Indian J Physiol Phannacoll994; 38(3): 185-188

CONTROL OF MELANOSOME MOVEMENTS IN ISOLATED SKINMELANOPHORES OF A CATFISH CLARIAS BATRACHUS (LINN.)

MOHAMMAD OVAlS

Department of Biosciences,Barkatullah University,Bhopal- 462026

( Received on February 4, 1992 )

Abstract: Adrenergic and cholinergic receptors have been studied in isolated skinmelanophores of a catfish Clarias batrachus. Catecholamines induced a strongaggregatory effect on the melanophores. Mclanosome aggregation induced byadrenaline and noradrenaline was partially blocked by alpha adrenergic receptorblockers and a beta receptor blocker. Cholinomimetic drugs .arouscd a significantdispersion of melanophroes. Atropine effectively blocked the dispersal, responsesof melanophores to acetylcholine and carbachol, while, hexamethonium blockedthe nocotinc induced dispersal responses of the melanophores.

Key words: fish melanophores adrenergic receptors cholinergic receptors

INTRODUCTION

Melanophores of some teleostean fishes have beenreported to show centripetal movement of melanosomesin response to catecholamine applications (1-4). Thiscentripetal movement of melanosomes has beenattributed mainly to the alpha nature of adrenergicreceptors in melanosomes (3-5). While in few fishspecies aggregation of melanosomes has been reportedto be due to activation of beta adrenergic receptors(2, 6, 7). The effects of cholinomimetic drugs on fishmelanophores are varied and only a few species respondwith a clear dispersion of melanosomes (1,4, 8).

The control of melanosome movements in catfishesstudied by Fujii and his group (9-11) reveals that inthese fish species the mechanism of dispersion andaggregation of melanosomes is variedly controlled. Insilurid catfishes, the melanosome aggregation has beenfound under the cholinergic control, while in othercatfishes it may be under adrenergic sympatheticpostganglionic control (11).

The present work is an attempt to elucidate thenature and control of melanosome movements in anIndian fresh water catfish Clarias batrachus.

METHODS

Pieces of skin (3-4 x 1O-l2 mm) were excised

from the lateral sides of a fresh water catfish Clariasbatrachus (8-18 cm in length) after decapitation. Theskin was immediately immersed in 0.7% NaCI solutionand smaller pieces of 2-3 mm? were cut. The pieces ofskin were equiliberated in saline solution for 10-15minutes. After equiliberation, the pieces of skin wereincubated (3-5 minutes) in various drugs freshlydissolved in saline medium. In experiments, whereantagonists were employed alongwith agonists, the piecesof skin were first incubated in the antagonist for 5minutes; then agonist was added and further incubationwas carried out for 3-5 minutes. After incubation pieceof skin was placed on a microscope slide covered by acover glass with incubation medium. The melanophoresin the skin were measured with the help of ocularmicrometer (Erma, Japan) and the mean melanophoresize index (MMSI) of the control as well as drug treatedskins was calculated, according to the method ofBhattacharyya et al (12). The experiments wereconducted on both the sexes during the months of April-June at the room temperature (25-30°C).

The following drugs were used : L-adrenalinebitartrate (C.H. Boehringer Sohn, Germany). L-noradrenaline bitartrate (Fluka, A.G.), Phenylephrinehydrochloride (C.H. Boehringer Sohn, Germany),Isoprenaline hydrochloride (Fluka, A.G.),Phenoxybenzamine (S.K. &. F., Philadelphia),

186 Ovais

Propranolol hydrochloride (Cipla, India), Phentolamine(CIBA-Geigy, India), Dihydroergotamine (Sandoz,India), Acetylcholine chloride (Merck, Germany),Carbachol (B. D.H., Bombay), Pilocarpine nitrate,(Sigma, U.S.A), Atropine sulphate (C.H. Boehringer,Sohn, Germany), Physostigmine sulphate (Mac FarlanSmith Ltd., Edinburgh), Nicotine (B.D.H., England),Hexamethonium bromide (Fluka, A.G.). Statisticalanalysis was carried out using students "t" test.

RESULTS

Adrenaline, noradrenaline, phenylephrine andisoprenaline all caused a marked aggregatory effect onthe dermal melanophores of Clarias batrachus(Table I). The threshold dose of these amines to elicita discernible response ranged between 1 x 10-7 - 1 X

10-6 g/ml, The higher dose of these drugs was selectedto get consistant results. The aggregation ofmelanophores caused by these amines was statistically

Indian J Physiol Phannacol 1994; 38(3)

significant in comparison to control values ofmelanophores in saline. In order to determine the siteof action of adrenaline and nor-adrenaline, specificalpha and beta adrenergic receptor antagonists wereemployed. Phenoxybenzamine partially blocked theaggregatory effects of adrenaline and noradrenaline.However, the blocking effect of phenoxybenzamine onthe responses to adrenaline was more pronounced thannoradrenaline. Phentolamine and dihydroergotaminealso blocked the aggregation of the melanophores inresponse to adrenaline treatment. The blocking effectof both the alpha adrenergic receptor antagonists toadrenaline Induced responses was significant.Phentolamine also partially blocked the responses ofmelanophores to noradrenaline. However, this was lessin comparison to adrenaline. Propranolol an unspecificbeta blocker also exerted a blocking effect on themelanophore responses to adrenaline and noradrenaline(Table I).

TABLE I : Effect of adrenergic drugs on the dermal melanophores of C. batrachus (Linn.).

DrugSNo. Experimental drug concentration MMSI±SE

in gtml

I. Control (0.7% saline) 1.074 ± 0.153 (9)

2. Adrenaline I x 10-5 0.466 ± 0.049 .•.•• (9)

3. Noradrenaline I x 10-5 0.400 ± 0.065 .••.• (1)

4. Phenylephrine I x 10-5 0.388 ± 0.034 .•.•.• (6)

5. Isoprenaline I x 10-5 0.320 ± 0.073 .•.•• (5)

6. Control (0.7% saline) 1.726 ± 0.259 (5)

7. Adrenaline I x 10-5 0.439 ± 0.056 .•.• (5)

8. Noradrenaline I x 10-5 0.384 ± 0.072'" (5)

9. Phenoxybenzamine + 5 x 10-5 0.880±0.153" (5)Adrenaline I x 10-5

10. Phenoxybenzamine + 5 x 10-5 0.464 ± 0.032* (5)Noradrenaline I x 10-5

II. Phentolamine + 5 x 10-5 1.032 ± 0.041" (5)Adrenaline 1 x 10-5

12. Phentolamine + 5 x 10-5 0.743 ± 0.037 .•.• (5)Noradrenaline 1 x 10-5

13. Dihydroergotamine + 5 x 10-5 1.379 ± 0.147* (6)Adrenaline 1 x 10-5

14. Propranolol + 2 x 10-5 0.805 ± 0.113* (5)Adrenaline 1 x 10-5

15. Propranolol + 2 x 10-5 0.885 ± 0.122" (5)Noradrenaline I x 10-5

.p < 0.05; .•.•p < 0.01; .•.•• p < 0.001

NS = Not significant

Numbers in parentheses indicate the number of experiments from different fishes.

187Autonomic Receptors in Fish MelanophoresIndian J Physiol Pharmacol 1994; 38(3)

TABLE II: Effect of cholinergic drugs on the dermal Melanophores of C. batrachus (Linn.).

DrugSNo. Experimental drug concentration J,;;.1S1± SE

in glml

I. Control (0.7% saline) 1.795 ± 0.086 (6)

2. Acetylcholine 5 x 10-5 2.3S4 ± 0.231* (6)

3. Carbachol 2.S x 10-5 2.602 ± 0.240" (6)

4. Pilocarpine 2.S x 10-5 2.504 ± 0.230* (6)

5. Control (0.7% saline) I.S82 ± 0.114 (5)

6. Atropine S x 10-5 0.806 ± 0.111" (S)

7. Atropine + S x 10-5 1.462 ± 0.294 (S) NSAcetylcholine 5 x 10-5

8. Atropine + 5 x 10-5 1.063 ± 0.040·* (S)Carbachol 2.S x 10-5

9. Physostigmine 2.S x 10-5 2.278 ± 0.IS6* (S)

10. Physostigmine + 2.5 x 10-5 3.2S7 ± 0.289*" (S)Acetylcholine S x 10-5

II. Control (0.7% saline) I.S34 ± 0.133 (S)

12. Nicotine I x 10-5 2.0S0 ± 0.081* (S)

13. Hexamethonium I x ID-" 0.S26 ± 0.07S*" (S»

14. Hexamethonium + Ix ID-" 1.706 ± 0.122 (S) NSNicotine I x 10-5

15. Hexamethonium + I x 10-4 0.932 ± 0.086** (S)Acetylcholine 5 x 10-5

*p < O.OS; "P < 0.01; "*p < 0.001

NS = Not significant

Numbers in parentheses indicate the number of experiments from different fishes.

responses of melanosomes to acetylcholine. However,hexamethonium per se caused highly significantaggregatory effect on the melanophores.

DISCUSSION

Acetylcholine, carbachol and pilocarpine allaroused a well marked dispersion of pigment in theC. batrachus melanophores (Table II). The melanophoreswere highly sensitive to carbachol. The order of potencywas Carbachol> pilocarpine> Acetylcholine (TableII). The latent period to elicit a discernible response ofthese drugs ranged between 10-30 seconds. Atropineper se elicited an aggregatory response in themelanophores (Table II). However, atropine almostcompletely blocked the dispersal effects of acetylcholineand carbachol on the melanophores. Physostigmine,the anticholinesterase agent per se elicited a significantdispersal effect on the C. batrachus melanophores.Pretreatment of the skin with physostigmine resultedin potentiation of dispersal response of melanophoresto acetylcholine. It is clear from the data presented inTable II that nicotine caused a significant dispersionof melanophores and this effect was completely blockedby hexamethonium. The pretreatment of pieces of skinin hexamethonium completely abolished the dispersal

Catecholamines induced a rapid and completeaggregation in melansomes of Clarias batrachus, whichwas partially blocked by alpha adrenergic receptorblocking agents (phenoxybenzamine, phentolamine anddihydroergotamine) and a beta adrenergic receptorblocker propranolol (Table 1). This indicates the partialinvolvement of both alpha and beta adrenergic receptorsin bringing about the melanosome aggregation of Clariasbatrachus. The view is further supported by the factthat both phenylephrine and isoprenaline which arespecific alpha and beta adrenergic receptor agonistsrespectively (13) induced strong melanosome aggregationin the present studies. Similar results have also beenreported from a few earlier studies on fish melanophores(2, 6, 7) which were not the catfishes. However, in

188 Ovais

catfishes belonging to the family Siluriidae viz.,Parasilurus and Kryptopterus, the aggregation ofmelanosomes is mediated through cholinergic muscarinicand dispersion through both alpha and beta adrenergicreceptors (9-11).

In the present studies, cholinomimetic drugsaroused a significant melanosome dispersion inC. batrachus melanophores. These results are inagreement with those of earlier studies on Ictalurid(catfish), Oryzias, Pleuronectes and Bathygobius (1, 2,4, 8). However, melanosomes of many fishes respondwith feeble dispersion or no effect at all to acetylcholine(3,5). It has also been demonstrated that in the catfishesbelonging to the family Siluriidae, the mclanosomeshave cholinergic muscarinic receptor of aggregatory innature (11).

The dispersal effect of acetylcholine in the presentstudy was effectively blocked by atropine and it waspotentiated by physostigmine an anticholinesteraseagent. These results indicate the presence of cholinergic

Indian J Physiol Phannacol 1994; 38(3)

muscariruc receptors of dispersing nature in themelanophores of C. batrachus. Nicotine also elicited asignificant dispersal response. which was effectivelyblocked by hexamethonium. Basing on this, it can besaid that cholinergic nicotinic receptors also takepart in bringing about the dispersal effect on thisfish melanophores. Thus aggregatory effects ofatropine and hexamethonium per se provide ampleevidence to the fact that both cholinergic muscarinicand nocotinic receptors take part in the dispersion ofthis fish melanophores. This is supported by theobservation that both these antagonists blocked theresponses of this fish melanophores to acetylcholineand nicotine. The negligible aggregatory effect ofacetylcholine in presence of hexamethonium is ratherdifficult to explain at the moment and need furtherinvestigations.

ACKNOWLEDGEMENTS

Thanks are due to Prof. Santosh Kumar. H.O.D.for necessary facilities.

REFERENCES

I. Watanabe M, Kobayashi M, Iwata KS. The action of certainautonomic drugs on the fish melanophores. Bioi J OkayamaUniv 1962; 8 : 103-114.

2. Fernando MM, Grove OJ. Melanophore aggregation in theplaice (Pleuronectes platessa L.) II. In vitro effects of adrenergicdrugs. Comp Biochem Physiol 1974; 48A : 723-732.

3. Fujii R, Miyashita Y. Receptor mechanisms in fishchromatophores-I. Alpha nature of adrenoceptors mediatingmelanophores aggregation in Guppy melanophores. CompBiochemPhysiol1976; 51C: 171-178.

4. Visconti MA, Castrucci AML. Evidences of adrenergic andcholinergic receptors in teleost melanophores. Comp BiochemPhysiol1981; 70C : 293-296.

5. Nagaishi H, Oshima N. Control of the pigment migration inmelanophores in the dorsal and ventral skin of the upsidedown catfish. Comp Biochem Physiol1989; 93C : 67-71.

6. Scott GT. Physiology and Pharmacology of colour change inthe sand flounder Scopthalmus aquosus. Limnol Oceanogr 1965;\0 : R230-R246.

7. Healey EG, Ross DM. The effects of drugs on the backgroundresponse of the minnow Phoxinus phoxinus L. Comp BiochemPhysioI1966; 19: 545-580.

8. Parker GI-I. Animal Colour Changes and their Neurohumours.Cambridge University Press, 1948 : p. 377.

9. Fujii R, Miyashita Y. Receptor mechanisms in fishchromatophores-IlI. Neurally contro\1ed melanosomeaggregation in a siluroid (Parasilurus asotus} is strangelymediated by Cholinoceptors. Comp Biochem Physiol 1976;55c : 43-49.

10. Fujii R, Miyashita Y, Fujii Y. Muscarinic cholinoceptorsmediate neurally evoked pigment aggregation in glasscatfish melanophores. J Neural Transmission 1982; 5429-39.

II. Kasukawa 1-1, Oshima N, Fujii R. A comparative surveyof the type of sympathetic cure-Melanophore transmissionin Catfishes. Comp B iochem P hysiol 1986 ; 85 C : 115-120.

12. Bhattacharyya SK, Parikh AK, Oas PK. Effects ofcatecholamines on the melanophores of frog Rana tigrina.Ind J Expt Bioi 1976; 14: 486-488.

13. Furchgou RF. The classification of adrenoceptors (Adrenergicreceptors) : An evaluation from the standpoint of receptortheory. In : Blashko, K. Muscholl, E. eds, Handbook ofExperimental Pharmacology. Berlin: Springer Verlag, 1972;283-335.