INTRODUCTION

Melanophores of lower vertebrates are specialized type of smooth muscles cells, derived from the neural crest and are present in the basal layers of the epidermis and dermis (Rawles, 1948), which possess thousands of black

ARTICLE INFORMATION:

*Corresponding Author Received 15th April, 2014

Accepted after revision 20th June, 2014 BBRC Print ISSN: 0974-6455 Online ISSN: 2321-4007

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melanin pigments, packed in melanosomes (Bagnara et al., 1986; Schliwa, 1976). These specialized pigment containing cells are derived from the neural crest, and are present in the epidermal and dermal layers of cold blooded vertebrates (Rawles, 1948; Arduini et al., 2008). The immense sensitivity of these cells to a variety of

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Swamy, Choudhary and Ayesha S. Ali

stimuli and their lineage with neurons of the sensory kind has made these cells a subject of great interest amongst researchers (Bagnara and Hadley, 1973; Fujii, 1993, Salim and Ali 2011, Ali et al., 2012).

Several extensive studies have been made on physi- ology and pharmacology of adrenergic and choliner- gic drugs on melanophores of fishes and amphibians (Abbott, 1968; Fujii & Hayashi, 1996; Ali, 1983; Ali and Ovais 1993; Ali et al., 1998). Though various earlier workers have extensively investigated the fish melano- phores, much attention has been directed towards endo- crine influences (Waring, 1963; Beaven, 1976; Tomita et al., 1988), unfortunately pharmacological charac- terization of the histamine receptors in fish melano- phores remains obscure and it is not yet clear whether histamine receptors plays a role in the physiology and pharmacology of fish melanophore responses. Hence in the present study the main objectives were to study the effects of the new modern and recently available spe- cific agonists of H1 and H2 histamine receptors along with their specific antagonists and potentiators on the isolated scale melanophores of the teleost fish, Rasbora elanga in order to find out the involvement and nature of histamine receptors at the cellular level.

MATERIAL AND METHODS

The teleostean fish Rasbora elenga has been selected for studying the effects of recent new class of histaminergic drugs on its isolated dorsal scale melanophores, in order to find the nature and role of receptors of histamin- ergic type in controlling skin pigmentation processes. The fish was selected because of its easy availability, sturdy nature as it can be kept live in laboratory con- ditions for long periods and the fact that its melano- phores are excellent model for in vitro studies, and no study has been done on them till now. They were caught with the help of fishermen from various water bodies and transported to the laboratory alive and they were kept in glass aquaria containing 100L of dechlorinated tap water. Experiments were performed in the labora- tory conditions having ambient temperature of 25-30° C with a pH of 7.2 to 7.4.

Prior to the experiments, the fish were allowed to acclimatize to laboratory conditions for 3 days. Dis- eased, injured, or lethargic fish were removed and only active, uniformly colored fish were used. For the in vitro studies, the fish scales were removed in accordance with the method of Spaeth, (1913) which included the removal of 20–25 scales from the dorso- lateral region of live R. elenga kept in a wet cloth, held loosely. The scales were removed by forceps from the dorsal lateral pigmented area. These were immediately

placed in 0.7% normal saline, containing 700 mg of sodium chloride in 100 mL of double distilled water. They were equilibrated in saline medium for 7–10 min with frequent shaking.

The responses of control as well as of those melano- phores that were incubated in 10 mL 0.7% fish saline containing various concentrations starting from 1×10-6 to 6.4×10−5 g/mL of Histamine, Pyridyl ethyl amine (Specific H1 agonist) and Amthamine (Specific H2 agonist) along with their specific antagonists, diphe- niramine and ranitidine (4×10-6 g/mL). Responses of the melanophores were measured in accordance with the method of Bhattacharya et al., (1976) based on Hog- ben and Slome (1931). In this method, actual diameter (length×breadth with the processes) of 10 randomly selected melanophores from each scale was measured using a Leitz Occulometer calibrated previously with stage micrometer. The value was then multiplied by the unit of the micrometer 15 μm. Thereafter, the arith- metical mean was calculated and this value was then divided by 100 to obtain the values. This was the mean melanophore size index (MMSI).

STATISTI AL ANALYSIS

Statistical data analyses are presented as mean ± stand- ard error of the mean (SEM) and n = 7, which repre- sents the number of individual experiments conducted with equal numbers of animals. Comparisons were made between treated and control groups by use of Student’s t-test. All data were analyzed using GraphPad Prism software (UK). P < 0.05 indicates statistically significant difference.

RESULTS AND DISSCUSION

Histamine is an important autacoid biogenic amine present in all biological tissues, and also regarded as a chemical mediator and neurotransmitter on broad spec- trum physiological level (Goodman and Gillman, 2006). It is contained in mast cells and basophiles found in all animal and mammalian tissues in both neural and non- neural compartments (Goodman and Gillman, 2006). In the present study histamine per se aggregated the dorsal skin melanophores of R. elenga in varying doses rang- ing from 1×10-6 g/ml to 6.4×10 10-5 g /ml. The MMSI decreased from the control value of 4.532±0.1282 to 0.9257±0.05455. After repeated washings and reim- mersion (RI) of the histamine treated melanophores in normal saline, it was found that the powerful melanin aggregation effects of the extract completely vanished, as the MMSI became 4.441 ± 0.1138, almost near the control values of 4.532 ± 0.1282 (Figure 1).

The present findings are in contradiction with those of Healy and Ross, (1966) that showed there was no effect of histamine on the melanophores of the Phoxi- nus phoxinus. Later on similar findings were reported by Bhide and Gupta, (1967) who found that histamine lib- erators like comp 48/80 caused blanching of R. tigerina skin via the indirect release of histamine. The workers had also found that histamine per se could produce mel- anin aggregation in the same species, but no receptor study was done by the workers. It was only in 1983, Ali described the presence of histamine receptors in a large number of lower vertibrates (Ali, 1983; Ali et al., 1993; Ali et al 1998; Peter et al., 1996, 2011).

Similarly, Pyridyl ethyl amine, a specific H1 receptor agonist, was also found to aggregate the fish melano- phores. Different concentrations of Pyridyl ethyl amine (1×10-6 g/ml to 6.4×10 10-5 g/ml) aggregated the skin melanophores, where MMSI decreased from a control value of 4.361 ± 0.05872 to 0.8871 ± 0.04980 as seen by the maximal concentration of 6.4×10-5 g/ml (Figure 2).

On other hands ammthamine, a specific H2 receptor agonist, also aggregate the melanophores of R. elanga in a dose dependent manner were the melanophore proc- ess had become disappeared, showed almost point shap morphology, leading to lightening of skin. In response to the maximal concentration of 6.4 × 10−5 g/mL of

ammthamine, the MMSI of the melanophores became 0.6429 ± 0.04433compared to the control value of 4.532

±0.1282. After repeated washings and reimmersion (RI) of the ammthamine treated melanophores in normal saline, it was found that the powerful melanin aggrega- tion effects of the extract completely vanished, as the MMSI became 4.441 ± 0.1138, almost near the control values of 4.532 ± 0.1282 (Figure 3).

In order to further validate the presence and role of

specific H1 and H2 histamine receptors, specific antago- nist dipheniramine and ranitidine were used to see the effect on the scale melanophores of R. elenga. In a series of experiments it was observed that the presence of dipheniramine completely abolished the melanophore aggregation effects of pyridyl ethyl amine. From the data presented in fig.1 it becomes clear that an even the highest dose of 6.4×10-5 g/ml of Pyridyl ethyl amine along with dipheniramine could not aggregate the tel- eost melanophores where the MMSI remained 4.404 ± 0.07311 which is almost near to the control value of 4.693 ± 0.06342, which without the presence of diphe- niramine was 0.8871 ± 0.0498 (Figure 1).

On the other hands pre-incubation of isolated scale fish melanophores in 2×10-6 g/ml of ranitidine was followed by the increasing concentrations series of ammathine (1×10-6 to 6.4×10-5 g/ml) resulted into com-

Swamy, Choudhary and Ayesha S. Ali

plete blockade of aggregating effects of ammathine (Fig- ure 3). Where the MMSI remained 5.194 ± 0.04264 in initial dose concentration of 1×10-6 g/ml of ammath- ine, which without the ranitidine was 4.179 ± 0.04546 (Figure 2). This trend continued further and even the concentration of 8×10-6 g/ml of ammathine could not aggregate the melanophores as the MMSI was 4.164 ± 0.03643 whereas, the same without the ranitidine was 1.695 ± 0.0566 (Fig. 2). Even the highest concentration i.e. 6.4×10-6 g/ml of ammathine along with ranitidine was not able to exert melanophores aggregation effects. The MMSI remained 2.829 ± 0.09289 at this concentra- tion, otherwise without the ranitidine the value of MMSI was 0.6429 ± 0.04433 (Figure 2). Thus, the present find- ing suggests that both dipheniramine and ranitidine completely blocked the aggregation effects of pyridyl ethyl amine and ammathine in all concentrations.

CONCLUSION

It is concluded that histamine, induced powerful, dose dependent, physiologically significant melanin aggre- gation effects in the isolated scale melanophores of R. elenga. The use of new specific receptor agonists and antagonists revealed that the skin pigmentary responses owing to histaminergic involvement are mediated by novel H1 and H2 subtype of receptors. It is further sug- gested that the stimulation of H1 and H2 receptors brings about pigment lightening. The pharmacological char- acterization of histamine receptors from our present study has revealed that these receptors are responsible for regulating the pigmentary responses in teleosts with subsequent lightening of skin.

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