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Studies on the neuropharmacology of papaverine —III. The inhibition of ganglionic transmission
Press.PrintedinGt.Britain. IVe~roph~rmarol~~Y, 1972,11,697-701Pergamon
STUDIES ON THE NEUROPHARMACOLOGY OF PAPAVERINE -111. THE INHIBITION OF GANGLIONIC TRANSMISSION V. BAUER*and R. CAPEK~ Institute of Pharmacology,
Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Accepted
24 January
1972)
Summary-The action of papaverine on the transmission in superior cervical ganglion of the cat was studied. The asynchronous discharge induced by close intra-arterial injection of nicotine, TMA, acetylcholine and pilocarpine and the action potentials evoked by preganglionic stimulation were recorded from postganglionic nerve fibres. Papaverine in low dose (0.5 mg i.a.) caused a slight inhibition of asynchronous postganglionic discharge evoked by nicotine agonists, whereas the action potential amplitude remained unchanged. The administration of papaverine in a moderate dose (1 .Omg) resulted in a decrease of the action potential amplitude which gradually returned to the original within 60 min. The asynchronous discharge evoked by nicotine agonists was completely blocked by this dose for 30 min. After the administration of the highest dose of papaverine (2.5 mg) nicotine agonists and preganglionic stimulation failed to evoke any response for several hours. Pretreatment with phentolamine (40 rg) did not change the inhibitory effect of papaverine. Physostigmine (90 pg) restored the papaverine blocked asynchronous discharge induced by acetylcholine. The restored response to acetylcholine was antagonized by atropine (1-2 pg) and was unchanged by hexamethonium (0.52.0 mg) or tubocurarine (0.4 mg). Similarly the asynchronous discharge evoked by pilocarpine after tetanic preganglionic stimulation was not markedly influenced by papaverine (2.5 mg) and hexamethonium (2.0 mg) but was blocked by atropine (2 pg). It suggested that the action of papaverine on ganglionic transmission might be due predominantly to its antinicotinic action, whereas an antimuscarinic effect was not observed.
PREVIOUSstudies from our laboratories on the neuromuscular junction (BAUERand CAPEK, 1971a,b) and on taenia coli of guinea pig (BAUERand KADLEC,1970) indicated that papaverine exerts an anticholinergic action, presumably an antinicotinic one. MINKER and KOLTAI (1961) showed that papaverine inhibits the contractions of nictitating membrane induced by preganglionic stimulation or by administration of ganglion stimulating agents. The aim of this paper was to find out whether the same effect of papaverine applies to ganglionic transmission as to the neuromuscular and whether it can be classified as nicotinic (LANGLEY,1918). The sympathetic superior ganglion is undoubtedly the test object of choice. This essentially cholinergic synapse contains a variety of receptors (e.g. PERRY, 1957; VOLLE,1966) and several experimental procedures have been described to distinguish between the effects of drugs on nicotinic or muscarinic receptors (ECCLESand LIBET, 1961 ; TAKESHIGEand VOLLE, 1962, 1963a,b, 1964; VOLLE,1969).
METHODS
The experiments were performed in 35 cats of both sexes, weighing 2.5-4.0 kg, anesthetized with 80 mg/kg of chloralose. The technique was essentially identical to that of VOLLE *Present address: Institute of Pharmacology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. tPresent address: Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada. 697
698
V. BAUEXand R. ~APEK
and KOELLE(1961). The sympathetic trunk was exposed, severed and placed on stimulating electrodes. A thin branch containing postganglionic fibers (n.carotis externus) going from the superior cervical ganglion to the carotid artery was dissected free from connective tissue and placed on recording electrodes. All branches of the carotid artery except that supplying the ganglion were tied and the lingual artery was cannulated for the close intra-arterial injections of drugs (TRENDELENBURG,1959). Two types of responses were recorded. The postganglionic action potential was evoked by supramaximal preganglionic electrical stimulation with square waves of O-5 msec duration. The postganglionic asynchronous discharge was evoked by intra-arterial injection of acetylcholine, nicotine, TMA or pilocarpine. The technique has been described in detail elsewhere (BAUERand CAPEK, 1969). Blood clotting in the cannulas was prevented by heparine (300 U/kg) administered intravenously immediately after surgery. Papaverine sulphate, acetylcholine hydrochloride, nicotine bitartarate, tetramethylammonium chloride (TMA), pilocarpine hydrochloride, atropine sulphate, hexamethonium dibromide, physostigmine salicylate, tubocurarine hydrochloride and phentolamine mesylate were dissolved in physiological saline and injected intra-arterially through the lingual artery to the ganglion. The volume administered never exceed 0.3 ml. All drugs except acetylcholine, TMA, nicotine and pilocarpine were flushed with the same volume of saline.
RESULTS
Efect on action potential Papaverine, in the dose of 0.5 mg, had no appreciable effect on the amplitude of the action potential evoked by preganglionic stimulation. A moderate dose of papaverine (1.0 mg) caused pronounced changes. After a delay of l-3 min, the action potential amplitude evoked by supramaximal preganglionic stimulation gradually decreased, the maximal effect being reached 7-12 min after papaverine administration, when the amplitude did not exceed 40% of the initial value. Then a slow recovery was observed; however even 60 min after the papaverine administration the initial amplitude was not reached (Fig. 1). A complete block of ganglionic transmission, as indicated by the disappearance of the action potential, was induced only by the highest dose (2.5 mg) of papaverine used. A pronounced depression of the action potential amplitude was observed after 1 min and a full disappearance 7-12 min after the drug administration. The observed block persisted for more than 60 min. The dose-dependence of the papaverine-induced action potential depression is shown by the dose-response curve plotted from values which were measured 12 min after the drug administration when the effect was maximal (Fig. 2). EfSect on asynchronous discharge A short-lasting decrease of the postganglionic asynchronous discharge evoked by intraarterial injection of 20 pg acetylcholine was observed after the administration of the lowest dose (0.5 mg) of papaverine. After 12 to 30 min the discharge recovered to its initial form. The moderate dose (1 a0 mg) of papaverine caused a decrease of the asynchronous discharge amplitude 3 min after the administration, to 50 % of the initial ones, and 12 min after the administration a complete blockade of the response to exogenous acetylcholine occurred. The blockade lasted 30 min and then a gradual recovery was observed 60-120 min after the administration of papaverine (Fig. 3).
FIG. 1. The effect of papaverine (1 .O mg La.) on the postgangl~onic action potential amplitude evoked by supramaximal pregangfionic stimulation. a: before; b: 3 min; c: 7 min; d: 12 min; e: 20 min and F: 60 min after the drug administration. The calibration represents 100 PV and IO msec respectively.
Nemo. f.p. 698
FIG. 3. Blockade of acetyicholine t (20 a&)-induced postganglionie asynchronous discharge by papaverine (1 .O mg i.a.) pretreatment. a: before, b: 3 min, c: 12 min and d: 30 min after papaverine administration. The calibration represents 10 pV and 2 set respectively.
postganglionic asynchronous discharge by FIG. 4. Blockade of TMA f (15 &induced papaverine (2.5 mg i.a.) pretreatment. a: before, b: 12 min, c: 30 min and d: 60 min after administration of papaverine. The calibration represents 10 yV and 2 set respectively.
FIG. 5. The effect of physostigmine (90 pg) on the papaverine (1.0 mg)-blocked asynchronous discharge. a: control response, b: 10 min after papaverine, c: 15 min after papaverine and 3 min after physostigmine, d: 25 min after papaverine and 13 min after physostigmine adt-administration of acetylcholine (20 pg). The calibration represents 10 PV and 2 ministration. set respectively.
FIG. 6. The effects of atropine and hexamethonium on physostigmine-restored asynchronous postganglionic discharge to acetyicholine $ (20 pg). a: 15 min after papaverine (1.0 mg) and 3 min after physostigmine (90 pg), b: 5 min after a and 2 min after hexamethonium (1.0 mg), c: the same as a from another experiment, d: 5 min after c and 2 min after atropine (2.0 pg) administration. The calibration represents 10 PV and 2 set respectively.
FIG. 7. The effect of papaverine (2.5 mg) and atropine (2.0 pg) on the asynchronous dischar ge evol ted by pilocarpine (40 pg) after repetitive preganglionic stimulation (30 Hz for 20 set). a:
cow trol response, b: 7 min after papaverine and c: 4 min after atropine administration. tion represents 10 PV and 2 set respectively.
Calibl *a-
Ganglionic effect of papaverine
699
FIG. 2. Dose-response curves of the papaverine effects on the postganglionic asynchronous discharge evoked by acetylcholine (AA) and on the amplitude of postganghonic action potentiaIs (O-O). Arithmetic means of 5 experiments are represented with their fiducial limits for P=O-05, lOO%=duration of asynchronous discharge or amplitude of action potentials before drug administration.
The highest dose (2.5 mg) of papaverine accelerated the appearance of the inhibitor and blocking effects. Three minutes after the administration, the amplitude was decreased to about 20 % of the initial value, and at 7 min a full block occurred which persisted for several hours. A cIear-cut dependence of the papaverine effect on dose is demonstrated by the dose-response curve (Fig. 2). The asynchronous postganglionic discharge evoked by other nicotine agonists, e.g. 15 pg TMA (Fig. 4) or 10 pg nicotine, was inhibited or blocked in the same way as the action of acetylcholine was, by I.0 or 25 mg of papaverine pretreatment. It was possible to overcome the blocking effects of papaverine against acetylcholine by physostigmine (90 pg) or by tetanic preganglionic stimulation (60 Hz for 20 set). The acetylcholine-induced discharge temporarily reappeared. The renewal of the response lasted IO-15 min when the block was induced by the moderate dose of papaverine (Fig. 5), but only 3-5 min after the administration of the highest dose. Hexamethonium (0.52.0 mg) or tubocurarine (0.4 mg) did not change the restored discharge by these procedures; however the discharge disappeared readily after the administration of atropine (1%2+0 pg) (Fig. 6). Pilocarpine (40 pg) was able to evoke postganglionic discharge when it was administered after repetitive preganglionic stimulation (30 Hz for 20 set). This discharge was not markedly influenced by hexamethonium (2.0 mg) but was blocked by atropine (2.0 pg). Papaverine in the highest dose used (2.5 mg) only slightly inhibited the action of piloca~ine (Fig. 7). Pretreatment with phentolamine (40 r)g) failed to produce any change in the effects of papaverine. The doses of papaverine used caused a decrease of blood pressure (20-40 mmHg) lasting l-3 min.
700
V. BAUER and R. CAPEK
DISCUSSION The changes described of both responses studied clearly indicated that papaverine causes an inhibition of ganglionic transmission. The circulatory effects of this substance cannot be responsible for the observed depression. Only slight and short-lasting lowering of blood pressure was noted which highly contrasted with the prolonged inhibition or even block of the ganglion. It was shown that papaverine produces hyperpolarization of the smooth muscle cell membrane (SHEVCHENKO, 1962; IMAI and TAKEDA,1967) and stimulates respiration presumably with the participation of adrenergic mechanisms (NIMS et al. 1953) and also increases muscle twitches in vivo during its first phase of action (BAUERand CAPEK, 1971b). All these stimulatory effects might be catecholamine-mediated. Due to their a-sympathomimetic effects, catecholamines have been shown to produce the hyperpolarization of the neuronal membrane resulting in the inhibition of ganglionic transmission (LUNDBERG,1952; DEGROAT and VOLLE, 1966a,b). However, it is highly unlikely that a-sympathomimetic effects of catecholamines are involved in the depression induced by papaverine, as it was not influenced by phentolamine. It is also obvious that a &sympathomimetic effect does not play a role in the phenomenon observed. No facilitation of ganglionic transmission which results from the &sympathomimetic action (DEGROATand VOLLE, 1966a,b) was observed in our experiments. In contrast to the ganglion blocking agents of the heaxamethonium type, which inhibit the action potentials evoked by preganglionic stimulation in smaller concentrations as compared to the asynchronous discharge caused by exogenous acetylcholine, papaverine blocked the responses to exogenous acetylcholine, nicotine and TMA in lower concentrations than the responses to electrical stimulation. This difference might be due to the fact that tubocurarine, hexamethonium and similar substances reduce the amount of acetylcholine released by incoming volleys due to their effect on the nerve terminals (MCKINSTRY and KOELLE,1967) while papaverine is devoid of this effect (HARRY, 1962) at least in other synapses. Also the existence of numerous cholinergic receptor sites on the ganglion cells might be responsible for the observed difference. They are localized not only at the subsynaptic membrane (PAPPANOand VOLLE, 1965) but also extrasynaptically (VOLLE, 1962). It is conceivable that papaverine might have higher affinity and/or better access to the extrasynaptic receptors which are activated by exogenous stimulants. Higher doses of papaverine are thus required to achieve binding with the subsynaptic receptors. By comparing blocking doses of papaverine with those of hexamethonium as reported by others in analogous experiments (e.g. VOLLE,1962) papaverine was on molar basis 2*53-O times less effective than hexamethonium in blocking the action potential evoked by preganglionic stimulation and had the same activity in blocking the asynchronous discharge evoked by nicotine agonists (acetylcholine, nicotine and TMA). Pretreatment with physostigmine or the tetanic stimulation of the ganglion resulted in considerable prolongation of the response to exogenous acetylcholine (TAKESHIGEand VOLLE, 1962). The asynchronous discharge consisted of two components. The initial nicotinic component was easily blocked by hexamethonium and the delayed one (muscarinic) was sensitive to atropine (TAKESHIGEand VOLLE1964). However after the blocking dose of papaverine, when the ganglion was subjected to these procedures, only the atropine-sensitive response appeared, though the threshold for the activation of atropine-sensitive sites by acetylcholine after physostigmine administration and/or tetanic stimulation is always lower than that for the activation of hexamethonium-sensitive ones (TAKESHIGE and VOLLE,
Ganglioniceffectof papaverine
701
1962). Similar to the mus~arinic effect of acety~cholin~, the postgang~ionic discharge evoked by pilocarpine after tetanic preganglionic stimulation was not markedly affected by papaverine. Our findings provide additional evidence that the anticholinergic effects of papaverine in the superior cervical ganglion of the cat are directed predominantly towards the effect of nicotine agonists. The data complements our previous reports (BAUER and KADLEC, 1970; BAUER and CAPEK, 1971a,b) providing an overall picture in two peripheral nicotinic synapses.
of the anticholinergic
effects of papaverine
REFERENCES BAUER,V. and CAPEK, R. (1969). The effect of 6-azauridine on the peripheral nervous system. I. The action on ganglionic transmission. Int. J. Neuropharmac. 8: 263-269. BAUER, V. and CAPEK, R. (1971a). Studies on the neuroph~ma~lo~ of papaverine. I. The curare-like action in isolated nerve-muscle preparation. Neurophar~cology 10: 499-506. BAUER,V. and CAPEK,R. (1971 b). Studies on the neuropharmacology of papaverine. II. The effect on neuromuscular transmission in the whole animal. Neuropharmacology 10: 507-Y 1. BAUER,V. and KADLEC, 0. (1970). Antinicotinic properties of papaverine in guinea-pig taenia coli. Experientia 26: 1331-1332. DEGROAT, W. C. and VOLLE,R. L. (1966a). The action of catecholamines on transmission in the superior cervical ganglion of the cat. J. Pharmac. exp. Ther. 154: 1-13. DEGROAT,W. C. and VOLLE,R. L. (1966b). Interactions between the catecholamines and ganglionic stimulating agents in sympathetic ganglia. J. P/tarmac. cxp. Ther. 154:200-215. ECCLES,R. M. and LIBET,B. (1961). Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiof. 157: 484-503. HARRY, J. (1962). Effect of cooling, locai anesthetic compounds and botulinum toxin on the responses of and acetylcholine output from the electrically transmurally stimulated isolated guinea-pig ileum. Br. J. Pharmac. Chemother. 19: 42-55. IMAI, S. and TAKEDA,K. (1967). Effect of vasodilatators upon the isolated taenia coli of the guinea pig. J. Pharmnc. exp. Ther. 156: 557-564. LANGLEY,J. N. (1918). On the stimulation and paralysis of nerve cells and nerve endings. II. Paralysis by curare, strichnine and brucine and its antagonism by nicotine. J. Physiol. 52: 247-266. LUNDBERG,A. (1952). Adrenaline and transmission in the sympathetic ganglion of the cat. Acta physiol. sea&. 26: 252-262. MCKINSTRY,D. N. and KOELLE,G. B. (1967). Effect of drugs on acetylcholine release from the cat superior cervical ganglion by carbachol and by preganglionic stimulation. J. Pharmac. exp. Ther. 157: 328-336. MINKER, E. and KOLTAI, M. (1961). Beitrlge zur Pharmakologie des Spasmolytica. Abstr. ZZ. Conf. Hung. Ther. Znv.Pharm. 434437. NIMS, R. G., SEVERINHAM, J. W. and COMROE,J. H. JR. (1953). Reflex hyperpnoe induced by procaine acting upon carotid and aortic bodies. J. Phurmac. exp. Ther. 109: 58-61. PAPPANO,A. J. and VOLLE,R. L. (1965). A comparison of the responses of sympathetic ganglia and chemoreceptors to cholinomimetic agents and sodium cyanide. J. Pharmac. exp. Ther. 147: 324-329. PERRY,W. L. M. (19.57). Transmission in autonomic ganglia. Br. med. Bull. 13: 220-226. SHEVCHENKO, A. 3. (1962). Vlijanie nekotorich farmakologit~h~kich vescestv na otvetnuju reakciju gladkoj mishci pri prjamom elektriczeskom rozdrazenii. B&H. Eksp. Biol. Med. 54: 57-60. TAKESHI~E,Ch. and VOLLE,R. J. (1962). Bimodal response of sympathetic ganglia to acetycholine following eserine or repetitive preganglionic stimulation. J. Pharmac. exp. Ther. 138: 66-73. TAKESHICE,Ch. and VOLLE,R. L. (1963a).Asynchronous postganglionic firing from the cat superior cervical sympathetic ganglion treated with neostigmine. Br. J. Pharmac. Chemuther. 20: 214-220. TAKESHIGE,Ch. and VOLLE, R. L. (1963b). Cholinoceptive sites in denervated sympathetic ganglion. J. Pharmac. exp. Ther. 141: 206-213. TAKESHIGE,Ch. and VOLLE,R. L. (1964). A comparison of ganglion potentials and block produced by acetylcholine and tetramethylammonium. Br. J. Pharmac. Chemother. 23: 80-89. TRENDELENBURG, U. (1959). Non-nicotinic g~glionstimulating substances. Fedn Proc. Fedn Am. &es exp. Biol. 18: 1~1-1~~. VOLLE,R. L. (1962). Enhancement of postganglionic responses to stimulating agents following repetitive preganglionic stimulation. J. Pharmac. exp. Ther. 136: 68-74. VOLLE,R. L. (1966). Modification by drugs of synaptic transmission in autonomic ganglia. Pharmac. Rev. 18: 839-869. VOLLE,R. L. (1969). Ganglionic tr~smission. A. Rev. Phurmac. 9: 135-146. VOLLE,R. L. and KOELL~,G. B. (1961). The physiological role of acetylchoiinesterase (AChE) in sympathetic ganglia. J. Pharmac. exp. Ther. 133: 223-240.
STUDIES ON THE NEUROPHARMACOLOGY OF PAPAVERINE -111. THE INHIBITION OF GANGLIONIC TRANSMISSION V. BAUER*and R. CAPEK~ Institute of Pharmacology,
Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Accepted
24 January
1972)
Summary-The action of papaverine on the transmission in superior cervical ganglion of the cat was studied. The asynchronous discharge induced by close intra-arterial injection of nicotine, TMA, acetylcholine and pilocarpine and the action potentials evoked by preganglionic stimulation were recorded from postganglionic nerve fibres. Papaverine in low dose (0.5 mg i.a.) caused a slight inhibition of asynchronous postganglionic discharge evoked by nicotine agonists, whereas the action potential amplitude remained unchanged. The administration of papaverine in a moderate dose (1 .Omg) resulted in a decrease of the action potential amplitude which gradually returned to the original within 60 min. The asynchronous discharge evoked by nicotine agonists was completely blocked by this dose for 30 min. After the administration of the highest dose of papaverine (2.5 mg) nicotine agonists and preganglionic stimulation failed to evoke any response for several hours. Pretreatment with phentolamine (40 rg) did not change the inhibitory effect of papaverine. Physostigmine (90 pg) restored the papaverine blocked asynchronous discharge induced by acetylcholine. The restored response to acetylcholine was antagonized by atropine (1-2 pg) and was unchanged by hexamethonium (0.52.0 mg) or tubocurarine (0.4 mg). Similarly the asynchronous discharge evoked by pilocarpine after tetanic preganglionic stimulation was not markedly influenced by papaverine (2.5 mg) and hexamethonium (2.0 mg) but was blocked by atropine (2 pg). It suggested that the action of papaverine on ganglionic transmission might be due predominantly to its antinicotinic action, whereas an antimuscarinic effect was not observed.
PREVIOUSstudies from our laboratories on the neuromuscular junction (BAUERand CAPEK, 1971a,b) and on taenia coli of guinea pig (BAUERand KADLEC,1970) indicated that papaverine exerts an anticholinergic action, presumably an antinicotinic one. MINKER and KOLTAI (1961) showed that papaverine inhibits the contractions of nictitating membrane induced by preganglionic stimulation or by administration of ganglion stimulating agents. The aim of this paper was to find out whether the same effect of papaverine applies to ganglionic transmission as to the neuromuscular and whether it can be classified as nicotinic (LANGLEY,1918). The sympathetic superior ganglion is undoubtedly the test object of choice. This essentially cholinergic synapse contains a variety of receptors (e.g. PERRY, 1957; VOLLE,1966) and several experimental procedures have been described to distinguish between the effects of drugs on nicotinic or muscarinic receptors (ECCLESand LIBET, 1961 ; TAKESHIGEand VOLLE, 1962, 1963a,b, 1964; VOLLE,1969).
METHODS
The experiments were performed in 35 cats of both sexes, weighing 2.5-4.0 kg, anesthetized with 80 mg/kg of chloralose. The technique was essentially identical to that of VOLLE *Present address: Institute of Pharmacology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. tPresent address: Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada. 697
698
V. BAUEXand R. ~APEK
and KOELLE(1961). The sympathetic trunk was exposed, severed and placed on stimulating electrodes. A thin branch containing postganglionic fibers (n.carotis externus) going from the superior cervical ganglion to the carotid artery was dissected free from connective tissue and placed on recording electrodes. All branches of the carotid artery except that supplying the ganglion were tied and the lingual artery was cannulated for the close intra-arterial injections of drugs (TRENDELENBURG,1959). Two types of responses were recorded. The postganglionic action potential was evoked by supramaximal preganglionic electrical stimulation with square waves of O-5 msec duration. The postganglionic asynchronous discharge was evoked by intra-arterial injection of acetylcholine, nicotine, TMA or pilocarpine. The technique has been described in detail elsewhere (BAUERand CAPEK, 1969). Blood clotting in the cannulas was prevented by heparine (300 U/kg) administered intravenously immediately after surgery. Papaverine sulphate, acetylcholine hydrochloride, nicotine bitartarate, tetramethylammonium chloride (TMA), pilocarpine hydrochloride, atropine sulphate, hexamethonium dibromide, physostigmine salicylate, tubocurarine hydrochloride and phentolamine mesylate were dissolved in physiological saline and injected intra-arterially through the lingual artery to the ganglion. The volume administered never exceed 0.3 ml. All drugs except acetylcholine, TMA, nicotine and pilocarpine were flushed with the same volume of saline.
RESULTS
Efect on action potential Papaverine, in the dose of 0.5 mg, had no appreciable effect on the amplitude of the action potential evoked by preganglionic stimulation. A moderate dose of papaverine (1.0 mg) caused pronounced changes. After a delay of l-3 min, the action potential amplitude evoked by supramaximal preganglionic stimulation gradually decreased, the maximal effect being reached 7-12 min after papaverine administration, when the amplitude did not exceed 40% of the initial value. Then a slow recovery was observed; however even 60 min after the papaverine administration the initial amplitude was not reached (Fig. 1). A complete block of ganglionic transmission, as indicated by the disappearance of the action potential, was induced only by the highest dose (2.5 mg) of papaverine used. A pronounced depression of the action potential amplitude was observed after 1 min and a full disappearance 7-12 min after the drug administration. The observed block persisted for more than 60 min. The dose-dependence of the papaverine-induced action potential depression is shown by the dose-response curve plotted from values which were measured 12 min after the drug administration when the effect was maximal (Fig. 2). EfSect on asynchronous discharge A short-lasting decrease of the postganglionic asynchronous discharge evoked by intraarterial injection of 20 pg acetylcholine was observed after the administration of the lowest dose (0.5 mg) of papaverine. After 12 to 30 min the discharge recovered to its initial form. The moderate dose (1 a0 mg) of papaverine caused a decrease of the asynchronous discharge amplitude 3 min after the administration, to 50 % of the initial ones, and 12 min after the administration a complete blockade of the response to exogenous acetylcholine occurred. The blockade lasted 30 min and then a gradual recovery was observed 60-120 min after the administration of papaverine (Fig. 3).
FIG. 1. The effect of papaverine (1 .O mg La.) on the postgangl~onic action potential amplitude evoked by supramaximal pregangfionic stimulation. a: before; b: 3 min; c: 7 min; d: 12 min; e: 20 min and F: 60 min after the drug administration. The calibration represents 100 PV and IO msec respectively.
Nemo. f.p. 698
FIG. 3. Blockade of acetyicholine t (20 a&)-induced postganglionie asynchronous discharge by papaverine (1 .O mg i.a.) pretreatment. a: before, b: 3 min, c: 12 min and d: 30 min after papaverine administration. The calibration represents 10 pV and 2 set respectively.
postganglionic asynchronous discharge by FIG. 4. Blockade of TMA f (15 &induced papaverine (2.5 mg i.a.) pretreatment. a: before, b: 12 min, c: 30 min and d: 60 min after administration of papaverine. The calibration represents 10 yV and 2 set respectively.
FIG. 5. The effect of physostigmine (90 pg) on the papaverine (1.0 mg)-blocked asynchronous discharge. a: control response, b: 10 min after papaverine, c: 15 min after papaverine and 3 min after physostigmine, d: 25 min after papaverine and 13 min after physostigmine adt-administration of acetylcholine (20 pg). The calibration represents 10 PV and 2 ministration. set respectively.
FIG. 6. The effects of atropine and hexamethonium on physostigmine-restored asynchronous postganglionic discharge to acetyicholine $ (20 pg). a: 15 min after papaverine (1.0 mg) and 3 min after physostigmine (90 pg), b: 5 min after a and 2 min after hexamethonium (1.0 mg), c: the same as a from another experiment, d: 5 min after c and 2 min after atropine (2.0 pg) administration. The calibration represents 10 PV and 2 set respectively.
FIG. 7. The effect of papaverine (2.5 mg) and atropine (2.0 pg) on the asynchronous dischar ge evol ted by pilocarpine (40 pg) after repetitive preganglionic stimulation (30 Hz for 20 set). a:
cow trol response, b: 7 min after papaverine and c: 4 min after atropine administration. tion represents 10 PV and 2 set respectively.
Calibl *a-
Ganglionic effect of papaverine
699
FIG. 2. Dose-response curves of the papaverine effects on the postganglionic asynchronous discharge evoked by acetylcholine (AA) and on the amplitude of postganghonic action potentiaIs (O-O). Arithmetic means of 5 experiments are represented with their fiducial limits for P=O-05, lOO%=duration of asynchronous discharge or amplitude of action potentials before drug administration.
The highest dose (2.5 mg) of papaverine accelerated the appearance of the inhibitor and blocking effects. Three minutes after the administration, the amplitude was decreased to about 20 % of the initial value, and at 7 min a full block occurred which persisted for several hours. A cIear-cut dependence of the papaverine effect on dose is demonstrated by the dose-response curve (Fig. 2). The asynchronous postganglionic discharge evoked by other nicotine agonists, e.g. 15 pg TMA (Fig. 4) or 10 pg nicotine, was inhibited or blocked in the same way as the action of acetylcholine was, by I.0 or 25 mg of papaverine pretreatment. It was possible to overcome the blocking effects of papaverine against acetylcholine by physostigmine (90 pg) or by tetanic preganglionic stimulation (60 Hz for 20 set). The acetylcholine-induced discharge temporarily reappeared. The renewal of the response lasted IO-15 min when the block was induced by the moderate dose of papaverine (Fig. 5), but only 3-5 min after the administration of the highest dose. Hexamethonium (0.52.0 mg) or tubocurarine (0.4 mg) did not change the restored discharge by these procedures; however the discharge disappeared readily after the administration of atropine (1%2+0 pg) (Fig. 6). Pilocarpine (40 pg) was able to evoke postganglionic discharge when it was administered after repetitive preganglionic stimulation (30 Hz for 20 set). This discharge was not markedly influenced by hexamethonium (2.0 mg) but was blocked by atropine (2.0 pg). Papaverine in the highest dose used (2.5 mg) only slightly inhibited the action of piloca~ine (Fig. 7). Pretreatment with phentolamine (40 r)g) failed to produce any change in the effects of papaverine. The doses of papaverine used caused a decrease of blood pressure (20-40 mmHg) lasting l-3 min.
700
V. BAUER and R. CAPEK
DISCUSSION The changes described of both responses studied clearly indicated that papaverine causes an inhibition of ganglionic transmission. The circulatory effects of this substance cannot be responsible for the observed depression. Only slight and short-lasting lowering of blood pressure was noted which highly contrasted with the prolonged inhibition or even block of the ganglion. It was shown that papaverine produces hyperpolarization of the smooth muscle cell membrane (SHEVCHENKO, 1962; IMAI and TAKEDA,1967) and stimulates respiration presumably with the participation of adrenergic mechanisms (NIMS et al. 1953) and also increases muscle twitches in vivo during its first phase of action (BAUERand CAPEK, 1971b). All these stimulatory effects might be catecholamine-mediated. Due to their a-sympathomimetic effects, catecholamines have been shown to produce the hyperpolarization of the neuronal membrane resulting in the inhibition of ganglionic transmission (LUNDBERG,1952; DEGROAT and VOLLE, 1966a,b). However, it is highly unlikely that a-sympathomimetic effects of catecholamines are involved in the depression induced by papaverine, as it was not influenced by phentolamine. It is also obvious that a &sympathomimetic effect does not play a role in the phenomenon observed. No facilitation of ganglionic transmission which results from the &sympathomimetic action (DEGROATand VOLLE, 1966a,b) was observed in our experiments. In contrast to the ganglion blocking agents of the heaxamethonium type, which inhibit the action potentials evoked by preganglionic stimulation in smaller concentrations as compared to the asynchronous discharge caused by exogenous acetylcholine, papaverine blocked the responses to exogenous acetylcholine, nicotine and TMA in lower concentrations than the responses to electrical stimulation. This difference might be due to the fact that tubocurarine, hexamethonium and similar substances reduce the amount of acetylcholine released by incoming volleys due to their effect on the nerve terminals (MCKINSTRY and KOELLE,1967) while papaverine is devoid of this effect (HARRY, 1962) at least in other synapses. Also the existence of numerous cholinergic receptor sites on the ganglion cells might be responsible for the observed difference. They are localized not only at the subsynaptic membrane (PAPPANOand VOLLE, 1965) but also extrasynaptically (VOLLE, 1962). It is conceivable that papaverine might have higher affinity and/or better access to the extrasynaptic receptors which are activated by exogenous stimulants. Higher doses of papaverine are thus required to achieve binding with the subsynaptic receptors. By comparing blocking doses of papaverine with those of hexamethonium as reported by others in analogous experiments (e.g. VOLLE,1962) papaverine was on molar basis 2*53-O times less effective than hexamethonium in blocking the action potential evoked by preganglionic stimulation and had the same activity in blocking the asynchronous discharge evoked by nicotine agonists (acetylcholine, nicotine and TMA). Pretreatment with physostigmine or the tetanic stimulation of the ganglion resulted in considerable prolongation of the response to exogenous acetylcholine (TAKESHIGEand VOLLE, 1962). The asynchronous discharge consisted of two components. The initial nicotinic component was easily blocked by hexamethonium and the delayed one (muscarinic) was sensitive to atropine (TAKESHIGEand VOLLE1964). However after the blocking dose of papaverine, when the ganglion was subjected to these procedures, only the atropine-sensitive response appeared, though the threshold for the activation of atropine-sensitive sites by acetylcholine after physostigmine administration and/or tetanic stimulation is always lower than that for the activation of hexamethonium-sensitive ones (TAKESHIGE and VOLLE,
Ganglioniceffectof papaverine
701
1962). Similar to the mus~arinic effect of acety~cholin~, the postgang~ionic discharge evoked by pilocarpine after tetanic preganglionic stimulation was not markedly affected by papaverine. Our findings provide additional evidence that the anticholinergic effects of papaverine in the superior cervical ganglion of the cat are directed predominantly towards the effect of nicotine agonists. The data complements our previous reports (BAUER and KADLEC, 1970; BAUER and CAPEK, 1971a,b) providing an overall picture in two peripheral nicotinic synapses.
of the anticholinergic
effects of papaverine
REFERENCES BAUER,V. and CAPEK, R. (1969). The effect of 6-azauridine on the peripheral nervous system. I. The action on ganglionic transmission. Int. J. Neuropharmac. 8: 263-269. BAUER, V. and CAPEK, R. (1971a). Studies on the neuroph~ma~lo~ of papaverine. I. The curare-like action in isolated nerve-muscle preparation. Neurophar~cology 10: 499-506. BAUER,V. and CAPEK,R. (1971 b). Studies on the neuropharmacology of papaverine. II. The effect on neuromuscular transmission in the whole animal. Neuropharmacology 10: 507-Y 1. BAUER,V. and KADLEC, 0. (1970). Antinicotinic properties of papaverine in guinea-pig taenia coli. Experientia 26: 1331-1332. DEGROAT, W. C. and VOLLE,R. L. (1966a). The action of catecholamines on transmission in the superior cervical ganglion of the cat. J. Pharmac. exp. Ther. 154: 1-13. DEGROAT,W. C. and VOLLE,R. L. (1966b). Interactions between the catecholamines and ganglionic stimulating agents in sympathetic ganglia. J. P/tarmac. cxp. Ther. 154:200-215. ECCLES,R. M. and LIBET,B. (1961). Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiof. 157: 484-503. HARRY, J. (1962). Effect of cooling, locai anesthetic compounds and botulinum toxin on the responses of and acetylcholine output from the electrically transmurally stimulated isolated guinea-pig ileum. Br. J. Pharmac. Chemother. 19: 42-55. IMAI, S. and TAKEDA,K. (1967). Effect of vasodilatators upon the isolated taenia coli of the guinea pig. J. Pharmnc. exp. Ther. 156: 557-564. LANGLEY,J. N. (1918). On the stimulation and paralysis of nerve cells and nerve endings. II. Paralysis by curare, strichnine and brucine and its antagonism by nicotine. J. Physiol. 52: 247-266. LUNDBERG,A. (1952). Adrenaline and transmission in the sympathetic ganglion of the cat. Acta physiol. sea&. 26: 252-262. MCKINSTRY,D. N. and KOELLE,G. B. (1967). Effect of drugs on acetylcholine release from the cat superior cervical ganglion by carbachol and by preganglionic stimulation. J. Pharmac. exp. Ther. 157: 328-336. MINKER, E. and KOLTAI, M. (1961). Beitrlge zur Pharmakologie des Spasmolytica. Abstr. ZZ. Conf. Hung. Ther. Znv.Pharm. 434437. NIMS, R. G., SEVERINHAM, J. W. and COMROE,J. H. JR. (1953). Reflex hyperpnoe induced by procaine acting upon carotid and aortic bodies. J. Phurmac. exp. Ther. 109: 58-61. PAPPANO,A. J. and VOLLE,R. L. (1965). A comparison of the responses of sympathetic ganglia and chemoreceptors to cholinomimetic agents and sodium cyanide. J. Pharmac. exp. Ther. 147: 324-329. PERRY,W. L. M. (19.57). Transmission in autonomic ganglia. Br. med. Bull. 13: 220-226. SHEVCHENKO, A. 3. (1962). Vlijanie nekotorich farmakologit~h~kich vescestv na otvetnuju reakciju gladkoj mishci pri prjamom elektriczeskom rozdrazenii. B&H. Eksp. Biol. Med. 54: 57-60. TAKESHI~E,Ch. and VOLLE,R. J. (1962). Bimodal response of sympathetic ganglia to acetycholine following eserine or repetitive preganglionic stimulation. J. Pharmac. exp. Ther. 138: 66-73. TAKESHICE,Ch. and VOLLE,R. L. (1963a).Asynchronous postganglionic firing from the cat superior cervical sympathetic ganglion treated with neostigmine. Br. J. Pharmac. Chemuther. 20: 214-220. TAKESHIGE,Ch. and VOLLE, R. L. (1963b). Cholinoceptive sites in denervated sympathetic ganglion. J. Pharmac. exp. Ther. 141: 206-213. TAKESHIGE,Ch. and VOLLE,R. L. (1964). A comparison of ganglion potentials and block produced by acetylcholine and tetramethylammonium. Br. J. Pharmac. Chemother. 23: 80-89. TRENDELENBURG, U. (1959). Non-nicotinic g~glionstimulating substances. Fedn Proc. Fedn Am. &es exp. Biol. 18: 1~1-1~~. VOLLE,R. L. (1962). Enhancement of postganglionic responses to stimulating agents following repetitive preganglionic stimulation. J. Pharmac. exp. Ther. 136: 68-74. VOLLE,R. L. (1966). Modification by drugs of synaptic transmission in autonomic ganglia. Pharmac. Rev. 18: 839-869. VOLLE,R. L. (1969). Ganglionic tr~smission. A. Rev. Phurmac. 9: 135-146. VOLLE,R. L. and KOELL~,G. B. (1961). The physiological role of acetylchoiinesterase (AChE) in sympathetic ganglia. J. Pharmac. exp. Ther. 133: 223-240.