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The antiarrhythmic activity of ±-opioid agonist RSD 939 is unrelated to ±-agonism Cheung, Pak Ho Paul 1994

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THE ANTIARRHYTHMIC ACTIVITY OF 1C-OPIOID AGONISTRSD 939 IS UNRELATED TO 1C-AGONISMbyPAK HO PAUL CHEUNGB.Sc.(Pharm) University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PHARMACOLOGY & THERAPEUTICSFACULTY OF MEDICINEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJune 1994© Pak Ho Paul CheungIn 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.Department of-‘-y J ‘77)The University of British ColumbiaVancouver, CanadaDate 7/DE-6 (2/88)11ABSTRACTCumulating evidence have indicated that various opioid agonistsand antagonists, especially kappa (ic) agonists, can exhibit a variety ofcardiovascular and antiarrhythmic actions. Two important questionsarise. First, are these cardiovascular and antiarrhythmic actions mediatedby the opioid receptors? Second, what is the underlying mechanism ofactions if these effects are not mediated by opioid receptors? Previousstudies have shown that some of the cardiac and cardiovascular actions ofic agonists are a result of direct actions on cardiac ionic channels and areindependent of ic agonism. RSD 939 is structurally related to ic agonistsand in binding studies appears to be a potent and selective ic agonist. Thepresent study is an attempt to study the involvement of opioid-receptorsin the cardiovascular and antiarrhythmic activity of ic agonists and todetermine the underlying mechanism of such activities using RSD 939.The cardiovascular and antiarrhythmic actions of RSD 939 wereinvestigated in a series of studies. Firstly, the initial profile of acutecardiovascular and toxic actions of RSD 939 were investigated. RSD 939was given as cumulative i.v. bolus doses to anaesthetised rats whoseblood pressure, heart rate and ECG were measured. It was found that at 8jimole/kg, RSD 939 decreased both blood pressure and heart rate by 25%.111At 5 tmole/kg, it also prolonged the P-R interval and increased RSh ofthe ECG. However, at a higher dose of 16 jtmole/kg, it also producedchanges in the Q-T interval.Since opioid receptors are found in the vagus nerve, in severalsympathetic ganglia as well as the heart, opioid peptides can influence thecardiovascular system both centrally and peripherally. Therefore, inorder to determine the direct cardiac effects of RSD 939, it is necessaryto examine the drug effects in the absence of neuronal and humoralinfluence on hearts. In isolated rat hearts, over the concentration range0.1 to 3.0 riM, RSD 939 concentration-dependently prolonged the P-R andQRS intervals of the ECG.The antiarrhythmic activity of RSD 939 was determined in terms ofits ability to prevent both electrically-induced and ischaemia-inducedarrhythmias. For electrical stimulation, two silver electrodes wereimplanted into the rat’s left ventricle and the ability of the drug to raisethe ventricular fibrillation threshold (VFt) was determined. In theischaemia model, the left anterior decending coronary artery (LAD) of therat was ligated. Occlusion of the LAD results in the production of acutemyocardial ischaemia and ventricular arrhythmias in a predictable andreproducible manner that mimics conditions found clinically inmyocardial ischaemia and infarction. At a dose of 1.5 jtmole/kg/min,ivRSD 939 significantly increased the threshold voltage needed to induceventricular fibrillo-flutter. At the same dose, the incidence of ventriculararrhythmias produced by occlusion was also significantly reduced(reduction of arrhythmia score from 7.0 in control groups to 3.2 in RSD939 group).Naloxone at a dose which had no cardiovascular or ECG actions,but blocked opioid receptors, was used to differentiate between opioidand nonopioid receptor-mediated actions of RSD 939. In a random anddouble-blind manner, either control vehicle or 8 j.imole/kg(1 tM in vitro)naloxone was given to rats or infused into isolated rat hearts. RSD 939 orcontrol vehicle was administered 5 mm later. The cardiovascular andantiarrhythmic actions of RSD 939 in naloxone pre-treated preparationsand untreated rats were also compared. It was found that the ECG andantiarrhythmic effects of RSD 939 were not antagonized by naloxone.The antiarrhythmic action of naloxone alone was also evaluated andcompared in the two groups. Naloxone alone had no effect on any of theECG variables except for P-R interval which was prolonged slightly.However, naloxone alone reduced the incidence and severity ofischaemia-induced arrhythmia. This action of naloxone was notsynergistic with RSD 939 since no difference in antiarrhythmic potencywas found between the naloxone pre-treated and nonnaloxone pre-treatedVgroups. From the above observations, it can be concluded that the cardiacand cardiovascular actions of RSD 939 were not mediated through opioidreceptors.Effects of RSD 939 on the ECG parameters such as P-R, QRS, RShand Q-T intervals and electrical stimulation parameters such as thresholdcurrent (iT), threshold duration (tT), and effective refractory period(ERP) were used to establish the underlying mechanism of actions of itsantiarrhythmic activities. RSD 939 dose-dependently prolonged P-R,QRS, RSIi, iT, and tT without significant effects on Q-T and ERP untilhigher doses. Since most sodium channels blockers will increase P-R,QRS, RSh, iT, and tT and most potassium channel blockers will prolongQ-T and ERP, we concluded that RSD 939 mediated its cardiac andcardiovascular effects by direct cardiac sodium and potassium channelblockade.1TABLE OF CONTENTSCHAPTER PAGEABSTRACT iiTABLE OF CONTENTS viLIST OF TABLES ixLIST OF FIGURES xiLIST OF ABBREVIATIONS xivDEDICATION xviiACKNOWLEDGEMENTS xviiiINTRODUCTION 11.1 Myocardial ischaemia and infarction 11.1.1 Overview 11.1.2 Electrophysiological changes caused bymyocardial ischaemia 21.1.3 Arrhytlunias in the early phase of myocardial ischaemia Reentry Abnormal automaticity Triggered automaticity 111.1.4 Pathophysiological progenitors of arrhythmias Potassium Calcium 161.1.5 Mechanisms of action of antiarrhythniic drugs 181.2 Experimental arrhythmogenesis 221.2.1 Arrhythmia models in general 221.2.2 Arrhythmia models in rats Overview Chemical model Electrical induction of arrhythmias Ischaemia and reperfusion 281.3 Opioid receptors in the heart 301.3.1 Classification of opioid receptors 311.3.2 Phannacological and physiological 33identification of cardiac opioid receptors1.3.3 Cardiac opioid binding sites 351.3.4 Actions of opioids in nerve and muscle 371.3.5 Opioids and arrhythmias 411.3.6 i-opioids and arrhythmias 44vii1.3.7 Mechanism of antiarrhytlmiic effects mediated by i agomsts 451.3.7.1 ic-opioid receptor mediated antiarrhythmic effects 451.3.7.2 Cardiac ion channel blockade 481.4 RSD939 501.5 Aims of studies 532 METHODS 552.1 Experimental plan 552.2 Opioid actions 562.2.1 Analgesia assays in vivo 562.2.2 Binding studies 572.3 Cardiovascular assessment 582.3.1 Surgical preparation 582.3.2 Experimental design 602.3.3 Data analysis 612.4 Isolated rat hearts 652.4.1 Perfusion apparatus 662.4.2 Preparation 672.4.3 Experimental design and data analysis 702.5 Electrical Stimulation 712.5.1 Experimental preparation 732.5.2 Experimental design 742.5.3 Experimental end-points 752.5.3.1 Threshold current 752.5.3.2 Threshold pulse width 762.5.3.3 Ventricular fibrillation threshold 762.5.3.4 Effective refractory period and 77maximum following frequency2.5.4 Data analysis 782.6 Myocardial ischaemia-induced arrhythmias 792.6.1 Experimental preparation 792.6.2 Experimental design 822.6.3 Data analysis 862.6.3.1 S-T segment and R-wave amplitude changes post-occlusion 86viii2.6.3.2 Analysis of arrhythmia 873. RESULTS 903.1 Opioid effects 903.2 Haemodynamic effects of RSD 939 in vivo 913.3 ECG effects of RSD 939 in vivo 953.4 In vitro effects of RSD 939 1023.5 Effects of electrical stimulation in vivo 1123.6 Antiarrhythmic actions of RSD 939 in vivo 1224. DISCUSSIONS 1274.1 Haemodyanmic effects of RSD 939 in vivo and in vitro 1274.2 Antiarrhythmic actions of RSD 939 1294.3 Non-opioid actions of RSD 939 1335. CONCLUSION 1386. REFERENCES 139ixLIST OF TABLESTable Page1 Potencies of RSD 939 in the presence and absence of 94naloxone pre-treatment with respect to haemodynamicresponses in vivo.2 Potencies of RSD 939 in the presence and absence of 101naloxone pre-treatment with respect to ECG responsesin vivo.3 Serum K levels in pentobarbital anaesthetized rats 120subjected to electrical stimulation.4 Potencies of RSD 939 in the presence and absence of 121naloxone pre-treatment with respect to sensitivity toelectrical stimulation in vivo.5 Potencies of RSD 939 in the presence and absence of 110naloxone pre-treatment with respect to cardiacfunctions in vitro.6 Potencies of RSD 939 in the presence and absence of 111naloxone pre-treatment with respect to ECG responsesin vitro.x7 Antiarrhythmic properties of RSD 939 in the presence 125and absence of naloxone pre-treatment againstischaemia-induced arrhythmias in pentobarbitalanaesthetized rats in early (0-0.5 hr) period followingcoronary artery occlusion.8 Occluded zone size and serum K levels in 126pentobarbital anaesthetized rats subjected to coronaryartery occlusion.9 Ionic composition of physiological salt solutions used 68for cardiac tissue in comparison with rat serum andinterstitial fluid.10 Binding studies 9011 Analgesia assays 90xiLIST OF FIGURESFigure Page1 Structure of RSD 939 522 A typical ECG from the rat and the variables which 64were measured.3 Effects of RSD 939 in the presence and absence of 92naloxone pre-treatmenth on mean arterial bloodpressure in pentobarbital anaesthetized rats.4 Effects of RSD 939 in the presence and absence of 93naloxone pre-treatmenth on heart rate in pentobarbitalanaesthetized rats.5 Effects of RSD 939 in the presence and absence of 96naloxone pre-treatmenth on P.R interval of ECG inpentobarbital anaesthetized rats.6 Effects of RSD 939 in the presence and absence of 97naloxone pre-treatmenth on QRS interval of ECG inpentobarbital anaesthetized rats.7 Effects of RSD 939 in the presence and absence of 98naloxone pre-treatmenth on Q-T1 interval ofECG inpentobarbital anaesthetized rats.xii8 Effects of RSD 939 in the presence and absence of 99naloxone pre-treatmenth on Q-T2 interval of ECG inpentobarbital anaesthetized rats.9 Effects of RSD 939 in the presence and absence of 100naloxone pre-treatmenth on RSh of ECG inpentobarbital anaesthetized rats.10 Effects of RSD 939 in the presence and absence of 115naloxone pre-treatmenth on ERP in pentobarbitalanae sthetized rats subjected to electrical stimulation.11 Effects of RSD 939 in the presence and absence of 116naloxone pre-treatmenth on iT in pentobarbitalanaesthetized rats subjected to electrical stimulation.12 Effects of RSD 939 in the presence and absence of 117naloxone pre-treatmenth on MFF in pentobarbitalanaesthetized rats subjected to electrical stimulation.13 Effects of RSD 939 in the presence and absence of 118naloxone pre-treatmenth on tT in pentobarbitalanaesthetized rats subjected to electrical stimulation.14 Effects of RSD 939 in the presence and absence of 119naloxone pre-treatmenth on VFt in pentobarbitalanaesthetized rats subjected to electrical stimulation.xiii15 Effects of RSD 939 in the presence and absence of 104naloxone pre-treatmenth on systolic ventricularpressure in isolated rat hearts.16 Effects of RSD 939 in the presence and absence of 105naloxone pre-treatmenth Ofl +dp/dtmax in isolated rathearts.17 Effects of RSD 939 in the presence and absence of 106naloxone pre-treatmenth on dP/dtmax in isolated rathearts.18 Effects of RSD 939 in the presence and absence of 107naloxone pre-treatmenth on heart rate in isolated rathearts.19 Effects of RSD 939 in the presence and absence of 108naloxone pre-treatmenth on P-R interval of ECG inisolated rat hearts.20 Effects of RSD 939 in the presence and absence of 109naloxone pre-treatmenth on QRS interval of ECG inisolated rat hearts.21 Occiuder placement for coronary occlusion 83xivLIST OF ABBREVIATIONSaction potential APaction potential duration APDarrhythmia score ASand &beta 13blood pressure BP2+calcium Cacentimetre cmdegree celciuselectrocardiogram ECGdose of drug producing 25%-or half-maximal response ED25,50effective refractory period ERPgram ghertz Hzhour(s) hrintraperitoneally i.p.intravenous i.v.kappa Kkilogram kgxvleft anterior descending LADless than <maximum following frequency MFFmicromolarmilligram per kilogram mg/kgmilligram per kilogram per mill mg/kg/mmmillimetre mmmillimetres of mercury mmHgmillisecond(s) msminute(s) mmmolecular weight MWmu p.nanomolar nMnon-spontaneously reverting VT NSVTnon-spontaneously reverting VF NSVFoccluded zone Ozpacemaker current ifpercentage %potassiumpremature ventricular contraction PVCsecond(s) sxvi+sodium Naspontaneously reverting VF SVFspontaneously reverting VT SVTstandard error of mean SEMthreshold current iTthreshold duration (threshold pulse width) tTventricular fibrillation VFventricular fibrillation threshold VFtventricular tachycardia VTxviiDedicationThis thesis is dedicated to my beloved parentsxviiiAchnowledgementsI wish to thank the members of my thesis committee Dr. Walker, Dr.Karim, and Dr. Sastry. I would also like to thank the following peoplefor their support and encouragement; Mike, Sandro, Eric, Ron, Tern,Jason, and Leon. As well I want to thank all the members of thedepartment (George, Christian, Jeff, Maureen, Elaine, Margaret, andJanelle) for all the little things which helped me complete my degree. Iam indebted to Rhythm Search Development and Medical ServiceFoundation who provided me with financial support. Finally, I would liketo thank my supervisor, Dr. M.J.A. Walker for offering me theopportunity to work with him on this project and for all the lessons hetaught me on the meanings of life.11. Introduction1.1 Myocardial ischaemia and infarction1.1.1 OverviewMyocardial ischaemia occurs when the oxygen supply to a portionof myocardium is insufficient. The underlying cause is coronaryarteriosclerosis and/or coronary artery spasm which impairs coronaryblood flow and results in insufficient supply of blood to the myocardium.The most common form of myocardial ischaemia is angina pectoris.Angina of effort is always produced by an increased demand on the heart(e.g., by exercise) and is usually due to atherosclerotic narrowing ofcoronary artery. Variant angina can occur at rest and is associated withcoronary artery spasm as a cause of reduced coronary flow. Angina isassociated with pain characteristically distributed in the chest, arm andneck. Relief of such pain is obtained by reducing sympathetic drive tothe heart with beta-adrenoceptor antagonists, or by dilating coronaryvessels with organic nitrites (Poole-Wilson, 1983).Myocardial infarction is defined as the necrotic and fibrous changesof the myocardial muscle resulting from maintained myocardial2ischaemia. Myocardial infarction is the commonest single cause of deathin many parts of the world; death usually results either from mechanicalfailure of the ventricle or most commonly from ventricular fibrillation(Oliver, 1982). The irreversible cellular damage (infarction) whichresults from vascular occlusion appears to be triggered by an increase inintracellular calcium concentration resulting from the impairment of twoATP-dependent processes, namely uptake of calcium by the sarcoplasmicreticulum and sodium extrusion from the cell which indirectly controlintracellular calcium concentration because of sodium-calcium exchange(Van der Vusse & Reneman, 1985).1.1.2 Electrophysiological changes caused by myocardialischaemiaNot unexpectedly myocardial ischaemia resulting from occlusion ofa coronary artery has a profound effect on the electrophysiologicalproperties of cardiac cells. Changes in resting membrane potential andinward and outward currents during the action potential lead to alterationsin conduction, refractoriness, and automaticity, all of which cancontribute to the occurrence of ventricular arrhythmias (Janse & Wit,1989).3Within minutes of experimental coronary artery occlusion, cellswithin the ischaemic region begin to depolarize, (i.e. resting membranepotential is reduced with a sigmoidal time course (Downar et al., 1977;Kieber, 1983). A major cause of the fall in the resting membranepotential of ischaemic cells is the altered potassium gradient across thecell membrane. This is the result of a net potassium efflux andextracellular potassium accumulation due to lack of blood flow (Hirche eta!., 1980). The reasons for ischaemic cells losing potassium are notcompletely established. In addition to the altered potassium gradient,other mechanisms for depolarization may contribute, such as an increasein intracellular calcium (Isenberg, 1983) and the effects oflipophosphoglycerides (LPG) (Clarkson & Teneick, 1983), producedduring early ischaemia, on the cell membrane.The extracellular fluid composition in ischaemic cardiac muscle hasa special composition that results from the lack of blood flow. Not onlyis extracellular potassium elevated but there is hypoxia, low pH, nosubstrates, high Pco2 and an accumulation of substances, such aslipophosphoglycerides (LPGs) and catecholamines (Janse & Wit, 1989).Each has an influence on membrane conductances while the variouscombinations may exert effects that are not predictable from the action ofeach substance alone. Together they cause changes in the action potential4including a reduction in action potential amplitude, upstroke velocity, andduration. The depressed upstroke, decreased amplitude, and decreasedvelocity of depolarization of action potentials in partially depolarizedmembrane potentials are primarily caused by the loss of a fast Na current(depressed fast response) and its replacement by a slow inward current(slow response) when the level of resting potential is reduced enough toinactivate the Na current (Cardinal et al., 1981).Due to the reduction in action potential duration, the effectiverefractory period (ERP) is expected to be abbreviated. However, this isnot necessarily so since ischaemic fibers may remain inexcitable evenafter complete repolarization. In partially depolarized fibers, recoveryfrom inactivation of both fast and slow inward current has been shown tobe markedly delayed until many milliseconds after completion ofrepolarization (Cranefield et al., 1972; Gettes & Reuter, 1974). This“postrepolarization refractoriness” is probably related to depolarization ofthe resting or maximum diastolic potential of the myocardial cell (Lazzaraet al., 1978). In the central ischaemic zone, the phenomenon ofpostrepolarization refractoriness causes the refractory period to lengthen,whereas in the border zone, refractory periods may become shorter thannormal. Inhomogeneity in recovery of excitability is largely caused byinhomogeneities in extracellular K (Kodama et al., 1984; Coronel et al.,51988), which in turn is caused partly by diffusion of K from theischaemic zone towards the normal zone and partly by the difference incoronary collateral perfusion between species. In species with multiplecoronary collaterals the ischaemic zone will not be homogeneous andtherefore extracelluar K may be variable. On the other hand in specieswithout collaterals (e.g. rats), changes in extracellular K are uniform.1.1.3 Arrhythmias in the early phase of myocardial ischaemiaIn patients with a diagnosis of acute myocardial ischaemia andinfarction, approximately one half will experience one or morecomplications. The type of complication encountered is often related tothe patients’s age and gender, and to the size and location of theinfarction. Conduction disturbances and cardiac arrhythmias are the mostcommon and fatal form of complications in the course of myocardialischaemia and infarction. More than 85% of myocardial infarctionsurvivors have some evidence of ventricular ectopy. In addition, asignificant number of patients have atrial arrrhythmias including eitheratrial flutter or fibrillation (Lovegrove & Thompson, 1978).The majority of deaths of cardiac origin are sudden and due tosevere tachyarrhythmias. The most frequent of such arrhythmias is6ventricular fibrillation although ventricular tachycardia and asystole canalso be a cause of sudden cardiac death (Gordon and Kannel, 1971). Themechanism of arrhythmias following acute infarction, whether enhancedautomaticity, or re-entry or both, appears to vary depending on the timeafter complete occlusion of a coronary artery. Early after the onset ofmyocardial ischaemia and infarction, ventricular fibrillation may resultfrom re-entrant mechanisms. During the later stages of infarctionenhanced automaticity may precipitate ventricular fibrillation (Pogwizd &Corr, 1987). ReentryArrhythmias may be generated by recirculating excitation incited byan initiating depolarization. Such arrhythmias, classified as reentrantarrhythmias, are self-sustained but are not self-initiated (Wit and Rosen,1983). Reentry results from a conduction disturbance whereby thecardiac propagating impulse may not die out after complete completeactivation of atria or ventricles but persists to re-excite the tissue afterthe end of its refractory period. The criteria that must be fulfilled todemonstrate that an arrhythmia is caused by reentry were formulated byMines as early as 1913 and 1914 and may be summarized as follows: 1)7an area of transient or unidirectional block of conduction must be present;2) there must be an anatomical or functional “barrier” to conduction suchthat activity must propagate along alternate pathways around the area ofblock, activate the tissue beyond the block with delay, and retrogradelytraverse the zone of block to reexcite the tissue proximal to the block; 3)interruption of the reentrant pathway must abolish the arrhythmia.Furthermore, the wavelength of the cardiac impulse in the reentrantcircuit, where the wavelength is the product of conduction velocity andrefractory period, must be shorter than the pathlength of the circuit sothat the tissue into which the impulse is reentering has time to recover itsexcitability. There are difficulties in attributing cardiac arrhythmias toreentry because, with a normal refractory period which is very long,normal conduction of cardiac impulse which is rapid would necessitatequite a long path since the reentrant pathways are reasonably short(Hoffman and Dangman, 1987). As a result of this crucial relationshipbetween pathlength, conduction velocity, and refractory period, it isnecessary that conduction velocity must be greatly slowed and refractoryperiod markedly shortened, or both.Reentry can occur randomly across the heart, as in fibrillation, orbe ordered and follow a fixed pathway. It has been suggested in past thatfibrillation might represent chaotic reentrant excitation or multiple8continually migrating activation wavefronts (Mines, 1913; Moe et a!.,1964). This type of reentry has been termed random reentry (as opposedto stable reentry based on a fixed anatomical path) where the path ofexcitation continously changes such that individual groups of fibers maybe repeatedly excited (Hoffman and Rosen, 1981). Micro-reentry hasbeen used to describe the small circuits such as might occur in the AVnode or distal Purkinje fibers. In this case the reentry circuit may beextremely short, perhaps only a few mm (Sasyniuk and Mendez, 1971).The refractory period in the anterograde pathway is also very shortallowing excitability to be quickly restored. Microreentry has beendetermined as a mechanism for ventricular tachycardia whereby smallepicardial conduction loops exit into non-refractory subendocardiuminitiating succeeding beats (Kramer et al., 1985). Alternatively, if thecircuit is long enough, there will be sufficient time for the anterogradepathway to recover excitability. This type of longer pathway circuit isreferred to as macro-reentry. Reflection is a form of reentry that isproduced through reflection in parallel unbranching fibers with depressedsegments (Antzelevitch et al., 1980; Jalife & Moe, 1981). If a delayedaction potential is caused by the electrotonic depolarization of a blockedimpulse and is distal to an inexcitable segment, then reflection occurs(Hoffman and Dangman, 1987). The delayed action potential then9reexcites the tissue proximal to the site of block. These reflectedimpulses can be modulated by changes in rate and rhythm.Since acute ischaemia is associated with areas of slow conductionand abbreviated action potential duration and/or prolonged refractoryperiods, reentry has been implicated for many years as an important causeof ischaemia-induced arrhythmias. Clinically, reentry is the usual causeof paroxysmal supraventricular tachycardia (Wellens et a!., 1974).Reentry in the His-Purkinje system is thought to be one cause of coupledventricular premature depolarizations and ventricular tachycardia, andventricular tachycardia may degenerate to ventricular fibrillation (Wit,1972). Abnormal AutomaticityNormally automaticity is found in sinoatrial node (SAN), subsidaryatrial fibers, fibers in and around the coronary sinus ostium, cardiacfibers in the tricuspid and mitral valve leaflets, the NH region of theatrioventricular junction, and the His bundle and Purkinje fiberramifications in the ventricle although, in the latter, automaticity is notnormally seen owing to the faster rate of the node (Hoffman andDangman, 1987; Wit et al., 1974). The faster intrinsic rate of impulse10initiation in the SAN overdrives the spontaneous depolarizations in otherregions of the heart, and thus it functions as the primary pacemaker(Rosen, 1988). However, when the rate of firing of the sinus node isslower than the intrinsic rate of the other sites in the specializedconducting system, the region with the higher rate might take over thepacemaker function as a result of overdrive suppression (Rosen, 1988).On the other hand, abnormal automaticity occuring in depolarizedventricular and/or Purkinje fibres is difficult to be suppress by overdrivepacing (Dangman & Hoffman, 1983). Abnormal automaticity simplymeans that heart tissue other than the sinus node has taken over thepacemaker role of the sinus node (Hoffman and Dangman, 1982;Sasyniuk, 1984), and it has been implicated as the causative agentresponsibles for a significant fraction of the arrrhythmias seen 24 hourafter myocardial infarction in human subjects (Wellens Ct al., 1974).Arrhythmias caused by augmented automaticity in normalspecialized conducting tissues are spontaneously generated and thereforedo not rely on a prior impulse (Hoffman and Rosen, 1981). One of thecurrents responsible for the spontaneous diastolic depolarization in SANis known as the pacemaker current (ii). In the sinus node the pacemakercurrent has recently been shown to result from the interaction of twocurrents, the decay of a delayed rectifier K current called ‘K, and the11activation of an inward current carried by Ca2, called ‘Ca (Shibata andGiles, 1985). Abnormal automaticity may occur as a result of anincreasing rate of activation in latent pacemaker tissue, or a shifting inthe voltage dependence for activation of ,j toward more positive(depolarized) values thus leading to a reduction in the threshold forgeneration of the (abnormal) propagated action potential (Hoffman andDangman, 1987). Triggered AutomaticityTriggered automaticity, by definition is the generation of one ormore impulses as a consequence of an exogenous source, and theexogenous source theoretically includes the normal wave ofdepolarization, particularly the previous impulse (Hoffman and Rosen,1981). Triggered activity is dependent upon oscillations of membranepotential following an action potential upstroke, i.e. early or delayedafterdepolarizations (Janse and Wit, 1989; Rosen, 1988). Thus it is notself-initiated, but triggered by a prior impulse. Early afterdepolarizations(EADs) are secondary depolarizations that occur before repolarization iscomplete, i.e. during Phase III repolarization. Early afterdepolarizationsare produced when repolarization is interrupted by secondary12depolarizations. Such responses can excite neighboring fibres and bepropagated (Rosen, 1988; Wit and Rosen, 1983). Trigered arrhythmiasfrom early afterdepolarizations are more likely to occur duringbradycardia, when the action potential duration is prolonged (Hoffmanand Dangman, 1987; Brachman et al., 1983). Therefore, earlyafterdepolarizations have been most closely identified as the cause oftorsades de pointes occuring in the setting of the long QT syndrome, andare the proarrhythmic mechanism of some antiarrhythmic drugs(Brachman et al., 1983). Delayed afterdepolarizations (DADs) aresecondary depolarization occuring early in diastole after fullrepolarization has been achieved. After terminal repolarization of anaction potential is achieved, membrane potential again transientlydepolarizes. If the transient or oscillatory depolarization reachesthreshold, a propagating response can occur (Rosen, 1988; Wit andRosen, 1983). Unlike early afterdepolarizations, arrhythmias induced bydelayed afterdepolarizations occur more readily when the precedingstimulation rate is rapid and will tend to increase in rate as the precedingdrive rate is increased (Monk and Rosen, 1984). Once the delayedafterdepolarizations are large enough to reach threshold, the triggeredaction potential can initiate a single premature depolarization. Thatpremature depolarization will be followed by a larger than usual delayed13afterdepolarization, and it in turn probably will attain threshold and resultin a second impulse (Hoffman and Dangman, 1987). In this way, delayedafterdepolarizations can cause either coupled extrasystoles or runs oftachyarrhythmias.It is important to recognize that although delayedafterdepolarizations and early afterdepolarizations are routinely classifiedtogether as causes of triggered arrhythmias their underlying cellularmechanisms appear to be different. Early afterdepolarizations have beenproduced in vitro by catecholamines (Hoffman & Cranefield, 1960),acidosis (Coraboeuf et al., 1980), low [K] (Carmeliet, 1961), low [Ca2](Sano and Sawanobori, 1972), hypoxia (Trautwein et al., 1954) andnumerous drugs (Schmidt, 1960; Dangman & Hoffman, 1981; Strauss etal., 1970; Gough et al., 1988; Levine et al., 1985; El-Sherif et al., 1988;Carlsson et al., 1990). The triggering mechanism of earlyafterdepolarizations, at least for those arising near the plateau, arethought to be the result of the interaction of surface membrane currentsystems leading to a time- and voltage-dependent recovery of L-type Ca2current which can carry depolarizing charge (January et al., 1991). Incontrast, the critical mechanism proposed to underlie delayedafterdepolarizations is overloading of cell myoplasm and the sarcoplasmicreticulum with Ca2, such as in reperfusion (Ferrier et al., 1985),14inhibition of Na/K ATPase by digitalis glycosides, hypokalemia andhypomagnesemia, etc. (Marriott & Conover, 1989). The transient rise inmyoplasmic Ca2 also induces a Ca2 sensitive inward current. This Ca2induced transient inward current (‘) is thought to be the actual mediatorof delayed afterdepolarizations (Lederer & Tsien, 1976; Kass et al., 1978;Matsuda et al., 1982).Abnormal automaticity might not responsible for arrhythmogenesisin acute ischaemia because abnormal automaticity is suppressed byelevated extracellular potassium concentration which occurred subsequentto coronary occlusion (Hoffman & Rosen, 1981; Hirsche et al., 1980).However, triggered activity may play a role in ischaemia inducedarrhythmia due to the possible presence of hypoxia (Trautwein et al.,1954), acidosis (Coraboeuf et al., 1953), elevated catecholamines (Brookset al., 1955), high extracellular potassium concentration, possiblemyocardial stretch (Pirzada et al., 1976), increased calcium concentration(Clusin et al., 1983), which may contribute to the generation ofoscillatory after depolarizations.1.1.4 Pathophysiological progenitors of arrhythmiasMany putative pathophysiological progenitors of arrhythmogenesis15have been proposed. In ischaemia these may include accumulation ofcatecholamines (Sheridan et al., 1980), lysophosphatides (Corr et al.,1984), thromboxane (Coker et al., 1981), and potassium ( Hill and Gettes,1980; Hirche et al., 1980). It remains unclear which of these substances,if any, plays the most important role in arrhythmogenesis. Other, as yetunidentified factors almost certainly play a role. Indeed, any substancewhose homeostasis is disturbed by ischaemia is a potential arrhythmogenuntil proven otherwise. PotassiumPotassium was one of the first substances to be suggested to play arole as an endogenous arrhythmogen in myocardial ischaemia. The roleof potassium in arrhythmogenesis is supported by evidence that (i)extracellular potassium concentration rises with a time-course similar tothe onset of ischaemia-induced arrhythmias (Hill and Gettes, 1980;Hirche et al., 1980) and (ii) regional elevation of extracellular potassiumconcentration in the absence of ischaemia produces electrophysiologicalchanges liable to precipitate arrhythmias. The latter include slowing ofconduction velocity (Schmitt and Erlanger, 1928), a decrease in restingmembrane potential (Morena et al., 1980) and a shortening of action16potential duration (Morena et al., 1980), effects similar to those producedby ischaemia itself (Janse et al., 1980; Inoue Ct a!., 1984; Janse, 1986).Furthermore, regional elevation of extracellular potassium in the absenceof ischaemia can cause re-entry (Schmidt and Erlanger, 1928), reduce thethreshold current necessary to elicit VF (Logic, 1973) and directlyprecipitate ventricular arrhythmias (Harris et al., 1958; Ettinger et al.,1973; Morena et al., 1980; Pelleg et a!., 1989). Thus a role of regionalhyperkalemia in arrhythmogenesis seems to be indicated. CalciumIt has been shown that intracellular free calcium [Ca2] is elevatedduring myocardial ischaemia in isolated perfused rat hearts (Steenbergenet al., 1987b) and in isolated multicellular neonatal rat ventricularmyocytes with an arrhythmia profile such as fibrillatory beating activity(Thandroyen et a!., 1991). The increase in [Ca2]1is a consequence of therelease of calcium from intracellular stores and influx of extracellularcalcium due to the altered cell permeability to calcium or incompletesodium-calcium exchange resulted from ischaemia-induced cell injury.The elevation of [Ca2]leads to a number of unfavourable consequenceswhich predispose to development of cardiac arrhythmias. Increase of17[Ca2], can provoke oscillatory depolarization of the cardiac membrane,triggering sustained action potential generation, and extrasystoles(Biliman, 1990). Ca2 overload is the proposed mechanism underlying thegeneration of delayed afterdepolarizations which is possibly theunderlying principal cause of triggered arrhythmias. The evidence is asfollows. Firstly, administration of BAPTA, a Ca2 chelator whichincreases cytoplasmic Ca2 buffering capacity, or administration ofryanodine which inhibits calcium release from SR (Sutko et al., 1979),suppresses the generation of delayed afterdepolarizations (Sutko andKenyon, 1983). Secondly, oscillations in cardiac membrane potential canbe abolished by caffeine (Shattock Ct al., 1991), a drug which enhancesthe release of calcium from the SR by impairing calcium reuptake by theSR (Lakatta et al., 1985). In addition to the activation of oscillatoryinward current flow that precipitates delayed afterdepolarizations, a2+number of mechanisms may also result in an elevation of [Ca ], leadingto arrhythmias. For instance, elevation of [Ca2]1 impairs intercellularcoupling between cells (DeMello, 1982; Pressler et al., 1982) and henceslows conduction and increases the likelihood of re-entry. Furthermore,an increase in [Ca2]1 can have deleterious metabolic effects whichpredispose to arrhythmogenesis. For example, high-energy phosphate(ATP) is spent in the sequestration of calcium into the cellular stores such18as SR and mitochondria. Moreover, transient or early elevation ofintracellular free calcium will activate a number of calcium-dependentmyocardial proteases within the cell so damaging the cytoskeleton andcell plasma membrane (Croall and DeMartino, 1983; Steenbergen et a!.,1987a). The development of defects in sarcolemma membrane integrityand early afterdepolarizations leads to an elevation of [Ca2]1 as aconsequence of an influx of calcium from extracellular fluid and hencefurther exaggerates the genesis of myocardial arrhythmias.1.1.5 Mechanisms of action of antiarrhythmic drugsNormalization of cardiac rhythm can be achieved if abnormalheterogeneity of excitation, conduction or repolarization caused byischaemia and infarction is prevented. This can be achieved with drugsthat block cardiac ionic channels. A classification of these, and related,drugs was introduced by Vaughan Williams (1970). The definition ofdrug classes has a strong historical background. Sodium channel blockerswere the first drugs to be shown to have antiarrhythmic activity. Theywere called Class I drugs. Later on, f3-adrenoceptor blocking drugs(Class II), potassium channel blockers (Class III) and, finally, calciumchannel blockers were developed (Class IV).19Based on the arrhythmogenic mechanisms discussed earlier(abnormal automaticity, triggered activity, and reentry), an agent effectiveagainst re-entrant arrhythmias should possess properties which will breaka re-entrant circuit. Theoretically, this can be achieved by (1) convertingareas of unidirectional block to bidirectional block, (2) prolongingrefractoriness in normal myocardium such that the fibers at the sites oforigin of the initiating impulses have not recovered their excitability atthe time of reentry, and (3) reducing the strength of slowly propagatingimpulses such that the wavefront dies out before completing the circuit(Winslow, 1984). The antiarrhythmic action of drugs on automaticrhythms could involve an inhibition of i, the time-dependent, pacemakerinward sodium current, a shifting of the maximum diastolic potential tomore negative values, or an increase in APD. These actions togetherlower the automatic focus firing rate (Davy et a!., 1988). Antiarrhythmicaction of drugs on triggered activity, on the other hand, could involvesuppression of the afterdepolarization by decreasing Ca2 or inward Nacurrents (Thale et a!., 1987).Class I antiarrhythmic agents (Class Ta: quinidine, disopyramide,and procainamide; Class Tb: lidocaine, tocainide, and mexiletine; ClassIc: encainide, flecainide, and lorcainide) are sodium channel blockersand thus reduce the fast inward sodium current. This results in reduced20maximum rate of rise (MRD), depressed conduction velocity, andprolongation of the effective refractory period (ERP). Class I agents alsoreduce spontaneous diastolic depolarization (Campbell, 1983b; Harrison1985). These agents would therefore be expected to be active againstarrhythmias involving reentry and abnormal automaticity. However,sodium channel blockers are almost without effect on abnormalpacemakers in which automaticity is maintained by slow inward currentand triggered activity caused by increase in Ca2, as in early and delayedafterdepolarizations.Class II antiarrhythmic agents (J3 adrenoceptor blockers) inhibitarrhythmic responses due to endogenous catecholamines (Nattel, 1991).Such adrenergic arrhythmias which occur during physical and mentalstress are suppressed most successfully by 13 adrenoceptor blockers. Longterm treatment with f3 adrenoceptor blockers also tends to prolong bothatrial and ventricular action potential duration (Vaughan Williams, 1978).Since re-entrant arrhythmias are favoured by slow conduction and shortrefractory periods such drugs (which increase refractoriness withoutslowing conduction) should be of value in treatment of re-entrantarrhythmias.Class III agents (amiodarone and (±)-sotalol) selectively prolongaction potential duration(APD) without slowing conduction velocity.21Prolonging APD delays recovery of voltage dependent Na channels,thereby increasing effective refractory periods (Singh & VaughanWilliams, 1970). Class III agents may be of value in preventing reentrant and abnormal automatic arrhythmias. However, the developmentof Class III antiarrhythmics has progressed slowly relative to developmentof Class I agents. This is due in part to their proarrhythmic potential incausing Torsade de Pointes (Dessertenne, 1966).The use of Class IV agents (verapamil and diltiazem) still remainscontroversial (Walker and Chia, 1989). It is known that theirantiarrhythmic actions depend on their ability to decrease the upstrokevelocity of action potentials in the AV node thus slowing AV conductionand increasing the refractory period of the AV node. Furthermore, sincepacemaker activity may arise solely from inward calcium currents(Borchard et al., 1989), Class IV agents may be effective in preventingarrhythmias arising from automatic mechanism and re-entrant mechanisminvolving the AV node as part of the re-entrant circle. Class IV agentsare also able to protect the cell against Ca2 overload and thus suppresstriggered activity such as afterdepolarization resulted from Ca2 overload.221.2 Experimental arrhythmogenesis1.2.1 Arrhythmia models in generalA large number of models have been designed to producearrhythmias. These have been reviewed periodically over the last decadeor so. For example, Szekeres (1971, 1979) very clearly outlined howarrhythmias may be induced in a variety of species by either electricalstimulation of the heart, administration of arrhythmogenic drugs andchemicals, or pathological damage to the heart via ischaemia orinfarction, local cooling, local warming, or mechanical injury. Inaddition, he listed several methods whereby arrhythmias can be inducedby means of electrical stimulation of the central nervous system.In 1984, Winslow carefully reviewed the methods available forproducing arrhythmias and assessing the antiarrhythmic actions of drugs.The methods in her review again involved electrical stimulation, chemicaladministration, and induction of arrhythmias by pathological means.Curtis and Walker (1988) reviewed in detail all of the models forinducing arrhythmias via myocardial ischaemia and infarction in the rat.The use of rat models has been further summarized by Walker et al.(1991). Except for the last two reviews, most authors specifically23discussed a number of different species in addition to the rat. In anydiscussion on the production of arrhythmias it is important, formechanistic and comparative purposes, to be able to classify the resultingarrhythmias.1.2.2 Arrhythmia models in rats1.2.2.1 OverviewThe rat is a common laboratory animal that has been used in manypharmacological, toxicological, biochemical, and pathophysiologicalstudies. It is perhaps the most accepted of all laboratory species and iswell understood in terms of anatomy, genetics, physiology, andbiochemistry (Carr and Krantz, Jr., 1949; Mitruka, 1976; Ringler andDabich, 1979; Petty, 1982). There are certain advantages anddisadvantages with the use of the rat as an experimental animal. Rats aremuch smaller than humans; their pharmacokinetics andpharmacodynamics are often quite different (Fox, 1967) and they have anumber of peculiarities with respect to biochemistry and physiology(Jorgensen, 1967).With regard to cardiac electrophysiology, the rat occupies a24somewhat special position in comparison with other laboratory species.This is due to in part to its high heart rate, but more particularly to amore rapid repolarization of cardiac action potentials (Hoffman andCranefield, 1960). The potassium channels responsible for cardiacpotential repolarization vary with species and cardiac tissue type(Carmeliet et al., 1987; Furukawa et al., 1992; Beatch et al., 1990), butare most different in the rat. Thus ventricular action potentials in ratsrely predominantly on the transient outward potassium currents (i10) forrepolarization (Josephson et al., 1984; Dukes and Morad, 1989).While the rat has distinct differences from man in terms of cardiacanatomy and electrophysiology, its advantages in the study of myocardialischaemia and arrhythmias may outweigh its disadvantages. The mainadvantages of rats are that they are small and easy to handle, inexpensive,and can be used in large numbers. In addition, a large variety of humandisease states have been modeled in the rat (Petty, 1982). Anothernotable advantage is the uniform lack of effective coronary collateralswhich results in reproducible occluded (ischaemic) zones (Curtis et al.,1987a). This is of prime importance since both ischaemia-inducedarrhythmias and infarct size depend upon the extent of collateralanastamoses (Curtis, 1986). Chemical modelA variety of arrhythmias can be readily produced in the rat byadministration of drugs and chemicals to the whole animal, or to isolatedhearts. In many species, cardiac glycosides (e.g., ouabain) are routinelyused to induce ventricular arrhythmias but this is not possible in the ratsince this species is insensitive to cardiac glycosides (Winslow, 1984).Some chemicals routinely used to produce arrhythmias (primarilyventricular) in the rat are acontine, calcium, and barium (Vargaftig andCoignet, 1969; Malinow et al., 1953; Ferrara et al., 1990). Normally,these compounds are given intravenously to the whole animal and are notroutinely used in isolated hearts. In larger species, such as the cat anddog, such chemical arrhythmogens can be applied locally (e.g., Nakayamaet al., 1971; Byrne et al., 1977; Winslow, 1981), but this is not usuallydone in the rat.One recently introduced ionic procedure for inducing arrhythmiasin isolated hearts is preferential perfusion of different coronary bed withsolutions containing an elevated potassium concentration as firstperformed in isolated rabbit hearts (Curtis, 1989b). Preferential perfusionof a single coronary bed can be achieved by use of an ingenious specialperfusion cannula situated within the aorta (Avkiran and Curtis, 1991). Electrical induction of arrhythmiasArrhythmias are induced routinely by electrical stimulation at avariety of cardiac sites such as atria, ventricles, and the atrioventricularnode in many species, including humans (Weissberg et a!., 1987). Thesmall size of the rat heart does not readily allow for a highly selectiveplacement of electrodes, and, as a result, the site chosen for electricalstimulation in rats is usually the right or left ventricle. Access to thesesites can be achieved by opening the chest and exposing the heart.However, it is easy to insert two electrodes, no more than 1-2 mm apart,by a transthoracic route using a 27-gauge needle (Howard and Walker,1990; Pugsley et a!., 1992). Suitable electrodes for this purpose can bereadily fashioned from teflon-coated stainless-steel wire.The types of arrhythmias that can be induced by electricalstimulation include single extrasystoles, tachycardia, and fibrillation.Extrasystoles can be induced by a single extra stimulus added to a chainof stimuli, or interposed during sinus rhythm. Ventricular tachycardiacan be induced by stimulating the heart to beat at a rate faster than thesinus rhythm. The rat heart can be driven at rates greater than twice thesinus rate. One variant of this technique is to continuously increase thefrequency of stimulation until the heart fails to follow, on a one to one27basis. The frequency of stimulation at which the heart fails to follow isknown as the maximum following frequency, an indirect measure ofeffective refractory period (Martinez and Crampton, 1981; Pugsley et a!.,1992).In order to induce ventricular fibrillation (actually a type offibrillo-flutter), super threshold square waves are applied at 50 Hz(Marshall et al., 1983). The ease of inducibility of ventricular fibrillationis assessed in terms of the threshold current. Characteristically, suchinduced ventricular fibrillation has a coarse type of ECG morphology andis best described perhaps as being more of a Torsade de Pointes type oftachycardia rather than true fibrillation. Furthermore, in over 95% ofcases this arrhythmia spontaneously reverts to normal sinus rhythmproviding that it is not continued for too long. Normally, sinus rhythmreappears on termination of stimulation.The above electrical stimulation procedures can be readily used inisolated hearts with the stimulating electrodes being placed anywherewithin, or upon, the heart. In addition, the atrioventricular node can beablated thereby freeing the ventricle of interfering impulses originating inthe atria. Ischaemia and ReperfusionA common pathological cause of arrhythmia in humans is theoccurrence of myocardial ischaemia and infarction, or reperfusion of apreviously ischaemic area of myocardium. These events can be readilyreproduced in both intact rats and in isolated hearts.The rat heart, in common with species such as pigs and primates,does not normally have extensive coronary collaterals (Johns and Olson,1954; Maxwell et al., 1987), i.e. rat coronary arteries are end arteries.Thus when a coronary artery is occluded tissue downstream to theobstruction is rendered uniformly ischaemic. Ischaemic is not absolutesince a residual 5% of flow is seen following complete ligation of anartery (Winkler et al., 1984; Maxwell et al., 1987). If occlusion of anartery is maintained for greater than 10 mm, irreversible damage occursand infarction results (Saint et al., 1992). If the occlusion and itsresulting ischaemia is permanent, the resulting infarct can occupy an areagreater than 80% of the original ischaemic zone. Any period of ischaemiacan be terminated by reperfusion but in rat hearts reperfusion will onlysuccessfully save all of the ischaemic zone from becoming infarcted ifreperfusion is insituted within 15 mm of the onset of ischaemia (Saint eta!., 1992). Reperfusion is a most powerful stimulus for inducing29arrhythmias (Manning and Hearse, 1984; Curtis and Hearse, 1987).However, the severity and intensiity of such reperfusion arrhythmiasdepends critically upon the duration of the preceding ischaemic period.The time-dependency effect is quite characteristic in that reperfusionarrhythmias are most severe after 5-10 mm ischaemia (Manning andHearse, 1984; MacLeod et a!., 1989). Reperfusion arrhythmias can beinduced in both intact and isolated hearts. Methods suitable for intactanimals have been described by Manning and Hearse (1984) and MacLeodet al., (1989) and those suitable for intact hearts by Curtis and Hearse(1989) and Lubbe et al. (1978).A number of methods have been described for producing coronaryligation or occlusion in intact rats, whether anaesthetized (Johns andOlson, 1954; Au et a!., 1979; Clarke et al., 1980) or conscious (Johnstonet al., 1983; Himori and Akihiro, 1989). In conscious rats, the variousresponses to occlusion can be recorded for hours and days after occlusion.Responses that have been measured include blood pressure, heart rate,ECG changes such as increases in R-wave height and S-T segmentelevation, mortality and ischaemic zone size as well as arrhythmias(Johnston et al., 1983). It has been shown in such preparations that theseverity and incidence of arrhythmias following occlusion of a coronaryartery is dependent upon the size of the occluded zone (Johnston et a!.,301983; Curtis et al., 1987) and serum potassium concentration (Curtis etal., 1987; Saint et al., 1992). Arrhythmia dependency upon extracellularpotassium concentration is also seen in isolated hearts (Curtis, 1989b;Curtis and Hearse, 1989).In order to obtain consistent results, it is important that factors asoccluded zone size and serum potassium concentration are measured. Tostandardize experimental design in arrhythmia studies, a series ofconventions (Lambeth Conventions) were established to improveuniformity and inter-laboratory comparisons (Walker et al., 1988).1.3 Opioid Receptors in the HeartAlthough the opioids have been, and continue to be, used primarilyas analgesic and general anaesthetics, cumulative evidences has indicatedthat many opioid agonists and antagonists are also involved in the genesisand prevention of cardiac arrhythmias arising during myocardialischaemia and infarction. The purpose of this chapter is to discuss ourlimited current knowledge about the cardiac effects of opioids.311.3.1 Classification of Opioid ReceptorsBased on the structural and steric specificity of analgesic action ofmorphine observed in early behavioural and clinical studies, the existenceof specific opioid receptors was suggested (Beckett and Casy, 1954;Portoghese, 1965). The identification of opioid receptor by receptorbinding assays (Pert and Snyder, 1973), together with the discovery ofenkephalins (Hughes, 1975; Hughes et al., 1975a; Hughes et al., 1975b)and endorphins (Bradbury et al., 1976; Li and Chung, 1976) acting asendogenous ligand for these receptors, it was believed that endogenousopioid peptides produce their effects by interaction with specificreceptors. The existence of more than one type of opioid receptors wasfirst postulated by Martin (1967) when he observed that nalorphine had adual mode of action, antagonizing the analgesic effect of morphine andyet itself possessing analgesic activity. The existence of three types ofopioid receptors was suggested based on the differential spectrum ofactions produced by different opioids and benzomorphan drugs in vivoand the finding that some opioids, but not all, are able to relievewithdrawal symptoms in morphine-dependent dogs (Gilbert and Martin,1976; Martin, 1976). These were designated as t-, ic- and a-receptor forwhich the prototypical agonists are morphine, ketazocine or32ethylketocyclazocine (EKC) and N-allylnormetazocine (SKF10047),respectively. Activation of each of these receptors by their respectiveagonists produce distinct pharmacological effects in whole animals. Forexample, morphine induces analgesia, bradycardia, hypothermia andmeiosis, whereas ketazocine induces meiosis, sedation and depression offlexor reflexes. SKF 10047 induces mydriasis tachypnoea, tachycardia,and mania. The sigma (a)-receptor is regarded by some as a non-opioidreceptor because other drugs, such as phencyclidine, also act via thisreceptor. However, it has also been suggested that receptor classificationbased on the measurements of responses in vivo may not be conclusive.In subsequent in vitro studies, employing bioassays with guinea-pigileum and mouse vas deferens to compare the rank order of potencies ofvarious opioids on guinea-pig ileum and mouse vas deferens, it was foundthat morphine is more potent on the guinea-pig than the mouse vasdeferens whereas met- and leu-enkephalin were more potent on mouse vasdeferens than on guinea-pig preparations. Furthermore, naloxone, a nonselective antagonist with preference to n-receptors, is less potent inantagonizing the actions of other opioids in mouse vas deferens thanmorphine. The opioid receptors in the guinea-pig, and mouse vasdeferens are therefore thought to be j.t- and 6-receptors, respectively. Thepresence of distinct morphine (i.t) and enkephalin (6) binding sites was33confirmed by displacement binding assays with 6- and jt- labelled ligandsthat were displaced more readily with their respective cold ligands. Thediscovery of a separate K-binding site was based on the observation thatselective i- and 6- agonists have low potency in displacing the binding of[3H]EKC (Kosterlitz et al., 1981). Furthermore, responsiveness to 6-, tand K- agonists or ligands can be selectively protected against alkylationby simultaneous incubation with their respective selective ligand whereasthe responsiveness to the others is either abolished or significantlyreduced. Such selective protection against alkylation provided directevidence that 6-, p.- and K-receptors are physically distinct and notinterconvertible (Robson and Kosterlitz, 1979; Smith and Simon, 1980;James et al., 1982). To date, it is generally accepted that opioid receptorsare classified into at least three main types, namely p.-, K- and 6-receptors.1.3.2 Pharmacological and Physiological identification ofcardiac opioid receptorsOpioid receptors have been shown to be widely distributed in thecentral nervous system and the periphery (Bloom, 1983; Wamsley, 1983)and are implicated in the regulation of many physiological functions in34addition to analgesia. It was demonstrated in the early 19th century thatmorphine and other opiate alkaloids possess potent cardiorespiratoryeffects. The discovery of endogenous opioid peptides and their receptorsin the brain nuclei such as nucleus tractus solitarius (De Jong et al.,1983), nucleus ambigus and dorsal vagal nucleus (Laubie et al., 1979;Lang et al., 1982) of the brain stem and hypothalamus, which areimportant for the modulation of cardiovascular control, implicateendogenous opioid systems in the regulation of cardiovascular functionthrough the central nervous system.The endogenous opioid system influences the cardiovascular systemnot only centrally, but also peripherally. Opioid peptides are found in thevagus nerve (Hughes et al., 1977; Lundberg et al., 1979), in severalsympathetic ganglia (Schultzberg et al., 1979), and in the heart (Lang etal., 1983; Spampinato and Goldstein, 1983; Weihe et al., 1983, 1985).Adrenal enkephalin has been shown to be involved in cardiovascularregulation. For instance, stimulation of splanchnic nerve in reserpinizeddogs causes a naloxone reversible hypotension as a result of enkephalinrelease from the adrenal gland (Hanbauer et al., 1982). Met-enkephalinproduces a naloxone reversible fall in perfusion pressure in isolated cathindlimb (Moore and Dowling, 1982).The heart is known to contain opioid peptides such as dynorphin35(Spampinato and Goldstein, 1983), met-enkephalin and leu-enkephalin(Lang et al., 1983). Direct effects of endogenous opioid on the heartwere demonstrated by Eiden and Ruth (1982) when they observed thatlow concentrations of enkephalins are able to antagonize chronotropicresponses to noradrenaline on isolated rat atria. Based on the findingthat opioids inhibit field-stimulated cardiac noradrenergic responses, butnot the effects of exogenous administration of noradrenaline, it wassuggested that opioids have effect on the release of noradrenaline fromthe nerve terminals of guinea-pig atria was suggested (Ledda andMantelli, 1982). In an attempt to identify the existence of opioidreceptors in the ventricular sympathetic nerve terminals, selective opioidagonists, dynorphin and [D-Ala2,D-Leu5]enkephalinamide, were shown toproduce a naloxone-reversible potentiation of contraction in fieldstimulated isolated guinea-pig ventricular strip (Mantelli et al., 1987),suggesting that the receptors are of K- and 8- types.1.3.3 Cardiac opioid binding sitesIn 1977, using radioligand binding assays with tritiated naloxoneand dihydromorphine, as well as non-selective opioid antagonists, asprobes, Simantov and his co-workers first demonstrated the existence of36selective opioid binding in crude membranes homogenates prepared fromthe whole hearts of guinea-pigs and rats. Unfortunately, the extent ofsaturable opioid binding, relative to the total binding, was very small,10% for hearts as compared to more than 60% for the brains.In a subsequent binding study, Burnie (1981) also detectedstereospecific opioid binding sites in cardiac papillary muscle from ratright ventricle using titrated diprenorphine, a non-selective opioidantagonist, as a probe. Unfortunately, the study did not provide furtherdetails on binding properties.Subsequent binding studies not only confirmed the existence ofopioid binding sites in the heart, but also provided evidence on the typeof opioid binding site. However, results were inconsistant. Usingindirect binding assays, Krumins et al., (1985) demonstrated that [3H}diprenorphine binding was displaced by DADLE (ö-agonist);ethylketocyclazocine (K-agonist) and levorphanol (universal opioidagonist), but not by DAGO, (j.t-agonist), suggesting that the binding siteswere of the - and K- but not p-type in the right atrium and ventricle ofthe rat heart. In their subsequent study with competition binding assaysto characterize the binding properties of dermorphin, a naturally occuring[D-A1a2]heptapeptide with potent opioid activity and binding selectivelyfor p-receptors (Westphal et al., 1985), it was found that37[3H]diprenorphine was displaced by increasing concentration ofdermorphin in the left atrial membranes of the rat heart, suggesting thatthe presence of .t-opioid binding sites. In the rat cardiac sarcolemmamembrane preparation Ventura et al. (1989) demonstrated the presence ofK- and 6- but not t- binding sites, in agreement with the finding ofKrumins et al. (1985). Further evidence of the properties and distributionof K-binding sites in the rat heart was provided by Tai et al. (1991) whofound that there are substantial specific[3H]U69593 (selective K-ligand)binding sites in rat heart.1.3.4 Actions of opioids in nerve and cardiac muscleIn general, activation of opioid receptors in nerve cells producedtwo direct effects-inhibition of neurotransmitter release, and reduction incell firing. These effects are achieved by affecting ionic conductances.Activation of pt-receptors increases potassium conductance inpreparations such as rat locus coeruleus cells (Yoshimura and North,1983), mouse dorsal root ganglion cells (Werz and Macdonald, 1983), ratsubstantia gelatinosa neurones (Yoshimura and North, 1983), guinea-pigmyenteric neurones (Morita and North, 1982) and guinea-pig locuscoeruleus neurones (Pepper and Henderson, 1980). Study on single-ion38channels suggest a direct interaction between the pt-receptor andpotassium channels (Miyake et al., 1989). Opening of potassium channelsby i-agonists can be potentiated by guanosine 5’-[y-thio]triphosphate(GTPyS), suggesting a G-protein involvement (North et al., 1987).In a similar manner 6-receptor activation reduces transmitter releaseby hyperpolarization of the neurones. This involves a shortening of theduration of the calcium action potential resulting from an increase inpotassium conductance of the mouse DRG cell membrane (Werz andMacdonald, 1983).Kappa-agonists such as dynorphin, tifluadom and U50,488H haveno effect on nerves of the rat locus coeruleus (Yoshimura and North,1983) and guinea-pig submucous plexus (Mihara and North, 1986) whichcontains - and 6- receptors, respectively. On the other hand, ic-agonistsshortens action potential duration in guinea-pig myenteric plexus(Cherubini and North, 1985), a tissue with i- and ic- opioid receptors.Shortening is still observed when the action potential is prolonged bycaesium, suggesting an effect mediated via a direct action on calciumconductance, rather than on the potassium channel. Direct reduction ofcalcium currents in mouse dorsal root ganglion cells by ic-agonists wasfirst postulated by Werz and Macdonald (1983). In a subsequent study,they demonstrated, using voltage clamp techniques, that ic- but not p.- and396- agonists reduces calcium currents in these nerve cells (Werz andMacdonald, 1984). On the other hand, recent electrophysiological studiesinto opioids actions on cultured dorsal-root ganglion cells revealed directexcitatory actions of opioids. Activation of 6-, i- and i- opioid receptorscan prolong action potential duration when opioids are applied atnanomolar concentrations (Shen and Cram, 1989). Prolongation of actionpotentials by 6- or p- and K-opioid receptor activation is due to adecrease in a voltage-sensitive potassium conductance and an increase involtage- sensitive calcium conductance of the membrane, respectively(Shen and Cram, 1989; Cram and Shen, 1990). The prolongation ofaction potentials is not affected by pertussis toxin. However, treatment ofthe DRG neurones with cholera toxin blocks the excitatory effects of theopioid, suggesting that the involvement of G-protein in the signaltransduction of the response (Cram and Shen, 1990).It has been shown that opioid peptides inhibits the release ofnorepinephrine (Gaddis and Dixon, 1982; Illes et al., 1985) andacetyicholine (Konishi et al., 1981; Wong-Dusting and Rand, 1987) fromsympathetic and parasympathetic nerve endings in various preparations.Presynaptic modulation of neurotransmitter release by opioids has beenshown to affect contractility in ventricular tissues (Mantelli et al., 1987).Interaction of opioids with neurotransmitters in the regulation of cardiac40function was reported by Kosterlitz and Taylor (1959) who demonstratedthat morphine reduces the cardiac slowing produced by vagal stimulation.The association of cardiac opioid receptor-mediated action with cellularcalcium was first demonstrated by Ruth et al. (1983). In an attempt tofurther clarify mechanism of enkephalin in attenuating noradrenalineinduced positive chronotropic effect in isolated and spontaneously beatingrat atria (Eiden and Ruth, 1982), they found that leu-enkephalin produces45 ++a naloxone-revesible antagonism of noradrenaline-induced Ca uptakein the isolated rat atrial slices. Interestingly, the same authorsdemonstrated in other studies that noradrenaline-induced positivechronotropy was augmented by leu-enkephalin. In association with this,leu-enkeplialin causes a further increase in noradrenaline-activated 45Ca++ uptake in isolated guinea-pig atria (Ruth et al., 1983). In addition toindirect action of opioids on cardiac functions, opioids have also beenshown to act directly on ventricular myocytes, which are devoid ofnervous influence (Laurent et al., 1985; Ventura et al., 1991). Therefore,the actions of opioids on cardiac muscle may via the autonomic nervoussystem or directly on muscle cells.Although activation of cardiac opioid receptors has been shown toincrease [Ca2+]j (Ventura et al., 1991). Knowledge of the effects ofcardiac opioid receptor stimulation on ionic fluxes across sarcolemmal41membrane is scanty. Meptazinol, a opioid partial agonist, has beenshown to increase action potential duration by 40% in rat papillarymuscle (Fagbemi et al., 1983). The opioid agonists, fentanyl andsufentanil, prolong action potential duration at 50% and 90%repolarization (APD50 and ADP9O) in canine Purkinje fibers, in a nonnaloxone-reversible manner, suggesting a direct membrane effect (Pruettet al., 1987; Blair et al., 1986). It has been reported that U50,488H at 105M reduces the slow inward current, a main current that maintains theplateau phase of the action potential in guinea-pig myocytes (duBell andLakatta, 1991). Whether the effects of the opioids on ionic fluxes inmuscle is mediated via opioid receptors have not been tested.1.3.5 Opioids and arrhythmiasIn addition to the effects on heart rate and contractility, cardiacopioid receptors have also been implicated in cardiac arrhythmogenesis.This was first demonstrated by Stein (1976) who showed that high dosesof morphine can induce cardiac arrhythmias including atrial fibrillationand atrio-ventricular block in conscious rats. Involvement of opioidreceptors in arrhythmias arising during myocardial ischaemia weresuggested when naloxone, a universal opioid antagonist, was42demonstrated to attenuate arrhythmias in both anaesthetized andconscious rats subjected to coronary artery ligation (Fagbemi et al.,1982). It was then generally believed that attenuation of responses bynaloxone was a pathognomonic involvement of opioid receptor. Insupport of the notion that opioid receptors were involved in ischaemicarrhythmogenesis, it was shown that the opioid antagonists, (-)-naloxone(Lee, 1992), posses antiarrhythmic activity while their pharmacologicallyinactive structural isomers did not. However, direct actions of naloxonecould also contribute to its antiarrhythmic activity. This is supported bythe finding that (+)-naloxone, which is 1,000-10,000 less potent as anopioid antagonist (lijima et al., 1978) is equipotent as an antiarrhythmicagent (Same et a!., 1988; Same et al., 1991).The use of isolated perfused rat heart preparations makes it possibleto determine directly whether cardiac opioid receptors are involved inarrhythmogenesis. Several lines of evidence suggest that activation ofcardiac opioid receptors are contributory to the genesis of cardiacarrhythmias arising during myocardial ischaemia and reperfusion. Firstly,naloxone attenuates the incidence of arrhythmias following myocardialischaemia and reperfusion in the isolated rat heart preparation (Zhan etal., 1985; Lee and Wong, 1986; l987a). In addition, naltrexone (Liu etal, 1988), another prototypical opioid antagonist, also possesses43antiarrhythmic activity. Isolated rat hearts of chronically morphine-treated rats exhibit less arrhythmias in response to dynorphin..3 (Wongand Lee, 1987), a phenomenon characteristic of receptor mediated event.The hearts of chronically morphine treated rats also exhibit lessarrhythmias in response to myocardial ischaemia and reperfusion (Wongand Lee, 1987), suggesting that ischaemia and reperfusion inducedarrhythmia may also involve cardiac opioid receptors. Further, aselective K receptor agonist, U50,488H was found capable of elicitingventricular arrhythmias in isolated rat hearts (Wong et al, 1990). Thesame drug has also been found to exacerbate ischaemia inducedarrhythmias in rats (Lee et al., 1992).However, there are also problems which make it difficult to ascribea pathophysiologically relevant arrhythmogenic role to opioid agonistsand an antiarrhythmic role to opioid antagonists. Firstly, there is noconvincing evidence that endogenous opioids actually accumulate in themyocardium during ischaemia or reperfusion although endogenous opioidpeptides were shown to have released following acute myocardialischaemia (Oldroyd et al., 1992). Secondly, data on arrhythmogenic andantiarrhythmic effects of opioid agonist and antagonist substances are notconsistent. Certain opioid agonists have been reported to reduce ratherthan increase the incidence and severity of arrhythmias elicited by44myocardial ischaemia (Pugsley et al., 1992a,b & 1993; Fagbemi et al.,1983; Boachie-Ansah et al., 1989). In contrast with Wong et al & Lee etal, U-50,488H was found to be pro-arrhythmic at low doses but wasantiarrhythmic at high doses (Pugsley et al., 1992a,b). Thirdly, evidencefor protection of arrhythmias by antiopioid agents is inconsistence.Bergey and Beil (1983) found that naloxone at varies doses wereineffective against ischaemia induced arrhythmias in pigs. Consistentwith this, Pruett et al (1991) found that naloxone at varies concentrationshad no effect on cardiac action potential configuration. Thus the questionof whether opioid agonists and antagonists are pro- or anti-arrhythmicstill has to be further investigated.1.3.6 i-opioids and arrhythmiasAlthough findings from previous studies have implicated theinvolvement of cardiac opioid receptors in arrhythmogenesis andantiarrhythmic activities during myocardial ischaemia and infarction, thetype opioid receptor(s) involved remains to be determined. In an attemptto identify the cardiac opioid receptor subtype(s) involved, Wong et a!(1990) has shown that U50,488H, a selective i-agonist, and MR2266, aselective ic-antagonist, have significant greater arrhythmogenic effects45and antiarrhythmic effects, respectively, than i- and a- opioid agonistsand antagonists such as DAGO, DPDPE, DADLE, and naloxone. Inagreement with the above finding, Sitsapesan and Parratt (1989) foundthat in the anaesthetized rat MR2266 is the most potent antiarrhytmicagent among the three types of opioid antagonists during ischaemia.Overall, the results of these studies suggest if opioid receptors areinvolved then the cardiac ic-receptors are the most likely receptor-subtypeinvolved in arrhythmogenesis or antiarrhythmic activities duringischaemia.1.3.7 Mechanism of antiarrhythmic effects mediated by icagonists1.3.7.1 ic-opioid receptor mediated antiarrhythmic effectsic-receptor mediated adenylate cyclase inhibition with the use ofdynorphin and U-50,488H have been reported in membranes from ratspinal cord neurons (Attali et al., 1989) and guinea pig cerebellum(Konkoy and Childers, 1989). Adenylate cyclase, which inhibited by thea-(GTP)subunit of the G protein, inhibits the formation of cAMP fromATP, and thus disrupts the protein phosphorlation step that controls a46variety of cellular activities. Opioid receptor mediated adenylate cyclaseinhibition can lead to attenuation of the cAMP-dependent protein kinaseactivity that mediated the phosphorylation (activation) of the voltage-dependent calcium channels. Modulation of calcium channels by cAMPhas been reported in several studies (Cachilin et a!., 1983; Chad et al.,1984; Kostyuk et al., 1981). Recent studies by Attali et al (1988) andGross et a! (1987) also showed that U50,488 and dynorphin decrease bothL and N-type calcium currents in dorsal root ganglion cells indirectly viaa cAMP-dependent mechanism. In addition, Gross et al (1990) and Northet al (1987) have reported the inhibitory effects of dynorphin andU50,488 on calcium conductance are direct effect to the voltage-dependent calcium channels via G-protein. Although these studies wereall done in neuronal tissue, it is acceptable to suggest that the secondmessenger system in the cardiac tissue would function in similar manner.Therefore, the antiarrhythmic activities mediated by K agonists orantagonists are possibly effects of cardiac calcium channel blockadesecondary to receptor binding.Another possible mechanism for the antiarrhythmic activitymediated by K agonists and antagonists is the change of intracellularcalcium concentration mediated via K agonists. Recent study by Misawaet a!. (1990) showed that binding of U50,488 to the K-receptors in the47guinea pig cerebellum inhibit GTP-stimulated phospholipase C (PLC)activity. PLC is important in the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol l,4,5-trisphosphate (1P3)which serve as an important intracellular second messengers for proteinkinase C activation and intracellular calcium mobilization. An opposingfinding is also reported by Periyasamy and Hoss (1990, 1991) whosuggested that U50,488, dynorphin, and ketocyclazocine (at highconcentrations) all stimulate phosphoinositol turnover in rat hippocampalslices by stimulating GTP-stimulated phospholipase C activity. 1P3 isknown to liberate Ca2 from cardiac SR (Nosek et al., 1986; Fabiato,1990; Kentish et al., 1990). Increase in 1P3 can lead to an increase in[Ca2] The increase or decrease in [Ca2]1resulted from stimulation ofprotein kinase C or PLC inhibition may play an important role in thearrhythmogenesis and antiarrhythmic activities of i agonists andantagonists since [Ca2]1 levels has been linked with myocardialarrhythmia occurrences as discussed earlier. However, at present, there isno evidence to support these 2 proposed antiarrhythmic mechanisms.481.3.7.2 Cardiac ion channel blockadeMany studies have shown that the antiarrhythmic actions of opioidsare independent of the opioid receptors but instead involve cardiac ionchannel blockade. Studies in rats by Pugsley et al (1992a,b, 1993) haveshown that the antiarrhythmic effects of ic agonists, U50,488H, and PD129290 were unaltered by naloxone pretreatment suggesting theantiarrhythmic actions are independent of ic and other opioid receptors.In addition to observing whether U50,488H and related compounds haveeffects on blood pressure and heart rate which are not attenuated bynaloxone, ECG observation is a useful indirect method for determiningthe effects of antiarrhythmic drugs on cardiac ion channels. ECG changesinduced by U50,488H can be interpreted as indicating ion channelblockade, particularly for the sodium channel (Class I activity). Thus,U50,488H produced P-R prolongation and QRS widening in rats (Pugsleyet a!., 1992b), together with elevation of “RSh,” an ECG index of sodiumchannel blockade in this species (Penz et al., 1992). These actions wereunaltered by naloxone. As with U50,488H, PD 129290 also showedsodium channel blocking action that are not blocked by naloxone. Theevidence for the above events being unrelated to the ic receptors wasstrengthened by findings with the R,R (+)-enantiomer of PD 129290, PD49129289 (Pugsley et al., 1993). Unlike PD 129290, PD 129289 has verylow affinity for ic receptors. Yet, both compounds increased the size ofthe P-R, QRS and RSh intervals of the ECO. However, it should also benoted that changes in the Q-T interval of the ECG seen at higher doses ofthese compounds suggest that a degree of potassium channel blockade(Class III activity) also occurred. It is possible that this action which isindependent of opioid receptors may also contribute to antiarrhythmicactions.Studies by Same et al (1989 & 1991) using a variety of opioidagonists and antagonists: naloxone and its non-opioid stereoisomer (+)-naloxone; Win 44,441-3 and its stereoisomer Win 44,441-2; levorphanoland its (+)-stereoisomer, dextrorphan, also suggested that theantiarrhythmic effects of these opioid drugs are direct effect on ioniccurrents in cardiac muscle and are not mediated by agonism orantagonism of the stereospecific opioid receptors. This is in agreementwith the previous finding that naloxone suppresses sodium and potassiumcurrents (Carratu and Mitolo-Chieppa, 1982; Brasch, 1986). Furtherstudies by Oldroyd et al (1993) have also shown that naloxone has both anon-opioid-receptor-mediated Class III antiarrhythmic effect on normalmyocardium and a Class I effect on ischaemic myocardium since bothracemic naloxone (active at opioid receptors) and d-naloxone (inactive)50prolonged action potential duration and effective refractory period innormally perfused rabbit interventricular septa, and enhanced the fall inmaximum upstroke velocity of action potential in partially depolarizedventricular myocardium. In addition, buprenorphine, the opioid whichpossesses both partial .t-agonist and K-receptor antagonist activity, andmeptazinol, a partial opioid agonist, have also been shown to beantiarrhythmic during myocardial ischaemia in the anaesthetised rats, byreducing the action potential height and maximum rate of depolarizationof phase zero (MRD) and prolonging the duration of the action potential,effects that resembled the actions of Class I and Class III antiarrhythmicagents (Boachie-Ansah et al., 1989; Fagbemi et al., 1983). These effects,indeed, may not be mediated via specific opioid receptors because theconcentrations required to confer protection against ischaemic arrhythmiawere much higher than the concentrations required for opioid receptorantagonism.1.4 RSD 939RSD 939 (Figure 1) was developed by Rhythm Search DevelopmentLtd. in an effort to discover a more effective and less toxic drug for thetreatment of cardiac arrhythmias. It has a pKa value of 8.3, the empirical51formula isC19H26NO1•HC1, and it has a molecular weight of 421.79g/mole.5200CICINFigure 1. The structure of RSD 939531.5 Aims of studiesIn myocardial ischemia or infarction, ventricular fibrillation isresponsible for the majority of deaths (approximately 90%). Class Iantiarrhythmic agents (sodium channel blockers) have a long history, bothin the treatment of a variety of arrhythmias and in their development bymodification of basic chemical structures. However, endeavours toimprove on prototypical class I agents have not met with success despitethe introduction of numerous newer agents. Therefore, there is acontinuing need to search for safer and more efficacious drugs (Podrid,1989). However, the route to new drugs is difficult due to the lack ofexact knowledge concerning the mechanisms underlying the genesis ofarrhythmias in the clinical population and the relative lack of knowledgeregarding pharmacological properties which confer antiarrhythmic activity(Botting et al., 1986).Recently, accumulating evidence has indicated that various opioidagonists and antagonists, especially K agonists, exhibit a variety ofcardiovascular and antiarrhythmic actions. Two important questionsarise. First, are these cardiovascular and antiarrhythmic actions mediatedby the opioid receptors? Second, what is the mechanism underlying theseactions if these effects are not mediated by opioid receptors? Previous54studies have shown that some of the cardiac and cardiovascular actions ofic agonists are a result of its direct action on cardiac sodium channels andare independent of opioid agonism. RSD 939 is structurally related to icagonists and it appears to be a potent and selective ic agonist in bindingstudies (unpublished data by Abraham et al). The present study is anattempt to study the involvement of opioid receptors in the cardiovascularand antiarrhythmic activity possessed by ic agonists and to determine theunderlying mechanism of these activities with the use of RSD 939.552 MethodsAll animal experiments conducted at U.B.C. were according toguidelines established by the U.B.C. Animal Care Committee.2.1 Experimental planThe overall goal of the laboratory investigation was to study theinvolvement of opioid receptors in the cardiovascular and antiarrhythmicactivity possessed by i agonists and to determine the underlyingmechanism of these activities with the use of RSD 939. In keeping withthis, the following studies were performed with RSD 939 in the presenceand absence of naloxone:1) Assays of antiarrhythmic efficacy against ischaemia-inducedarrhythmias in rats.2) Assays of antiarrhythmic efficacy against electrical inductionof fibrilloflutter in rats.3) Characterization of the pharmacological profile anddetermination of possible mechanisms of underlyingantiarrhythmic efficacy through analysis of effects on ECG,56haemodynamic data, and electrical stimulation parameters inrats and in isolated rat hearts.2.2 Opioid actions2.2.1 Analgesia assays in vivoAnalgesic properties of RSD 939 were measured in mice by the useof the tail pinch method. This involved pinching the tail of the mousewith a guarded arterial clip for a time not greater than 5-10 seconds. Theduration of analgesia was not followed, but the fact of its presence orabsence within 5 to 15 mm of injection. Injection of RSD 939 was fromthe distal part of the tail via the tail veins using a 1 cc syringe and a 27.5gauge needle.Determination of ED50, a dose that causes analgesia in 50% of thepopulation studied, was based on the following protocol: Four groups ofanimals composed of 4 mice per group, were injected with different dosesof RSD 939. This gave us 4 dose levels for these groups. Among these 4groups, at least 2 groups should show partial analgesia. ED50 wasdetermined by probit analysis of the dose-analgesia curve in whichpercentage of animal experiencing analgesia was plotted against log-dose.572.2.2 Binding studiesOpioid receptor binding studies were conducted by Dr. S.Abrahamt. The experimental procedure used is given below. It is amodification of procedures used by other groups (Millan, 1990; Rothmanet al, 1989).Guinea pig were sacrificed by decapitation and their brains weredissected free of membrane. Brains (excluding cerebella, for jt receptors)or cerebella only (for i receptors) were homogenized with a polytronhomogenizer in 20 volumes of 50 mM Tris HC1 (pH 7.5) buffer andcentrifuged at 30000 x g for 15 mm. The pellet was rehomogenized andcentrifuged 2 addition times. Membranes were then suspended in TrisHC1 buffer to a tissue concentration of 20 mg wet weight per ml.Tissue homogenates (0.5m1) were incubated at 25°C with[3HJDAGO (0.5 nM) or[3H]U-69593 (0.5 nM) and 9 concentrations ofRSD 939 in a total volume of 1 ml Tris HC1 buffer. The reactionmixtures were incubated for 90 or 60 minutes, for t or i receptors,respectively. All assays were performed in duplicate and two separatedeterminations were performed for each observation. Nonspecific bindingwas determined in the presence of 10 tM of morphine or the unlabelledDepartment of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel.58ligand U-69593. The reaction was terminated by filtration through glassfiber filters (Whatman GF/B). Radioactivity bound to the filters wasmeasured by lipid scintillation spectrometry.1C50 values were determined by linear regression of the log-probability plots of the displacement curve obtained with each sample.2.3 Cardiovascular assessmentIn this study, RSD 939 was given as cumulative i.v. bolus doses toanaesthetized rats whose blood pressure, heart rat and ECG weremeasured. This study gave an initial profile of the acute cardiovascularand toxic actions of RSD 939, and data from these studies was used toestablish the dose range to be used in subsequent isolated heart, electricalstimulation and myocardial ischaemia assays. The involvement of opioidreceptor in mediating responses was determined by the administration ofnaloxone, at a dose which had no cardiovascular or ECG actions butblocked opioid receptors.2.3.1 Surgical preparationMale Sprague-Dawley rats (Charles River Laboratories, Montreal,59Quebec) weighing 200-300 g were subjected to preparative surgery undera large loading dose of sodium pentobarbitone anaesthesia (65 mg/kg,i.p.). Supplemental doses of diluted pentobarbitone (1/10 dilution) weregiven i.v. when, and if, necessary to ensure an adequate level ofanaesthesia. Body temperature was monitored by a rectal thermometerand maintained between 36-37 °C with a heating lamp.The right external jugular vein was cannulated for intravenousinjections of drugs, while the left carotid artery was cannulated formeasurement of mean arterial blood pressure through a calibratedpressure transducer connected to a Grass Polygraph (Model 79D).Animals were placed in a supine position, a tracheotomy performed and ablunt 15 gauge needle was secured in the trachea to prevent obstruction ofthe airway. However, the animal was not ventilated and was allowed tobreathe spontaneously in order to allow the cause of death to be recorded.In order to obtained the best ECG signal in rat, a modified lead IIconfiguration was used. Three ECG needle limb leads were placedsubcutaneously along the suspected anatomical axis of the heart (rightatrium to apex) as determined by palpation. The superior electrode wasplaced at the level of the right clavicle, approximately 0.5 cm from themidline of the trachea, and the inferior electrode was placed on the leftside of the thorax, approximately 0.5 cm from the midline at the level of60the ninth and tenth ribs (Penz et al., 1992). The ECG records wereobtained on a Grass Polygraph using a 7P1F low level preamplifier andassociated driver amplifiers at a bandwidth of 0.1-40 Hz. Both bloodpressure and ECG measurements were made directly from Grasspolygraph records recorded at chart speed of 100 mm sec’.2.3.2 Experimental DesignIn vivo dose-response curves were constructed for the effects ofRSD 939 at a cumulative dose of 1.0, 2.0, 4.0, 8.0, 16.0 iimole kg’ i.v. oruntil death occured in pentobarbitone anaesthetized rats (n 5). In aseparate group of animal (n 5), the effects of naloxone on thecardiovascular and ECG effects produced by RSD 939 were alsodetermined.In this study, rats were selected at random from a single group.After surgical preparation, they were allowed 30 mm to recover beforedrug administration. In a random and double-blind manner rats weregiven an initial injection of either saline or 8 itmole kg’ naloxone, a dosewhich is much higher than the pA2 (Martin, 1983) but shown to have nocardiovascular or ECG actions (Pugsley et al., 1992). This dose could beexpected to effectively block any opioid receptor-dependent effects of61RSD 939, even when given at the highest doses. The injection of vehicle,or the first dose of RSD 939 was randomly and blindly given 5 mm later.This resulted in four groups of animals (n = 5 each group). Group I wasnaloxone pretreated rats tested with RSD 939. Group II was naloxonepretreated rats tested with vehicle. Group III and IV were salinepretreated rats tested with RSD 939 and vehicle, respectively. All doseswere injected i.v. over 30 sec and blood pressure, heart rate and ECGwere recorded 0.5, 1, 2, 4, and 8 mm after and immediately prior toaddition of the next cumulative dose. The vehicle was 22% ethyl alcoholand 78% distilled water. The maximun volume of vehicle or drug givento the rat was 1.0 ml / lOOg body weight. The cause of death in eachanimal was recorded as being due either to arrhythmias (very uncommon),an irreversible decline in blood pressure, or respiratory failure.2.3.3 Data AnalysisMean blood pressure, heart rate, and ECG variables were recordedusing a Grass Polygraph as described above. The ECG variables weremeasured manually with a micrometer from recordings made at a chartspeed of 100 mm/sec (see Figure 2). The mean blood pressure was takenas an approximate average of systolic and diastolic pressures. Heart rate62was calculated by dividing the R-R distance between two beats in mminto 6000 mm/mm (recording was made at 100 mm/sec = 6000 mm/mm)to get the number of beats per minute. ECG intervals were defined asfollows:P-R interval - measured from the beginning of the P wave to a linedrawn from the peak of the R wave to the isoelectric line following thecurvature of the paper.QRS complex - measured from the beginning of the R wave to theend of the S wave allowing for curvature of the paper.QT1 interval - measured from the beginning of the R wave to thepeak of the T wave (i.e. to a line drawn down from the peak of the T waveto the isoelectric line following the curvature of the paper).QT2 interval - measured from the beginning of the R wave to thepoint of inflection of the downstroke of the T wave (again following thecurvature of the paper).RSh - measured from the peak of the R wave to the base of the Swave, again following the curvature of the trace. This new measure,termed “RSh” or RS-height, is a more sensitive measure of sodiumchannel blockade than conventional measures such as QRS complexwidening and P-R interval prolongation (Penz et al., 1992). Penz et al,using various Class I sodium channel blockers, have shown that changesin RSh occurred before changes in QRS or P-R.6364Figure 2: A typical ECG from the rat. Also shown are the variablessRSh0-PQsQ—T 10—T2which are measured.65Differences in effects of blood pressure, heart rate, and ECGintervals between vehicle-control and drug treatment groups wereexamined by unweighted means ANOVA, and if the sources of variancesignificant (p < 0.05), were followed by Duncan’s multiple range testusing NCSS computer statistical packages (Hintze, 1987). Since this is acumulative dose study, the above two statistical tests were also used totest all doses of RSD 939 to determine if drug effects on the variablesmeasured were statistically significant. Thus all data were also comparedbetween pre-drug and dosage level.2.4 Isolated rat heartsOpioid receptors are found in the vagus nerve, in severalsympathetic ganglia as well as the heart, thus opioid peptides caninfluences the cardiovascular system not only centrally but alsoperipherally. Since myocardial tissue was the tissue in whichantiarrhythmic actions of RSD 939 were expressed, it was of interest toevaluate the cardiac pharmacology of RSD 939 in the absence of neuronaland humoral innervationorganism so conventional studies were performedon unpaced Langendorff perfused rat hearts. The Langendorff rat heartmodel was first devised by Langendorff in 1895. It has many advantages66for the investigation of the actions of drugs on both the mechanical(contractile force) and electrical (ECG) activity. Yet we also perceived anumber of deficiencies of this model. In the Langendorff heart, a filteredKrebs-Henseleit solution is used to replace the blood. This results ingradual failure with edema. In addition, only the ventricles are filledwith solution. Measurements of coronary flow by simply collecting theoutflow is also subject to considerable error. However, the use of fivehearts for each determination tended to reduce such sources of varianceand allowed sufficient accuracy for observing pharmacological effects onthe myocardium which might explain any antiarrhythmic actions of RSD939. Other limitations to the use of this method such as the low oxygen-carrying capacity of the perfusate and the ease of damage of the aorticvalves which allowed perfusion fluid to enter and distend the leftventricle have been minimized by the use of a modified perfusionapparatus developed by our laboratory (Curtis et al., 1986a).2.4.1 Perfusion apparatusExperiments were performed using the modified perfusionapparatus designed and constructed in our laboratory to study the actionsof drugs on the mechanical and electrophysiological behavior of hearts67from small animals (e.g., rat, guinea pig) hearts (Curtis et al., 1986a).This apparatus consists of nine chambers (each of 250 ml capacity)connected to the aortic perfusion cannula of the Langendorff perfusedheart via separate silastic tubes was designed to allow a low volume dead-space for rapid switching between different solutions in order to permitthe rapid generation of dose-response data. Perfusates were kept constantat 37 °C by circulating warm water heated by an external thermoregulatedheater. 100% 02 was used to oxygenate each solution and to pressurizethe chambers (at 100 mmHg) to drive perfusate through the coronarycirculation.2.4.2 PreparationThe perfusing solution was formulated in our laboratory to allowmaximal oxygen carrying capacity and optimal pH stability. It consistedof (in mM) NaC1, 1.23; KC1, 3.35; MgSO4.7H20, 1.18; D-Glucose, 11.1;CaCl2H0, 2.52; PIPES (Piperazine-N,N’-bis[2-ethanesulfonic acid]),14.34 and NaOH titrated to pH 7.4 (Table 9). This newly formulatedbuffer together with our modified apparatus improved the oxygen deliveryto the heart because this system reduces loss of oxygen to theTable9.IoniccompositionofphysiologicalsaltsolutionsusedforcardiactissueincomparisonwithratserumandinterstitialfluidCanonsAnionsGas (%)SolutionNaKCaMgClHCO3H2P04SO4OHUreaGlucosePIPES02CO2Total ionconcentrationinplasma1523.72.71.0611426.51.70.697.05.8lonizedionconcentrationinpiasma1503.61.60.7611225.71.60.697.05.8Ionizedionconcentrationininterstitialfluid1473.51.50.7211526.31.70.737.05.8Krebs-Henseleitbuffer1435,92.5012824.91.181.6405.6955P]PESbuffer1533.42.51.18131001.186.0011.114.31000TheconcentrationsofanionsandcationsaregiveninmM.Notethat2.0mlvisodiumpyruvateisaddedtoKrebs-HenseleitsolutionsPIPESPiperazine-N,N’bis[2-ethanesulfonicacid].OHinPIPESbuffersolutionisfromNaOHaddedduringtitration.0069dead space between the reservoir and the heart, and because the apparatusoxygenates preheated buffer, rather than heating pre-oxygenated buffer.Male Sprague-Dawley rats weighing 300 to 400 g were sacrificedby a blow to the base of the skull and exsanguinated before hearts wererapidly excised from the chest cavity. Hearts were immediatelyretrogradely perfused with 10 ml of ice-cold PIPES buffer solution andthen mounted on the perfusion apparatus via an aortic cannula. Heartsimmediately were perfused with an oxygenated PIPES buffer solution at37 °C and pH 7.4. Within seconds, the heart began beating in sinusrhythm. The left atrium was then removed in order to insert a smallcompliant, but non-elastic balloon made of plastic wrapping film (“SaranWrap”) into the left ventricle for ventricular pressure measurements(Curtis et a!., 1986a). For maximal ventricular contractility, the pressurewithin the balloon was adjusted to give an left ventricular end-diastolicpressure of 10 mmHg. The aortic root perfusion pressure controlled bythe oxygenating gas (100% 02) was kept constant at 100 mmHg to mimicthe normal perfusion pressure of the coronary arteries in vivo. Bothperfusion pressure and ventricular pressure were measured by pressuretransducers while the contractility or the maximal rate of intraventricularpressure development (+dpldtmax) and maximal rate of intraventricularrelaxation (dp/dtmax) were obtained by differentiating left ventricular70pressure using a Grass Polygraph differentiator (model 7P20C). Specialatraumatic, silver-ball electrodes were designed for ECG recording fromthe epicardial surface of the heart (Curtis, 1986) using a Grass Polygraph(model 7D) at a bandwidth of 0.1-40 Hz. Electrodes were placed in anapproximately Lead II configuration thus one electrode was placed on theright atrium to allow recording of a large P wave, and the second on theleft ventricle. Measurements of mean coronary flow perfusate was doneby collecting effluent at one minute intervals in a graduated cylinder.2.4.3 Experimental design and data analysisTo assess direct effects of RSD 939 on rat cardiac tissue in vitrodose-response curves were constructed for the effects of RSD 939 at acumulative dose of 0.1, 0.3, 1.0, and 3.0 j.tM on isolated rat hearts (n5). In a separate group of hearts (n = 5), the effects of naloxone on thecontractility and ECG effects produced by RSD 939 was also determined.For 15 minutes, hearts were perfused with PIPES buffer solutionalone and measurements of heart rate, contractility, and ECG were takenevery minute for a minimum of 15 minutes, or until stable control valueswere obtained. For the dose-response study, RSD 939 at concentrationsof 0.1 to 3.0 LLM were administered cummulatively for a period of 371minutes at each concentration. Recordings were made at 0.5, 1, 2, and 3minutes interval. A 5 minutes wash period then followed. Experimentswith naloxone were performed by adding naloxone to the perfusate for aperiod of 5 minutes before the co-administration of RSD 939 andnaloxone. Two sets of control experiments were done. The first one usedonly the vehicle for RSD 939. The second one used both vehicle andnaloxone. The pre-drug period also act as the control values for theexperiments.Heart rate, systolic ventricular pressure, contractility (+dp/dtmax anddP/dtmax), P-R interval, and QRS complex were measured. However, Q-Twas not measured as a result of the difficulty in determining the T-wavein isolated rat hearts. Statistical analysis were performed as in theprevious studies.2.5 Electrical stimulaltionElectrical stimulation was designed to further define theelectrophysiological actions of RSD 939 in intacts rats. It involvedcumulative i.v. infusions of RSD 939 to anaesthetised rats, with implantedleft ventricular electrodes, in order to test for responsiveness to electricalstimulation. Electrical stimulation adds further information regarding72effects on blood pressure, heart rate, ECG and toxicity. A program ofelectrical stimulation allows assessment of possible effects on myocardialionic channels such as sodium and/or potassium channels since sodiumand potassium channel blocking drugs have clear profiles of action insuch test. It has been well established that drugs which decrease sodiumcurrents increase threshold current & threshold pulse width for capture ofsingle beats (iT-ji.A & tT-ms) and ventricular fibrillation threshold (VFt1iA) (Wiggers & Wegria, 1940). On the other hand, pure potassiumchannel blockers might not affect thresholds for capture yet suppress VFinduction by making the heart refractory to the fractionating wavefront(Winslow, 1984). However, a pure potassium channel blocker would beexpected to prolong the effective refractory period (ERP-ms) and decreasethe maximum following frequency (MFF-Hz) to square wave stimuli(Vaughan-Williams, 1970; 1975). Thus, by testing the drugs for theirinfluence on iT, tT, VFt, ERP, and MFF, we hoped to establish an indexof their sodium vs. potassium blocking actions. In addition, electricalstimulation was also useful in the assessment of the potency and efficacyof RSD 939 in protecting against electrically-induced arrhythmias.732.5.1 Experimental preparationMale Sprague Dawley rats weighing 250 to 350 g were prepared ina manner similar to that used for cardiovascular assessment studies, withone exception. A tracheotomy was performed and rats were artificiallyventilated using room air with a Palmer small animal respirator at a strokevolume of 10 mi/kg and rate of 60 strokes/mm to ensure appropricateblood-gas levels (MacLean and Hiley, 1988).The skin above the the level of the heart was removed and palpationperformed to determine the position of the left ventricle. Stimulatingelectrodes were made from Teflon coated silver wire by removing 1-2 mmsegment of insulation from the end of the wire which was passed throughthe lumen of a 27 gauge needle. The desheathed tip of the wire was bentback to form a barb. The needle were passed into the thoracic cavity andthe electrode lodged in the left ventricular apical free wall. This processwas repeated for a second electrode 1-2 mm apart from the first one. Thepositioning of the electrodes were confirmed at the end of the experimentby dissection. This technique allowed rapid insertion of stimulatingelectrodes 1-2 mm apart with minimal trauma. Stimulation of the leftventricle with square wave pulses was with Grass SD9 Stimulators(Howard and Walker, 1990). Subcutaneous ECG electrodes in a Lead II74configuration were used. The ECG and BP were recorded on a GrassPolygraph (model 79D), and a delayed loop oscilloscope (HoneywellModel E for M) was used for continuous assessment of the ECG.2.5.2 Experimental designIn vivo dose-response curves were constructed for the effects ofRSD 939 at infusions of 1.0, 2.0, 4.0, 8.0, 16.0 tmole/kg/min i.v. (n = 5).In a separate group of animal (n = 5), the effects of naloxone onresponses to RSD 939 were determined.In this study, rats were selected at random from a single group.After surgical preparation, they were allowed 30 mm to recover beforedrug administration. In a random and double-blind manner rats weregiven an initial injection of either saline or 8 pmole kg1 naloxone. Theinfusion of vehicle, or the first dose of RSD 939 was randomly andblindly given 5 mm later. This resulted in four groups of animals (n = 5each group). Group I was naloxone pretreated rats tested with RSD 939.Group II was naloxone pretreated rats tested with vehicle. Group III andIV were saline pretreated rats tested with RSD 939 and vehicle,respectively. Each dose was infused i.v. over 3 minutes and electricalstimulation variables were measured in triplicate after 2 minutes of75infusion. Each animal also acted as its own control since prior to druginfusion, control values of electrical stimulation variables weredetermined every five minutes until stable control values were obtained.The last set of values was taken as pre-drug values.Prior to determination of electrical stimulation variables and at theend of the experiment, a 1 ml sample of blood was withdrawn from thecarotid artery line and the initial and final [K] was determined (loneticsPotassium Analyzer).2.5.3 Experimental end-pointsSquare waves stimulation was used and discrimination of the endpoints were made on an oscilloscope. Each end-point (iT, tT, VFt, ERP,and MFF) was determined in triplicate 2 minutes after commencing eachinfusion step. The mean value of three measurements were used. Theprocedures for the end-points measurement has been described by Curtiset al. (1984, 1986). Threshold currentThreshold current (iT) is the minimum current required for capture.76The heart is captured when it follows the pulses generated by thestimulator and is easily observed. With capture, the following wereobserved:i) an increase in signal size.ii) a regular rhythm at a fast rate of 7.5 Hz.iii) a slight but sudden drop in blood pressure.The threshold current was determined at 7.5 Hz, approximately 100beats/mm above the sinus rate, and a pulse width of 1 ms. Thesholdcurrent usually falls within the range of 20 to 100 j.tA. Threshold pulse widthThreshold pulse width for capture (tT) was the minimum duration tocapture the heart. It was determined according to the criteria formeasuring the threshold current and at twice current threshold. Averagethreshold pulse widths were 0.3 ms. Ventricular fibrillation thresholdVentricular fibrillation threshold was defined as the currentnecessary to produce fibrillation and a precipitous drop in blood pressure.77Periods of stimulation of approximately 4 seconds duration were requiredfor each determination. The end point was determined by increasing thecurrent strength (at 50 Hz and twice the threshold pulse width) untilfibrillation occurred. The characteristic were generally non-sustainedfibrillo-flutter. Therefore, fibrillation threshold measurements alloweddetermination of the effectiveness of RSD 939 against a non-damagingtype of arrhythmia. Effective refractory periodand maximum following frequencyEffective refractory period (ERP) in the ventricle was defined asthe shortest interval between two stimuli to which the ventricle responds.It was determined by the extra-stimulus method. In this method the heartis paced at a baseline frequency of 7.5 Hz, twice the threshold current andtwice the threshold pulse width. A single extra stimulus of the samefrequency, current strength, and pulse width was applied at a variabledelay after the pacing stimuli. The minimum delay at which an extrastimulus resulted in a extra-systole was taken as the effective refractoryperiod.Maximum following frequency (MFF) was defined as the frequency78at which the heart failed to follow, on a 1:1 basis, a steadily increasingfrequency of stimulation from a baseline of 5 Hz. It was determined by attwice threshold current and pulse width. The frequency of stimulationwas rapidly increased until the heart was unable to follow as determinedfrom the blood pressure, which had been reduced by the increasingtachycardia with increasing rate, suddenly showing a large escape beat.In addition, there was a missing beat on the ECG. The maximumfollowing freqency is reciprocally related to the effective refractoryperiod and interventions which increase effective refractory period couldbe expected to decrease the maximum following frequency.2.5.4 Data analysisElectrical stimulation end-points were examined at baseline and 2minutes after beginning each infusion. These end-points were comparedwith control values and baseline values using analysis of variance,ANOVA followed by Duncan’s multiple range test (NCSS package,Hintze, 1987).792.6 Myocardial ischaemia-induced arrhythmiasThis set of experiments involved estimation of the antiarrhythmicactivity of RSD 939 in an anaesthetized rat model of coronary occlusion.Occlusion was made of the coronary artery in the presence of an infusionof RSD 939. The dependency of opioid receptors on the antiarrhythmicactivity of RSD 939 was determined with the use of naloxone.2.6.1 Experimental preparationMale Sprague Dawley rats (250-350 g) were subjected topreparative surgery similar to those in electrical stimulation studies butwith a left coronary artery occiuder implanted. The occiuder,manufactured from polyethylene, was first described by Au et al. (1979)and Johnston et al. (1983). Its design and manufacture have beenextensively described by Curtis et al. (1986). In brief, a 5.0 gaugeatraumatic polypropylene suture (Ethicon) was threaded through thepolyethylene guide such that the needle end of the suture appeared at theflared end of the guide.The surgical procedure used was a modification of techniquesdescribed by John and Olsen (1954), and they were the same as those80employed by Au et al. (1979) and Paletta et al. (1989). After tracheotomyand cannulation of carotid artery and jugular vein, an incision was madethrough the skin at the base of the sternum, using blunt scissors. The skinwas loosened from the underlying muscle mass using blunt dissection tothe base of the neck. The skin was then cut from the base of the sternumto the neck and peeled back, revealing the chest musculature. Artificialrespiration was applied at this time using 100% oxygen to ensureadequate oxygenation. Fine pointed scissors were used to make a 1 cmskin incision over the 4th to 6th ribs on the left thorax and this wasenlarged by blunt dissection. The forceps were then inserted under thepectoralis muscle, which was gently separated from the underlying rectusabdominus, exposing the Intercostal muscles beneath. The 5th or 6thintercostal space was then punctured and this incision was enlarged byblunt dissection. If the heart was exposed unfavourably for placement ofthe occiuder, then a second intercostal incision was made. This wasnecessary in less than 5% of preparations. Retractors were used to widenthe intercostal incision and hold the chest wall back, and blunt forcepswere used to open the pericardium. By inserting the retractors’ tipsthrough the small pericardial tear, such that the pericardium was includedwith the retracted tissue, the heart was lifted toward the intercostalincision and a pericardial cradle that facilitated subsequent surgery was81created.The procedures for ligation of the left coronary artery has beendescribed in detail by Johnston et al. (1983) and Curtis et al. (1986). Theleft anterior descending coronary artey (LAD) is predominant andsupplies the left ventricle. There is no true circumflex artery in the rat.The coronary arteries lie beneath the epicardium and sometimes can beseen at operation in the intact beating heart as tiny red streaks beneath thesurface of the heart. In our experiments, the LAD was located using thehighly visible coronary veins as landmarks. In the rat the main leftcoronary artery can be ligated at a point just beneath the left auricularappendage. Occasionally, branching will have already begun under theleft atria. In this situation it is necessary to ligate several branches at thesame time in order to obtain a good-sized area of infarct. A good-sizedarea of infarct is important since it has been shown that the incidence andseverity of arrhythmia correlates with the size of the infarct (Johnston etal., 1983, 1983a).In the experiments, the left atria was lifted with wetted cotton stick(Q-tips). The needle of the polypropylene suture of the occluder, held instraight hemostats, was looped under the LAD. The suture was then sewnthrough the flared end of the guide tubing and was cut off and melteddown to form a small ball. A loose occluder was thus implanted in the82ventricular muscle, with the needle entered and left the myocardiumapproximately 2 mm either side of the artery, to ensure that the artery wasoccluded. Occasionally, there was a minor bleeding, amounting to lessthan 1 ml of blood. Any bleeding was stopped by allowing the blood toclot and the thoracic cavity was cleared of excess blood. The chest wasclosed with silk sutures and negative pressure was applied inserting alength of PE9O polythene tubing as the chest was closed to preventpneumothorax.ECG and blood pressure recordings were made as in previouscardiovascular assessment and electrical stimulation studies.2.6.2 Experimental designThe antiarrhythmic actions of RSD 939 at a high dose of 1.5tmo1e/kg/min i.v., a dose chosen from the previous dose-response studiesas one that produced significant, but not maximal ECG, heart rate, andblood pressure changes, and at a low dose of 0.5 .tmo1e/kg/min, a dosewhich had minimal effects on ECG, heart rate and blood pressure butnoticeable effect on RSh, were examined in pentobarbitone anaesthetizedrats subjected to occlusion of the left anterior descending83Figure 21. Diagram of the rat heart showing approximate placement ofthe occiuder around the left anterior descending coronary artery (LAD).84coronary artery. In a separate group of animal, the effects of naloxone onthe ischaemia induced arrhythmias reduced by RSD 939 was alsodetermined.The animal was allowed to recover for 30 minutes prior to drugadministration. A blood sample of approximately 1 ml was drawn tomeasure serum potassium levels prior to drug administration and at theend of the experiment if the animal survived. Drug were administered ina double blind randomized design as described earlier in cardiovascularassessment studies and electrical stimulation studies.In a random and double-blind manner rats were given an initialinjection of either saline or 8 j.imole kg1 naloxone. The infusion ofvehicle, or RSD 939 was randomly and blindly given 5 mm later. Thisresulted in four groups of animals (n 5 each group). Group I wasnaloxone pretreated rats tested with RSD 939. Group II was naloxonepretreated rats tested with vehicle. Group III and IV were salinepretreated rats tested with RSD 939 and vehicle, respectively. Bloodpressure and ECG were recorded 5 minutes after begining of infusion.Thereafter the occluder was pulled so as to produce coronary arteryocclusion. ECG, arrhythmias, blood pressure, heart rate, and mortalitywere monitored for 30 minutes after occlusion, and recordings at fastpaper speed (100 mm/sec) were taken every minutes for the first 585minutes and every 5 minutes thereafter. Arrhythmias during themonitoring period were diagnosed from the oscilloscope screen and noteddirectly on the chart for analysis, as described below. The criteria forexclusion have been detailed by Curtis (1986). In the event of exclusionof a rat by these criteria the treatment was immediately repeated inanother rat before continuation.All rats surviving 30 minutes were sacrificed by an overdose ofpentobarbitone. After death, the size of the occluded zone was measuredby perfusing the hearts by the Langendorff technique with PIPESsolution. Blood quickly washed out of all areas except the infarct. Thiswas followed by perfusion with PIPES solution containing 1 mg/miindocyanine (Fast green dye. BDH) for 60 sec. The perfused tissuestained dark green and the ischaemic area (occluded zone) remain red.The occluded zone was then cut out and weighed as the percent of thewhole ventricular weight.862.6.3 Data analysis2.6.3.1 S-T segment and R-wave amplitude changes post-occlusionCoronary occlusion produces a rapid increase in ECG signal,characterized by a large increase in R-wave amplitude and an initialdepression of S-T segment. The increase in R-wave amplitude graduallyreturns towards baseline (pre-occiusion) with time (Johnston et al., 1981).R-wave height was measured from the isoelectric baseline to the peak ofthe positive deflection and was expressed in mV. Follows an initialdecrease, the S-T segment elevates and is maintained for the duration ofthe experiment. The S-T segment elevation was expressed as a percentageof the R-wave amplitude, where the S-T segment is defined as the heightof the S wave position above the isoelectric baseline. The isoelectricbaseline was defined as the voltage at the foot of the P wave of thepreceding beat. Although these effects were produced by occlusion, andpresumably myocardial ischaemia, they were not the primary concern ofthe present study.872.6.3.2 Analysis of arrhythmiaIschaemia-induced arrhythmias appear in a biphasic time-dependentmanner corresponding to early arrhythmias (0-0.5 hr) and late arrhythmias(0.5-4.0 hr) (Johnston et al., 1981). In these experiments, theantiarrhythmic actions of RSD 939 were only studied in the earlyarrhythmia phase.Arrhythmias were analyzed according to the guidelines establishedby the Lambeth conventions (Walker et al., 1988) and Curtis (1986) aspremature ventricular contractions (PVC), ventricular tachycardia (VT) orventricular fibrillation (VF). The arrhythmia history of each rat wasexpressed as an arrhythmia score (AS) (Curtis and Walker, 1988).PVC were defined as extrasystoles with QRS complexes occuringindependently of the P wave. They were generally accompanied by atransient drop in aortic blood pressure. Only singlets, doublets (bigemini)and triplets were counted as PVCs. Runs of 4 or more consecutiveextrasystoles were recorded as VT. Singlets, doublets, and triplets werenot classified as distinct arrhythmia but rather were pooled implying thatthey were one and the same arrhythmia.VT was defined as a run of 4 or more consecutive extrasystoleswith a clearly distinguishable QRS and were not subclassified according88to rate. VT was subdivided into spontaneously reverting VT (SVT),lasting less than 10 sec, and non-spontaneously reverting VT (NSVT),which lasted more than 10 sec or was irreversible. A drop in bloodpressure was also seen with VT.VF was defined as a disordered ECG accompanied by a precipitousfall in blood pressure. As opposed to VT, VF has a chaotic ECG patternwith no identifiable QRS complex and a blood pressure of less than 10mmHg. As in VT, any VF lasted less than 10 sec was defined asspontaneously reverting VF (SVT), and any VF lasted more than 10 sec orirreversible was defined as non-spontaneously reverting VF (NSVF).An arrhythmia score, an arbitrary numerical grading of the severityof ventricular arrhythmias with time post-occlusion, was used tosummarize the arrhythmia profile of each animal. There are manypossible different scoring systems (Curtis and Walker, 1988) but thefollowing scoring system was used.0 = 0-49 PVCs1 = 50-499 PVCs2 >499 PVCs and/or 1 episode of spontaneously revertingVTorVF3 = >1 episode of VT or VF or both (<60 sec total combinedduration)894 = VT or VF or both (60-119 sec total combined duration)5 = VT or VF or both (>119 sec total combined duration)6 = fatal VF starting at >15 mm after occlusion7= fatal VF starting at between 4 mm and 14 mm 59 sec afterocclusion8 = fatal VF starting at between 1 mm and 3 mm 59 sec afterocclusion9 = fatal VF starting <1 mill after occlusion9033.1ResultsOpioid effectsTable 10. Binding studiesj.t opioid receptor K opioid receptor1C50 1.0 iiM 0.006 iiMTable 11. Analgesia assaysTail PinchED50 1.5 imo1e/kgIn the above two assays, it was found that RSD 939 binded 1000times more selective to K receptor, and it produced analgesia at an ED50of 1.5 p.mole/kg (Table 10 & 11).913.2 Haemodynamic effects of RSD 939 in vivoIn intact pentobarbitone anaesthetized rats, dose-response curves(Figure 3 & Figure 4) and ED25 values (Table 1) for effects of RSD 939on blood pressure and heart rate in the presence and absence of naloxonepre-treatment were obtained. RSD 939 dose-dependently lowered bloodpressure and heart rate in a statistically significant manner after 1 to 2tmole/kg as compared with the vehicle control. The ED25, for 25 %changes from pre-treatment values, for both blood pressure and heart ratewere 8.0 .tmole/kg. All tested animals died from an irreversible declinein blood pressure after 32 jimole/kg. Effects of RSD 939 on bloodpressure and heart were reduced by pre-treatment with naloxone andsignificant depression of blood pressure and heart rate was not observeduntil after 4 to 8 imole/kg in the naloxone pre-treated group. In thisgroup, ED25 for blood pressure and heart rate were 12.0 pmole/kg and15.0 .tmole/kg, respectively. In the saline vehicle control group, bothblood pressure and heart rate were stable over the measurement period.Naloxone alone produced no statistical significant haemodynamic effects.92• Vehicle 0 RSD939 • Naloxone V 939+NaI150TT• • -r10050**0— I I IControl 1 2 4 8 16Dose (i.imollkg)Figure 3. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on mean arterial blood pressure in pentobarbitalanaesthetized rats. Intravenous doses were given every 8 minutes.Measurements were made 0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drugadministration, and the peak effects of these measurements used. Thegroups indicated are: • = saline pre-treated vehicle control; 0 =cumulative doses of RSD 939; . = naloxone pre-treated vehicle control (8imole/kg); v cumulative doses of RSD 939 with naloxone pretreatment. Controls are the pre-drug values or the post-naloxonetreatment values for the naloxone pretreated groups. * indicates p<0.05as compared to the vehicle control group. t indicates significantdifference between RSD 939 in the presence and absence of naloxone pretreatment at p<O.OS (ANOVA and Duncan’s range test). All values areexpressed as mean ± S.E.M. with n = 5 per group.93• Vehicle 0 RSD939 • Naloxone V 939+Nal500,..1‘ 400E .r*. *Va * - *300 **I ::IControl 1 2 4 8 16Dose (prnoljkg)Figure 4. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on heart rate in pentobarbital anaesthetized rats. Intravenousdoses were given every 8 minutes. Measurements were made 0.5, 1.0,2.0, 4.0, and 8.0 minutes after drug administration, and the peak effectsof these measurements used. The groups indicated are: • = saline pretreated vehicle control; 0 = cumulative doses of RSD 939; • = naloxonepre-treated vehicle control (8 .tmole/kg); V = cumulative doses of RSD939 with naloxone pre-treatment. Controls are the pre-drug values or thepost-naloxone treatment values for the naloxone pre-treated groups. *indicates p<0.OS as compared to the vehicle control group. t indicatessignificant difference between RSD 939 in the presence and absence ofnaloxone pre-treatment at p<O.O5 (ANOVA and Duncan’s range test). Allvalues are expressed as mean ± S.E.M. with n = 5 per group.94Table 1. Potencies of RSD 939 in the presence and absence ofnaloxone pre-treatment with respect to haemodynamicresponses in vivo.Group Blood Pressure Heart RateED25 (j.imolelkg)RSD 939 7.7 8.0RSD 939 + Nal 12.0 15.0The potencies of RSD 939 in the presence and absence of naloxone pretreatment with respect to blood pressure (BP), and heart rate (HR) inpentobarbital anaesthetized rats. Nal = naloxone (8jimole/kg) pretreatment. Values are expressed as the dose required to produce a 25%change from the pre-drug values, i.e. ED25953.3 ECG effects of RSD 939 in vivoECG measures in intact rats were also influenced in a dose-relatedmanner by RSD 939 both in the presence and absence of naloxone pretreatment (Figure 5 to Figure 9). No statistical significant difference wasfound between the RSD 939 group and the naloxone pre-treated RSD 939group. The P-R, QRS, and RSh measure from the ECG were significantlyincreased at doses of 1 jimole/kg or 2 j.imole/kg. Although significantprolongation of QRS interval occurred at 2 tmole/kg, RSD 939 was not aspotent in increasing the QRS duration as compared with P-R interval andRSh. The ED25 value for QRS interval was 26.0 jimole/kg, 5 times higherthan the ED25 values for P-R interval (4.6) and RSII (6) (Table 2). TheECG measurements least influenced by RSD 939 were QT1 and QT2.These intervals were not statistically significantly increased until 4imolefkg. ED25 values for QT2 and QT1 were 11.0 and 16.0 .tmole/kg,respectively. Vehicle and naloxone pre-treatment alone had nostatistically significant effects on the ECO measures except for a slightincrease in P-R interval after naloxone.96• VehIcle 0 RSD939 • Naloxone V 939+NaI100 -*90- * ——80-*———70 —--- j!. 60 L50-0.1C 40-30-20 -10 -0- I IControl 1 2 4 8 16Dose (imoIJkg)Figure 5. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on P-R interval of ECG in pentobarbital anaesthetized rats.Intravenous doses were given every 8 minutes. Measurements were made0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drug administration, and the peakeffects of these measurements used. The groups indicated are: • = salinepre-treated vehicle control; 0 cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 imole/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. Controls are the pre-drugvalues or the post-naloxone treatment values for the naloxone pre-treatedgroups. * indicates p<O.05 as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<0.05 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.97• Vehicle 0 RSD939 I Naloxone V 939+Nal40 -**T T* — *____z.-.**0 ..... -- -H- zzJ- -I20 -0C0)10 -0— I I IControl 1 2 4 8 16Dose (pmol/kg)Figure 6. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on QRS interval of ECG in pentobarbital anaesthetized rats.Intravenous doses were given every 8 minutes. Measurements were made0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drug administration, and the peakeffects of these measurements used. The groups indicated are: • salinepre-treated vehicle control; 0 = cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 jimole/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. Controls are the pre-drugvalues or the post-naloxone treatment values for the naloxone pre-treatedgroups. * indicates p<0.05 as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<O.05 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.98• Vehicle 0 RSD939 • Naloxone V 939+Nal8050400300200100—I I IControl 1 2 4 8 16Dose (pmollkg)Figure 7. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on Q-T1 interval of ECG in pentobarbital anaesthetized rats.Intravenous doses were given every 8 minutes. Measurements were made0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drug administration, and the peakeffects of these measurements used. The groups indicated are: • = salinepre-treated vehicle control; 0 = cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 imo1e/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. Controls are the pre-drugvalues or the post-naloxone treatment values for the naloxone pre-treatedgroups. * indicates p<0.O5 as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<0.05 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.99• Vehicle 0 RS0939 • Naloxone V 939+Nal8070 *— *60 ** __.—-—-;, 50400.4;:; 3oI.0 20100I I I I IControl 1 2 4 8 16Dose (pmol/kg)Figure 8. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on Q-T2 interval of ECG in pentobarbital anaesthetized rats.Intravenous doses were given every 8 minutes. Measurements were made0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drug administration, and the peakeffects of these measurements used. The groups indicated are: • = salinepre-treated vehicle control; 0 = cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 jimole/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. Controls are the pre-drugvalues or the post-naloxone treatment values for the naloxone pre-treatedgroups. * indicates p<0.O5 as compared to the vehicle control group. 1indicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<O.OS (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.100E.C,)1.501.000.500.00• Vehicle 0 RSD939 • Naloxone V 939+NalFigure 9. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on RSh of ECG in pentobarbital anaesthetized rats.Intravenous doses were given every 8 minutes. Measurements were made0.5, 1.0, 2.0, 4.0, and 8.0 minutes after drug administration, and the peakeffects of these measurements used. The groups indicated are: • = salinepre-treated vehicle control; 0 = cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 jimole/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. Controls are the pre-drugvalues or the post-naloxone treatment values for the naloxone pre-treatedgroups. * indicates p<O.OS as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<O.OS (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = S pergroup.****I—Control 1 2 4 8 16Dose (pmol!kg)101Table 2. Potencies of RSD 939 in the presence and absence ofnaloxone pre-treatment with respect to ECG responses invivo.Group P-R QRS Q-Tl Q-T2 RShED25 (j.imole/kg)RSD 939 4.6 26.0# 16.0 11.0 6.0RSD 939 7.0 28.0’ 17.0” 11.0 8.5+NalThe potencies of RSD 939 in the presence and absence of naloxone pretreatment with respect to P-R interval, QRS interval, Q-T1 interval, Q-T2interval, and RSh in pentobarbital anaesthetized rats. Nal = naloxone(8pmole/kg) pre-treatment. Values are expressed as the effective dosenecessary to produce 25% change from the pre-drug values, ED25. #indicates the values of QRS interval and Q-T 1 interval are extrapolatedfrom the extended portion of the dose-response curve.1023.4 In vitro effects of RSD 939Figure 15,16,17, and 20 shows the effects of RSD 939, in thepresence and absence of naloxone, on heart rate, peak systolic leftventricular pressure, and contractility in isolated perfused rat hearts.Corresponding changes in ECG intervals recorded from isolated perfusedrat hearts are shown in Figure 18 and Figure 19.In contrast with the haemodynamic effects observed with RSD 939in vivo, concentration-related bradycardia and reduction in ventricularpressure were not seen in isolated perfused rat hearts. RSD 939 producedno significant changes in heart rate, peak systolic left ventricularpressure, maximum rate of intraventricular pressure development(+dp/dtmax), and maximal rate of intraventricular relaxation (dp/dtmax) atthe tested dose range of 0.1 to 3 jiM in vitro. ED25 values could nottherefore be estimateabled (Table 5). The only notable difference in thedose-response curves were a tendency for higher peak systolic pressure,+dp/dtmax, and dp/dtmax, and a slight decrease in heart rate in the presenceof RSD 939.RSD 939 concentration-dependently prolonged both P-R intervaland QRS duration. The P-R and QRS prolongation were statisticallysignificant at 1 p.M when compared to vehicle control. As with effects103observed in intact rats, RSD 939 had a much lesser effect on QRS. Thepotency of RSD 939 on QRS duration was 10 times lower than its potencyon P-R interval. The ED25 values for P-R interval and QRS duration werefound to be 1.0 .tM and 10.0 jiM, respectively (Table 6).All of the above effects were still present in the presence of 1.0 jiMnaloxone. No significant difference on the above measures were foundbetween the naloxone pre-treated RSD 939 group and RSD 939 group.The effects of naloxone on isolated hearts were not statistically differentfrom those of vehicle control. The slight increase in P-R interval afternaloxone treatment observed in intact rats were also seen in isolatedperfused rat hearts though to a lesser degree.Figure 15. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on systolic ventricular pressure in isolated rat hearts.Hearts were perfused with PIPES buffer solution containing eithervehicle, RSD 939, naloxone, or RSD 939 plus naloxone. Each dose ofdrug was infused for 3 minutes, and the steady state values at 3 minutesused in the analysis. The groups indicated are: Veh = vehicle control;939 = cumulative concentrations of RSD 939; Nal = naloxone (1.0 j.tM);939N = cumulative concentrations of RSD 939 with naloxone. Controlsare the pre-drug values. * indicates p<O.05 as compared to the vehiclecontrol group. indicates significant difference between RSD 939 in thepresence and absence of naloxone pre-treatment at p<O.05 (ANOVA andDuncan’s range test). All values are expressed as mean ± S.E.M. with n =5 per group.104Veh 939 I I Nal 939NEE0I000II00‘-4-C0>004-0,0200 r1000Control 0.1 0.3 1 3Dose (pM)10540003000200010000Veh 939 I .INaI 939NFigure 16. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on a measure of contractility, the maximal rate ofintraventricular pressure development (+dP/dtmax) in isolated rat hearts.Hearts were perfused with PIPES buffer solution containing eithervehicle, RSD 939, naloxone, or RSD 939 plus naloxone. Each dose ofdrug was infused for 3 minutes, and the steady state values at 3 minutesused in the analysis. The groups indicated are: Veh = vehicle control;939 = cumulative concentrations of RSD 939; Nal = naloxone (1.0 jiM);939N cumulative concentrations of RSD 939 with naloxone. Controlsare the pre-drug values. * indicates p<O.O5 as compared to the vehiclecontrol group. indicates significant difference between RSD 939 in thepresence and absence of naloxone pre-treatment at p<O.05 (ANOVA andDuncan’s range test). All values are expressed as mean ± S.E.M. with n5 per group.a.0EEE4-0.V+Control 0.1 0.3 1 3Dose (pM)10640003000200010000Veh ... 939 Nal 939NFigure 17. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on a measure of contractility, the maximal rate ofintraventricular relaxation (dp/dtmax) in isolated rat hearts. Hearts wereperfused with PIPES buffer solution containing either vehicle, RSD 939,naloxone, or RSD 939 plus naloxone. Each dose of drug was infused for3 minutes, and the steady state values at 3 minutes used in the analysis.The groups indicated are: Veh vehicle control; 939 = cumulativeconcentrations of RSD 939; Nal = naloxone (1.0 tM); 939N = cumulativeconcentrations of RSD 939 with naloxone. Controls are the pre-drugvalues. * indicates p<0.05 as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<0.O5 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.a0U)xEE‘CEVControl 0.1 0.3 1 3Dose (pM)Figure 18. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on heart rate in isolated rat hearts. Hearts wereperfused with PIPES buffer solution containing either vehicle, RSD 939,naloxone, or RSD 939 plus naloxone. Each dose of drug was infused for3 minutes, and the steady state values at 3 minutes used in the analysis.The groups indicated are: Veh = vehicle control; 939 = cumulativeconcentrations of RSD 939; Nal = naloxone (1.0 jiM); 939N = cumulativeconcentrations of RSD 939 with naloxone. Controls are the pre-drugvalues. * indicates p<O.05 as compared to the vehicle control group. tindicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<O.05 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.107Veh 939 Ii Nal 939NEI0.0,-I0,.00,h.0,x250200150100500Control 0.1 0.3 1 3Dose (pM)108Figure 19. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on P-R interval of ECG in isolated rat hearts. Heartswere perfused with PIPES buffer solution containing either vehicle, RSD939, naloxone, or RSD 939 plus naloxone. Each dose of drug was infusedfor 3 minutes, and the steady state values at 3 minutes used in theanalysis. The groups indicated are: Veh vehicle control; 939 =cumulative concentrations of RSD 939; Nal = naloxone (1.0 i.M); 939N =cumulative concentrations of RSD 939 with naloxone. Controls are thepre-drug values. * indicates p<0.05 as compared to the vehicle controlgroup. t indicates significant difference between RSD 939 in thepresence and absence of naloxone pre-treatment at p<O.05 (ANOVA andDuncan’s range test). All values are expressed as mean ± S.E.M. with n =5 per group.Veh 939 I I Nal 939N*C,0E04-C1TTT100500Control 0.1 0.3 1 3Dose (pM)109Figure 20. Concentration-response effects of RSD 939 in the presenceand absence of naloxone pre-treatment, effects of vehicle control, andeffects of naloxone on QRS interval of ECG in isolated rat hearts. Heartswere perfused with PIPES buffer solution containing either vehicle, RSD939, naloxone, or RSD 939 plus naloxone. Each dose of drug was infusedfor 3 minutes, and the steady state values at 3 minutes were in theanalysis. The groups indicated are: Veh vehicle control; 939 =cumulative concentrations of RSD 939; Nal = naloxone (1.0 jtM); 939N =cumulative concentrations of RSD 939 with naloxone. Controls are thepre-drug values. * indicates p<0.0S as compared to the vehicle controlgroup. t indicates significant difference between RSD 939 in thepresence and absence of naloxone pre-treatment at p<O.05 (ANOVA andDuncan’s range test). All values are expressed as mean ± S.E.M. with n =5 per group.Veh 939 tI Nat 939N*000E04-CCoa403020100Control 0.1 0.3 1 3Dose (pM)110Table 5. Potencies of RSD 939 in the presence and absence ofnaloxone pre-treatment with respect to cardiac functions invitro.SystolicGroup Heart Rate Ventricular +dp/dtmax dP/dtmaxPressureED25 (jiM)RSD 939 >3 >3 >3RSD 939 >3 >3 >3:+NalThe potencies of RSD 939 in the presence and absence of naloxone pretreatment with respect to heart rate, systolic ventricular pressure,contractility (+dp/dtmax & dP/dtmax) in isolated rat hearts. Nal = naloxone(1.0 jiM). Values are expressed as the effective dose necessary toproduce 25% change from the pre-drug values, ED25. indicates ED25are non-estimateable at the tested dose range (changes from pre-drugvalues are less than 25% at the highest tested dose).111Table 6. Potencies of RSD 939 in the presence and absence ofnaloxone pre-treatment with respect to ECG responses invitro.Group P-R QRSED25 (jiM)RSD 939 1.0 10.0#RSD939+Nal 1.5 8.0’The potencies of RSD 939 in the presence and absence of naloxone pretreatment with respect to P-R interval, and QRS interval in isolated rathearts. Nal = naloxone (1.0 jiM). Values are expressed as the effectivedose necessary to produce 25% change from the pre-drug values, ED25. #indicates the values of QRS interval are extrapolated from the extendedportion of the dose-response curve.1123.5 Effects of electrical stimulation in vivoFigure 10 to Figure 14 illustrates the effects of RSD 939 in thepresence and absence of naloxone pre-treatment on sensitivity toelectrical stimulation in intact rats. In a clearly dose-related manner RSD939 increased threshold current (iT), threshold pulse width (tT), andventricular fibrillation threshold (VFt). In addition RSD 939 dose-dependently lengthened the effective refractory period (ERP) whilereducing the closely related but different variable, maximum followingfrequency (MFF). Statistically significant difference on iT, VFt, MFF,and ERP occurred at doses of 2 imole/kg/min and at a dose of 4jimole/kg/min for tT when compared to vehicle control. However, after 4ji.mole/kg/min RSD 939, the characteristic ventricular fibrillo-fluttercould not be induced by electrical stimulation despite the use of themaximum current of 1000 tA produced by the stimulator. Instead aventricular tachycardia was all that could be achieved by this “burstpacing” method (50 Hz, 1.0 ms). The potency of RSD 939 on ERP (ED25= 2.3 imole/kg/min) was slight lower than its potencies on iT (ED25 2.0tmole/kg/min) and VFt (ED25 = 1.5 tmole/kg/min) (Table 4). Thepotency of RSD 939 on tT (ED25 = 4.6 imole/kg/min) was much lower113than iT, suggesting that RSD 939 was less potent in preventingextrasystoles induced by stimulus with long pulse width. ERP and MFFare reciprocally related such that it might be expected that MFF =l000/ERP. However, the potency of RSD 939 on MFF was much lowerthan on ERP. The ED25 for MFF was found to be 3.6 jimole/kg/min ascompared to of 2.3 g.tmole/kg/min for ERP.Naloxone had no statistically significant effects on electricalstimulation, although iT and VFt showed a slight increase after naloxonetreatment. Naloxone pre-treatment did not influence the changes inducedby RSD 939. However, the dose-dependent reduction in MFF wasaccentuated by pre-treatment with naloxone though the responses werenot markedly different from the RSD 939 group. Data for vehicle controlrats showed no changes with time over the experimental period.Serum K levels increased from a control values of 3.4 ± 0.1 to 3.6± 0.2 and 3.3 ± 0.1 to 3.6 ± 0.1 at the end of the experimental period forthe vehicle control and the naloxone pre-treated control group,respectively (Table 3). For the RSD 939 treated group, and the naloxonepre-treated RSD 939 group, serum K increased from a control value of3.3 ± 0.1 to 3.9 ± 0.2 and 3.4 ± 0.1 to 3.9 ± 0.1, respectively, by the endof the experimental period. The changes of K prior to and afterexperiment for all 4 groups were statistically significant but no114statistically significant differences were seen for comparison betweengroups.• Vehicle 0 RSD939 • Naloxone V 939+Nal115Dose (pmol/kg!min)Figure 10. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on effective refractory period, ERP as determined by theextra stimulus method in pentobarbital anaesthetized rats subjected toelectrical stimulation of the left ventricle, are shown as changes from predrug values. Each dose of drug or vehicle were infused for 3 minutes.Measurements of the electrical stimulation variables were madetriplicately at 2 minutes of each infusion. The average of the 3measurements were used. The groups indicated are: • = saline pretreated vehicle control; 0 = cumulative doses of RSD 939; • = naloxonepre-treated vehicle control (8 imole/kg); v = cumulative doses of RSD939 with naloxone pre-treatment. * indicates p<O.O5 as compared to thevehicle control group. t indicates significant difference between RSD939 in the presence and absence of naloxone pre-treatment at p<O.O5(ANOVA and Duncan’s range test). All values are expressed as mean ±S.E.M. with n = 5 per group.9080700o 609..E0.40w.s 3020100-10-20*//V*VV *———.* --I1 2 4 8——* —* ------...-tJ2 4 8Dose (pmollkg!mln)Figure 11. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on threshold current, iT in pentobarbital anaesthetized ratssubjected to electrical stimulation of the left ventricle, are shown aschanges from pre-drug values. Each dose of drug or vehicle were infusedfor 3 minutes. Measurements of the electrical stimulation variables weremade triplicately at 2 minutes of each infusion. The average of the 3measurements were used. The groups indicated are: • = saline pretreated vehicle control; 0 = cumulative doses of RSD 939; • = naloxonepre-treated vehicle control (8 jimole/kg); v = cumulative doses of RSD939 with naloxone pre-treatment. * indicates p<O.O5 as compared to thevehicle control group. indicates significant difference between RSD939 in the presence and absence of naloxone pre-treatment at p<O.O5(ANOVA and Duncan’s range test). All values are expressed as mean ±S.E.M. with n = 5 per group.116• Vehicle 0 RSD939 • Naloxone V 939+NaI*300250200 /‘1*//1//150 //•/ I/// /1OO / /7 /IC0C500-501117• Vehicle 0 RSD939 • Naloxone V 939+Nai5-—NU.•.5.C.)*-10 I I 1*1 2 8Dose (pmoilkgj’min)Figure 12. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on maximum following frequency, MFF in pentobarbitalanaesthetized rats subjected to electrical stimulation of the left ventricle,are shown as changes from pre-drug values. Each dose of drug or vehiclewere infused for 3 minutes. Measurements of the electrical stimulationvariables were made triplicately at 2 minutes of each infusion. Theaverage of the 3 measurements were used. The groups indicated are: • =saline pre-treated vehicle control; 0 = cumulative doses of RSD 939; • =naloxone pre-treated vehicle control (8 tmole/kg); v = cumulative dosesof RSD 939 with naloxone pre-treatment. * indicates p<O.O5 as comparedto the vehicle control group. t indicates significant difference betweenRSD 939 in the presence and absence of naloxone pre-treatment at p<O.O5(ANOVA and Duncan’s range test). All values are expressed as mean ±S.E.M. with n = 5 per group.*40.40• Vehicle 0 RSD939 • Naloxone V 939+NaI118Dose (pmol/kglmln)Figure 13. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on threshold duration, tT in pentobarbital anaesthetized ratssubjected to electrical stimulation of the left ventricle, are shown aschanges from pre-drug values. Each dose of drug or vehicle were infusedfor 3 minutes. Measurements of the electrical stimulation variables weremade triplicately at 2 minutes of each infusion. The average of the 3measurements were used. The groups indicated are: • = saline pretreated vehicle control; 0 = cumulative doses of RSD 939; • = naloxonepre-treated vehicle control (8 imole/kg); V = cumulative doses of RSD939 with naloxone pre-treatment. * indicates p<O.O5 as compared to thevehicle control group. 1 indicates significant difference between RSD939 in the presence and absence of naloxone pre-treatment at p<O.O5(ANOVA and Duncan’s range test). All values are expressed as mean ±S.E.M. with n = 5 per group.a00ta2EI-4-C0C.)0.350.300.*//// */--/-----V------ z* *------ --1 2 4 8• Vehicle 0 RSD939 • Naloxone V 939+Nal119--—V2 4 8Dose (iimol/kg!mln)Figure 14. Dose-response effects of RSD 939 in the presence andabsence of naloxone pre-treatment, effects of vehicle control, and effectsof naloxone on the induction of ventricular fibrillation threshold, VFt inpentobarbital anaesthetized rats subjected to electrical stimulation of theleft ventricle, are shown as changes from pre-drug values. Each dose ofdrug or vehicle were infused for 3 minutes. Measurements of theelectrical stimulation variables were made triplicately at 2 minutes ofeach infusion. The average of the 3 measurements were used. Thegroups indicated are: • = saline pre-treated vehicle control; 0 =cumulative doses of RSD 939; • naloxone pre-treated vehicle control (8.tmole/kg); v cumulative doses of RSD 939 with naloxone pretreatment. * indicates p<O.05 as compared to the vehicle control group.t indicates significant difference between RSD 939 in the presence andabsence of naloxone pre-treatment at p<O.05 (ANOVA and Duncan’srange test). All values are expressed as mean ± S.E.M. with n = 5 pergroup.At 8 pmole/kg/min RSD 939 both in the presence and absence ofnaloxone, only ventricular tachycardia can be induced with the maximumcurrent output of 1000 tA in all the rats.>C0C0C.,8007006005004003002001000-1001Ia// ////-/---7---.120Table 3. Serum K levels in pentobarbital anaesthetized rats subjectedto electrical stimulation.Group Serum K levels (mM)Pre-electrical Post-electricalstimulation stimulationVehicle control 3.3 ± 0.2 3.7 ± 0.1*Naloxone 3.2 ± 0.2 3.8 ± 0.3*RSD 939 3.3 ± 0.1 3.9 ± 0.2*RSD 939 + Nal 3.4 ± 0.2 3.9 ± 0.2*Values are expressed as means ± S.E.M.. Na! = naloxone at 8pmole/kg.* indicates significant difference between pre- and post-electricalstimulation values. t indicates p<0.O5 versus vehicle control.121Table 4. Potencies of RSD 939 in the presence and absence ofnaloxone pre-treatment with respect to sensitivity to electricalstimulation in vivo.Group iT tT VFt ERP MFFED25 (pmole/kg/min)RSD 939 2.0 4.6 1.5 2.3 3.6RSD 939 1.8 5.0 1.5 2.5 3.0+NalThe potencies of RSD 939 in the presence and absence of naloxone pretreatment with respect to threshold current (iT), threshold pulse width(tT), ventricular fibrillation threshold (VFt), effective refractory period(ERP), and maximum following frequency (MFF) in pentobarbitalanaesthetized rats. Nal = naloxone (8tmo1e/kg). Values are expressed asthe effective dose necessary to produce 25% change from the pre-drugvalues, ED25.1223.6 Antiarrhythmic actions of RSD 939In the antiarrhythmic study for ischaemic arrhythmias, the twoinfusion doses of RSD 939 used were chosen on the basis of dose-response effects seen with RSD 939 in the electrical stimulation study(data not shown). The higher dose (1.5 tmole/kg/min) of RSD 939 hadsignificant, but sub-maximal effects on heart rate, blood pressure, andECG. The lower dose (0.5 jtmole/kg/min) minimally changed heart rate,blood pressure and ECG but had noticeable effect on RSh. Both doses ofRSD 939 statistically significantly reduced the incidence of arrhythmiasinduced by coronary artery occlusion (Table 7). The high dose (1.25tmole/kg/min) was more antiarrhythmic than the low dose (0.5mole/kg/min). The high dose of RSD 939 reduced the incidence ofNSVT by 40%, the incidence of NSVF by 60%, and mortality by 100%when compared with the vehicle control group. The low dose reduced theincidence of NSVF by 40%, mortality by 60%, but did not reduce theincidence of NSVT. Although both doses statistically significantlyreduced AS, the reduction in AS by the high dose RSD 939 was muchhigher than with the low dose. The AS of high dose RSD 939 was 3.2 ±0.2 while the AS of low dose was 5.4 ± 0.2 which indicated low doseRSD 939 almost failed to influence arrhythmias.123Naloxone alone also reduced the incidence of NSVF as exemplifiedby the reduction in AS, but did not reduce the incidence of NSVT. Theincidence of NSVT and NSVF were not different between high dose RSD939 alone or when naloxone was given prior to RSD 939. Naloxoneappeared to have no effect on the antiarrhythmic effects of high dose RSD939 since no statistical significant difference were found. On the otherhand, while low dose RSD 939 alone had limited effects on arrhythmias,when combined with naloxone it reduced arrhythmias to a statisticallysignificant extent. The AS of low dose RSD 939 was reduced to 4.0 ± 0.2when combined with naloxone pre-treatment. This reduction was mostlikely resulted from naloxone treatment rather than from RSD 939treatment.The antiarrhythmic activity of RSD 939 and naloxone could not beascribed to changes in either the size of the occluded zone (zone-at-risk)or serum potassium concentrations. The group mean occluded zone sizedid not differ statistically significantly between groups (Table 8). In asimilar manner serum potassium concentrations before and after occlusionwere also not differ statistically significantly between groups althoughthey were significantly increased after occlusion in the high dose RSD939 treatment group.124Statistical test was not performed to determine the significantdifference in the group incidences of NSVT or NSVF because the 1-tailedchi2 test referenced by Mainland’s contingency tables (Mainland et al.,1956) required the minimum group size to be 9 in order to be able toreveal a 50% reduction in NSVT and NSVF.125Table 7. Antiarrhythmic properties of RSD 939 in the presence andabsence of naloxone pre-treatment against ischaemia-inducedarrhythmias in pentobarbital anaesthetized rats in the early(0-0.5 hr) period following coronary artery occlusion.Group Incidence of VT and VF (%) Mortality ASNSVT (%)SVT SVF NSVT NSVF and/orNSVFVehicle 100 0 100 100 100 100 7.0 ±0Nal 100 0 100 40 100 0 3.4 ±0.2*RSD939 100 20 60 40 60 0 3.2 ± 0.2*(H)RSD939 100 0 80 20 80 0 3.0 ±0.3*(H) + NalRSD939 100 0 100 60 100 40 5.4 ± 0.2*t(L)RSD939 100 20 100 40 100 0(L) + NalThe antiarrhythmic actions of 1.5 j.tmole/kg/min (H) and 0.5.tmo1e/kg/min(L) RSD 939, alone or in the presence of naloxone pre-treatment, as wellas naloxone, are expressed in terms of the percent of animalsexperiencing one or more episodes of the particular arrhythmias. Nal =naloxone (8 jimole/kg). NSVT non-spontaneously reverting ventriculartachycardia. NSVF = non-spontaneously reverting ventricular fibrillation.SVT = spontaneously reverting ventricular tachycardia. SVF =spontaneously reverting ventricular fibrillation. AS = arrhythmia score.Calculations to determine AS were discussed in the methods and allvalues represent mean ± S.E.M.. % = % animal per group (n=5). *indicates significant difference from vehicle control at p<O.O5. tindicates p<0.O5 between RSD 939 and RSD 939 + Naloxone.126Table 8. Occluded zone size and serum K levels in pentobarbitalanaesthetized rats subjected to coronary artery occlusion.Group OZ (%) Serum K levels (mM)Pre-occiusion Post-occlusionVehicle 37.5 ± 1.5 3.9 ± 0.2 4.0 ± 0.2Nal 36.0 ± 1.0 3.8 ± 0.1 4.1 ± 0.2RSD 939 (H) 37.0 ± 1.0 3.8 ± 0.1 4.4 ± 0.2*RSD939 (H) + 38.0 ± 1.5 3.8 ± 0.1 4.2 ± 0.2*NalRSD 939 (L) 38.0 ± 1.5 3.9 ± 0.2 4.1 ± 0.2RSD939 (L) + 37.0 ± 1.0 3.8 ± 0.2 4.2 ± 0.2NalRSD 939 (H) = 1.5 imole/kg/min. RSD 939 (L) = 0.5 tmo1e/kg/min.Values are means ± S.E.M.. Nal = naloxone (8 pmo1e/kg/min). OZ =weight % of the occluded zone in ventricle. * indicates significantdifference between pre- and post-occlusion serum [K] values. tindicates p<O.05 versus vehicle control for serum [K] levels or OZ.1274. Discussions4.1 Haemodynamic effects of RSD 939 in vivo and in vitroIt has been proposed that opiate mechanisms play a role in centraland/or peripheral cardiovascular regulation (Holaday, 1983). 1 agonists,ethylketocyclazocine (EKC), U50,488H, and spiradoline, have shown toinduce decreases in blood pressure and heart rate via either autonomicnervous system regulation or direct peripheral actions at K sites locatedon the heart (Laurent and Schmitt, 1983; Wu and Martin, 1983; Hall etal., 1988). These effects were mediated by K receptors and werecompletely reversed by the opioid receptor antagonist naloxone. In ourstudies, RSD 939 (over a range of doses from 1 to 16 tmole/kg i.v.) dose-dependently reduced blood pressure and heart rate in pentobarbitoneanaesthetized rats. However, these effects could only be partiallyantagonized by naloxone. Naloxone reduced the actions of low dosesRSD 939 on heart rate and blood pressure but not at higher doses. Theseactions were remarkably similar to those of high doses of U50,488, PD129289, and PD 129290, which our laboratory has tested previously undersimilar conditions (Pugsley et al., 1992a,b & 1993). In contrast, over theconcentration range 0.1 to 3.0 p.M. RSD 939 produced no significant128changes on heart rate, systolic ventricular pressure, and dp/dt in isolatedrat heart studies, although a noticeable decrease in heart rate and anincrease in systolic ventricular pressure as well as dp/dt were observed athigher doses. These responses were not prevented by naloxone. Fromsuch results, it is reasonable to suggest that at least part of the effects ofRSD 939 on heart rate are mediated by receptor-dependent effects in thecentral nervous systme (vagal system and/or sympathetic nervous system)since there is a significant reduction in the effects on heart rate in theisolated heart preparations. The fall in blood pressure could have beendue partly to a possible decrease in cardiac output which may result fromthe bradycardia. Our results also suggest that the haemodynamic effectsof RSD 939, particularly at higher doses, are not mediated via ic opioidreceptors since the effects were naloxone-resistant. In addition, thereduction in heart rate seen with isolated heart preparations may not berelated to the direct peripheral actions of RSD 939 on 1 opioid receptorslocated in the heart since the effects were insensitive to naloxone. Theinfluence of opioid agonists and antagonists on cardiac muscle contractileforce has been examined in a variety of preparations with a variety ofresults, including positive, negative, and biphasic (both positive andnegative) inotropic effects (Rendig et al., 1980; Laurent et al., 1985;Goldberg & Padget., 1969; Strauer, 1972). The increase in systolic129ventricular pressure and contractility with RSD 939 in isolated heart isnot related to opioid receptor but may be the results of increased Ca2loading subsequent to an increase in action potential duration asdiscussed later.4.2 Antiarrhythmic actions of RSD 939Opioid agonists and antagonists had been shown to be effectiveagainst a variety of experimental arrhythmias in isolated heart and intactanimal studies (Alzheimer et al., 1990; Boachie-Ansah et al., 1989;Brasch, 1986; Frame et al., 1985; Fagbemi et al., 1982 & 1983; Helgesenet a!., 1987; Huang et a!., 1986; Lee et a!., 1986; MacKenzie et al., 1986;Parratt et al., 1986; Pruett et al., 1991; Pugsley et a!., 1992a,b & 1993;Same et al., 1988 & 1991; Wong et al., 1990). In this study, we foundthat low dose (0.5 p.mole/kg/min) RSD 939 offered only very minimumprotection against arrhythmias induced by myocardial ischaemia but at ahigh dose (1.5 p.mole/kg/min), RSD 939 reduced the incidence ofarrhythmias significantly. At the same dose, RSD 939 also increasedresistance to electrical induction of ventricular fibrillo-flutter. A muchhigher dose of 16 p,mole/kg/min, RSD 939 completely prevented theelectrical induction of ventricular fibrillo-flutter as only ventricular130tachycardia could be induced despite the use of maximum current of 1000ptA. These results suggested the antiarrhythmic actions of RSD 939 couldbe dose-related. However, the antiarrhythmic activities againstischaemia-induced arrhythmia at this dose range were not investigatedbecause of marked depression effect on blood pressure. In neither of theprevious studies was the actions of RSD 939 antagonized by naloxonegiven at a dose which has been shown to antagonize the actions of Kagonists (Same et al., 1991). This finding is in agreement with ourprevious studies in which K agonists, U50,488H and PD 129290 wereshown to have antiarrhythmic activities unrelated to opioid receptors(Pugsley et al., 1992a,b & 1993). We thus suggest that such non-opioidactions of RSD 939, like those of U50,488H and PD 129290 are also theresult of cardiac ion channel blockade.While naloxone did not antagonize the antiarrhythmic effects ofRSD 939, naloxone itself possessed slight antiarrhythmic properties sincethe incidence of ischaemia-induced arrhythmias were significantlyreduced by pre-treatment with 8 mole/kg naloxone. The mechanismunderlying the antiarrhythmic action of naloxone has been the subject ofseveral investigations with many proposing that its antiarrhythmic actionis unrelated to opioid receptors (Same et al., 1988 & 1991; Brasch, 1986;Oldroyd et al., 1993). In our study, naloxone might have been131responsible for the increase in antiarrhythmic effectiveness of low doseRSD 939. However, naloxone appeared to have no synergistic effect withthe high dose RSD 939 since no difference in arrhythmia score was foundbetween the naloxone treated and untreated group.There was a statistically significant elevation of serum Kconcentrations post-occlusion associated with administration of RSD 939(Table 8), particularly at the high dose. Elevations in serum areknown to occur following acute surgery, and this may contribute to theresults of this study. Elevation of serum K is associated with a fall inthe incidence of VF in patients with acute myocardial infarction(Nordrehaug and von der Lippe, 1983; Solomon, 1984) andexperimentally in rats (Saint et al., 1992). In isolated perfused hearts,elevation of the K concentration of the perfusate drastically reducesarrhythmias induced by coronary occlusion (Lubbe et al., 1978;Daugherty et al., 1981). The mechanism by which hyperkalaemia protectsagainst ischaemia-induced arrhythmias might be due, at least in part tothe elevation of the threshold for electrical excitation resulted from highserum K. Thus the normal myocardium would be protected frominvasion by aberrant impulses emanating from ischaemic tissue. In ourstudy, the serum K levels with high dose RSD 939 were 4.4 ± 0.2 mMpost-occlusion. According to the results obtained by Curtis et al (1985),132the incidence of VF was reduced by 23% in groups of rats whose serumK were in the range of 4.0 - 4.9 mM. However, in our study, we havebeen able to induce VF in 100% of the animals in the control group,which has a serum K of 4.0 ± 0.2 mM post-occlusion. The serum K incontrol group was not significantly different from the RSD 939 group.Therefore, we believed that the slight increase in serum K was notenough to protect against ischaemia-induced arrhythmias.According to previous studies in our laboratory, arrhythmias(incidence and duration) depend on the size of the ischaemic (occluded)zone (OZ) such that arrhythmia score (AS) is linearly correlated withsquare root of the OZ (Johnston et al., 1983 a). This implies that it is notdirectly the amount of ischaemic tissue, but the presence of both normaland ischaemic tissue which is necessary for arrhythmogenesis since thesite of arrhythmogenesis is the interface area between the ischaemic andnormal tissue (Brofman et a!., 1956; Beck, 1958; Janse et a!., 1979&1980). Therefore, OZ size should be measured in all rats in order toverify that a proper occlusion has taken place. From our pastexperiences, the optimal OZ size for arrhythmogenesis in rats should bein the range of 25-45% (Curtis, 1984; Johnston et al., 1983a). Since theOZ size in our study average at 37% and the variance for OZ size issmall, antiarrhythmic activity could not be ascribed to changes in OZ in133this study.4.3 Non-opioid actions of RSD 939A variety of electrophysiological studies have shown that opioidagonists and antagonists have actions independent of opioid receptors andcan produce electrophysiologic changes consistent with those seen withClass I antiarrhythmic agents (Blari et al., 1986; Pruett et al., 1987;Oldroyd et al., 1993; Carratu and Mitolo-Chieppa, 1982; Brasch, 1986;Boachie-Ansah et al., 1989; Fagbemi et al., 1983; Alzheimer andBruggencate, 1990; Pugsley et al., 1993). In additional toelectrophysiological studies, ECG observation and responses to electricalstimulation are also useful in determining the effects of antiarrhythmicdrugs on cardiac ion channels. The ECG effects of the 4 classes ofantiarrhythmic agents have been summarized by Botting et al (1986) andPenz et al (1992). Class Ia Na channel blockers slow conductionvelocity at high concentrations thus widen the QRS duration, prolong P-Rintervals, and increase RSh. They also widen the AP thus prolonged theQ-T intervals. Class lb agents demonstrate limited (if any) effect on P-Rinterval, QRS duration, RSH, and conduction while shortening the Q-Tinterval and APD. Class Ic agents slow conduction at low concentrations134thus prolong P-R interval, increase RSh, and widen QRS complex, buthave little effect on repolarization and APD. Class II drugs, f3-blockers,can widen P-R intervals in vivo if AV nodal conduction has beenenhanced by a significant degree of sympathetic tone beforeadministration, as the P-R interval reflects the conduction time throughthe AV node. Class III drugs, K channel blockers, delay repolarizationof the cardiac action potential. Since the T-wave of the surface ECGreflects the repolarization phase in the ventricle (Einthoven, 1912; Katz,1928), selective Class III drugs should widen the Q-T intervals of theECG with no other effects on QRS or P-R intervals due to a lack of effecton conduction velocity in atrial, nodal, or ventricular tissue. Class IVdrugs, Ca2 channel blockers, can widen P-R intervals if given insufficient doses to inhibit the slow inward current, is,. In intact hearts,RSD 939 prolonged the P-R interval and elevated the RSh at low doseswhile Q-T interval and QRS duration were not affected until higher doses.This is in agreement with the results obtained from isolated hearts wherethe drug was shown to be more potent in prolonging the P-R interval thanQRS duration. These evidences suggested that RSD 939 acted directly oncardiac tissue and might have Class I actions at lower doses. However,the effects on Q-T intervals at higher doses could either be achieved byAPD prolongation (Class III) or was accompanied by conduction slowing135(Class Ta). The ECG effects also slightly resemble those of Class lbcompounds which have limited action on QRS duration at normal sinusbeating rates due to their high frequency dependency (Campbell, 1983b;Courtney, 1987). Since P-R interval widening is seen with both Na andCa2 channel blockers, we can not rule out the possibility of calciumchannel blockade. However, by studying the responses to electricalstimulation, it is proved that sodium channel blockade is most likely thecause of P-R interval prolongation.In addition to the ECG evidence, the effects of RSD 939 on iT, tT,and VFt were consistent with sodium channel blockade. It has been wellestablished that Na channel blockers increase iT, tT, and VFt in responseto electrical stimulation (Wiggers & Wegria, 1940; Beatch et al., 1988;Hodess et al., 1979; Marshall et al., 1983; Yoon et a!., 1974). On theother hand, unlike Class I drugs which dose dependently elevated iT, tT,and VFt, K channel blockers might not affect iT and tT but might renderthe heart completely resistant to VF at a high enough dose. This resultedwhen the refractoriness was prolonged to such an extent that multiplefractionations of induced reentrant wave fronts were not possible(Sugimoto et al., 1989; Winslow, 1984). At low doses, VFt was graduallyincreased dose-dependently. Yet, at 8 jimole/kg/min, RSD 939 suddenlyand completely suppressed the induction of ventricular fibrillo-flutter,136thus again suggested the involvement of potassium channel blockade atsuch high dose.The recovery of excitability after a preceding impulse is determinedmainly by the availability of sodium channels, which are voltagedependent and thus AP widening can prolong refractoriness. Thusprolongation of ERP can be expected to occur with Class Ia sodiumchannel blockers and Class III antiarrhythmics (Vaughan-Williams, 1970& 1975). In the present study with RSD 939, ERP was increased as wasQ-T interval, findings associated with Class Ia and/or potassium channelblockade. ERP and MFF are related such that it might be expected thatMFF(Hz) = 1000/ERP(ms). However, although the two are similar theyare sufficiently different to warrant reporting both. ERP, as measured, isa reasonable measure of effective refractory period. MFF is more ameasure of relative refractory period, and ventricular functionalrefractory period, and thus can exhibit a different sensitivity of drugsfrom ERP. The process of determination of MFF, namely a steadilyincreasing frequency of stimulation, can be associated with accumulationof extracellular K (see Table 3) thereby adding an extra component towhat would otherwise be another measure of ERP. Thus MFF and ERPare not equally sensitive to frequency-dependent sodium or potassiumchannel blockers (Walker & Beatch, 1988). Comparisons between 1/MFF137and ERP allow us to ascertain the frequency dependency of refractorinessincrease with RSD 939. In our study, RSD 939 treatments did not changethe ratio of l/MFF to ERP values, thus rule out the possibility offrequency dependent sodium channel blockade (Class Ib).The ECG effects and the responses to electrical stimulation seenwith RSD 939 were unrelated to ic opioid receptors since these actionsoccurred at doses and concentrations above those required for 1-agonism,and naloxone did not abolish nor reduce any of these effects statisticallysignificantly. However, the dose of naloxone used have caused aminimum degree of sodium channel blockade as indicated by the slightincrease in RSh, P-R interval, iT, and tT.1385 ConclusionIn conclusion, the present study provides evidence that RSD 939, apotent and selective ic agonist, possess antiarrhythmic activity againstischaemia-induced and electrically-induced arrhythmias. 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