In lower doses, the catecholamines promoted a melano- some dispersion, blocked by propranolol. However, isoproterenol was unable to induce pigment disper-.
Comp. Biochem. Physiol. Vol. 70C, pp. 293 to 296, 1981
0306-4492/81/060293-04102.00/0 Pergamon Press Ltd
Printed in Great Britain
EVIDENCE OF ADRENERGIC AND CHOLINERGIC RECEPTORS IN TELEOST MELANOPHORES MARIA A. VISCONTI and ANA M. DE L. CASTRUCCI Departamento de Fisiologia Geral, Instituto de Bioci~ncias, Universidade de S~o Paulo, C.P. 11.176, S~o Paulo, Brazil
(Received 2 April 1981) Abstract--1. Melanophores of Bathygobius soporator aggregated in the presence of high concentrated catecholamines, antagonized by phentolamine. In lower doses, the catecholamines promoted a melanosome dispersion, blocked by propranolol. However, isoproterenol was unable to induce pigment dispersion. 2. Melanosome dispersion was obtained with cholinester and alkaloid treatments, inhibited by atropine and d-tubocurarine. 3. It is assumed the presence of two different types of receptors: (a) adrenoceptors, uncharacterized in respect to alpha or beta nature; (b) cholinoceptors, undifferentiated in relation to muscarinic or nicotinic nature.
INTRODUCTION
It is well established that dermal melanophores of teleostean fishes are mainly under the control of the autonomic nervous system and that the sympathetic post-ganglionic fibers promote the centripetal movement of pigment granules (Bagnara & Hadley, 1973). A number of studies denotes the adrenergic nature of these fibers, which probably release norepinephrine at the nerve endings. This neurotransmitter binds to a post-synaptic ~ adrenoceptor, inducing granule aggregation (Fujii & Novales, 1969a; Fernando & Grove, 1974; Fujii & Miyashita, 1975; Iga, 1979). Epinephrine, active on both ~ and fl adrenoceptors, and isoproterenol, remarkably effective on fl one, have also been reported to aggregate melanosomes (Fernando & Grove, 1974; Fujii & Miyashita, 1975). Despite the fact that some authors have questioned that melanosome dispersion could also be promoted by nerve fibers (Iwata & Fukuda, 1973), this process both in vivo and in vitro has been reported to be so rapid in some species that this hypothesis cannot be discarded. It has been suggested that dispersing postganglionic fibers are parasympathetic and the neurotransmitter is acetylcholine (Parker, 1948). More recently the sympathetic nature of dispersing fibers was shown through catecholamine action on beta adrenoceptors (Miyashita & Fujii, 1975; Fujii & Miyashita, 1976; Iga, 1980). The present work is an attempt to verify the occurrence and to characterize the receptors in Bathygobius melanophores.
tubocurarine chloride (all from Sigma), phentolamine methanosulphate (Ciba-Geigy); isoproterenol sulphate (Boehringer); propranolol chloridrate (Ayerst). All drugs were diluted in saline (prior to use) and the experiments were performed at room temperature (1828°C). Dermal melanophore responses of Bathygobius were recorded using the time to reach full aggregation or dispersion as the measurable parameter. The results (10 experiments for each drug) were compared statistically employing Student's t-test (for P < 0.05). RESULTS
Effects of catecholamines and specific antagonists As expected, both 10-5 M epinephrine and 10-5 M norepinephrine promoted a complete melanosome aggregation, the latter more rapidly than the former (46 +_6.14 and 89 +_ 15.95see, respectively). Both were antagonized by phentolamine, a competitive et-blocking agent (Fig. 1). The calculated pAs0 values
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MATERIALS AND METHODS
Scales of the marine teleost Bathygobius soporator (Gobiidae) were excised from the middorsal region of live Bathygobius and immediately immersed in physiological solution (Fujii & Novales, 1969b). The following drugs were used: norepinephrine and epinephrine hydrochloride; acetyl-, propionyl-, carbamyl, butyryl-, benzoyl- and acetyl-fl-methylcholine chloride; pilocarpine; arecoline; nicotine; atropine sulphate; d293
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Fig. 1. Effects of phentolamine on the melanosome aggregation response to 10-SM epinephrine (O--O; pAso = 10 -715) and 10-5M norepinephrine (A--A; paso = 10- 7.o).
MARIAA. VISCONTIand ANA M. D~ L. CASrRUCC~
294
slightly differed: 10 -7.0 for norepinephrine and 10 -715 for epinephrine. Isoproterenol at 10 -4 and 10 -6 M) did not bring about aggregation of pigment granules. Epinephrine and norepinephrine applied in lower doses (at 10 -9 M) after epinephrine aggregation had a dispersing effect (Table 1), which was significantly delayed by 10-SM propranolol. If the aggregation was brought about by norepinephrine, both catecholamines were uneffective. Isoproterenol, a beta sympathomimetic drug, did not exhibit a dispersing effect on melanosomes, at any of the dilutions employed (10 -3, 10 -5, 1 0 - 7 and 1 0 - 9 M).
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pilocarpine. As the recorded orders of potency of cholinesters and cholinomimetic alkaloids did not help to learn on the nature of the cholinoceptor, 10-5 M atropine, an antimuscarinic agent, and 10-SM d-tubocurarine, an antinicotinic one, were assayed. The inhibiting effect of these drugs was checked only against three of the most potent drugs which dispersed the melanosomes after the use of aggregating medium, namely: pilocarpine, acetylcholine, nicotine (epinephrine) and nicotine, acetyl-/~-methylcholine and arecoline (norepinephrine), all of them at 10 -5 M. In both cases, inhibition by atropine and d-tubocurarine showed to be competitive, since the time required to reach full dispersion with these drugs was significantly higher.
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