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A potent factor in extracts of the skin of the Australian frog, PSeudophryne coriacea—III
kurophnnnacology Vol. 25, No. 8, pp. 807-814, 1986 Printed in Great Britain
0028-3908/86 S3.00 + 0.00 Pergamon Journals Ltd
A POTENT FACTOR IN EXTRACTS OF THE SKIN OF THE AUSTRALIAN FROG, PSEUDOPHR YNE CORIACEA-III POTENTIATION MAMMALIAN BY DIRECT
OF CONTRACTIONS ELICITED IN AVIAN AND ISOLATED SKELETAL MUSCLE PREPARATIONS AND INDIRECT ELECTRICAL STIMULATION
G. FALCONIERIERSPAMER,V. ER~PAMERand P. MELCHIORRI Institutes of Pharmacology and Pharmacognosy and Medical Pharmacology III, First University of Rome, Citta Universitaria, 00100 Rome, Italy (Accepted 8 November 1985) Summary-Extracts of the skin of the Australian frog Pseudophryne coriucea displayed a striking potentiating effect on contractions evoked in isolated skeletal muscle preparations of mammals (phrenic nerve diaphragm) and birds (chick biventer cervicis and semispinalis muscles) by indirect and direct electrical stimulation. There was both a conspicuous increase in the amplitude of the twitch, up to IO-fold, and a remarkable prolongation of the duration of the twitch. The effect was dose- and frequencydependent. In the presence of the extract, fusion of twitches after tetanic stimulation occurred earlier. No tachyphylaxis upon repeated stimulation by the extract was observed and the response to large doses persisted, declining slowly, for hours. These effects must be ascribed to an alkaloid related in structure to pumiliotoxin B. Response to the extract of Pseudophrynecoriacea by indirectly-stimulated preparations was potentiated by physostigmine and blocked by tubocurarine and a-bungarotoxin, demonstrating that in these preparations the extract acted pre-synaptically to facilitate the release of acetylcholine from motor nerve endings. However, the extract of Pseudophrynecoriaceu displayed equally potent effects in directly stimulated preparations, insensitive to physostigmine and to blockers of nicotinic acetylcholine receptors, indicating a direct action on the skeletal muscle. It is suggested that, like pumiliotoxin B, the Pseudophryne coriaceu alkaloid may interfere in the regulation of calcium channels in both nerve and muscle fibres. Key words: Pseudophryne coriacea skin, pumiliotoxin B-like alkaloid, skeletal muscle preparations, direct stimulation, indirect stimulation, acetylcholine release, calcium channels.
A preceding paper (Erspamer, Falconieri Erspamer, Melchiorri and Mazzanti, 1985) showed that methanol extracts of the skin of some Australian frogs, belonging to the genus Pseudophryne (Myobatrachidae family), displayed potent stimulant effects on isolated preparations of intestine and on electrically-driven vas deferens preparations. The most thoroughly studied Pseudophryne species was
to an alkaloid related in structure to the pumiliotoxins (Daly, 1982), but different from any of the known pumiliotoxin alkaloids (Daly, personal communication). Elucidation of the structure of the Pseudophryne coriacea alkaloid is in progress, the main obstacle to reach the goal being represented by the great difficulty in collecting frog material. This paper describes the actions of a semi-purified Ps. coriacea extract of the skin of Ps. coriacea in several isolated skeletal-muscle and nerve-skeletal muscle preparations of mammals and birds. It will be seen that the extract potently reinforced and conspicuously prolonged the muscle twitch elicited both by direct and indirect electrical stimulation.
Pseudophryne coriacea.
On the basis of results obtained with antagonists and blocking agents it was suggested that Pseudophryne coriacea had a pre-synaptic, neurogenic point of attack and that it acted to facilitate release of transmitters from the nerve endings. Acetylcholine was considered the most important agent involved in the response to Pseudophryne coriacea by the intestinal muscle and noradrenaline in the response by vas deferens preparations. It seems highly probable that the striking activity of extracts of Pseudophryne coriucea must be ascribed
Dedicated to Egidio Meneghetti, Professor of Pharmacology at the University of Padua, Italy, on the 25th anniversary of his death.
METHODS Amphibian material
The amphibian material used in this study was the semi-purified extract of the skin of Ps. coriucea, described in the preceding paper. This semi-purified material was free of biologically active contaminants, as demonstrated by HPLC experiments showing a single peak of activity. Concentrations of the extract, expressed in terms of pg/ml,
807
808
G. FALCONIERIERSPAMERer al.
always refer to the amount of dried skin from which the semi-purified extract was obtained. On the basis of the Dragendorff and Reinecke salt reactions, it has been calculated that the biologically active pumiliotoxin-like alkaloid had a tissue concentration not exceeding 200-400 pg/g dry skin. Bioassay
The extract of Ps. coriacea was assayed on the following isolated nerve-skeletal muscle and skeletal muscle preparations: (1) Phrenic nerve-diaphragm from mice, rats, hamsters and guinea-pigs, suspended in a 30-50ml bath of Krebs solution, at 38°C. (2) Chick biventer cervicis muscle isolated and prepared as described by Ginsborg and Warriner (1960), and suspended in a 20-30 ml bath of Krebs-Henseleit solution at 39°C. (3) Chick semispinalis muscle, isolated and prepared as described by Child and Zaimis (1960), same solution and temperature. All preparations were aerated with 95% 0, + 5% COz. The phrenic nerve-diaphragm and the chick biventer cervicis muscle were stimulated both indirectly and directly; the chick semispinalis muscle only directly. Single stimulations (Electric Stimulator Digit 3T, Lace Elettronica, Pisa) were carried out with rectilinear pulses of 0.5 msec duration, at 0.05-0.2 Hz, using supramaximal voltage. Tetanic stimulation was performed, at 1 min intervals and for 1 set, at frequencies of 10, 20, 30, 40, 50 and 70 Hz. The mechanical activity of all muscle preparations was recorded isotonically (7006 isotonic transducer) or isometrically (DY2) by a strain-gauge transducer and displayed on a recording microdynamometer (Unirecord 7050, Basile, Milan). Two simple electrode assemblies were used in this study, one for direct, and the other for indirect stimulation. The assembly for direct stimulation of the muscle consisted of a 5 mm diameter plastic tube, 200 mm long, bent at one end. The electrode of stainless steel emerged from the bent end and terminated in a hook designed to hold the preparation. The counter electrode was a stainless-steel tube collar, 30mm long, round the plastic tube. The hook was embedded as far as possible in one extremity of the muscle, with little or no wire left exposed. The assembly for nerve stimulation consisted of two platinum leads, embedded in a vertical plastic bar, connected to two perforated platinum discs, similarly embedded in a plastic protrusion at the lower end of the bar. The phrenic nerve or the upper tendon of the chick biventer cervicis muscle (encapsulating nerve) was gently passed through the hole. This was narrow for the phrenic nerve, somewhat larger for the tendon (Ginsborg and Warriner, 1960). A similar, but more simple version of the above assembly consisted of two small perforated platinum discs embedded in a pip of light-dentistry resin
(OrthocrylX) connectd to the stimulator by very thin wires. The system floated freely in the nutrient bath and any stretching of the nerve was avoided. This assembly was elaborated by Mr Fortuna. Istituto Superiore di Sanitl, Rome, and always gave excellent results. In the present experiments the duration of twitch was the time elapsing between the start of contraction and return to the base-line; contraction time, the time required for the tension to rise from the base-line to peak; 90% relaxation time, the time required for the tension to fall from the peak to a point of the falling phase equal to 10% of the twitch amplitude. Drugs
Drugs used were as follows: nifedipine (mol. wt 346; Bayer, Leverkusen), acetylcholine bromide (mol. wt 226), tubocurarine chloride.5H,O (mol. wt 785), tetrodotoxin (mol. wt 3 19), suxamethonium chloride. 2H,O (mol. wt 397) physostigmine salicylate (mol. wt 413), EGTA [ethylene glycol bis(aminoethylether) N,N,N’,N’-tetraacetic acid, mol. wt 3801, a-bungarotoxin (Sigma, St Louis, Missouri).
RESULTS
Chick biventer cervicis muscle
The responses of this preparation were almost identical, both upon indirect and direct stimulation, and will be described together. Threshold concentrations of the extract of Ps. coriacea varied, in 35 experiments, between 8 and 25 pg dried skin/ml bathing solution. Response to the extract consisted in an increase in base-line tension with simultaneous prolongation of the duration of twitch, followed by potentiation of the amplitude of twitch. Increase in base-line tension, had its earliest manifestation in the disappearance of the fly-back below the base-line, caused by the return spring of the writing pen. In turn, prolongation of the duration of the twitch was mainly due to lengthening of relaxation time (Fig. 1). Prolongation of the duration of twitch and 90% relaxation time were approximately the same at any concentration of extract above 2-fold the threshold, and were clearly correlated with the frequency of stimulation, the greatest prlongation occurring at the lowest rate of stimulation. As measured in 5 experiments at 0.1 Hz stimulation, the duration of the twitch increased from 140-170 to 1200-1800msec, and the 90% relaxation time from 40-80 to 1000-1300 msec. Potentiation of the amplitude of the twitch was clearly dose-dependent: preceded by a latency of 3-8 min for threshold concentrations of the extract, it was prompt for larger concentrations. Generally, doses 3-5-fold the threshold produced a maximum twitch increase, up to 600-1000% of control. Washing caused rapid return to basal conditions at small concentrations of extract; large concentrations required repeated or
Pse~dop~ry~ corticeu extract on skeletal muscle
809
continuous washing for 5-20 min. ~~eversibi~ity of Tubocurarine, in concentrations of more than the effect was never seen, 2-3 PM, produced a more or less rapid but complete Moderate or large concentrations of the extract, inhibition of basal twitch and a total or partial left in the organ bath without washing, produced a inhibition of extract-potentiated twitch. There was a clear-cut reciprocal antagonism between tubopotent increase in the amplitude of the twitch lasting curarine and extract. Thus, neuromuscular biock for hours, with slow decline. In one experiment produced by 1.5 and 3 PM tubocurarine could be response to 50 pg dried skin/ml (5-fold the threshold) partially reversed by I5 and 30 pg/ml of extract, was recorded for 11 hr. The muscle was stimulated at respectively. After 20 FM tubocurarine, blockade of 0.05 Hz. Maximum poientiation reached 800% of control and at the end of the experiment it was still response induced by the extract was complete up to con~ntrations of 150 pg/ml of extract (IO-fold the 160%. In a further series of experiments, the muscle was threshold). At this point, washing caused, after a successively stimulated at I-min intervals and for latency of a few minutes, a formidable, gradual 1 set, at 10, 20, 30, 40, 50 and 70 Hz. Under control after-potentiation of the twitch (500X), which then conditions, the amplitude of the first twitch (or of declined very slowly, upon repeated washing. This tetanic contraction) increased progressively, from 10 indicates that binding of the extract to its receptors to 70 Hz and fusion of twitches occurred only at was more tenacious than that of tubocurarine. 30 Hz. After exposition of the muscle for 5min to a-Bungarotoxin, in concentrations of 2-5 gg/ml, 25 ag/ml of extract the response was conspicuously completely abolished the basal twitch and the repotentiated, with a maximum nearly identical at sponse elicited by any concentration of extract (up to 20-fold the threshold] or suxamethonium. Blockade any frequency of stimulation, and fusion of the single was definitive and could not be twitches was anti~pated, being complete at 20 Hz by a-bungarotoxin removed either by continuous washing or repeated (Fig. 2). a~i~stration of the extract. ft is well known that the chick biventer cervicis muscle, owing to its content of both slow and fast Nif~pine, a wmpound believed to bfock calcium fibres, responds to depolarizing agents acting on channels, produced a clear-cut reduction of the nicotinic receptors of the end-plate with a contracture potentiation of the twitch induced by the extract, at and shift of the base-line, masking the effect on concentrations ranging from 15 to 20 PM. This effect twitch. Given during the spasm provoked by could always be removed by additional amounts of suxamethonium (0.1-0.2 p M), the extract reduced extract. However, it should be noted that the active the contracture and simultaneously caused the re- concentration of nifedipine here was 100 times larger appearance of vigorous twitches of increasing amplithan that required to inhibit both spontaneous and motility of the isolated intestinal tude. To obtain this effect 20, 40 and lOOpg/ml of extract-induced extract were required, against suxamethonium con- smooth muscle (Erspamer ef al., 1985). centrations of 0.25, 0.40 and 1.2 FM, respectively Chick sern~~p~a~i~ m~cle (Fig. 1). The extract was completely inactive in Response to the extract of Ps. coriacea by directlynon-stimulated preparations. However, in concentrations of 20-40 pg/ml it caused a small, but con- stimulated preparations (6 experiments) was similar to that observed in the biventer cervicis muscle. stant increase (IO-30%) in the response to both of the frog extract were acetylcholine and suxamethonium, given 5 min Threshold concentrations later. Tetrodotoxin (0.02-0.2 p M) regularly caused a of the same order and there was always a good conspicuous or total blockade of both basal and dose-response relationship, with no tachyphylaxis. extract-potentiated twitch, regardless of the mode Rat, mouse, guinea-pig and hamster diaphragm and of stimulation. Total blockade of the twitch by phrenic-nerve diaphragm preparations 0.15-0.2 @M tetrodotoxin was unsurmountable by Direct stimulation was carried out on diaphragm the extract. However, after large concentrations of frog extract, a clear-cut after-potentiation of the preparations from 10 rats, 3 mice, 5 guinea-pigs and 7 hamsters and indirect stimulation on 7 rat, 4 amplitude of the twitch was sometimes seen upon guinea-pig and 5 hamster phrenic ne~e-diaphm~ washing. Owing to their mechanism of action, neither of the extract tu~curarine and a-bugarotoxin (reversible or ir- preparations. Threshold con~ntrations for all preparations, regardless of their mode of reversible blockade of the nicotinic acetylchoIine stimulation, ranged between 8 and 2Opg dried receptors on the end-plate) nor physostigmine (inhibition of cholinesterase) affected, the response to skin/ml. However the supramaximal voltage required was less in the case of indirect stimulation. As in the the extract by the directly stimulated chick biventer preparations. Conversely, the above drugs were chick biventer cervicis muscle preparations, the response consisted of an increase in the base-line potently active on the indirectly-stimulated preparatension, increase in the amplitude of twitch and tions (Fig. 3). Physostigmine (0.1-0.15 gM) halved prolongation of the duration of twitch. The extract the threshold concentration of extract and conspicuwas completely inactive, at any concentration level, ously potentiated response to larger concentrations.
810
G. FALCONIERIERSPAMERet al.
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Fig. 2. Chick biventer cervicis muscle preparation. Effect of the extract of the Ps. coriacea on the tetanus elicited by I-see stimulations, at different Hz, given at I-min intervals. Under control conditions, the amplitude of the tetanic response increased with the frequency and fusion of the twitches which occurred at 30 Hz. After exposure to the extract (expressed as pg dried skin/ml) for 10 min there was a conspicuous increase in the amplitude of the tetanic response, which was the same at 10, 20 and 30 Hz, and an anticipation of complete fusion of the twitches (20 Hz). Relaxation after the extract was slowed down,
and fly-back below the base-line was lacking.
in non-stimulated preparations. Prolongation of the duration of the twitch for the diaphragm, was mainly due to the prolongation of the relaxation time. In experiments carried out with 2-3-fold the threshold concentration of extract, the total duration of the twitch rose from 80-140 to 1X@-25OOmsec, the contraction time increased from 30-40 to lOO-2OOmsec and the 90% relaxation time from SO-60 to 1000-l 500 msec. Values are approximate, as calculated from polygraphic records. Potentiation of the twitch produced by small concentrations of the extract was rapidly removed by washing; that elicited by large concentrations required repeated or continuous washing for S-20 min. An irreversibility of the effect was never seen, even at concentrations of the extract up to 20-fold the threshold. Moderate or large concentrations of the
frog extract left in the organ bath without washing produced a potentiation of the twitch lasting for hours, with a very slow decline (Fig. 4). Tetrodotoxin (0.1-0.2 PM) caused an inhibition of both basal and extract-stimulated twitch in all preparations. One to three micromoles of tubocurarine, 2WO FM suxamethonium and 24 pg/ml a -bungarotoxin caused a complete, unsurmountable blockade of both basal and potentiated twitch in indirectly-stimulated preparations, and 0.05-0.1 PM physostigmine, in turn, produced a conspicuous potentiation of the response to the extract. The effect of the drugs described above, acting either on nicotinic acetylcholine receptors in the end-plate or on acetylcholinesterase was either lacking or, more frequently, partial, never exceeding a maximum of IO-30% blockade, in the directly-
Figures I and 3 on facing page Fig. 1. Chick biventer cervicis muscle, two preparations (indirect and direct electrical stimulation at 0.1 Hz). *Washing. The effect of graded concentrations of extract of Ps. coriaceu (PSC; expressed as fig dried skin/ml). Note the good dose-response relationship and prompt return to basal conditions after washing. The first response to threshold doses of extract (7-10 pg/ml) was an increase in basal tension, simultaneous with prolongation of the duration of the twitch. Increase in the amplitude of the twitch appeared somewhat later (see lower tracing). Spasm elicited by suxamethonium (sux) was overcome by the extract with re-appearance of twitch contractions of increasing amplitude. Fig. 3. Chick biventer cervicis muscle (two preparations). *Washing. Upper tracing: indirect electrical stimulation (0.1 Hz). Large asterisk, adjustment of the base-line. Physostigmine (PHYS) produced a conspicuous potentiation of the response to the extract @g dried skin/ml); tubocurarine (TUB) and a-bungarotoxin (BTX) abolished both the basal twitch and the response to the extract. Washing caused the appearance of a powerful rebound potentiation of twitch after tubocurarine, whereas standstill caused by a-bungarotoxin was unsurmountable. Lower tracing: predominant direct electrical stimulation. Both tubocurarine and a-bungarotoxin only slightly affected the basal twitch and response. to the extract. Tetrodotoxin (TTX), at increasing concentrations, progressively reduced the twitch potentiated by the extract until abolition.
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812
Pseudophryne coriaceu extract on skeletal muscle
stimulated diaphragm preparations (Fig. 5). This may be explained by the fact that direct stimulation was nearly always accompanied by some indirect, field stimulation of the nerve terminals present inside the musculature of the diaphragm. The response to the extract by the indirectlystimulated phrenic nerve-diaphragm preparation of the rat was progressively reduced, in comparison to control, until unresponsiveness, after exposure of the preparation to calcium-free bath solution plus 1 mM sodium EGTA or to normal bath solution plus 5 mM EGTA, for 30 min. At this point, addition of calcium up to 4 and 8 mM, respectively, promptly and completely restored the stimulant effect of the extract of Ps. coriacea. DISCUSSION
The experiments reported in this paper show that extracts of Ps. coriacea produced a marked potentiation of contractions elicited by indirect and direct electrical stimulation in all preparations of nerveskeletal muscle and skeletal muscle examined. Preceded by increase in the baseline tension, both the amplitude and duration of the twitch were increased several times, with an optimum at lower frequencies of stimulation. The effect was proportional to the dose of extract and there was no sign of tachyphylaxis. Upon washing, the return to basal contraction was immediate for small doses of the frog extract, but took 5-15 min for large doses. In numerous experiments, the threshold concentrations of the extract showed a great constancy, ranging generally from 10 to 20 pg dry skin/ml, i.e. 2-8 ng/ml of active alkaloid. Concentrations of extract 3-4 times the threshold produced a maximum stimulation which lasted, slowly declining in amplitude, for hours. This description is nearly completely superimposable on that of Albuquerque, Wamick, Maleque, Kauffman, Tamburini. Nimit and Daly (1981) concerning the pharmacological effects of pumiliotoxin B on the skeletal muscles of the frog, rat and crayfish. In the case of indirect stimulation (phrenic nervediaphragm preparations, chick nerve-cervical biventer muscle preparation), experiments with physostigmine, tubocurarine and a-bungarotoxin strongly
813
suggest that the extract of P.S. coriacea acts on motoneurones causing a release of acetylcholine from the terminals. In fact, the effect of the extract was potentiated by inhibition of cholinesterase and abolished, reversibly or irreversibly, by blockade of the nicotinic acetylchohne receptors on the end-plate. However, the extract also elicited a similar potentiation on contractions induced by direct muscular stimulation. In this case, the response to the frog extract was not modified either by physostigmine or by tubocurarine and a-bungarotoxin, indicating that the alkaloid acted directly on the muscle fibres. As expected, regardless of the mode of stimulation, tetrodotoxin abolished both basal and the extractpotentiated twitch. The mechanism of action of the extract of Ps. coriacea remains to be elucidated. However, in this regard two important points may be considered as’ firmly established: (1) The extract acts through a stimulus-dependent mechanism. The extract was completely inactive in non-stimulated preparations. (2) Both in its chemical structure and pharmacological effects the active alkaloid appeared to be strictly related to pumiliotoxin B. This has clearly emerged not only from preliminary results of the structural analysis by Daly, but also from pharmacological studies carried out by Albuquerque et al. (1981) the Mensa-Dwumah and Daly (1978) on pumiliotoxin B as well as from the results of a parallel bioassay of pumiliotoxin B and extract carried out in this laboratory. In eight test systems (guinea-pig large intestine and ileum, rat and chick large intestine, guinea-pig and rabbit vas deferens; chick biventer cervicis muscle, rat phrenic nerve-diaphragm) pumihotoxin B and the extract presented the same ratio of potency (1 mg Ps. coriacea dried skin = 7-12 pg pumihotoxin B) and responses were nearly indistinguishable from each other. However, it may be calculated, that the absolute potency of the alkaloid was at least 30 times greater than that of pumiliotoxin B. On the basis of their studies on rat and frog skeletal muscle Albuquerque et al. (1981) suggest that pumiliotoxin B potentiates and prolongs the muscle twitch by (a) facilitating the release of calcium from
Figures 4 and 5 on facing page Fig. 4. Rat phrenic nerve-diaphragm preparation (indirect electrical stimulation at 0.05 Hz). *Washing. The frog extract (PSC, expressed as fig dried skin/ml) produced a prompt, conspicuous increase in twitch amplitude (320%) lasting, with a slow decline, over 2 hr. Fig. 5. Upper tracing: rat phrenic nerve-diaphragm preparation (indirect electrical stimulation, 0.1 Hz). *Washing. Physostigmine (PHYS) produced a remarkable potentiation of the response to the extract (expressed as pg dried skin/ml); tubocurarine (TUB) reduced (first dose) and then abolished (second dose) this response, until standstill. Washing caused a moderate after-potentiation. Lower tracing: hamster diaphragm preparation (direct electrical stimulation, 0.1 Hz). Note the ineffectiveness of both tubocurarine and z-bungarotoxin (BTX) on the twitch stimulated by the extract. Washing promptly restored the basal twitch.
G. FALCONIERI ERSPAMERef al.
814
storage sites within the sarcoplasmatic reticulum, (b) mobilizing calcium from extracellular sites and (c) blocking the reuptake of calcium by calciumdependent adenosine t~phosphata~. Some evidence supports the view that the effect of the extract of Ps. coriaceu on the muscle twitch was also dependent in part on calcium. First, both directly and indirectly stimulated preparations showed in the presence of EGTA, a calcium chelating agent, a progressive depression of potent&ion of the twitch by the extract until ~responsiveness occurred. Addition of calcium at this point promptly restored the response to the extract. Second, nifedipine, a compound considered to block calcium channels, produced a clear-cut reduction of the potentiation of the twitch induced by the extract, at concentrations ranging from 15 to 30 FM. This inhibitor effect coufd always be antagonized by raising the concentration of the frog extract. However, owing to the very large concentrations required, it cannot be excluded that the effect of nifedipine was, at least in part, tmspecific. Finally, Raiteri, Marchi, Maura, Meichio~ and Erspamer (1984) found that the resting release of a variety of transmitters from superfused synaptosome preparations by the extract was strongly reduced in the absence of calcium. Acknowledgements-This
from the Consiglio
work was
Nazionaie
supported by grants
delle Ricerche (Progetto
Chimica Fine e Secondaria), Rome. We are indebted to Miss M. Casali for typing the manuscript. REFERENCES
Albuquerque E. X., Warnick J. E., Maieque M. A.. Kauffman F. C.. Tamburini R.. Nimit Y. and Dalv J. W. (1981) The pharmacology of pumiliotoxin-B. Interaction with calcium sites in the sarcoplasmatic reticulum of skeletal muscle. yolec. Pharmac. 19: 411424. Child K. J. and Zaimis E. (1960) A new biological method for the assay of depolarizing substances using the isolated semispinalis muscfe of the chick. Br. J. Pharmac. 15: 412-416. Daly J. W. (1982) Alkaloids of neotropical poison frogs (Dendrobatidae). Prog. Chem. Organ. Nat. Prod. 41: 205-340.
Erspamer V., Falconieri Erspamer G., Melchiorri P. and Mazzanti G. (1985) A potent factor in extracts of the skin of the Australian frog, P~euduphryne coriaeea. Apparent facilitation of transmitter release in isolated smooth muscle preparations. Neuropharmacology 24: 783-792. Ginsborg B. L. and Warringer J. (1960) The isolated chick biventer cervicis nerve-muscle preparation. Br. J. Phnrmac. 18: 410-411.
Mensah-Dwumah M. and Daly J. W. (1978) Pharmacological activity of alkaloids from poison-dart frogs ~~endrubu~jd~).
Toxicon 16: 189-194.
Raiteri M., Marchi M., Mama G., Melchiorri P. and Erspamer V. (1984) Stimulation of neurotransmitter release from brain synaptosomes by an alkaloid substance extracted from the skin of the amphibian Pseudoohryne coriaeea. In: XXII Congress of the-Italian Pharm&&gy Society, Bologna, October 10-13, 1984, Abstr. p. 377.
Negri, Bologna.
0028-3908/86 S3.00 + 0.00 Pergamon Journals Ltd
A POTENT FACTOR IN EXTRACTS OF THE SKIN OF THE AUSTRALIAN FROG, PSEUDOPHR YNE CORIACEA-III POTENTIATION MAMMALIAN BY DIRECT
OF CONTRACTIONS ELICITED IN AVIAN AND ISOLATED SKELETAL MUSCLE PREPARATIONS AND INDIRECT ELECTRICAL STIMULATION
G. FALCONIERIERSPAMER,V. ER~PAMERand P. MELCHIORRI Institutes of Pharmacology and Pharmacognosy and Medical Pharmacology III, First University of Rome, Citta Universitaria, 00100 Rome, Italy (Accepted 8 November 1985) Summary-Extracts of the skin of the Australian frog Pseudophryne coriucea displayed a striking potentiating effect on contractions evoked in isolated skeletal muscle preparations of mammals (phrenic nerve diaphragm) and birds (chick biventer cervicis and semispinalis muscles) by indirect and direct electrical stimulation. There was both a conspicuous increase in the amplitude of the twitch, up to IO-fold, and a remarkable prolongation of the duration of the twitch. The effect was dose- and frequencydependent. In the presence of the extract, fusion of twitches after tetanic stimulation occurred earlier. No tachyphylaxis upon repeated stimulation by the extract was observed and the response to large doses persisted, declining slowly, for hours. These effects must be ascribed to an alkaloid related in structure to pumiliotoxin B. Response to the extract of Pseudophrynecoriacea by indirectly-stimulated preparations was potentiated by physostigmine and blocked by tubocurarine and a-bungarotoxin, demonstrating that in these preparations the extract acted pre-synaptically to facilitate the release of acetylcholine from motor nerve endings. However, the extract of Pseudophrynecoriaceu displayed equally potent effects in directly stimulated preparations, insensitive to physostigmine and to blockers of nicotinic acetylcholine receptors, indicating a direct action on the skeletal muscle. It is suggested that, like pumiliotoxin B, the Pseudophryne coriaceu alkaloid may interfere in the regulation of calcium channels in both nerve and muscle fibres. Key words: Pseudophryne coriacea skin, pumiliotoxin B-like alkaloid, skeletal muscle preparations, direct stimulation, indirect stimulation, acetylcholine release, calcium channels.
A preceding paper (Erspamer, Falconieri Erspamer, Melchiorri and Mazzanti, 1985) showed that methanol extracts of the skin of some Australian frogs, belonging to the genus Pseudophryne (Myobatrachidae family), displayed potent stimulant effects on isolated preparations of intestine and on electrically-driven vas deferens preparations. The most thoroughly studied Pseudophryne species was
to an alkaloid related in structure to the pumiliotoxins (Daly, 1982), but different from any of the known pumiliotoxin alkaloids (Daly, personal communication). Elucidation of the structure of the Pseudophryne coriacea alkaloid is in progress, the main obstacle to reach the goal being represented by the great difficulty in collecting frog material. This paper describes the actions of a semi-purified Ps. coriacea extract of the skin of Ps. coriacea in several isolated skeletal-muscle and nerve-skeletal muscle preparations of mammals and birds. It will be seen that the extract potently reinforced and conspicuously prolonged the muscle twitch elicited both by direct and indirect electrical stimulation.
Pseudophryne coriacea.
On the basis of results obtained with antagonists and blocking agents it was suggested that Pseudophryne coriacea had a pre-synaptic, neurogenic point of attack and that it acted to facilitate release of transmitters from the nerve endings. Acetylcholine was considered the most important agent involved in the response to Pseudophryne coriacea by the intestinal muscle and noradrenaline in the response by vas deferens preparations. It seems highly probable that the striking activity of extracts of Pseudophryne coriucea must be ascribed
Dedicated to Egidio Meneghetti, Professor of Pharmacology at the University of Padua, Italy, on the 25th anniversary of his death.
METHODS Amphibian material
The amphibian material used in this study was the semi-purified extract of the skin of Ps. coriucea, described in the preceding paper. This semi-purified material was free of biologically active contaminants, as demonstrated by HPLC experiments showing a single peak of activity. Concentrations of the extract, expressed in terms of pg/ml,
807
808
G. FALCONIERIERSPAMERer al.
always refer to the amount of dried skin from which the semi-purified extract was obtained. On the basis of the Dragendorff and Reinecke salt reactions, it has been calculated that the biologically active pumiliotoxin-like alkaloid had a tissue concentration not exceeding 200-400 pg/g dry skin. Bioassay
The extract of Ps. coriacea was assayed on the following isolated nerve-skeletal muscle and skeletal muscle preparations: (1) Phrenic nerve-diaphragm from mice, rats, hamsters and guinea-pigs, suspended in a 30-50ml bath of Krebs solution, at 38°C. (2) Chick biventer cervicis muscle isolated and prepared as described by Ginsborg and Warriner (1960), and suspended in a 20-30 ml bath of Krebs-Henseleit solution at 39°C. (3) Chick semispinalis muscle, isolated and prepared as described by Child and Zaimis (1960), same solution and temperature. All preparations were aerated with 95% 0, + 5% COz. The phrenic nerve-diaphragm and the chick biventer cervicis muscle were stimulated both indirectly and directly; the chick semispinalis muscle only directly. Single stimulations (Electric Stimulator Digit 3T, Lace Elettronica, Pisa) were carried out with rectilinear pulses of 0.5 msec duration, at 0.05-0.2 Hz, using supramaximal voltage. Tetanic stimulation was performed, at 1 min intervals and for 1 set, at frequencies of 10, 20, 30, 40, 50 and 70 Hz. The mechanical activity of all muscle preparations was recorded isotonically (7006 isotonic transducer) or isometrically (DY2) by a strain-gauge transducer and displayed on a recording microdynamometer (Unirecord 7050, Basile, Milan). Two simple electrode assemblies were used in this study, one for direct, and the other for indirect stimulation. The assembly for direct stimulation of the muscle consisted of a 5 mm diameter plastic tube, 200 mm long, bent at one end. The electrode of stainless steel emerged from the bent end and terminated in a hook designed to hold the preparation. The counter electrode was a stainless-steel tube collar, 30mm long, round the plastic tube. The hook was embedded as far as possible in one extremity of the muscle, with little or no wire left exposed. The assembly for nerve stimulation consisted of two platinum leads, embedded in a vertical plastic bar, connected to two perforated platinum discs, similarly embedded in a plastic protrusion at the lower end of the bar. The phrenic nerve or the upper tendon of the chick biventer cervicis muscle (encapsulating nerve) was gently passed through the hole. This was narrow for the phrenic nerve, somewhat larger for the tendon (Ginsborg and Warriner, 1960). A similar, but more simple version of the above assembly consisted of two small perforated platinum discs embedded in a pip of light-dentistry resin
(OrthocrylX) connectd to the stimulator by very thin wires. The system floated freely in the nutrient bath and any stretching of the nerve was avoided. This assembly was elaborated by Mr Fortuna. Istituto Superiore di Sanitl, Rome, and always gave excellent results. In the present experiments the duration of twitch was the time elapsing between the start of contraction and return to the base-line; contraction time, the time required for the tension to rise from the base-line to peak; 90% relaxation time, the time required for the tension to fall from the peak to a point of the falling phase equal to 10% of the twitch amplitude. Drugs
Drugs used were as follows: nifedipine (mol. wt 346; Bayer, Leverkusen), acetylcholine bromide (mol. wt 226), tubocurarine chloride.5H,O (mol. wt 785), tetrodotoxin (mol. wt 3 19), suxamethonium chloride. 2H,O (mol. wt 397) physostigmine salicylate (mol. wt 413), EGTA [ethylene glycol bis(aminoethylether) N,N,N’,N’-tetraacetic acid, mol. wt 3801, a-bungarotoxin (Sigma, St Louis, Missouri).
RESULTS
Chick biventer cervicis muscle
The responses of this preparation were almost identical, both upon indirect and direct stimulation, and will be described together. Threshold concentrations of the extract of Ps. coriacea varied, in 35 experiments, between 8 and 25 pg dried skin/ml bathing solution. Response to the extract consisted in an increase in base-line tension with simultaneous prolongation of the duration of twitch, followed by potentiation of the amplitude of twitch. Increase in base-line tension, had its earliest manifestation in the disappearance of the fly-back below the base-line, caused by the return spring of the writing pen. In turn, prolongation of the duration of the twitch was mainly due to lengthening of relaxation time (Fig. 1). Prolongation of the duration of twitch and 90% relaxation time were approximately the same at any concentration of extract above 2-fold the threshold, and were clearly correlated with the frequency of stimulation, the greatest prlongation occurring at the lowest rate of stimulation. As measured in 5 experiments at 0.1 Hz stimulation, the duration of the twitch increased from 140-170 to 1200-1800msec, and the 90% relaxation time from 40-80 to 1000-1300 msec. Potentiation of the amplitude of the twitch was clearly dose-dependent: preceded by a latency of 3-8 min for threshold concentrations of the extract, it was prompt for larger concentrations. Generally, doses 3-5-fold the threshold produced a maximum twitch increase, up to 600-1000% of control. Washing caused rapid return to basal conditions at small concentrations of extract; large concentrations required repeated or
Pse~dop~ry~ corticeu extract on skeletal muscle
809
continuous washing for 5-20 min. ~~eversibi~ity of Tubocurarine, in concentrations of more than the effect was never seen, 2-3 PM, produced a more or less rapid but complete Moderate or large concentrations of the extract, inhibition of basal twitch and a total or partial left in the organ bath without washing, produced a inhibition of extract-potentiated twitch. There was a clear-cut reciprocal antagonism between tubopotent increase in the amplitude of the twitch lasting curarine and extract. Thus, neuromuscular biock for hours, with slow decline. In one experiment produced by 1.5 and 3 PM tubocurarine could be response to 50 pg dried skin/ml (5-fold the threshold) partially reversed by I5 and 30 pg/ml of extract, was recorded for 11 hr. The muscle was stimulated at respectively. After 20 FM tubocurarine, blockade of 0.05 Hz. Maximum poientiation reached 800% of control and at the end of the experiment it was still response induced by the extract was complete up to con~ntrations of 150 pg/ml of extract (IO-fold the 160%. In a further series of experiments, the muscle was threshold). At this point, washing caused, after a successively stimulated at I-min intervals and for latency of a few minutes, a formidable, gradual 1 set, at 10, 20, 30, 40, 50 and 70 Hz. Under control after-potentiation of the twitch (500X), which then conditions, the amplitude of the first twitch (or of declined very slowly, upon repeated washing. This tetanic contraction) increased progressively, from 10 indicates that binding of the extract to its receptors to 70 Hz and fusion of twitches occurred only at was more tenacious than that of tubocurarine. 30 Hz. After exposition of the muscle for 5min to a-Bungarotoxin, in concentrations of 2-5 gg/ml, 25 ag/ml of extract the response was conspicuously completely abolished the basal twitch and the repotentiated, with a maximum nearly identical at sponse elicited by any concentration of extract (up to 20-fold the threshold] or suxamethonium. Blockade any frequency of stimulation, and fusion of the single was definitive and could not be twitches was anti~pated, being complete at 20 Hz by a-bungarotoxin removed either by continuous washing or repeated (Fig. 2). a~i~stration of the extract. ft is well known that the chick biventer cervicis muscle, owing to its content of both slow and fast Nif~pine, a wmpound believed to bfock calcium fibres, responds to depolarizing agents acting on channels, produced a clear-cut reduction of the nicotinic receptors of the end-plate with a contracture potentiation of the twitch induced by the extract, at and shift of the base-line, masking the effect on concentrations ranging from 15 to 20 PM. This effect twitch. Given during the spasm provoked by could always be removed by additional amounts of suxamethonium (0.1-0.2 p M), the extract reduced extract. However, it should be noted that the active the contracture and simultaneously caused the re- concentration of nifedipine here was 100 times larger appearance of vigorous twitches of increasing amplithan that required to inhibit both spontaneous and motility of the isolated intestinal tude. To obtain this effect 20, 40 and lOOpg/ml of extract-induced extract were required, against suxamethonium con- smooth muscle (Erspamer ef al., 1985). centrations of 0.25, 0.40 and 1.2 FM, respectively Chick sern~~p~a~i~ m~cle (Fig. 1). The extract was completely inactive in Response to the extract of Ps. coriacea by directlynon-stimulated preparations. However, in concentrations of 20-40 pg/ml it caused a small, but con- stimulated preparations (6 experiments) was similar to that observed in the biventer cervicis muscle. stant increase (IO-30%) in the response to both of the frog extract were acetylcholine and suxamethonium, given 5 min Threshold concentrations later. Tetrodotoxin (0.02-0.2 p M) regularly caused a of the same order and there was always a good conspicuous or total blockade of both basal and dose-response relationship, with no tachyphylaxis. extract-potentiated twitch, regardless of the mode Rat, mouse, guinea-pig and hamster diaphragm and of stimulation. Total blockade of the twitch by phrenic-nerve diaphragm preparations 0.15-0.2 @M tetrodotoxin was unsurmountable by Direct stimulation was carried out on diaphragm the extract. However, after large concentrations of frog extract, a clear-cut after-potentiation of the preparations from 10 rats, 3 mice, 5 guinea-pigs and 7 hamsters and indirect stimulation on 7 rat, 4 amplitude of the twitch was sometimes seen upon guinea-pig and 5 hamster phrenic ne~e-diaphm~ washing. Owing to their mechanism of action, neither of the extract tu~curarine and a-bugarotoxin (reversible or ir- preparations. Threshold con~ntrations for all preparations, regardless of their mode of reversible blockade of the nicotinic acetylchoIine stimulation, ranged between 8 and 2Opg dried receptors on the end-plate) nor physostigmine (inhibition of cholinesterase) affected, the response to skin/ml. However the supramaximal voltage required was less in the case of indirect stimulation. As in the the extract by the directly stimulated chick biventer preparations. Conversely, the above drugs were chick biventer cervicis muscle preparations, the response consisted of an increase in the base-line potently active on the indirectly-stimulated preparatension, increase in the amplitude of twitch and tions (Fig. 3). Physostigmine (0.1-0.15 gM) halved prolongation of the duration of twitch. The extract the threshold concentration of extract and conspicuwas completely inactive, at any concentration level, ously potentiated response to larger concentrations.
810
G. FALCONIERIERSPAMERet al.
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Fig. 2. Chick biventer cervicis muscle preparation. Effect of the extract of the Ps. coriacea on the tetanus elicited by I-see stimulations, at different Hz, given at I-min intervals. Under control conditions, the amplitude of the tetanic response increased with the frequency and fusion of the twitches which occurred at 30 Hz. After exposure to the extract (expressed as pg dried skin/ml) for 10 min there was a conspicuous increase in the amplitude of the tetanic response, which was the same at 10, 20 and 30 Hz, and an anticipation of complete fusion of the twitches (20 Hz). Relaxation after the extract was slowed down,
and fly-back below the base-line was lacking.
in non-stimulated preparations. Prolongation of the duration of the twitch for the diaphragm, was mainly due to the prolongation of the relaxation time. In experiments carried out with 2-3-fold the threshold concentration of extract, the total duration of the twitch rose from 80-140 to 1X@-25OOmsec, the contraction time increased from 30-40 to lOO-2OOmsec and the 90% relaxation time from SO-60 to 1000-l 500 msec. Values are approximate, as calculated from polygraphic records. Potentiation of the twitch produced by small concentrations of the extract was rapidly removed by washing; that elicited by large concentrations required repeated or continuous washing for S-20 min. An irreversibility of the effect was never seen, even at concentrations of the extract up to 20-fold the threshold. Moderate or large concentrations of the
frog extract left in the organ bath without washing produced a potentiation of the twitch lasting for hours, with a very slow decline (Fig. 4). Tetrodotoxin (0.1-0.2 PM) caused an inhibition of both basal and extract-stimulated twitch in all preparations. One to three micromoles of tubocurarine, 2WO FM suxamethonium and 24 pg/ml a -bungarotoxin caused a complete, unsurmountable blockade of both basal and potentiated twitch in indirectly-stimulated preparations, and 0.05-0.1 PM physostigmine, in turn, produced a conspicuous potentiation of the response to the extract. The effect of the drugs described above, acting either on nicotinic acetylcholine receptors in the end-plate or on acetylcholinesterase was either lacking or, more frequently, partial, never exceeding a maximum of IO-30% blockade, in the directly-
Figures I and 3 on facing page Fig. 1. Chick biventer cervicis muscle, two preparations (indirect and direct electrical stimulation at 0.1 Hz). *Washing. The effect of graded concentrations of extract of Ps. coriaceu (PSC; expressed as fig dried skin/ml). Note the good dose-response relationship and prompt return to basal conditions after washing. The first response to threshold doses of extract (7-10 pg/ml) was an increase in basal tension, simultaneous with prolongation of the duration of the twitch. Increase in the amplitude of the twitch appeared somewhat later (see lower tracing). Spasm elicited by suxamethonium (sux) was overcome by the extract with re-appearance of twitch contractions of increasing amplitude. Fig. 3. Chick biventer cervicis muscle (two preparations). *Washing. Upper tracing: indirect electrical stimulation (0.1 Hz). Large asterisk, adjustment of the base-line. Physostigmine (PHYS) produced a conspicuous potentiation of the response to the extract @g dried skin/ml); tubocurarine (TUB) and a-bungarotoxin (BTX) abolished both the basal twitch and the response to the extract. Washing caused the appearance of a powerful rebound potentiation of twitch after tubocurarine, whereas standstill caused by a-bungarotoxin was unsurmountable. Lower tracing: predominant direct electrical stimulation. Both tubocurarine and a-bungarotoxin only slightly affected the basal twitch and response. to the extract. Tetrodotoxin (TTX), at increasing concentrations, progressively reduced the twitch potentiated by the extract until abolition.
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812
Pseudophryne coriaceu extract on skeletal muscle
stimulated diaphragm preparations (Fig. 5). This may be explained by the fact that direct stimulation was nearly always accompanied by some indirect, field stimulation of the nerve terminals present inside the musculature of the diaphragm. The response to the extract by the indirectlystimulated phrenic nerve-diaphragm preparation of the rat was progressively reduced, in comparison to control, until unresponsiveness, after exposure of the preparation to calcium-free bath solution plus 1 mM sodium EGTA or to normal bath solution plus 5 mM EGTA, for 30 min. At this point, addition of calcium up to 4 and 8 mM, respectively, promptly and completely restored the stimulant effect of the extract of Ps. coriacea. DISCUSSION
The experiments reported in this paper show that extracts of Ps. coriacea produced a marked potentiation of contractions elicited by indirect and direct electrical stimulation in all preparations of nerveskeletal muscle and skeletal muscle examined. Preceded by increase in the baseline tension, both the amplitude and duration of the twitch were increased several times, with an optimum at lower frequencies of stimulation. The effect was proportional to the dose of extract and there was no sign of tachyphylaxis. Upon washing, the return to basal contraction was immediate for small doses of the frog extract, but took 5-15 min for large doses. In numerous experiments, the threshold concentrations of the extract showed a great constancy, ranging generally from 10 to 20 pg dry skin/ml, i.e. 2-8 ng/ml of active alkaloid. Concentrations of extract 3-4 times the threshold produced a maximum stimulation which lasted, slowly declining in amplitude, for hours. This description is nearly completely superimposable on that of Albuquerque, Wamick, Maleque, Kauffman, Tamburini. Nimit and Daly (1981) concerning the pharmacological effects of pumiliotoxin B on the skeletal muscles of the frog, rat and crayfish. In the case of indirect stimulation (phrenic nervediaphragm preparations, chick nerve-cervical biventer muscle preparation), experiments with physostigmine, tubocurarine and a-bungarotoxin strongly
813
suggest that the extract of P.S. coriacea acts on motoneurones causing a release of acetylcholine from the terminals. In fact, the effect of the extract was potentiated by inhibition of cholinesterase and abolished, reversibly or irreversibly, by blockade of the nicotinic acetylchohne receptors on the end-plate. However, the extract also elicited a similar potentiation on contractions induced by direct muscular stimulation. In this case, the response to the frog extract was not modified either by physostigmine or by tubocurarine and a-bungarotoxin, indicating that the alkaloid acted directly on the muscle fibres. As expected, regardless of the mode of stimulation, tetrodotoxin abolished both basal and the extractpotentiated twitch. The mechanism of action of the extract of Ps. coriacea remains to be elucidated. However, in this regard two important points may be considered as’ firmly established: (1) The extract acts through a stimulus-dependent mechanism. The extract was completely inactive in non-stimulated preparations. (2) Both in its chemical structure and pharmacological effects the active alkaloid appeared to be strictly related to pumiliotoxin B. This has clearly emerged not only from preliminary results of the structural analysis by Daly, but also from pharmacological studies carried out by Albuquerque et al. (1981) the Mensa-Dwumah and Daly (1978) on pumiliotoxin B as well as from the results of a parallel bioassay of pumiliotoxin B and extract carried out in this laboratory. In eight test systems (guinea-pig large intestine and ileum, rat and chick large intestine, guinea-pig and rabbit vas deferens; chick biventer cervicis muscle, rat phrenic nerve-diaphragm) pumihotoxin B and the extract presented the same ratio of potency (1 mg Ps. coriacea dried skin = 7-12 pg pumihotoxin B) and responses were nearly indistinguishable from each other. However, it may be calculated, that the absolute potency of the alkaloid was at least 30 times greater than that of pumiliotoxin B. On the basis of their studies on rat and frog skeletal muscle Albuquerque et al. (1981) suggest that pumiliotoxin B potentiates and prolongs the muscle twitch by (a) facilitating the release of calcium from
Figures 4 and 5 on facing page Fig. 4. Rat phrenic nerve-diaphragm preparation (indirect electrical stimulation at 0.05 Hz). *Washing. The frog extract (PSC, expressed as fig dried skin/ml) produced a prompt, conspicuous increase in twitch amplitude (320%) lasting, with a slow decline, over 2 hr. Fig. 5. Upper tracing: rat phrenic nerve-diaphragm preparation (indirect electrical stimulation, 0.1 Hz). *Washing. Physostigmine (PHYS) produced a remarkable potentiation of the response to the extract (expressed as pg dried skin/ml); tubocurarine (TUB) reduced (first dose) and then abolished (second dose) this response, until standstill. Washing caused a moderate after-potentiation. Lower tracing: hamster diaphragm preparation (direct electrical stimulation, 0.1 Hz). Note the ineffectiveness of both tubocurarine and z-bungarotoxin (BTX) on the twitch stimulated by the extract. Washing promptly restored the basal twitch.
G. FALCONIERI ERSPAMERef al.
814
storage sites within the sarcoplasmatic reticulum, (b) mobilizing calcium from extracellular sites and (c) blocking the reuptake of calcium by calciumdependent adenosine t~phosphata~. Some evidence supports the view that the effect of the extract of Ps. coriaceu on the muscle twitch was also dependent in part on calcium. First, both directly and indirectly stimulated preparations showed in the presence of EGTA, a calcium chelating agent, a progressive depression of potent&ion of the twitch by the extract until ~responsiveness occurred. Addition of calcium at this point promptly restored the response to the extract. Second, nifedipine, a compound considered to block calcium channels, produced a clear-cut reduction of the potentiation of the twitch induced by the extract, at concentrations ranging from 15 to 30 FM. This inhibitor effect coufd always be antagonized by raising the concentration of the frog extract. However, owing to the very large concentrations required, it cannot be excluded that the effect of nifedipine was, at least in part, tmspecific. Finally, Raiteri, Marchi, Maura, Meichio~ and Erspamer (1984) found that the resting release of a variety of transmitters from superfused synaptosome preparations by the extract was strongly reduced in the absence of calcium. Acknowledgements-This
from the Consiglio
work was
Nazionaie
supported by grants
delle Ricerche (Progetto
Chimica Fine e Secondaria), Rome. We are indebted to Miss M. Casali for typing the manuscript. REFERENCES
Albuquerque E. X., Warnick J. E., Maieque M. A.. Kauffman F. C.. Tamburini R.. Nimit Y. and Dalv J. W. (1981) The pharmacology of pumiliotoxin-B. Interaction with calcium sites in the sarcoplasmatic reticulum of skeletal muscle. yolec. Pharmac. 19: 411424. Child K. J. and Zaimis E. (1960) A new biological method for the assay of depolarizing substances using the isolated semispinalis muscfe of the chick. Br. J. Pharmac. 15: 412-416. Daly J. W. (1982) Alkaloids of neotropical poison frogs (Dendrobatidae). Prog. Chem. Organ. Nat. Prod. 41: 205-340.
Erspamer V., Falconieri Erspamer G., Melchiorri P. and Mazzanti G. (1985) A potent factor in extracts of the skin of the Australian frog, P~euduphryne coriaeea. Apparent facilitation of transmitter release in isolated smooth muscle preparations. Neuropharmacology 24: 783-792. Ginsborg B. L. and Warringer J. (1960) The isolated chick biventer cervicis nerve-muscle preparation. Br. J. Phnrmac. 18: 410-411.
Mensah-Dwumah M. and Daly J. W. (1978) Pharmacological activity of alkaloids from poison-dart frogs ~~endrubu~jd~).
Toxicon 16: 189-194.
Raiteri M., Marchi M., Mama G., Melchiorri P. and Erspamer V. (1984) Stimulation of neurotransmitter release from brain synaptosomes by an alkaloid substance extracted from the skin of the amphibian Pseudoohryne coriaeea. In: XXII Congress of the-Italian Pharm&&gy Society, Bologna, October 10-13, 1984, Abstr. p. 377.
Negri, Bologna.