European Journal of Pharmacology, 2111(1992) 45-51
45
c~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52211
The electrophysiological effects of opioid receptor-selective antagonists on sheep Purkinje fibres Marie McIntosh, Kathleen Kane and James Parratt Department of Physiology and Pharmacology, Uni~'ersi~of Strathclyde, 204 George Street, Glasgow G I 1XIE U.K Received 28 March 1901, revised MS received 17 September 1991, accepted 15 October 1991
The cardiac electrophysiological effects of 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexonc, which are relatively specific opioid antagonists for 3, K and p. receptors, respectively, were studied in paced (1.5 Hz) sheep Purkinjc fibres in vitro. M8008 (1 ng ml-~-10 #g ml 1) caused a concentration-dependent reduction in the maximum rate of depolarisation of phase 0 (MRD) and in the action potential duration measured at 50% repolarisation, APDs0. Neither NBT (10 ng ml 1-10/zg ml t) nor naltrexone (1 ng ml ~-10/xg ml 1) produced any significant effect on the cardiac action potential. In the presence of a physiological salt solution modified to mimic some of the changes that occur during myocardial ischaemia (i.e. hypoxia, acidosis, hyperkalaemia), M8008 caused a more marked reduction in MRD and prolonged rather than shortened APDs0. These results suggest that the reported antiarrhythmic activity of M8008. but not NBT or naltrexone, may be. at least in part, explained by a direct cardiac electrophysiological action. Cardiac electrophysiology; Ischaemia; Opioid receptor antagonists; Purkinjc fibres
1. Introduction
In the accompanying manuscript (McIntosh et at., 1991) it was shown that the opioid receptor antagonists, 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexone which are specific for & K and /~ receptors respectively, at certain concentrations, were all able to protect the heart against ischaemia-induced arrhythmias. There are two likely explanations for this protective effect. Firstly, the opioid receptor antagonists may be blocking the effects of endogenously released opioid peptides; secondly, the antagonists may possess directly mediated electrophysiological effects on the cardiac muscle action potential. Other opioid receptor antagonists, which are antiarrhythmic in vivo, have been shown to exhibit direct electrophysiological effects in cardiac tissue in vitro. Naloxone (Frame and Argentieri, 1985; Brasch, 1988; Cerbai et al., 19891 and buprenorphine (Boachie-Ansah et al., 1989) both have class I antiarrhythmic activity (i.e. they reduce the maximum rate of depolarisation of phase 0) and class II1 activity (i.e. they prolong the action potentiaD. In addition, the class I action of
Correspondence to: K.A. Kane, Department of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow G1 IXW, U.K. Tel. 44.41.552 4400, ext. 2620.
buprenorphine has been shown to be potentiated by conditions simulating myocardial ischaemia (BoachieAnsah et al., 1989). However, the electrophysiological effects of more specific antagonists such as M8008 NBT and naltrexone, have not been examined either under normal or simulated ischaemic conditions. The aims of this study were, therefore, to determine the direct electrophysiological effects of M8008, NBT and naltrexone on normal sheep Purkinje fibres in vitro. Additionally the effects of M8008 were examined on Purkinje fibres superfused with a physiological solution modified to simulate some of the changes that occur during myocardial ischaemia, namely, hypoxia, acidosis and hyperkalaemia.
2. Materials and methods
2.1. Tissue preparation Experiments were performed on sheep cardiac Purkinje fibres. Sheep hearts were brought from the slaughter house, within around 30 rain of excision, to the laboratory in cool oxygenated physiological salt solution. Purkinje fibres were excised from the left ventricle with a small piece of ventricular muscle attached. Suitable fibres were selected and pinned via the attached muscle pieces to the silastic base of the
46 recording chamber. Tissues were initially superfused at a rate of 4 ml min ~ with a normal physiological salt solution, equilibrated with 95% 0 2 - 5 % CO 2. Preparations were electrically stimulated at a frequency of 1.5 Hz using rectangular pulses of 0.5 (or 1.0) ms in duration and at least twice threshold voltage. Stimuli were delivered via a bipolar silver electrode connected to a simulator (Grass Model $88) and an isolation unit (Grass Model SIU5). The perfusion chamber temperature was maintained at 36.5 +_ 0.5 ° C using a heating lamp underneath, which was controlled by an HSE T e m p e r a t u r e Regulator (Hugo Sachs Electronik, Model 319). The oxygen tension in the perfusion chamber was recorded routinely throughout the experimental period using an oxygen electrode (Strathkelvin Instruments) immersed into the chamber.
2.2. Action potential measurements Glass microelectrodes filled with 3 M KCI (resistance 10-30 MS2) were used to measure transmembrane resting and action potentials via a preamplifier and the rate of rise of the upstroke via an electronic differentiating circuit (Hugo Sachs). The action potential and the differentiated signal were recorded from the dual beam storage oscilloscope (Tektronix) on to photographic film using an oscilloscope camera (Nihon Kohden Model RLG-6201). The records were then measured by using a bitpad and a computer. The variables measured were as follows: resting m e m b r a n e potential (RMP); action potential amplitude (APA); maximum rate of depolarization of phase 0 (MRD); and action potential duration at 50 and 90% repolarization levels (APDs0 and APD,~0).
2.3. Experimental conditions Experiments were performed in which the Purkinje fibres were superfused with a normal physiological salt solution (PSS) or with a salt solution, the composition of which was modified in order to mimic some of the in vivo conditions of myocardial ischaemia (i.e. hypoxic, hyperkalaemic acidotic). Thus the composition of the normal Tyrode solution was as follows (mM): NaCI 125: N a H C O 3 25; N a H 2 P O 4 1.2; MgC12 1.0; KC1 5.4; CaCI~ 1.8; glucose 5.5. The normal PSS was bubbled with 95% 0 2 - 5 % CO 2 to yield a bath PO 2 = 610_+ 25 mm Hg and pH = 7.4. The modified salt solution had the same composition as normal Tyrode solution with the exceptions of (mM): KC1 8.(I; N a H C O 3 8.5 and NaC1 141.5. The altered physiological salt solution was equilibrated with 95% N 2 - 5 % CO 2 to yield bath PO 2 and p H values of 22 + 1 mm Hg and 6.8, respectively. In order to achieve this PO x, the PSS was pre-equilibrated with 95% N 2 - 5 % CO 2 in a closed reservoir.
Furthermore stainless steel tubing was used for the perfusion system to reduce uptake of oxygen from the atmosphere. In each set of experiments, an equilibration period of about 1-2 h in normal PSS was allowed before the beginning of the experimental protocol.
2.4. Experimental protocol To observe drug effects under normal conditions, 6-10 action potentials were recorded before, and 30-40 min after, cumulative additions of the drug, dissolved in reservoirs of gassed normal physiological salt solution, to obtain the final bath concentrations used. In some of the experiments the effects of a 30 min or 1 h washout, with drug-free normal Tyrode solution, were examined at the end of the last drug exposure period. The effects of the modified PSS alone were studied by recording 6-10 action potentials before (i.e. in normal Tyrode solution) and at 30, 60, 90, 120 and 150 min following exposurc to the modified PSS. To observe drug-induced effects in the presence of the modified PSS, 6-10 action potentials were recorded before (i.e. in normal Tyrode solution) and 3(I-40 min after superfusion with the modified salt solution alone and subsequently after the administration of cumulative concentrations of drug dissolved in this solution. Action potentials were recorded 30-40 min after the addition of each concentration of the drug. 'Control' and 'test' action potentials werc obtained i~rom multiple impalements in a pre-selected area of each fibre. Multiple rather than a single maintained impalement was used because of the difficulty of maintaining one impalement throughout the experimental protocol. All three experimental protocols were performed using different sets of Purkinje fibres.
2.5. Analysis of data and statistics Action potential measurements from each set of 6-10 impalements were meaned and the mean values used to represent the data from each preparation. Data are expressed as either mean absolute values or mean percentage changes from control values (derived from 4 to 10 experiments) together with the S.E.M. In the presence of the modified PSS, drug-induced effccts were expressed as a % change from the values obtained after 30 min of superfusion with the modified PSS alone. Multiple treatment and control means were analysed firstly using one-way analysis of variance and where appropriate, if the F value permitted further analysis, individual treatment means were compared with respective control values by a modified t-test.
47
When comparing drug-induced effects, either under normal or 'ischaemic' conditions, a Student's unpaired t-test was employed. P < 0.05 was considered to be statistically significant in all tests.
M8008 were dissolved in Tyrode solution. Stock solutions of M8008 were prepared first in distilled water and a few drops of dilute NaOH (0.1 N) added; the pH was then adjusted to neutral with dilute HCI (0.1 N).
2.6. Drugs 3. Results The following drugs and sources were used. 16Methylcyprenorphine hydrochloride M8008, (Reckitt & Colman), norbinaltrophimine hydrochloride, NBT, (Reckitt & Colman), naltrexone hydrochloride (Reckitt & Colman). All drugs with the exception of
3.1. Control data A comparison of the control values of the variables given in tables 1-4 shows that in the fibres treated with
TABLE 1 The effects of M8008 on action p o t e n t i a l c h a r a c t e r i s t i c s of s h e e p Purkinje fibres u n d e r n o r m a l conditions. V a l u e s are given as m e a n s + S.E.M. of six e x p e r i m e n t s . " P < 0.05 i n d i c a t e s significantly d i f f e r e n t from the w d u e in the control group. In this and s u b s e q u e n t tables: resting m e m b r a n e p o t e n t i a l ( R M P ) : m a x i m u m rate of d e p o l a r i s a t i o n of p h a s e zero ( M R D ) : action p o t e n t i a l a m p l i t u d e (APA): o v e r s h o o t (OS): action p o t e n t i a l d u r a t i o n at 50 and 90c)k r e p o l a r i s a t i o n respectively (APD>0 and APD,~q~). Drug
RMP (mY)
MRD (Vs ])
APA (mV)
OS (mV)
APDsll (ms)
APD,,II (Ins)
Control M8008 1 ngml i M8008 I #gml ~ M8008 10 u g ml ~ Washout
- 86.9 _4-0.4
487.5 + 20.0
116.4 + 1.1
29.4 + 1
174.9 + 10.7
289.3 4__18.6
-86.2+0.2
467.2-+ 17.4
113.8+ 1.2
27.6_+ 1.2
157.3-+ 13.6a
288.6+25.8
-8e~.7+0.2
4 3 4 . 5 + 10.6
114.1+1.4
2 7 . 3 + 1.5
151.3+ 12.0 ~'
289.4+21).6
-86.2+0.4 85.2 4__0.8
288.2_+34.4 a 286.9 + 37.6 ~'
96.0_+5.4 :' 1t)2.6 + 3.6 "
9.8_+5.4 " 17.4 _+ 2.9 :'
139.2_+ 9.8 a 109.6 + 19.8 ~'
311.2_+24.5 331.0 + 28. I
TABLE 2 The effect of N B T on n o r m a l s h e e p Purkinje action p o t e n t i a l characleristics. V a l u e s are e x p r e s s e d as m e a n s + S.E.M. of six e x p e r i m e n t s . A b b r e v i a t i o n s as m table I. Drug Control NBT 10ngrnl ~ NBT 1 /xgml i NBT 10 p.g m l ~ Washout
RMP (mY)
MRD (Vs i)
APA (mY)
OS (mV)
APDso (ms)
APD,~o (ms)
442.9 + 12.6
112.8 + 1.3
24.7 4__0.7
158.4 ± 6.3
249.2 + 14.8
-87.9+0.6
4 3 0 . 4 + 19.4
110.6+ 1.7
22.8_+ 1.6
152.7+6.7
2 5 1 . 4 + 16.1
-87.8_+0.6
396.0_+20.2
111.6_+1.5
2 3 . 8 + 1.7
155.0+6.8
262.2+21.0
88.1 _+0.8 - 87.8 + 0.6
389.0+29.5 469.2 + 42.0
110.4+2.4 111.8 + 11.4
22.2+2.4 24.0 _4_0.4
150.2+7.8 136.0 _4_6.8
258.9+23.4 237.0 _+ 7.1
88.1 ± 0.9
TABLE 3 The effect of n a l t r e x o n e on the cardiac action p o t e n t i a l s of n o r m a l s h e e p Purkinje fibres. V a l u e s are e x p r e s s e d as m e a n s + S . E . M , of four e x p e r i m e n t s . A b b r e v i a t i o n s as in table 1. Drug Control
APD,~q~
RMP (mV)
MRD (Vs i)
APA (mV)
OS (mV)
APD~ (ms)
(ms)
- 87.3 + 0.6
478.6 + 45.0
117,2 + 1.1
29.8 + 0.5
194.0 + 9.0
275.3 + 10.8
-86.2+1.1
443.0+36.9
115.6+_0.9
29.3+0.8
185.9+10.2
271.9+14.4
86.2+0.8
468.1 + 3 9 . 6
115.2-+0.8
29.0+0.2
182.9+
8.4
2 7 2 . 4 + 13.2
85.8_+1.7
451.1+38.7
114.1-+1.4
28.2+0.8
17().4_+14.0
280.4_+ 13.6
Nahrexone
I ngml t Naltrexone 1 #gml t Naltrexone 10#gml ~
48
NBT, the A P A and overshoot values were slightly but significantly less than those recorded in the other treatment groups.
T I M E (rain) 0
IJU
3.2. Effects of M8008, NBT and naltrexone on normal sheep Purkinje fibres
-20
~
"
30 I'
~.~ \
\
~
60 i
120 I
150 I
•
~
~
RMP
~
~
APDo °
*
z "' ~ l-
90 I
-40
o
Table 1 describes the effects of M8008 on the action potential characteristics of normal sheep Purkinje fibres. M8008 at a concentration of 10/xg ml ~ caused a reduction in the M R D without any effect on RMP. A concomitant decrease in action potential amplitude and overshoot was also produced by M8008. The action potential duration at 50% repolarisation (APDs0) was reduced significantly by all doses of M8008 studied (1 ng ml -] to l0 kLg ml ~). However, no statistically significant effect APDg0 was observed. At the highest dose level, however, there was a tendency for M8008 to lengthen APDg0. These effects were not reversed by a 1 h washout period in drug free Tyrode solution (table 1). Table 2 shows that although NBT (10 ng m l - l - 1 0 /xg m l - l ) tended to reduce M R D the changes were not statistically significant. NBT had no other effect on the cardiac action potentials. Similarly, naltrexone (1 ng m l - ~ - 1 0 / x g ml ~) had no statistically significant effect on any of the action potential variables (table 3).
3.3. Effects of simulated ischaemia on the action potential character±tics Figure 1 shows the % changes in action potential variables induced by simulated ischaemia (hypoxia, acidosis and hyperkalaemia) over a 2.5 h exposure period. Within 30 rain of exposure to the modified PSS all of the measured variables were significantly reduced. The depressant effects on R M P and M R D were accompanied by a reduction in A P A and overshoot. These changes were maximal by 30 min of exposure. APDg0
-6o
J-
APDso MRD
I ~
-80
Fig. 1. The % changes in action potential characteristics induced by a solution simulating ischaemia. Significant differences from zero have been omitted for the sake of clarity. * P < 0.05 denotes significantly different from % changes induced at 30 rain of exposure to ischaemic solution.
and APDs0, however, continued to fall slightly until 90 min post-exposure. The modified PSS also exerted depressant effects on excitability of the preparations. In about 50% of them simulated ischaemia increased the threshold voltage by a factor of about 2. In these instances the stimulation voltage was maintained at twice threshold. This change in stimulation voltage did not significantly modify the measurements of MRD.
3.4. Effect of M8008 under simulated ischaemic conditions In the presence of simulated myocardial ischaemia, M8008 (30 ng m l - I - 1 0 /zg ml 1) caused a significant concentration dependent reduction in M R D (table 4). Associated with this, was a reduction in A P A and overshoot but no effect on RMP. Since APDg0 and APD50 values were not stable during the period when the effects of M8008 were examined, i.e. the 30-150 min exposure period, the M8008 induced changes in these variables were expressed relative to those induced by the modified PSS alone. M8008 dose depen-
TABLE 4 The effects of M8008 on action potential characteristics of sheep Purkinje fibres exposed to simulated ischaemic conditions. Values are expressed as m e a n s + S . E . M , of six experiments. ~' P < 0.05 indicates significantly difference from values at 30 rain post-ischaemia. Statistically significant changes from pre-ischaemia are not shown for the sake of clarity. Abbreviations as in table 1. Drug
RMP (mV)
MRD (Vs i )
APA (mV)
APDs~ ~ (ms)
APD,~ (ms)
Control (30 rain postischaemia) M8008 1 ngml I M8008 30 ng ml ~ M8008 / 1,agml t M8008 10 ,ag ml ~
- 87.9 _+0.6
468.4 +_ 17.4
116.4 ± 0.6
189.9 + 6.6
285.5 ± 10.8
- 77.6 _+0.5
175.5 ± 23.2
80.8 + 3.4
113.8 ± 11.9
187.4 ± 14.1
-78.7±0.4
151.3+ 19.8
80.9+3.8
95.7±13.0
169.8+14.1
-78.6+0.4
133.4±20.3 ~'
78.8+3.0
93.2_+ 10.9
174.8± 12.8
-78.6±0.4
93.7_+20.0 a
71.9+4.1 ~
99.2+111.8
186
-79.2+0.4
54.2± 6.9 ~'
58.9+3.8 ~'
12(I.6± 12.2
±15.1
227.9_+ 18.5
49 MBO00 1nllml"1
lpgml-I
lOpgm1-1
-20 -40 t -60 -00
40 20 0 -20 -40
40 g
T
20
a. o
.LI
~ £
-20 Fig. 2. The % change in action potential characteristics produced by M8008 under both normal ([]) and simulated ischaemic ( • ) conditions. * P < 0.05 indicates significantly different from change under normal conditions.
dently reversed the 'ischaemia'-induced shortening in APDs0 and prolonged APDg0. The lowest concentration of M8008 studied, i.e. 1 ng m l - l had no statistically significant effect on the action potential characteristics under ischaemic conditions. In all concentrations, M8008 had a greater depressant effect on M R D in the presence of the modified compared to normal PSS (fig. 2). Figure 2 also illustrates that under ischaemic conditions M8008 prolonged APDs0 but shortened it in normal PSS. However, there were no statistically significant differences between the effects of M8008 on APDg0 under normal and simulated ischaemic conditions (fig. 2).
4. D i s c u s s i o n
4.1. Effects of the opioid receptor antagonists on normal sheep Purkinje fibres These results have demonstrated that the 6 opioid receptor antagonist, M8008, has marked electrophysio-
logical effects on paced normal sheep Purkinje fibres. M8008 caused a concentration-dependent reduction in MRD APA and APDs~ ~ without significantly altering RMP or APDso. The ionic mechanisms that underly these effects of M8008 on the cardiac potential have not been studied. It is likely, however, that M8008 reduces the upstroke of the action potential by blocking the sodium channels responsible for the rapid inward sodium current which depolarises the cell membrane. Such an action has been classified by Vaughan Williams (1970) as a class I antiarrhythmic effect. In Purkinje tissue, the action potential plateau is known to be partially maintained by a slowly inactivating component of inward sodium current (Attwell et al., 1979). M80(J8's ability to shorten the APDs~ ~ may, therefore, be due to a blocking effect on this current. Indeed M8008 may preferentially block this population of sodium channels, which have larger opening times than those responsible for phase 0 depolarisation (Wasserstrom and Salata, 1988). This would explain why, in this study, M8008 significantly reduced APDs0 at all concentrations studied, whereas MRD was affected by the highest concentration only. However, it is also possible that M8008 may have an action on other currents, such as the inward calcium current and outward potassium currents that influence the action potential duration. These elcctrophysiological effects of M8008 were observed at concentrations at which the drug has been shown to block ~ or tx opioid receptors in vitro (Smith, 1987). This might suggest that these effects could be opioid receptor-mediated, although there is no evidence to suggest that opioid peptides are released under these experimental conditions. Moreover, the 6 opioid agonist [D-Ala2,D-LeuS]enkephalin (DADLE), over a concentration range up to 100 ng ml-J has no electrophysiological effects on sheep Purkinje fibres (Mclntosh, 1991). Thus, it would seem that the electrophysiological effects of M8008 are more likely to be a consequence of some other 'direct' effect on the cardiac cell membrane. In accordance with these findings, other opioid receptor antagonists with selectivity for receptor types other than for 6 receptors, have also, been shown t o have class 1 actions. Naloxone (Frame and Argentieri 1985; Brasch 1986; Cerbai et al., 1989), buprenorphine (Boachie-Ansah et al., 1989) and nalmafene (Oldroyd et al., 1990) have all been demonstrated to exhibit class I activity either on Purkinje or ventricular tissue. In addition, it has been reported that the ( + ) and ( - ) isomers of naloxone induce comparable class I effects (Brasch, 1986) supporting the suggestion that these electrophysiological effects are not opioid receptor-mediated. Over a similar concentration range to M8008, NBT and naltrexone, which have a greater selectivity at K and Ix receptors respectively, had no marked effect on
511
normal sheep Purkinje fibres. It is, however, possible that with concentrations greater than 10 p,g m l - I of these drugs a direct electrophysiological effect could be obtained.
plained by such an action. It is possible, however, that NBT and naltrexone may exhibit electrophysiological effects under ischaemic, but not normal, conditions, This possibility was not examined in the present study.
4.2. Electrophysiological ef[ects of M8008 during simulated ischaemia
Acknowledgements
The electrophysiological effects of the hypoxic, acidotic and hyperkalaemic salt solution were similar to those previously described in in vitro preparations (Gilmour and Zipes, 1980; Kimura et al., 1984). These changes, i.e. depolarisation, reduced MRD and abbreviated action potential duration are also similar to the electrophysiological derangements recorded from intact hearts during a period of myocardial ischaemia following coronary artery ligation (Downar et al., 1977; Russell et al., 1979). Previous work with clinically used antiarrhythmic drugs have shown that such simulated ischaemic conditions can potentiate their class I activity (Kimura et al., 1982; Evans et al., 1984). The results of the present study have now shown that the class I effect of M8008 is also potentiated by conditions mimicking myocardial ischaemia. As has been suggested for lignocaine (Hondeghem and Katzung 1977; Wasserstrom and Salata, 1988) this may be due to its greater affinity for inactivated sodium channels than for rested sodium channels. Since simulated ischaemia causes depolarisation, a greater proportion of the sodium channels will be in the inactivated form with the consequence that drugs with a greater affinity for this channel form will exhibit a more marked effect. In the presence of simulated ischaemia, M8008 no longer abbreviated the plateau phase of the action potential. The action potential is markedly abbreviated by simulated ischaemia and this could in part be caused by a depolarisation-induced inactivation of the slowly inactivating current. Thus if M8008 abbreviated the normal action potential plateau by reducing this current, simulated ischaemia would attenuate or abolish such an action. In addition to losing the ability to shorten APDs~ , M8008 slightly prolonged action potential duration during ischaemia, suggesting that it may also block outward K + conductance under these conditions. Voltage clamp experiments would need to be carried out to examine these postulated ionic effects of M8008. In conclusion, this study has shown that M8008, but not NBT or naltrexone, has marked electrophysiological effects on sheep Purkinje fibres. The ability of M8008 to reduce MRD under normal conditions and the potentiation of this effect by ischaemia, could be the basis of its antiarrhythmic activity in vivo. Since neither NBT nor naltrexone had significant direct electrophysiological effects under normal conditions their antiarrhythmic efficacy in vivo is unlikely to be ex-
M. Mclntosh was an SERC CASE award student with Rcckitt & Colman. We thank them for their financial support and Dr. C. Smith (formerly of Reckitt & Colman, now with ICI) for his advice.
References Attwelk D., I. Cohen, D. Eisner, M. Ohba and C. Ojeda, 1979, The steady state TTX-sensitive (window) sodium current in cardiac Purkinje fibres, Pfliigers Arch. 379, 137. Boachic-Ansah, G., R. Sitsapesan, K.A. Kane and J.R. Parratt, 1989, The antiarrhythmic and cardiac clectrophysiological effects of buprenorphinc, Br. J. Pharmacol. 97. 801. Brasch, H. 1986, lnfluence of the optical isomers ( + } and ( - )-naloxone on beating frequency, contractile force and action potentials of guinea-pig isolated cardiac preparations, Br. J. Pharmacol. 88, 733. Cerbai, E., P.B. Cawdcabo, I. Masini, F. Porciatti and A. Mugelli, 1989, Antiarrhythmic properties of naloxone: an clectrophysiological study on sheep cardiac Purkinje fibres, Eur. J. Pharmacol. 162, 491. Downar, E.M.J. Janse and D. Durrer, 1977, The effect of acute c o r o n a ~ artery occlusion on subepicardial transmembrane potentials in the intact porcine heart, Circulation 56, 217. Evans, J.J., R.F. Gilmour and D.P. Zipes, 1984, The effects of lignocainc and quinidine on impulse propagation across the canine Purkinje muscle junction during combined hypcrkalaemia. hypoxia and acidosis. Circ. Res. 55, 185. Frame, L.H. and T.M. Argcntieri, 1985, Naloxone has local anaesthetic effects on canine cardiac Purkinje fibres, Circ. Res. 72, 34. Gilmour, R.F. and D.P. Zipes, 1980, Different electrophysiological responses of canine endocardium and epicardium to combined hyperkalaemia, hypoxia and acidosis, Circ. Res. 46, 814. ttondeghem, L. and B.G. Katzung, 1977, Time and voltage dependent interactions of antiarrhythmic drugs with cardiac sodium channels, Biochim. Biophys. Acta 172, 373. Kimura, S., H. Nakaya and M. Kanno, 1982, Effects of verapamil and lignocaine on changes in action potential characteristics and conduction time induced by combined hypoxia, hyperkalaemia and acidosis in canine ventricular myocardium, J. Cardiovasc. Pharmacol. 4, 658. Mclntosh, M.A., 1991, The Role of Opioid Peptides and Their Receptors During Acute Myocardial Ischaemia (Ph.D. Thesis, University of Strathclyde, Glasgow). Mclntosh, M.A., K. Kane and J. Parratt, 1991, Effects of selective opioid receptor agonists and antagonists during myocardial ischaemia, Eur. J. Pharmacol. 210, 37. Oldroyd, K., M.N. [licks and S.M. Cobbe, 19911, The class Ill antiarrhythmic effects of opioid receptor ligands are not receptor mediated, J. Mol. Cell Cardiol. 22 (Suppl. lIl), $82. Russell, D.C., J.H. Smith and M.F. Oliver, 1979. T r a n s m e m b r a n e potential changes and ventricular fibrillation during repetitive myocardial ischaemia in the dog, Br. Heart J. 42, 88. Smith, C.F.C., 1987, 160-Mecyprenorphine (RX 8008M): a potent opioid antagonist with some ,~ selectivity, Life Sci. 4/), 267.
51 Vaughan Williams, E.M., 1979, Classification of antiarrhythmic drugs, in: Symposium on Cardiac Arrhythmias, eds. E. Sandoe, E. Flendsted-Jensen and K. Olssen (Sweden AB Astra, Sodertalje) p. 449.
Wasserstrom, J.A. and J.J. Salata, 1988, Basis for tetrodotoxin and lignocaine effects on action potentials in dog ventricular myocytes, Am. J. Physiol. 254, 111157.
45
c~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52211
The electrophysiological effects of opioid receptor-selective antagonists on sheep Purkinje fibres Marie McIntosh, Kathleen Kane and James Parratt Department of Physiology and Pharmacology, Uni~'ersi~of Strathclyde, 204 George Street, Glasgow G I 1XIE U.K Received 28 March 1901, revised MS received 17 September 1991, accepted 15 October 1991
The cardiac electrophysiological effects of 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexonc, which are relatively specific opioid antagonists for 3, K and p. receptors, respectively, were studied in paced (1.5 Hz) sheep Purkinjc fibres in vitro. M8008 (1 ng ml-~-10 #g ml 1) caused a concentration-dependent reduction in the maximum rate of depolarisation of phase 0 (MRD) and in the action potential duration measured at 50% repolarisation, APDs0. Neither NBT (10 ng ml 1-10/zg ml t) nor naltrexone (1 ng ml ~-10/xg ml 1) produced any significant effect on the cardiac action potential. In the presence of a physiological salt solution modified to mimic some of the changes that occur during myocardial ischaemia (i.e. hypoxia, acidosis, hyperkalaemia), M8008 caused a more marked reduction in MRD and prolonged rather than shortened APDs0. These results suggest that the reported antiarrhythmic activity of M8008. but not NBT or naltrexone, may be. at least in part, explained by a direct cardiac electrophysiological action. Cardiac electrophysiology; Ischaemia; Opioid receptor antagonists; Purkinjc fibres
1. Introduction
In the accompanying manuscript (McIntosh et at., 1991) it was shown that the opioid receptor antagonists, 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexone which are specific for & K and /~ receptors respectively, at certain concentrations, were all able to protect the heart against ischaemia-induced arrhythmias. There are two likely explanations for this protective effect. Firstly, the opioid receptor antagonists may be blocking the effects of endogenously released opioid peptides; secondly, the antagonists may possess directly mediated electrophysiological effects on the cardiac muscle action potential. Other opioid receptor antagonists, which are antiarrhythmic in vivo, have been shown to exhibit direct electrophysiological effects in cardiac tissue in vitro. Naloxone (Frame and Argentieri, 1985; Brasch, 1988; Cerbai et al., 19891 and buprenorphine (Boachie-Ansah et al., 1989) both have class I antiarrhythmic activity (i.e. they reduce the maximum rate of depolarisation of phase 0) and class II1 activity (i.e. they prolong the action potentiaD. In addition, the class I action of
Correspondence to: K.A. Kane, Department of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow G1 IXW, U.K. Tel. 44.41.552 4400, ext. 2620.
buprenorphine has been shown to be potentiated by conditions simulating myocardial ischaemia (BoachieAnsah et al., 1989). However, the electrophysiological effects of more specific antagonists such as M8008 NBT and naltrexone, have not been examined either under normal or simulated ischaemic conditions. The aims of this study were, therefore, to determine the direct electrophysiological effects of M8008, NBT and naltrexone on normal sheep Purkinje fibres in vitro. Additionally the effects of M8008 were examined on Purkinje fibres superfused with a physiological solution modified to simulate some of the changes that occur during myocardial ischaemia, namely, hypoxia, acidosis and hyperkalaemia.
2. Materials and methods
2.1. Tissue preparation Experiments were performed on sheep cardiac Purkinje fibres. Sheep hearts were brought from the slaughter house, within around 30 rain of excision, to the laboratory in cool oxygenated physiological salt solution. Purkinje fibres were excised from the left ventricle with a small piece of ventricular muscle attached. Suitable fibres were selected and pinned via the attached muscle pieces to the silastic base of the
46 recording chamber. Tissues were initially superfused at a rate of 4 ml min ~ with a normal physiological salt solution, equilibrated with 95% 0 2 - 5 % CO 2. Preparations were electrically stimulated at a frequency of 1.5 Hz using rectangular pulses of 0.5 (or 1.0) ms in duration and at least twice threshold voltage. Stimuli were delivered via a bipolar silver electrode connected to a simulator (Grass Model $88) and an isolation unit (Grass Model SIU5). The perfusion chamber temperature was maintained at 36.5 +_ 0.5 ° C using a heating lamp underneath, which was controlled by an HSE T e m p e r a t u r e Regulator (Hugo Sachs Electronik, Model 319). The oxygen tension in the perfusion chamber was recorded routinely throughout the experimental period using an oxygen electrode (Strathkelvin Instruments) immersed into the chamber.
2.2. Action potential measurements Glass microelectrodes filled with 3 M KCI (resistance 10-30 MS2) were used to measure transmembrane resting and action potentials via a preamplifier and the rate of rise of the upstroke via an electronic differentiating circuit (Hugo Sachs). The action potential and the differentiated signal were recorded from the dual beam storage oscilloscope (Tektronix) on to photographic film using an oscilloscope camera (Nihon Kohden Model RLG-6201). The records were then measured by using a bitpad and a computer. The variables measured were as follows: resting m e m b r a n e potential (RMP); action potential amplitude (APA); maximum rate of depolarization of phase 0 (MRD); and action potential duration at 50 and 90% repolarization levels (APDs0 and APD,~0).
2.3. Experimental conditions Experiments were performed in which the Purkinje fibres were superfused with a normal physiological salt solution (PSS) or with a salt solution, the composition of which was modified in order to mimic some of the in vivo conditions of myocardial ischaemia (i.e. hypoxic, hyperkalaemic acidotic). Thus the composition of the normal Tyrode solution was as follows (mM): NaCI 125: N a H C O 3 25; N a H 2 P O 4 1.2; MgC12 1.0; KC1 5.4; CaCI~ 1.8; glucose 5.5. The normal PSS was bubbled with 95% 0 2 - 5 % CO 2 to yield a bath PO 2 = 610_+ 25 mm Hg and pH = 7.4. The modified salt solution had the same composition as normal Tyrode solution with the exceptions of (mM): KC1 8.(I; N a H C O 3 8.5 and NaC1 141.5. The altered physiological salt solution was equilibrated with 95% N 2 - 5 % CO 2 to yield bath PO 2 and p H values of 22 + 1 mm Hg and 6.8, respectively. In order to achieve this PO x, the PSS was pre-equilibrated with 95% N 2 - 5 % CO 2 in a closed reservoir.
Furthermore stainless steel tubing was used for the perfusion system to reduce uptake of oxygen from the atmosphere. In each set of experiments, an equilibration period of about 1-2 h in normal PSS was allowed before the beginning of the experimental protocol.
2.4. Experimental protocol To observe drug effects under normal conditions, 6-10 action potentials were recorded before, and 30-40 min after, cumulative additions of the drug, dissolved in reservoirs of gassed normal physiological salt solution, to obtain the final bath concentrations used. In some of the experiments the effects of a 30 min or 1 h washout, with drug-free normal Tyrode solution, were examined at the end of the last drug exposure period. The effects of the modified PSS alone were studied by recording 6-10 action potentials before (i.e. in normal Tyrode solution) and at 30, 60, 90, 120 and 150 min following exposurc to the modified PSS. To observe drug-induced effects in the presence of the modified PSS, 6-10 action potentials were recorded before (i.e. in normal Tyrode solution) and 3(I-40 min after superfusion with the modified salt solution alone and subsequently after the administration of cumulative concentrations of drug dissolved in this solution. Action potentials were recorded 30-40 min after the addition of each concentration of the drug. 'Control' and 'test' action potentials werc obtained i~rom multiple impalements in a pre-selected area of each fibre. Multiple rather than a single maintained impalement was used because of the difficulty of maintaining one impalement throughout the experimental protocol. All three experimental protocols were performed using different sets of Purkinje fibres.
2.5. Analysis of data and statistics Action potential measurements from each set of 6-10 impalements were meaned and the mean values used to represent the data from each preparation. Data are expressed as either mean absolute values or mean percentage changes from control values (derived from 4 to 10 experiments) together with the S.E.M. In the presence of the modified PSS, drug-induced effccts were expressed as a % change from the values obtained after 30 min of superfusion with the modified PSS alone. Multiple treatment and control means were analysed firstly using one-way analysis of variance and where appropriate, if the F value permitted further analysis, individual treatment means were compared with respective control values by a modified t-test.
47
When comparing drug-induced effects, either under normal or 'ischaemic' conditions, a Student's unpaired t-test was employed. P < 0.05 was considered to be statistically significant in all tests.
M8008 were dissolved in Tyrode solution. Stock solutions of M8008 were prepared first in distilled water and a few drops of dilute NaOH (0.1 N) added; the pH was then adjusted to neutral with dilute HCI (0.1 N).
2.6. Drugs 3. Results The following drugs and sources were used. 16Methylcyprenorphine hydrochloride M8008, (Reckitt & Colman), norbinaltrophimine hydrochloride, NBT, (Reckitt & Colman), naltrexone hydrochloride (Reckitt & Colman). All drugs with the exception of
3.1. Control data A comparison of the control values of the variables given in tables 1-4 shows that in the fibres treated with
TABLE 1 The effects of M8008 on action p o t e n t i a l c h a r a c t e r i s t i c s of s h e e p Purkinje fibres u n d e r n o r m a l conditions. V a l u e s are given as m e a n s + S.E.M. of six e x p e r i m e n t s . " P < 0.05 i n d i c a t e s significantly d i f f e r e n t from the w d u e in the control group. In this and s u b s e q u e n t tables: resting m e m b r a n e p o t e n t i a l ( R M P ) : m a x i m u m rate of d e p o l a r i s a t i o n of p h a s e zero ( M R D ) : action p o t e n t i a l a m p l i t u d e (APA): o v e r s h o o t (OS): action p o t e n t i a l d u r a t i o n at 50 and 90c)k r e p o l a r i s a t i o n respectively (APD>0 and APD,~q~). Drug
RMP (mY)
MRD (Vs ])
APA (mV)
OS (mV)
APDsll (ms)
APD,,II (Ins)
Control M8008 1 ngml i M8008 I #gml ~ M8008 10 u g ml ~ Washout
- 86.9 _4-0.4
487.5 + 20.0
116.4 + 1.1
29.4 + 1
174.9 + 10.7
289.3 4__18.6
-86.2+0.2
467.2-+ 17.4
113.8+ 1.2
27.6_+ 1.2
157.3-+ 13.6a
288.6+25.8
-8e~.7+0.2
4 3 4 . 5 + 10.6
114.1+1.4
2 7 . 3 + 1.5
151.3+ 12.0 ~'
289.4+21).6
-86.2+0.4 85.2 4__0.8
288.2_+34.4 a 286.9 + 37.6 ~'
96.0_+5.4 :' 1t)2.6 + 3.6 "
9.8_+5.4 " 17.4 _+ 2.9 :'
139.2_+ 9.8 a 109.6 + 19.8 ~'
311.2_+24.5 331.0 + 28. I
TABLE 2 The effect of N B T on n o r m a l s h e e p Purkinje action p o t e n t i a l characleristics. V a l u e s are e x p r e s s e d as m e a n s + S.E.M. of six e x p e r i m e n t s . A b b r e v i a t i o n s as m table I. Drug Control NBT 10ngrnl ~ NBT 1 /xgml i NBT 10 p.g m l ~ Washout
RMP (mY)
MRD (Vs i)
APA (mY)
OS (mV)
APDso (ms)
APD,~o (ms)
442.9 + 12.6
112.8 + 1.3
24.7 4__0.7
158.4 ± 6.3
249.2 + 14.8
-87.9+0.6
4 3 0 . 4 + 19.4
110.6+ 1.7
22.8_+ 1.6
152.7+6.7
2 5 1 . 4 + 16.1
-87.8_+0.6
396.0_+20.2
111.6_+1.5
2 3 . 8 + 1.7
155.0+6.8
262.2+21.0
88.1 _+0.8 - 87.8 + 0.6
389.0+29.5 469.2 + 42.0
110.4+2.4 111.8 + 11.4
22.2+2.4 24.0 _4_0.4
150.2+7.8 136.0 _4_6.8
258.9+23.4 237.0 _+ 7.1
88.1 ± 0.9
TABLE 3 The effect of n a l t r e x o n e on the cardiac action p o t e n t i a l s of n o r m a l s h e e p Purkinje fibres. V a l u e s are e x p r e s s e d as m e a n s + S . E . M , of four e x p e r i m e n t s . A b b r e v i a t i o n s as in table 1. Drug Control
APD,~q~
RMP (mV)
MRD (Vs i)
APA (mV)
OS (mV)
APD~ (ms)
(ms)
- 87.3 + 0.6
478.6 + 45.0
117,2 + 1.1
29.8 + 0.5
194.0 + 9.0
275.3 + 10.8
-86.2+1.1
443.0+36.9
115.6+_0.9
29.3+0.8
185.9+10.2
271.9+14.4
86.2+0.8
468.1 + 3 9 . 6
115.2-+0.8
29.0+0.2
182.9+
8.4
2 7 2 . 4 + 13.2
85.8_+1.7
451.1+38.7
114.1-+1.4
28.2+0.8
17().4_+14.0
280.4_+ 13.6
Nahrexone
I ngml t Naltrexone 1 #gml t Naltrexone 10#gml ~
48
NBT, the A P A and overshoot values were slightly but significantly less than those recorded in the other treatment groups.
T I M E (rain) 0
IJU
3.2. Effects of M8008, NBT and naltrexone on normal sheep Purkinje fibres
-20
~
"
30 I'
~.~ \
\
~
60 i
120 I
150 I
•
~
~
RMP
~
~
APDo °
*
z "' ~ l-
90 I
-40
o
Table 1 describes the effects of M8008 on the action potential characteristics of normal sheep Purkinje fibres. M8008 at a concentration of 10/xg ml ~ caused a reduction in the M R D without any effect on RMP. A concomitant decrease in action potential amplitude and overshoot was also produced by M8008. The action potential duration at 50% repolarisation (APDs0) was reduced significantly by all doses of M8008 studied (1 ng ml -] to l0 kLg ml ~). However, no statistically significant effect APDg0 was observed. At the highest dose level, however, there was a tendency for M8008 to lengthen APDg0. These effects were not reversed by a 1 h washout period in drug free Tyrode solution (table 1). Table 2 shows that although NBT (10 ng m l - l - 1 0 /xg m l - l ) tended to reduce M R D the changes were not statistically significant. NBT had no other effect on the cardiac action potentials. Similarly, naltrexone (1 ng m l - ~ - 1 0 / x g ml ~) had no statistically significant effect on any of the action potential variables (table 3).
3.3. Effects of simulated ischaemia on the action potential character±tics Figure 1 shows the % changes in action potential variables induced by simulated ischaemia (hypoxia, acidosis and hyperkalaemia) over a 2.5 h exposure period. Within 30 rain of exposure to the modified PSS all of the measured variables were significantly reduced. The depressant effects on R M P and M R D were accompanied by a reduction in A P A and overshoot. These changes were maximal by 30 min of exposure. APDg0
-6o
J-
APDso MRD
I ~
-80
Fig. 1. The % changes in action potential characteristics induced by a solution simulating ischaemia. Significant differences from zero have been omitted for the sake of clarity. * P < 0.05 denotes significantly different from % changes induced at 30 rain of exposure to ischaemic solution.
and APDs0, however, continued to fall slightly until 90 min post-exposure. The modified PSS also exerted depressant effects on excitability of the preparations. In about 50% of them simulated ischaemia increased the threshold voltage by a factor of about 2. In these instances the stimulation voltage was maintained at twice threshold. This change in stimulation voltage did not significantly modify the measurements of MRD.
3.4. Effect of M8008 under simulated ischaemic conditions In the presence of simulated myocardial ischaemia, M8008 (30 ng m l - I - 1 0 /zg ml 1) caused a significant concentration dependent reduction in M R D (table 4). Associated with this, was a reduction in A P A and overshoot but no effect on RMP. Since APDg0 and APD50 values were not stable during the period when the effects of M8008 were examined, i.e. the 30-150 min exposure period, the M8008 induced changes in these variables were expressed relative to those induced by the modified PSS alone. M8008 dose depen-
TABLE 4 The effects of M8008 on action potential characteristics of sheep Purkinje fibres exposed to simulated ischaemic conditions. Values are expressed as m e a n s + S . E . M , of six experiments. ~' P < 0.05 indicates significantly difference from values at 30 rain post-ischaemia. Statistically significant changes from pre-ischaemia are not shown for the sake of clarity. Abbreviations as in table 1. Drug
RMP (mV)
MRD (Vs i )
APA (mV)
APDs~ ~ (ms)
APD,~ (ms)
Control (30 rain postischaemia) M8008 1 ngml I M8008 30 ng ml ~ M8008 / 1,agml t M8008 10 ,ag ml ~
- 87.9 _+0.6
468.4 +_ 17.4
116.4 ± 0.6
189.9 + 6.6
285.5 ± 10.8
- 77.6 _+0.5
175.5 ± 23.2
80.8 + 3.4
113.8 ± 11.9
187.4 ± 14.1
-78.7±0.4
151.3+ 19.8
80.9+3.8
95.7±13.0
169.8+14.1
-78.6+0.4
133.4±20.3 ~'
78.8+3.0
93.2_+ 10.9
174.8± 12.8
-78.6±0.4
93.7_+20.0 a
71.9+4.1 ~
99.2+111.8
186
-79.2+0.4
54.2± 6.9 ~'
58.9+3.8 ~'
12(I.6± 12.2
±15.1
227.9_+ 18.5
49 MBO00 1nllml"1
lpgml-I
lOpgm1-1
-20 -40 t -60 -00
40 20 0 -20 -40
40 g
T
20
a. o
.LI
~ £
-20 Fig. 2. The % change in action potential characteristics produced by M8008 under both normal ([]) and simulated ischaemic ( • ) conditions. * P < 0.05 indicates significantly different from change under normal conditions.
dently reversed the 'ischaemia'-induced shortening in APDs0 and prolonged APDg0. The lowest concentration of M8008 studied, i.e. 1 ng m l - l had no statistically significant effect on the action potential characteristics under ischaemic conditions. In all concentrations, M8008 had a greater depressant effect on M R D in the presence of the modified compared to normal PSS (fig. 2). Figure 2 also illustrates that under ischaemic conditions M8008 prolonged APDs0 but shortened it in normal PSS. However, there were no statistically significant differences between the effects of M8008 on APDg0 under normal and simulated ischaemic conditions (fig. 2).
4. D i s c u s s i o n
4.1. Effects of the opioid receptor antagonists on normal sheep Purkinje fibres These results have demonstrated that the 6 opioid receptor antagonist, M8008, has marked electrophysio-
logical effects on paced normal sheep Purkinje fibres. M8008 caused a concentration-dependent reduction in MRD APA and APDs~ ~ without significantly altering RMP or APDso. The ionic mechanisms that underly these effects of M8008 on the cardiac potential have not been studied. It is likely, however, that M8008 reduces the upstroke of the action potential by blocking the sodium channels responsible for the rapid inward sodium current which depolarises the cell membrane. Such an action has been classified by Vaughan Williams (1970) as a class I antiarrhythmic effect. In Purkinje tissue, the action potential plateau is known to be partially maintained by a slowly inactivating component of inward sodium current (Attwell et al., 1979). M80(J8's ability to shorten the APDs~ ~ may, therefore, be due to a blocking effect on this current. Indeed M8008 may preferentially block this population of sodium channels, which have larger opening times than those responsible for phase 0 depolarisation (Wasserstrom and Salata, 1988). This would explain why, in this study, M8008 significantly reduced APDs0 at all concentrations studied, whereas MRD was affected by the highest concentration only. However, it is also possible that M8008 may have an action on other currents, such as the inward calcium current and outward potassium currents that influence the action potential duration. These elcctrophysiological effects of M8008 were observed at concentrations at which the drug has been shown to block ~ or tx opioid receptors in vitro (Smith, 1987). This might suggest that these effects could be opioid receptor-mediated, although there is no evidence to suggest that opioid peptides are released under these experimental conditions. Moreover, the 6 opioid agonist [D-Ala2,D-LeuS]enkephalin (DADLE), over a concentration range up to 100 ng ml-J has no electrophysiological effects on sheep Purkinje fibres (Mclntosh, 1991). Thus, it would seem that the electrophysiological effects of M8008 are more likely to be a consequence of some other 'direct' effect on the cardiac cell membrane. In accordance with these findings, other opioid receptor antagonists with selectivity for receptor types other than for 6 receptors, have also, been shown t o have class 1 actions. Naloxone (Frame and Argentieri 1985; Brasch 1986; Cerbai et al., 1989), buprenorphine (Boachie-Ansah et al., 1989) and nalmafene (Oldroyd et al., 1990) have all been demonstrated to exhibit class I activity either on Purkinje or ventricular tissue. In addition, it has been reported that the ( + ) and ( - ) isomers of naloxone induce comparable class I effects (Brasch, 1986) supporting the suggestion that these electrophysiological effects are not opioid receptor-mediated. Over a similar concentration range to M8008, NBT and naltrexone, which have a greater selectivity at K and Ix receptors respectively, had no marked effect on
511
normal sheep Purkinje fibres. It is, however, possible that with concentrations greater than 10 p,g m l - I of these drugs a direct electrophysiological effect could be obtained.
plained by such an action. It is possible, however, that NBT and naltrexone may exhibit electrophysiological effects under ischaemic, but not normal, conditions, This possibility was not examined in the present study.
4.2. Electrophysiological ef[ects of M8008 during simulated ischaemia
Acknowledgements
The electrophysiological effects of the hypoxic, acidotic and hyperkalaemic salt solution were similar to those previously described in in vitro preparations (Gilmour and Zipes, 1980; Kimura et al., 1984). These changes, i.e. depolarisation, reduced MRD and abbreviated action potential duration are also similar to the electrophysiological derangements recorded from intact hearts during a period of myocardial ischaemia following coronary artery ligation (Downar et al., 1977; Russell et al., 1979). Previous work with clinically used antiarrhythmic drugs have shown that such simulated ischaemic conditions can potentiate their class I activity (Kimura et al., 1982; Evans et al., 1984). The results of the present study have now shown that the class I effect of M8008 is also potentiated by conditions mimicking myocardial ischaemia. As has been suggested for lignocaine (Hondeghem and Katzung 1977; Wasserstrom and Salata, 1988) this may be due to its greater affinity for inactivated sodium channels than for rested sodium channels. Since simulated ischaemia causes depolarisation, a greater proportion of the sodium channels will be in the inactivated form with the consequence that drugs with a greater affinity for this channel form will exhibit a more marked effect. In the presence of simulated ischaemia, M8008 no longer abbreviated the plateau phase of the action potential. The action potential is markedly abbreviated by simulated ischaemia and this could in part be caused by a depolarisation-induced inactivation of the slowly inactivating current. Thus if M8008 abbreviated the normal action potential plateau by reducing this current, simulated ischaemia would attenuate or abolish such an action. In addition to losing the ability to shorten APDs~ , M8008 slightly prolonged action potential duration during ischaemia, suggesting that it may also block outward K + conductance under these conditions. Voltage clamp experiments would need to be carried out to examine these postulated ionic effects of M8008. In conclusion, this study has shown that M8008, but not NBT or naltrexone, has marked electrophysiological effects on sheep Purkinje fibres. The ability of M8008 to reduce MRD under normal conditions and the potentiation of this effect by ischaemia, could be the basis of its antiarrhythmic activity in vivo. Since neither NBT nor naltrexone had significant direct electrophysiological effects under normal conditions their antiarrhythmic efficacy in vivo is unlikely to be ex-
M. Mclntosh was an SERC CASE award student with Rcckitt & Colman. We thank them for their financial support and Dr. C. Smith (formerly of Reckitt & Colman, now with ICI) for his advice.
References Attwelk D., I. Cohen, D. Eisner, M. Ohba and C. Ojeda, 1979, The steady state TTX-sensitive (window) sodium current in cardiac Purkinje fibres, Pfliigers Arch. 379, 137. Boachic-Ansah, G., R. Sitsapesan, K.A. Kane and J.R. Parratt, 1989, The antiarrhythmic and cardiac clectrophysiological effects of buprenorphinc, Br. J. Pharmacol. 97. 801. Brasch, H. 1986, lnfluence of the optical isomers ( + } and ( - )-naloxone on beating frequency, contractile force and action potentials of guinea-pig isolated cardiac preparations, Br. J. Pharmacol. 88, 733. Cerbai, E., P.B. Cawdcabo, I. Masini, F. Porciatti and A. Mugelli, 1989, Antiarrhythmic properties of naloxone: an clectrophysiological study on sheep cardiac Purkinje fibres, Eur. J. Pharmacol. 162, 491. Downar, E.M.J. Janse and D. Durrer, 1977, The effect of acute c o r o n a ~ artery occlusion on subepicardial transmembrane potentials in the intact porcine heart, Circulation 56, 217. Evans, J.J., R.F. Gilmour and D.P. Zipes, 1984, The effects of lignocainc and quinidine on impulse propagation across the canine Purkinje muscle junction during combined hypcrkalaemia. hypoxia and acidosis. Circ. Res. 55, 185. Frame, L.H. and T.M. Argcntieri, 1985, Naloxone has local anaesthetic effects on canine cardiac Purkinje fibres, Circ. Res. 72, 34. Gilmour, R.F. and D.P. Zipes, 1980, Different electrophysiological responses of canine endocardium and epicardium to combined hyperkalaemia, hypoxia and acidosis, Circ. Res. 46, 814. ttondeghem, L. and B.G. Katzung, 1977, Time and voltage dependent interactions of antiarrhythmic drugs with cardiac sodium channels, Biochim. Biophys. Acta 172, 373. Kimura, S., H. Nakaya and M. Kanno, 1982, Effects of verapamil and lignocaine on changes in action potential characteristics and conduction time induced by combined hypoxia, hyperkalaemia and acidosis in canine ventricular myocardium, J. Cardiovasc. Pharmacol. 4, 658. Mclntosh, M.A., 1991, The Role of Opioid Peptides and Their Receptors During Acute Myocardial Ischaemia (Ph.D. Thesis, University of Strathclyde, Glasgow). Mclntosh, M.A., K. Kane and J. Parratt, 1991, Effects of selective opioid receptor agonists and antagonists during myocardial ischaemia, Eur. J. Pharmacol. 210, 37. Oldroyd, K., M.N. [licks and S.M. Cobbe, 19911, The class Ill antiarrhythmic effects of opioid receptor ligands are not receptor mediated, J. Mol. Cell Cardiol. 22 (Suppl. lIl), $82. Russell, D.C., J.H. Smith and M.F. Oliver, 1979. T r a n s m e m b r a n e potential changes and ventricular fibrillation during repetitive myocardial ischaemia in the dog, Br. Heart J. 42, 88. Smith, C.F.C., 1987, 160-Mecyprenorphine (RX 8008M): a potent opioid antagonist with some ,~ selectivity, Life Sci. 4/), 267.
51 Vaughan Williams, E.M., 1979, Classification of antiarrhythmic drugs, in: Symposium on Cardiac Arrhythmias, eds. E. Sandoe, E. Flendsted-Jensen and K. Olssen (Sweden AB Astra, Sodertalje) p. 449.
Wasserstrom, J.A. and J.J. Salata, 1988, Basis for tetrodotoxin and lignocaine effects on action potentials in dog ventricular myocytes, Am. J. Physiol. 254, 111157.
