantthan the negative inotropic effect produced by 1 11M carbachol. Increasing the dose ofpertussis toxin to 1 µg/kg did not result in any greater inhibition of the ability of 1 iiMcarbachol to promote the 86 Rb-efflux, but the negative inotropic response of left atria to1 ptM carbachol was further attenuated. These data are consistent with those obtainedwith 4-aminopyridine and further suggest that some mechanism in addition to increasesin the potassium-conductance contributes to the direct negative inotropic response of leftatria to muscarinic receptor stimulation. No further decrease in the tension was observedin presence of 10 1.1.M carbachol in atria from the pertussis toxin-treated rabbits, although150this concentration of carbachol produced a much greater increase in the 86Rb-efflux-rate-constant than 1 tM carbachol. Instead, 10 ptM carbachol had a tendency to reversethe negative inotropic response observed in the presence of 1 i.tM carbachol in atria fromboth saline and pertussis toxin pre-treated rabbits. Increasing the concentration ofcarbachol to 100 i.tM produced an even greater reversal of the negative inotropic responseto carbachol. It is well established that muscarinic agonists in concentrations of 10 f.t.Mand higher can promote phosphoinositide turnover and exert a positive inotropic responseby a pertussis toxin-insensitive mechanism (Tajima et al, 1987; see Pappano, 1990). It ispossible that this effect might have contributed to the reversal of the negative inotropicresponse seen with 10 and 100 I_IM carbachol in the present investigation.The mechanism by which carbachol could produce a potassium channel-independent direct negative inotropic response is not known. Previous studies have ruledout the involvement of cAMP (Endoh et al., 1985; MacLeod, 1986; Ray and MacLeod,1990) and cGMP (MacLeod and Diamond, 1986) in this process. It is possible that thepertussis toxin- and 4-aminopyridine- insensitive component of the direct negativeinotropic response to carbachol may be related to the ability of muscarinic agonists toinhibit the calcium current directly (Ten Eick et al, 1976; Cerbai et al, 1988).4.2 INTERACTION OF CARBACHOL WITH PHElVYLEPHRINE ANDISOPROTERENOLCarbachol completely reversed the positive inotropic response of rabbit left atriato phenylephrine. 4-Aminopyridine produced a concentration-dependent antagonism ofthe negative inotropic response to carbachol in the presence of phenylephrine, which may151suggest that the muscarinic receptor-induced activation of potassium current may explainthe reversal by carbachol of the positive inotropic response to phenylephrine.When the ability of carbachol to promote the 86 Rb-efflux was monitored in thepresence of phenylephrine, carbachol produced an increase in the 86Rb-efflux-rateconstant and a reduction in tension in the presence of phenylephrine. 4-Aminopyridine(50 and 500 ).1M) attenuated the negative inotropic responses to carbachol in the presenceof phenylephrine but were not very effective in blocking the increases in the carbachol-stimulated 86 Rb-efflux-rate-constant. The reason why the carbachol-stimulated 86Rb-efflux was inhibited to a lesser extent in the presence of the combination of 4-aminopyridine and phenylephrine than in the presence of 4-aminopyridine alone isunknown.The same concentration of pertussis toxin which had little effect on the increasesin the 86Rb-efflux-rate-constant in response to 10 .tM carbachol alone, completelyblocked the increases in the rate-constant of 86Rb-efflux produced by 10 1.IM carbacholin the presence of phenylephrine. This was associated with almost complete loss of thereversal by carbachol of the positive inotropic responses to phenylephrine. These resultsare consistent with a role for the increases in the potassium-efflux in the inotropicresponses to carbachol in the presence of phenylephrine.4-Aminopyridine was found to exert a greater attenuating effect on the responseto carbachol in the presence of phenylephrine than on either the direct negative inotropicresponse to carbachol or the negative inotropic responses to carbachol in the presence ofisoproterenol, forskolin or IBMX (see below). A similar observation was also made in thepresent as well as in an earlier study (Ray and MacLeod, 1990) concerning the inhibitoryeffect of pertussis toxin on the negative inotropic response to carbachol in the presence of152phenylephrine. The explanation for this observation is unclear at present. However, itcould be related to the ability of phenylephrine to block the outward potassium current(Apkon and Nerbonne, 1988; Braun et al., 1990; Fedida and Bouchard, 1992) which isbelieved to contribute to the a-adrenoceptor-mediated positive inotropy. The greaterinhibitory effect of pertussis toxin and 4-aminopyridine on the responses to carbachol inthe presence of phenylephrine may be due to an additive effect between pertussis toxin or4-aminopyridine and phenylephrine, resulting in greater antagonism of the carbachol-stimulated potassium-efflux.Carbachol also antagonized the positive inotropic responses of left atria toisoproterenol in a concentration-dependent manner. The response to carbachol in thepresence of isoproterenol was only slightly inhibited by 4-aminopyridine. It is wellestablished that carbachol can inhibit the isoproterenol-stimulated adenylate cyclaseactivity and cAMP generation (Endoh et al., 1985; Sorota et al., 1985; MacLeod, 1986;Ray and MacLeod, 1992; also the present study). Our results suggest that because theinhibitory effect of carbachol on the isoproterenol-stimulated cAMP generationcontributes to the functional interaction between isoproterenol and carbachol, carbacholretained its inhibitory effect on the positive inotropic response to isoproterenol even inthe presence of the potassium channel blockade.4.3. EFFECTS OF PINACIDIL AND CROMAKALIMUsing a different approach, the effects of potassium channel openers on theability of phenylephrine and isoproterenol to exert positive inotropic responses werestudied. It was argued that if carbachol antagonized the effects of these positive inotropic153agents by opening potassium channels, then potassium channel agonists should alsoantagonize the development of positive inotropic responses to isoproterenol andphenylephrine. Pinacidil and cromakalim, two known agonists of the ATP-dependentpotassium channel (Cook, 1988), were used in the study. Both pinacidil and cromakalimantagonized the phenylephrine-induced positive inotropic response in a concentration-dependent manner. This, in agreement with our 4-aminopyridine data, suggests thatactivation of the potassium current contributes to the negative inotropic responses of leftatria to carbachol in the presence of phenylephrine. In contrast, neither pinacidil norcromakalim had any effect on the positive inotropic responses to isoproterenol,suggesting that inhibition of the isoproterenol-stimulated adenylate cyclase activity bycarbachol plays a more dominant role in this interaction.4.4. INTERACTION OF CARBACHOL WITH FORSKOLIN AND IBMXResults from this (MacLeod and Diamond, 1986; Ray and MacLeod, 1992) andother laboratories (Brown, 1979; Biegon et al., 1980; Pappano et al., 1982; Lindemannand Watanabe, 1985; Schmied and Korth, 1990) have shown that muscarinic agonistseither do not inhibit the forskolin- or IBMX- induced increases in cAMP levels or thatmuscarinic antagonism of the positive inotropic responses of left atria to forskolin andIBMX occurs independently of changes in the forskolin- or IBMX- induced increases incAMP levels. However, others have demonstrated that muscarinic agonists can reducethe forskolin- and IBMX- stimulated adenylate cyclase activity and cAMP levels in theatrial myocardium (Brown et al 1980; Sulakhe et al., 1985). It has also been argued thatcarbachol may only reduce adenylate cyclase activity and cAMP levels elevated by154forskolin in a small compartment of the heart, linked to the inotropic response, which isnot detectable when total tissue levels of cAMP or adenylate cyclase activity aremeasured (Hartzell, 1988). In order to rule out the possible involvement of muscarinicinhibition of adenylate cyclase in the functional interaction of carbachol with forskolinand IBMX, muscarinic receptors were uncoupled from adenylate cyclase using pertussistoxin. In rabbits pre-treated with 2.2 jig/kg pertussis toxin, carbachol lost its inhibitoryeffect on the isoproterenol-induced increases in adenylate cyclase activity and cAMPlevels, suggesting that muscarinic receptors were completely uncoupled from adenylatecyclase. Under this circumstance, consistent with previous reports (Endoh et al., 1985;Ray and MacLeod, 1992), pertussis toxin pre-treatment of rabbits resulted in apronounced attenuation of the negative inotropic response to carbachol in the presence ofisproterenol. At the same time, however, pertussis toxin had relatively little attenuatingeffect on the inhibition by carbachol of the positive inotropic responses to forskolin andIBMX, and pertussis toxin did not have any inhibitory effect on the direct negativeinotropic response to carbachol. This, in agreement with a previous report from thislaboratory (Ray and MacLeod, 1992), may suggest that inhibition of the forskolin- orIBMX- induced increases in cAMP levels does not play an important role in thefunctional interaction of carbachol with these positive inotropic agents.The mechanism of this cAMP-independent component of the negative inotropicresponse to carbachol in the presence of forskolin and IBMX was investigated using 4-aminopyridine in saline and pertussis toxin pre-treated rabbits. 4-Aminopyridineproduced a concentration-dependent attenuation of the response to carbachol in thepresence of forskolin in atria from saline-treated animals. The magnitude of theinhibitory effect of 4-aminopyridine on both the direct negative inotropic response to155carbachol and on the response to carbachol in the presence of forskolin was very similarin atria from saline and pertussis toxin-treated animals. This is consistent with a role foractivation of potassium channels in mediating the response to carbachol in the presenceof forskolin. The response of atria from saline-treated rabbits to carbachol in thepresence of IBMX appeared to be more resistant to inhibition by 4-aminopyridine thaneither the direct negative inotropic response to carbachol or the response to this agonist inthe presence of forskolin. The reason for this is not clear. However, it is possible that aninhibitory effect of 4-aminopyridine was at least partially masked by the effectiveness ofcarbachol in reversing the response to IBMX, since the majority of atria (7/13 or 54%)from saline-treated rabbits stopped beating in the presence of the maximum concentrationof carbachol plus IBMX. The fact that only 2 out of 7 atria (29%) stopped beating inresponse to carbachol in the presence of IBMX following treatment of atria with 50 ii,M4-aminopyridine suggests that this may be the case. This is further supported by the dataobtained in atria from pertussis toxin-treated rabbits, in which carbachol was still veryeffective in reversing the response to IBMX, but arrested only one atrium out of a total ofseven atria used in the study. Under these circumstances, 4-aminopyridine exerted amore marked inhibitory effect on the response to carbachol in the presence of IBMX,similar in magnitude to the inhibitory effect of 4-aminopyridine on the direct negativeinotropic response to carbachol. These data are also consistent with a role for themuscarinic receptor-mediated activation of potassium current in the reversal by carbacholof the positive inotropic response to IBMX.However, it should be noted that the higher concentration of 4-aminopyridineused did not attenuate completely the response to carbachol in the presence of IBMX andforskolin. This suggests that some mechanism in addition to potassium channel156activation, eg. activation of protein phosphatase (Ahmad et al., 1989) and calciumchannel inhibition (TenEick et al., 1976; Cerbai et al., 1988), may also contribute to theseresponses to carbachol.It is not clear how pertussis toxin attenuated the negative inotropic responses tocarbachol in the presence of forskolin and IBMX, although it did not have any inhibitoryeffect on the direct negative inotropic response to carbachol. There could be severalpossible explanations. Firstly, it is possible that a small uncoupling of muscarinicreceptors from potassium channels might have been sufficient to attenuate the negativeinotropic responses to carbachol in the presence of IBMX and forskolin but wasinadequate to attenuate the direct negative inotropic response to carbachol. A secondpossibility could be that some effector other than adenylate cyclase and potassiumchannels, eg. phosphatase, might have been uncoupled from muscarinic receptorsresulting in a greater attenuation of the inhibitory effect of carbachol in the presence offorskolin and IBMX. Lastly, it is possible that uncoupling of adenylate cyclase frommuscarinic receptors in a compartment not detectable in the whole tissue cAMP assaymight have contributed to this greater attenuation of the inhibitory responses to carbacholin the presence of forskolin and IBMX.In contrast to forskolin or IBMX, the negative inotropic responses to carbachol inthe presence of isoproterenol was affected very modestly by 4-aminopyridine. This isagain consistent with our data discussed in the previous section and the pertussis toxindata discussed in this section, and suggests a dominant role of muscarinic inhibition ofadenylate cyclase in the interaction. However, in the present investigation pertussistoxin, although completely uncoupling muscarinic receptors from adenylate cyclase, didnot completely inhibit the ability of carbachol to reverse the positive inotropic response157to isoproterenol. The response to carbachol in the presence of isoproterenol thatremained in pertussis toxin-treated tissues was further inhibited, in a concentration-dependent manner, by 4-aminopyridine. This suggests that although inhibition of theisoproterenol-stimulated cAMP generation plays an important role in the functionalinteraction of isoproterenol and carbachol, some of the negative inotropic response tocarbachol in the presence of isoproterenol may depend on the ability of carbachol to openpotassium channels. This is in agreement with previous reports that inhibition of TT) C J3-adrenoceptor-stimulated increases in adenylate cyclase activity is not sufficient tocompletely explain the inhibition by muscarinic agonists of the positive inotropicresponse to P-adrenoceptor stimulation (Brown et al., 1980; Endoh et al., 1985;MacLeod, 1986).4.5. DIFFERENTIAL EFFECTS OF PERTUSSIS TOXINWe have reported in an earlier study (Ray and MacLeod, 1992) that injection ofrabbits with pertussis toxin resulting in complete loss of the ability of carbachol toinhibit the isoproterenol-stimulated increases in cAMP levels and positive inotropy onlypartially affected the cAMP-independent responses to carbachol such as the directnegative inotropic effect and the inhibitory effect on the forskolin-induced positiveinotropy. In the present study, a similar result was obtained when it was observed that 2.2lig/kg pertussis toxin uncoupled muscarinic receptors from adenylate cyclase but had noinhibitory effect on the direct negative inotropic response to carbachol and only partiallyinhibited the ability of carbachol to inhibit the forskolin- and IBMX- induced positiveinotropy. Since our data suggests that these cAMP-independent responses to carbachol158are related, at least in part, to the ability of carbachol to open potassium channels, weinvestigated the possible differential uncoupling of muscarinic receptors from potassiumchannels and adenylate cyclase in the atrial myocardium. We had also found that a doseof pertussis toxin which completely attenuated the inhibitory effect of carbachol on theisoproterenol-stimulated increases in cAMP levels and positive inotropy in the atrialmyocardium did not have any inhibitory effect on the negative inotropic response tocarbachol in the presence of isoproterenol in the rabbit right ventricular papillary muscle(Ray and MacLeod, unpublished observations). This prompted us to investigate if themuscarinic inhibition of adenylate cyclase in the atrium is more sensitive to theuncoupling effect of pertussis toxin than the muscarinic receptor-mediated inhibition ofadenylate cyclase in the ventricle. We have compared the effect of carbachol onadenylate cyclase activity in the presence and absence of isoproterenol in atrial andventricular homogenates from saline and pertussis toxin pre-treated rabbits. Theuncoupling effect of pertussis toxin on the carbachol-inhibited adenylate cyclase activitywas compared with the effect of pertussis toxin on the ability of carbachol to promote theefflux of 86 Rb from the left atrium of rabbits. The attenuating effects of pertussis toxinon the carbachol-stimulated 86 Rb-efflux-rate-constant and on carbachol-mediatedinhibition of adenylate cyclase activity in atria and ventricles were correlated with thedegree of ADP-ribosylation of G-proteins in these tissues.In the present study, carbachol did not have any effect on the basal adenylatecyclase activity but inhibited the enzyme activity stimulated by isoproterenol in the atrialhomogenates. This is in agreement with previous reports from this and other laboratoriesthat muscarinic agonists can inhibit the 13-adrenoceptor agonist-stimulated adenylatecyclase activity and cAMP generation but not the basal enzyme activity or cAMP levels159in atria (Sorota et al., 1985; MacLeod, 1986; Ray and MacLeod, 1990). Carbachol alsostimulated the rate constant of 86 Rb efflux in rabbit atrial strips (discussed in theprevious section). Pre-treatment of rabbits with 0.5 and 1 1.1g/kg pertussis toxin, whichADP-ribosylated approximately 60 - 70% of the atrial G-proteins, only partiallyattenuated the ability of carbachol to increase the rubidium-efflux-rate-constant.However, the same dose of pertussis toxin completely blocked the inhibition bycarbachol of the isoproterenol-stimulated adenylate cyclase in the atrium.The identity of the pertussis toxin-sensitive G-proteins present in the rabbit hearthas not been established, but studies have demonstrated the presence of four differentpertussis toxin-sensitive G-proteins, the 3 isoforms of Gi, Gil, Gi2 and GB, and Go inhearts of other mammalian species. Neither the identity of the G-protein(s) nor themechanism by which the G-protein(s) couple muscarinic receptors to adenylate cyclaseand potassium channels invivo are known. One possible explanation for our findings isthat two or more different G-proteins, which are not equally susceptible to ADP-ribosylation by pertussis toxin, may link muscarinic receptors to adenylate cyclase andpotassium channels. On the other hand, it is also possible that different subunits of thesame G-protein couple muscarinic receptors to adenylate cyclase and potassium channelsin the atrium, with the a-subunit directly activating the potassium channels and the flysubunits inhibiting the isoproterenol-stimulated adenylate cyclase by quenching the a-subunit of the stimulatory G-protein, as suggested by Brown (1990) and Robishaw andFoster (1990). This possibility was also proposed by Pappano and Mubagwa (1992) toexplain the differential desensitization they observed of the carbachol-activatedpotassium current and carbachol-mediated inhibition of the isoproterenol-stimulated slowinward calcium current in the guinea pig atrial myocardium. Recently, Matesic et al.160(1991) showed that muscarinic receptors in the rat atrium associate preferentially withGo , supporting the hypothesis that a single G-protein, Go , couples muscarinic receptorsto both adenylate cyclase and potassium channels in this tissue. If this is the case, thedifferential sensitivity of muscarinic responses in the atrium to pertussis toxin that weobserved could arise because on partial ADP-ribosylation of G o, the number of 137subunits produced is less than is required to promote the quenching of Gsa, whilesufficient a-subunits are still available to activate potassium channels. However, at thistime we cannot rule out the possibility of two different G-proteins coupling muscarinicreceptors to potassium channels and adenylate cyclase in the atrium.In the ventricle, carbachol inhibited both basal and isoproterenol-stimulatedadenylate cyclase activity in a concentration-dependent and atropine-sensitive manner, inagreement with previous reports in rabbit (Jakobs et al. 1979) and rat (Sulakhe et al.1985) ventricles. However, the muscarinic receptor-mediated inhibition of adenylatecyclase in the ventricle was less sensitive than that in the atrium to inhibition by pertussistoxin. There were no significant differences in the extent of invivo ADP ribosylation inatria and ventricles from rabbits injected with either 0.5 and 1 g.tg/kg pertussis toxin.However, carbachol completely lost its ability to inhibit the isoproterenol-stimulatedadenylate cyclase in atria from rabbits treated with 0.5 pg/kg pertussis toxin, which ADP-ribosylated 68% of G-proteins. In contrast, pre-treatment of rabbits with 1 µg/kg pertussistoxin, which ADP-ribosylated 60 % of the ventricular G-proteins, did not have any effecton the inhibition by carbachol of either basal or isoproterenol-stimulated adenylatecyclase. Increasing the dose of pertussis toxin to 3 jig/kg, which resulted in completeADP-ribosylation of the ventricular G-proteins, abolished the ability of carbachol toinhibit adenylate cyclase in the ventricle.161Matesic et al (1991) have demonstrated that in the rat ventricle, in contrast to theatrium, muscarinic receptors couple to two different G-proteins, Gil and Go. Both the a-subunit of Gi2 and the Py subunits of both Gi and G o could potentially contribute to themuscarinic receptor-mediated inhibition of adenylate cyclase in the ventricle. Thus, thedifferential sensitivity of the muscarinic receptor-mediated inhibition of adenylatecyclase found between the atrium and ventricle could arise because two different G-proteins and/or different subunits of the same G-protein convey the message ofmuscarinic receptor occupancy in the atrium and ventricle.The observation that muscarinic agonists can inhibit the basal adenylate cyclaseactivity in the ventricle but not in the atrium suggests that the mechanism of muscarinic-inhibition of adenylate cyclase may differ between the two tissues. We have furtherinvestigated this possibility by comparing the inhibitory effects of carbachol on theforskolin- and GTPyS- stimulated adenylate cyclase activity in the atrium and ventricle.Consistent with our observation on the basal adenylate cyclase activity, carbacholinhibited both forskolin- and GTPyS- stimulated adenylate cyclase activity in theventricle but not in the atrium. Watanabe and group (Fleming et al., 1987) havesuggested that muscarinic inhibition of the myocardial adenylate cyclase is indirect innature and occurs only when the enzyme activity is stimulated by a 0-adrenoceptoragonist. According to this model, Py subunits released from the inhibitory G-proteinssequester the G sa subunit and prevent it from stimulating the adenylate cyclase.However, the results of the present study suggest that Watanabe's model may be true inrabbit atria but not in the ventricle, where we cannot rule out the possibility of a directinhibition of the enzyme by either the a- or Py- subunits of the Gi proteins.162It is not clear why 2 µg/kg pertussis toxin did not attenuate the inhibitory effect ofcarbachol on the isoproterenol-stimulated adenylate cyclase activity in the atrium. Thislack of effect cannot be attributed to the lack of ADP-ribosylating effect of pertussistoxin, because in these same rabbits pertussis toxin ADP-ribosylated nearly 95% of G-proteins. It is well established in the heart that there are at least three different Gi and oneGo protein, all of which can potentially link muscarinic receptors to adenylate cyclase.The functional significance of all of these G proteins has not been clearly elucidated andwe cannot rule out a complicated interaction between different G proteins activated as aresult of muscarinic receptor stimulation. It is possible that ADP-ribosylation of G-proteins by pertussis toxin might have unmasked the inhibitory effect of some G-proteinwhose effects are otherwise not expressed.In summary, the results of the present study demonstrate that the muscarinicreceptor-mediated inhibition of adenylate cyclase in the atrium is more sensitive touncoupling by pertussis toxin than either the muscarinic receptor-mediated activation ofpotassium-efflux in the atrium or inhibition of adenylate cyclase in the ventricle. Inaddition, we have also observed that in the atrial myocardium carbachol can inhibit onlythe isoproterenol-stimulated adenylate cyclase, whereas in the ventricle carbachol inhibitsnot only the isoproterenol-stimulated but also basal, forskolin- and GTPyS- stimulatedadenylate cyclase activity. This suggests that there are differences in the coupling ofmuscarinic receptors with the adenylate cyclase and potassium channels in the atrium andventricle.163SUMMARY AND CONCLUSIONS5.1. SUMMARY1. The direct negative inotropic response to carbachol was attenuated in aconcentration-dependent manner by 4-aminopyridine, suggesting a role for themuscarinic receptor-activated potassium current in this process. However, the highestconcentration of 4-aminopyridine (500 p.M) used in the study did not completely inhibitthe direct negative inotropic response to carbachol.2. Carbachol promoted the 86 Rb-efflux from left atrial strips in an atropine-, 4-aminopyridine- and pertussis toxin-sensitive manner. However, although 500 .tM 4-aminopyridine completely attenuated the ability of carbachol to increase the rate-constantof 86 Rb-efflux, the direct negative inotropic response to carbachol was only partiallyinhibited by the same concentration of 4-aminopyridine. Similarly, pre-treatment ofrabbits with pertussis toxin (0.5 and 1 tg/kg) shared almost complete attenuation of the86 Rb-efflux-rate-constant increased by 1 .tM carbachol, but the negative inotropicresponse to 1 .tM carbachol was only partially attenuated. This suggests that only a partof the negative inotropic response to carbachol in rabbit left atrium is related to theability of carbachol to open potassium channels.3. The negative inotropic response to carbachol in the presence of phenylephrinewas also attenuated in a concentration-dependent manner by 4-aminopyridine. Carbacholincreased the rate constant of 86 Rb efflux in the presence of phenylephrine. Although 4-aminopyridine attenuated in a concentration-dependent manner the negative inotropicresponse to carbachol in the presence of phenylephrine, only 500 1.1M 4-aminopyridineslightly reduced the carbachol-induced increases in the 86 Rb-efflux-rate-constant in thepresence of phenylephrine. In contrast, pre-treatment of rabbits with pertussis toxin (0.5164and 1 µg/kg) resulted in complete loss of the ability of carbachol to increase the 86 Rb-efflux-rate-constant and inhibit the positive inotropic responses of left atria tophenylephrine. Similarly, the potassium channel openers, pinacidil and cromakalim,attenuated in a concentration-dependent manner the positive inotropic responses of leftatria to phenylephrine. Overall, the results suggest that the ability of carbachol to openpotassium channels contributes to the negative inotropic response to carbachol in thepresence of phenylephrine.4. The negative inotropic responses of left atria to carbachol in the presence ofisoproterenol was attenuated very modestly by 4-aminopyridine. Neither pinacidil norcromakalim had any inhibitory effect on the isoproterenol-induced positive inotropy.This suggests that inhibition by carbachol of the isoproterenol-stimulated cAMPgeneration plays a dominant role in the functional interaction of carbachol withisoproterenol.5. Injection of rabbits with 2.2 lig/kg pertussis toxin resulted in completeattenuation of the inhibitory effect of carbachol on the isoproterenol-stimulated adenylatecyclase activity and cAMP generation. Under this conditions, the negative inotropiceffects of carbachol in the presence of forskolin and IBMX were only partiallyattenuated. Pertussis toxin pre-treatment, however, had a much greater inhibitory effecton the negative inotropic response to carbachol in the presence of isoproterenol. Thissuggests that inhibition of cAMP generation may play an important role in theisoproterenol-carbachol interaction, but not in the functional interaction of carbachol withforskolin and IBMX.6. 4-Aminopyridine attenuated in a concentration-dependent manner the negativeinotropic responses of left atria to carbachol in the presence of forskolin and IBMX,165suggesting that the ability of carbachol to open potassium channels contributes to theinteraction of carbachol with forskolin and IBMX.7. In atrial homogenates, carbachol inhibited the isoproterenol-stimulatedadenylate cyclase in a concentration-dependent manner and this inhibition was sensitiveto blockade by atropine. Carbachol did not have any inhibitory effect on the basal,forskolin- or GTPyS- stimulated adenylate cyclase activity. In contrast, in the ventriclecarbachol inhibited not only the isoproterenol-stimulated but also basal, forskolin- andGTPyS- stimulated enzyme activity. This suggests that the mechanism of muscarinicinhibition of adenylate cyclase may differ between the atrium and ventricle.8. In atrial homogenates from rabbits pre-treated with 0.5 and 1 .tg/kg pertussistoxin, which ADP-ribosylated 68 - 70 % of G-proteins, carbachol lost its inhibitory effecton the isoproterenol-stimulated adenylate cyclase. The same dose of pertussis toxinshifted the carbachol-concentration-response curves for the 86 Rb-efflux to the rightwithout inhibiting the maximum response. On the other hand 1 14/kg pertussis toxin,which ADP-ribosylated 60 % of G-proteins in the ventricle, did not have any inhibitoryeffect on either basal or isoproterenol-stimulated adenylate cyclase activity in thepresence of carbachol. These results suggest that inhibition of adenylate cyclase in atriaby carbachol is more sensitive to uncoupling by pertussis toxin than either the sameresponse to carbachol in ventricles or the ability of carbachol to open potassium channelsin left atria.5.2. CONCLUSIONSThe results obtained in the present study suggest that:166(a) the ability of carbachol to open potassium channels in left atria contributes tothe cAMP-independent component of the negative inotropic response to carbachol in thepresence of the cAMP-elevating and cAMP-independent positive inotropic agents.(b) The direct negative inotropic response to carbachol in rabbit left atria isrelated, in part, to the ability of carbachol to open potassium channels. 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