Inhibition of 5-hydroxytryptamine-evoked autonomic transmitter release by apomorphine

European Journal of Pharmacology, 81 (1982)469-477 Elsevier Biomedical Press

469

INHIBITION OF 5-HYDROXYTRYPTAMINE-EVOKED AUTONOMIC TRANSMITTER RELEASE BY A P O M O R P H I N E SUSAN R. CARR * and JOHN R. FOZARD ** Department of Pharmacology, Materia Medica & Therapeutics, University of Manchester, MI3 9PT, U.K.

Received 27 November 1981, revised MS received 2 March 1982, accepted 13 April 1982

S.R. CARR and J.R. FOZARD, Inhibition of 5-hydroxytryptamine-evoked autonomic transmitter release by apomorphine, European J. Pharmacol. 81 (1982) 469-477. Apomorphine inhibited chronotropic responses of the isolated rabbit heart to 5-HT by 40% at 1.17/tM and by 90% at 4.68 #M and strongly inhibited the outflow of 3H following preloading of hearts with [3H]-(--)-noradrenaline. Apomorphine, 4.68/~M, had no significant effects on transmitter release evoked by DMPP or tyramine but inhibited the responses to SNS at frequencies up to 3.2 Hz. The inhibitory effects of apomorphine on 5-HT were resistant to blockade by chlorpromazine, 1.4 #M, haloperidol, 1.6/~M, spiroperidol, 2.5 #M, or yohimbine, 2.8 #M. In contrast, the inhibitory effects of apomorphine on low frequency SNS were abolished by yohimbine. On the guinea-pig ileum treated with methysergide, apomorphine, 1.17-4.68 /xM, blocked the indirect cholinergic responses to 5-HT less markedly than it blocked the indirect sympathomimetic responses to 5-HT on the rabbit heart. Moreover, the effects were non-selective since responses to DMPP and transmural stimulation of the intramural cholinergic nerves were similarly reduced. Modification of 5-HT receptor function is the most likely explanation for the action of apomorphine with the differential effect on 5-HT in heart and ileum reflecting differences in the receptors a n d / o r post receptorial events at the two sites. Neuronal tryptamine receptors

5-HT

Apomorphine

Dopaminoceptor antagonists

1. Introduction 5 - H y d r o x y t r y p t a m i n e (5-HT) stimulates transmitter release from the sympathetic nerves of the perfused rabbit heart by a mechanism which is calcium dependent and mediated by specific receptor sites F o z a r d and Mwaluko, 1976; Fozard and M o b a r o k All, 1978a). The order of potency of a n u m b e r of 5 - H T and tryptamine analogues in evoking transmitter release from the heart is similar to that obtained in guinea-pig ileum where these agents release acetylcholine (Fozard and M o b a r o k Ali, 1978a), suggesting similarities be* Present address:" Department of Pharmacology, School of Pharmacy and Pharmacology, University of Bath, BA2 7AY, U.K. ** Present address and address for correspondence: Centre de Recherche Merrell International 16 rue d'Ankara, 67084 Strasbourg Cedex, France. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

Yohimbine

tween the receptors on the cardiac sympathetic nerves and the 'M'-receptor of the intramural cholinergic nerves ( G a d d u m and Picarelli, 1957). Several c o m p o u n d s of diverse pharmacological actions have been reported to inhibit selectively the receptor-mediated, indirect sympathomimetic effect of 5 - H T in the perfused rabbit heart, including ( - ) - c o c a i n e and a n u m b e r of its analgoues (Fozard, 1979; Fozard et al., 1979), procaine but not lignocaine or tetracaine (Fozard et al., 1979) and metoclopramide (Fozard and M o b a r o k Ali, 1978b). We now report that apomorphine is a selective antagonist of transmitter release evoked by 5-HT from the sympathetic nerves of the perfused rabbit heart. Moreover, the mechanism is novel in that it appears not to involve activation of presynaptic d o p a m i n e receptors in this tissue (Fuder and Muscholl, 1978). Some of these data were presented previously at

470 a Meeting of the British Pharmacological Society (Carr and Fozard, 1977).

2. Materials and methods

2.1. Perfused rabbit heart Rabbits of either sex weighing 1.9-2.9 kg were given heparin 500 units/kg into a marginal ear vein. Two to five min later, they were stunned by a blow to the head and bled. Hearts were removed rapidly, some with the right sympathetic nerves attached (Hukovi6 and Muscholl, 1962), and perfused by the Langendorff technique with modified Tyrode solution (composition in g/l: NaC1 8.0; KC1 0.2; CaC12 0.2; MgC12 0.1; N a H C O 3 1.0; N a H 2 P O 4 0.05; glucose 1.0 and ascorbic acid 0.01), gassed with a mixture of 95% O 2 and 5% CO 2. Perfusion pressure was maintained at approximately 60 cm water. Atropine, 1.4 /~M, was routinely added to the perfusion fluid to prevent interference from indirect cholinergic activity. Right ventricular tension and rate and atrial ~ension were recorded as described by Fozard and Muscholl (1971).

2.2. Guinea-pig ileum Segments of ileum were removed from freshly killed guinea-pigs and set up for recording changes in longitudinal tension in, the modified Tyrode solution described above. Methysergide, 2.8 #M, was routinely added to the bathing fluid to eliminate the smooth muscle or 'D'-receptor component of the response to 5-HT (Fozard and Mobarok Ali, 1978a). The tissue was stimulated transmurally using steel electrodes 1 cm apart, and a Grass SD9 square-wave stimulator.

was usually 5 min. Effects of modifying drugs were evaluated by introducing them into the perfusion fluid for 15 min prior to and during establishment of a second dose response curve. In most instances, a third dose response was obtained 15 min after return to drug-free Tyrode solution. Experiments with guinea-pig ileum were essentially similar. Successive dose response curves were constructed at 15 min intervals, modifying drugs being added between curves. In all cases, control curves constructed in the absence of modifying drugs were reproducible (Carr, 1979).

2.4. 3H-overflow experiments The procedure for these experiments was based on that described by Starke (1971). ( - ) [7-3H]noradrenaline, sp. act. 7.2-9 C i / m m o l was diluted with unlabelled ( - ) - n o r a d r e n a l i n e and infused into the heart to give a final concentration of 5.0 × 10-SM and activity of 43-50 nCi/ml. The loading period was 12 min and a washout period of 25 min was allowed before the experiment proceeded. The flow rate of perfusion fluid was maintained at 25 m l / m i n by a Watson-Marlow roller pump. One ml samples of the perfusate were taken for radioassay as detailed in the Results section, dispersed in 10 ml of scintillant (composition: 0.1 g dimethyl POPOP, 5.5g diphenyloxazole, 333 ml Triton X-100, 667 ml toluene) and counted (Packard Tricarb Scintillation Spectrometer). Results were corrected for counting efficiency and quenching.

2.5. Statistical analysis Results are presented as means and standard errors. Student's t-test was used to assess the significance of the differences between mean values. Number of observations is denoted by n.

2.3. Design of experiments 2.6. Drugs used In the perfused heart, dose-effect curves to 5HT, dimethylphenylpiperazinium (DMPP) and noradrenaline were established using bolus injections of drug in volumes of 50/xl or less delivered rapidly (less than 0.5 s) into the Tyrode solution perfusing the heart. The interval between doses

Apomorphine hydrochloride (MacfarlanSmith); atropine sulphate (Koch-Light); chlorpromazine hydrochloride (May & Baker); dimethylphenylpiperazinium iodide (Sigma); 5-hydroxytryptamine creatinine sulphate (Koch-Light); halo-

471

l a). Concomitant atrial and ventricular tension changes were similarly inhibited by apomorphine and all of these effects could be at least partially reversed by a 15 min washout period (results not shown). Fig. 1 also shows the e~ffect of the higher concentration of apomorphine (4.68 #M) on responses evoked by the nicotine receptor agonist, DMPP, by SNS, and by noradrenaline. Responses to DMPP were somewhat depressed by apomorphine, but the changes could not be demonstrated to be significant. SNS was inhibited principally at the lowest frequencies of stimulation, and the effects were significant at 1 and 3.2 Hz. These effects of apomorphine are likely to be presynaptic and a reflection of reduced transmitter release since heart rate responses to bolus injections of noradrenaline were unaffected by perfusion of hearts with apomorphine (4.68/tM) (fig. ,'ld).

peridol (Searle); methysergide bimaleate (Sandoz); [3H]noradrenaline hydrochloride (Radiochemical Centre, Amersham); (-)-noradrenaline bitartrate (Koch-Light); tyramine hydrochloride (KochLight); spiroperidol (Janssen Pharmaceuticals); yohimbine hydrochloride (Merck, Darmstadt).

3. Results

3.1. Effects of apomorphine on responses of rabbit hearts to 5-HT, DMPP, electrical stimulation of the cardiac sympathetic nerves (SNS) and noradrenaline In control experiments, three successive cardiac rate response curves, carried out at 15 min intervals, and using either 5-HT, DMPP, SNS or noradrenaline were found to be reproducible (results not illustrated, but see Carr, 1979). The maximum increases in cardiac rate were close to 100 beats/min for each stimulation procedure which represents an increase of 75-85% over the mean resting rate (fig. 2 and Carr, 1979). Apomorphine (1.17 and 4.68 ~tM) caused a concentration-dependent inhibition of the responses of cardiac rate to bolus doses of 5-HT (fig.

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Fig. 2. Effect of apomorphine on 3H-overflow from hearts preloaded with [3H]noradrenaline evoked by 5-HT, tyramine and dimethylphenylpiperazinium (DMPP). O, control (no apomorphine); O, apomorphine, 4.68/~M, present throughout. Note different vertical axis for DMPP. The histograms represent the maximum changes in cardiac rate obtained following drug administration. [~, control values; [], values in the presence of apomorphine, 4.68 ~M. Values represent means + S.E.M. of 4 individual experimental observations.

indeed accompanied by a reduction in transmitter overflow was obtained using hearts preloaded with [3H]noradrenaline. In these experiments, single injections of three stimulants, 5-HT (180 nmol), tyramine (440 nmol) and D M P P (210 nmol) chosen for their similar effects on cardiac rate, were given at 15 min intervals to hearts perfused with normal Tyrode solution or with solution containing apomorphine (4.68 /~M) introduced to the hearts 15 min before the end of the washout period following loading. Apomorphine markedly inhibited the overflow of 3H induced by 5-HT and the accompanying chronotropic response (fig. 2a). In contrast, 3H-release induced by tyramine was little affected although some reduction in release was evident during the first 3 min after injection (fig. 2b). Consistent with its effects on the chronotropic response to DMPP, apomorphine caused a small inhibition in the 3H-overflow evoked by this agent (fig. 2c).

3.3. Effects of dopamine receptor antagonists and yohimbine on apomorphine-induced inhibition of responses to 5 - H T in isolated rabbit hearts In an attempt to define the mechanism of action of apomorphine, the dopamine receptor antagonists, chlorpromazine, haloperidol, and spiroperidol, and the az-adrenoceptor antagonist, yohimbine, were tested for their ability to prevent the inhibitory effect of apomorphine on transmitter release evoked by 5-HT. The compounds were present in the perfusion fluid for 15 min prior to establishing the first curve to 5-HT and maintained to the end of the third curve. Apomorphine, 1.17 /~M, was applied 15 min before and during establishment of the second curve. The results are presented in fig. 3. Comparison of the first curves in the presence of the antagonists with the control curves included in fig. 1 discloses minimal effects of these com-

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Fig. 3. Effect of dopamine receptor antagonists and yohimbine on apomorphine-induced inhibition of chronotropic responses on isolated rabbit hearts induced by 5-HT. The receptor antagonists were present from 15 min before the first curve and for the duration of the second curve. (a) Chlorpromazine (1.4 laM); (b) haloperidol (1.6 jaM); (c) spiroperidol (2.5 jaM); (d) yohimbine (2.8 jaM). O, first (control) curve; A, second curve obtained in the presence of apomorphine, 1.17 jaM. Points represent mean values+--S.E.M, of 3-4 individual experiments.

a b s e n c e of a p o m o r p h i n e , b u t i n p r e s e n c e o f a n t a g o n i s t , r e s u l t e d in d e p r e s s a n t effects of a p o m o r p h i n e l u s t r a t e d , b u t see Carr, 1979). I n direct c o n t r a s t to its effects

p o u n d s p e r se o n the r e s p o n s e s to 5 - H T . Similarly, n o m a r k e d a n t a g o n i s m of the i n h i b i t o r y effect of a p o m o r p h i n e b y a n y of these c o m p o u n d s was n o t e d ( c o m p a r e fig. 3 a - d with fig. la). I n all cases, p e r f u s i o n of the h e a r t s for a f u r t h e r 15 rain in the

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474

inhibitory effect of apomorphine on the response to low frequency nerve stimulation was abolished by perfusion of the hearts with yohimbine, 2.8/~M (see Carr, 1979). 3.4. Effect of apomorphine on the responses of guinea-pig ileum to 5-HT, DMPP, transmural nerve stimulation and acetylcholine Methysergide, 2.8 t~M, was included in the Tyrode solution to exclude any contribution to the observed responses arising through activation of 5-HT D-receptors (Fozard and Mobarok Ali, 1978a). Under these conditions, dose/frequency response curves established, then repeated twice at 15 min intervals, were reproducible (Fozard et al., 1979). Apomorphine was added to the Tyrode solution at concentrations of 1.17 and 4.68 /~M during the second and third dose-response curves respectively. The results shown in fig. 4 indicate that apomorphine is not as effective in inhibiting responses mediated through neuronal tryptamine receptors in the ileum as it is on the perfused rabbit heart (compare fig. 4a with fig. l a). Moreover, unlike in the heart, inhibition was not manifested selectively against 5-HT. Thus, responses to D M P P and particularly those to stimulation of the intramural parasympathetic nerves were also inhibited by apomorphine (fig. 4b, c). These effects of apomorphine are, however, clearly presynaptic since responses to added acetylcholine remained close to control levels (fig. 4d).

4. Discussion

The principal finding of the present work is that apomorphine inhibits transmitter release arising from stimulation of 5-HT receptors on the postganglionic sympathetic nerves of the perfused rabbit heart whilst leaving responses to DMPP, tyramine and SNS little altered. It had been assumed that the effect would arise through stimulation of dopamine a n d / o r a2-adrenoceptors which are located presynaptically on the terminal fibres of the cardiac sympathetic nerves and which reduce transmitter overflow evoked by depolarizing

stimuli when activated (Starke, 1977; Westfall, 1977; Fuder and Muscholl, 1978; Langer, 1980; Starke, 1981). In fact, the evidence clearly indicates that presynaptic dopamine receptors are unlikely to be involved in the inhibitory effect of apomorphine on 5-HT since neither chlorpromazine, haloperidol, nor spiroperidol in concentrations demonstrably able to block the presynaptic dopamine receptor (Hope et al., 1977; Starke et al., 1978; Steinsland and Hieble, 1978) prevented the effect of apomorphine (fig. 3a-c). Similarly, although affinity of apomorphine for presynaptic a2-adrenoceptors has been demonstrated in vitro (Gibson and Samini, 1979), it seems unlikely that activation of these sites can explain the inhibitory effects of apomorphine against 5-HT. Thus, yohimbine, used at a concentration more than adequate to ensure presynaptic a2-adrenoceptor blockade (Starke et al., 1975; Borowski et al., 1977; Wikberg, 1979; Carr and Fozard, 1981), did not block the inhibitory effects of apomorphine (fig. 3d). Furthermore, clonidine itself, used at a concentration which caused maximal inhibition of low-frequency sympathetic nerve stimulation failed to modify transmitter release evoked from the rabbit heart by 5-HT (Carr and Fozard, 1981). On the other hand, the inhibitory effects of apomorphine on low frequency SNS were abolished by yohimbine which suggests a different mechanism of inhibition to that operating against 5-HT. The observation is consistent with apomorphine activating presynaptic c~2adrenoceptors in this tissue as has previously been described for the mouse vas deferens (Gibson and Samini, 1979). It is conceivable that apomorphine would share with morphine agonist activity at the mu opioid receptor which mediates inhibition of transmitter release at a number of autonomic neuroeffector junctions probably through hyperpolarization of the neuronal membrane (North and Williams, 1977; Henderson et al., 1979; Hughes, 1981). However, in marked contrast to ,apomorphine, morphine had only weak, non-selective inhibitory effects against 5-HT in the perfused rabbit heart (Fozard and Mwaluko, 1976), whereas it potently inhibited responses to 5-HT in the ileum (Gaddum and Picarelli, 1957; Waterfield et al., 1977).

475

In seeking other potential explanations for the inhibitory effect of apomorphine against 5-HT in the heart, a local anaesthetic or 'membrane stabilizing' effect of the compound acting to counteract a depolarizing stimulus is theoretically tenable. The suggestion is perhaps particularly pertinent in view of the observation that 5-HT is a much weaker stimulant of 3H-overflow than DMPP (fig. 2) despite the effects on cardiac rate being similar (fig. 1). DMPP is known to evoke a greater maximum depolarization of the cell bodies of postganglionic sympathetic neurones than 5-HT (Haefely, 1974a; 1974b) and this could be the basis of the differences in evoked transmitter release from the terminals (see also Fozard, 1978; Fozard and Mobarok Ali, 1978c). Thus, the question arises as to whether apomorphine is selective only in that it can reduce transmitter release evoked by a relatively weak depolarizing stimulus (5-HT) to a greater extent than the more powerful DMPP. The possibility seems unlikely for two reasons. First, there is little evidence that apomorphine has local anaesthetic or 'membrane stabilizing' activity even at concentrations many times higher than those used in the present experiments (see Simon and Van Maanen, 1976; Ennis et al., 1979). Moreover, a number of compounds with recognized and powerful local anaesthetic activities are consistently more potent inhibitors of responses to D M P P on the rabbit heart than of responses to 5-HT (Fozard et al., 1979). Perhaps, the simplest explanation for the selective inhibition of 5-HT by apomorphine on the heart would be that apomorphine blocked the 5-HT receptors of the cardiac sympathetic nerves which mediate transmitter release evoked by 5-HT and are distinct from the neuronal nicotine receptors (Fozard and Mwaluko, 1976; Fozard and Mobarok Ali, 1978a, c: Fozard et al., 1979; G/Sthert and Diihrsen, 1979). Such an explanation would, however, necessarily imply differences between the 5-HT receptors of the adrenergic nerves of the heart and those of the cholinergic nerves of the ileum, since apomorphine showed no selectivity as an antagonist of 5-HT on the ileum (fig.4). Although there are undoubtably similarities between the receptors at the two sites (for instance, the order of potency of a number of agonists was

similar in the two tissues (Fozard and Mobarok Ali, 1978a) as was the susceptibility of the receptors at each site to blockade by ( - ) - c o c a i n e and (+)-pseudococaine (Fozard et al., 1979) more recent experiments have disclosed differences between the two receptor populations. Thus, 5methoxytryptamine is a potent agonist at the 5-HT receptor present on the cholinergic nerves of the ileum yet is devoid of effects on the heart (Fozard and Mobarok Ali, 1978a). Moreover, ( - ) norcocaine is a significantly more potent antagonist of 5-HT on the heart than on the ileum (Fozard, 1980). Given that differences exist between the two receptor populations, it is certainly possible to envisage apomorphine as a selective, although clearly non-surmountable (fig. l a), antagonist at the sympathetic neuronal tryptamine receptors of the rabbit heart. Whilst differences between receptors provide a logical explanation of the selective inhibition of 5-HT by apomorphine in heart but not in ileum, there may also be differences beyond the receptor level in the mechanism by which 5-HT releases transmitter from the cholinergic or adrenergic nerves. It is known that acetylcholine release evoked from the ileum by 5-HT is inhibited by tetrodotoxin and hence dependent on an inward sodium current (Gershon, 1967; Costa and Furness, 1979; Huidobro-Toro and Foree, 1980). In contrast, the release of noradrenaline from the rabbit heart evoked by 5-HT is resistant to blockade by tetrodotoxin (Fozard and Mwaluko, 1976). However, the same situation holds for DMPP (Fozard and Mwaluko, 1976; Costa and Furness, 1979), whose effects are not selectively inhibited by apomorphine on heart or ileum, suggesting selective depression of 5-HT responses in the heart by apomorphine is unlikely to reflect simply a resistance to blockade of a tetrodotoxin-sensitive mechanism. Tetrodotoxin-sensitive transmitter release is, however, a common feature of the indirect cholinergic responses of the ileum to 5-HT, DMPP and transmural nerve stimulation and may have a bearing on the mode of action of apomorphine in this tissue. From the literature, apomorphine inhibits electrically evoked transmitter release from the guinea-pig ileum at concentrations between

476

10 6 and 10-SM through activation of presynaptic dopaminoceptors a n d / o r a2-adrenoceptors (Ennis et al., 1979; Tayo, 1979). For the sympathetic nervous system, persuasive evidence exists that restriction of impulse propagation in the terminal fibres is a major factor in the attenuation of transmitter release following stimulation of a : adrenoceptors (Stj~irne, 1978; Alberts et al., 1981). Activation of a similar mechanism by apomorphine within the parasympathetic nerve plexus of the ileum would not only explain why responses to 5-HT are reduced but also why responses to DMPP and transmural stimulation, which also release transmitter through a tetrodotoxin-sensitive mechanism, are similarly affected. In conclusion, inhibition by apomorphine of transmitter release evoked by activation of sympathetic neuronal 5-HT receptors in the rabbit heart cannot be explained by activation of either presynaptic dopamine receptors or a2-adrenoce ptors. Similarly, it is unlikely to occur as a result of stimulation of mu opioid receptors, by 'membrane stabilization' or by selective suppression of excitation-secretion coupling arising from tetrodotoxinresistant depolarization. Blockade of neuronal 5-HT receptors may be a feasible explanation for the action of apomorphine with the differential effect on 5-HT in heart and ileum reflecting differences in the receptors a n d / o r post post-receptorial events at the two sites (Fozard, 1980).

Acknowledgments This work was supported by a grant to S.R.C. from the Medical Research Council. We are grateful to Janssen Pharmaceuticals and Searle for generous gifts of drugs.

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