J. Biosci., Vol. 18, Number 1, March 1993, pp 83–91. © Printed in India. Role of calcium in melanosome aggregation within Labeo melanophores

SHASHI PATIL and AJAI K JAIN* Pigment Cell Biology Unit, School of Studies in Zoology, Jiwaji University, Gwalior 474 011, India MS received 30 December 1991; revised 10 August 1992 Abstract. In isolated scale melanophores of Labeo rohita the melanosome aggregating effect of K+ was inhibited in Ca2+ deprived medium. Moreover, the Ca2+-antagonists, verapamil and lanthanum inhibited the action of K+ in concentration dependent manner. The elevation of extracellular Ca2+ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca2+ appeared to be required only for K+ -induced aggregation and not melanosome aggregation per se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca2+ free medium. Keywords. Verapamil; lanthanum; melanophore; melanosome aggregation; melanosome dispersion; Ca2+ , K+.

1. Introduction Because of its unique properties, calcium is the most crucial cation in terms of diversity of function. Though most of the calcium in the body (95%) is sequestered in bone, this reservoir is in dynamic equilibrium with the remaining pools of calcium serving to maintain a highly coordinated mechanism for calcium homeostasis. The cell is surrounded by fluid whose composition is kept constant by neural and endocrine mechanisms. There are large concentration differences between inside and outside the cell which are maintained by uphill movements, and the large electrochemical gradient enables the free calcium concentration of the cell to be increased rapidly and dramatically.

As reviewed by Blaustein (1985), the aspects of Ca2+ metabolism that enable this cation to serve as an effective second messenger include: Ca2+ entry into the cells via voltage-regulated or chemical transmitter-activated channels, through other (leakage) pathways or "reverse mode" Na+-Ca2+ exchange. Intracellular Ca2+ is buffered by cytoplasmic binding proteins and can be sequestered in mitochondria or endoplasmic reticulum. In addition, Ca2+ can also be triggered to enter the cytosol from endoplasmic reticulum while Ca2+ extrusion takes place either via Na+-Ca2+ exchange or a Ca2+-ATPase.

In relation to the nervous system controlling fish chromatophores Fujii and Fujii (1965) first reported that Ca2+ is required for catecholamine release from thesympathetic nerve terminals in the goby Chasmichthys gulosus. In erythrophores of squirrel fish Holocentrus ascensionis (Luby-Phelps and Porter 1982), melanophores of medaka Oryzias latipes (Negishi and Obika 1985) and melanophores and * Corresponding author.

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84 Shashi Patil and Ajai Κ Jain erythrophores of platyfish Xiphophorus maculatus (Oshima et al 1988) the experimental elevation of the cytoplasmic Ca2+ concentration induced pigment aggregation of melanosomes/erythrosomes. There are a few reports which demonstrate that pigment aggregation is coupled with a decrease in the intracellular Ca2+ level. In the medaka Oryzias injection of the Ca2+ precipitant sodium oxalate resulted in aggregation of melanosomes (Ishibashi 1957). Novales and Novales (1965) reported aggregation of tissue cultured melanophores in Ambystoma and other amphibian species in Ca2+ free medium. In guppy melanophores saponin in Ca2+ deprived medium produced a slight aggregation of pigment within the melanophores (Miyashita and Fujii 1978). Such contradictory observations point that detailed study is required to understand the role of Ca2+ in the motile activities of the melanosomes. The present study has been designed with the intention of elucidating the action of Ca2+ in controlling the activity ofmelanophores in the fresh-water Indian major carp, Labeo rohita commonly known as, rohu (which exhibits a bluish black colouration along the back and lateral surfaces and silvery along the ventral surface) in response to Κ+ stimulation. The effect of Ca2+ antagonists on the aggregation response to K+ has also been studied. 2. Materials and methods 2.1 Animals Specimens of Labeo rohita of either sex ranging 7–11 cm were used for experiments. The animals were procured from Govt. Fish Farm, Gwalior (MP) and were stocked in transparent glass aquaria under natural photoperiodic conditions. 2.2 Procedure Scales were isolated from the anterodorsal region of the trunk and placed in perfusion chamber for microscopic observations (Jain and Patil 1990). The stock solution of lanthanum was prepared in distilled water and diluted with Ca2+ free Ringer (CFR) as and when required. Verapamil was obtained in ampoules. The Ca2+ free Ringer had the following composition (mM): NaCl, 128·3; KCl. 2·68; glucose, 5·6; Hepes-NaOH, 5·0 (pH 7·4); EDTA, 0·2. The Ca2+ -free K+ Ringer (CFK+) was prepared by adding equimolar KCl substituted for NaCl in CFR. All experiments were carried at room temperature ranging 20–26oC. Results are expressed as mean ± SD. 2.3 Recording of melanophore responses Hogben and Slome (1931) developed a so-called melanophore index (MI) that is in use since the earliest studies of melanophore responses to melanotropins. In this microscopic assay, the degree of melanosome dispersion is determined according to a 5-point scale, to yield a melanophore index that ranges between 1 and 5. This method employs 5 distinct stages representing different degrees of pigment dispersalwithin the melanophore; the most aggregated state is designated as stage 1 and the

Role of calcium in melanophore regulation 85 most dispersed as stage 5. Stages 2, 3 and 4 represent intermediate degrees of pigment dispersal. As pointed out by Bagnara and Hadley (1973), by using this system, an index of the relative dispersal of melanosomes can be obtained for quantitative use or for graphic representation. Based on this system, the degree of melanosome dispersion was determined by comparison of individual cells to the chart prepared for the fish, L rohita depicting 5 distinct stages of melanophore index (figure 1). This method is presently in use by many investigators throughout the world to study the melanophore responses of teleosts, amphibians and reptiles to various chemical stimuli including drugs and melanotropic peptides.

Teleostean melanophores attain the equilibrium state of nearly full dispersion of melanosomes (MI = 5) when they are under no stimulation and placed in an isotonic physiological saline solution (in vitro). Agents like norepinephrine and melanin-concentrating hormone cause a centripetal movement of the melanosomes towards the. cell centre (MI= 1). 2.4 Chemicals Verapamil hydrochloride (Boehringer-Knoll Ltd., Bombay), lanthanum nitrate (Loba Chemie, Wien-Fischamend), EDTA-ethylene diamine tetraacetic acid, disodium salt (Sarabhai M Chemicals, Baroda). All other chemicals used were of analytical grade. 3. Results 3.1 Effect of Ca2+ on K+

action The isolated scale melanophores equilibrated in physiological saline (20 min) attained full dispersed state. Subsequent incubation in CFR containing EDTA (0.2 mM) did not alter the dispersed state. CFK+ containing EDTA (0·2 mM) caused melanosome aggregation in these melanophores (figures 2 and 3, control) but this was followed by reversal of the effect during continued incubation in the same saline (CFK+).

3.2 Effect of Ca2+ antagonists The effect of verapamil (V), a Ca2+ influx blocker on the K+ induced aggregation, is depicted in figure 2. The inhibitory action of verapamil depended on its concentration (5 × l0–6M to 10–4 M, 40 min incubation) in the perfusing medium. The inhibitory effect was discernible at 5 × l0-5M, with 10-5 M and5 × 10-5M the inhibition was partial and the melanosome aggregation induced by K+ was completely inhibited by the drug at 10-4M.

The response of melanophores to CFK+ was also affected by Ca2+ antagonist lanthanum (La3+) known to substitute for Ca2+ at Ca2+ binding sites onmelanophore membrane. The melanophores were treated with various concentrations of La3+ (10-4M to 10-3M) for 40min. The inhibition of CFK+ action was incomplete at these concentrations of La3+ (figure 3). Higher concentrations of La3+ could not be tried and thus it is not certain whether it can bring about complete inhibition of CFK+ induced melanosome aggregation.

86 Shashi Patil and Ajai Κ Jain

Role of calcium in melanophore regulation 8 7

Figure 2. The effect of different concentrations (5×!0–6M to 10–4M) of Ca2+ antagonist-V on the action of Κ + in Ca 2+ free medium. Control (—o—) represents the response of CFR-equilibrated melanophores to CFK +. Each point is the mean of 25 measurements (5 animals). Perpendiculars drawn indicate SD.

3.3 Effect of elevated Cα2+ concentration The inhibitory effect of verapamil was overcome by increasing the extracellular Ca2+ concentration as shown in figure 4. The concentration of verapamil was kept constant (10–5 M) and Ca2+ concentration added to Ca2+ -free medium ranged from 0·5 mM to 1·5 mM. The inhibitory effect of verapamil was reversed in a graded manner with increase in Ca2+ concentrations. 4. Discussion The results of the present study presented as figures 2–4 show that Κ+ -induced pigment aggregation was inhibited in Ca2+ -deprived medium. Further the Ca2+ antagonists verapamil and lanthanum counteracted the effect of CFK+ in a concentration dependent manner and increase in exogenous Ca2+ reversed the inhibitory action of verapamil.

In our earlier study (Patil and Jain 1989) we have demonstrated that in Labeo high [K+]0 induced rapid and reversible pigment aggregation in innervated but not in denervated melanophores. It was speculated that high [K+]0 action initially induces a presynaptic membrane depolarization leading to an increase in Ca2+ influx, finally resulting in the release of neurotransmitter from the adrenergic nerve terminals.

88 Shashi Patil and Ajai Κ Jain

Figure 3. The effect of different concentrations (10–4 Μ to 10–3 'M) of Ca2+ antagonist La3+ on the action of K+ in Ca2+ free medium. Control (–o–) represents the response of CFR equilibrated melanophores to CFK+.

In the present study CFK+ produced aggregation of melanosomes but it was

rapidly followed by the reversal of the effect in the same medium. This may be due to the withdrawal of Ca2+ which interferes with the release of the chemical transmitter from sympathetic nerves controlling fish melanophores (Fujii and Fujii 1965; Kasukawa and Fujii 1984).

The motile responses of the melanophores remained unaffected on treatment with agents such as adrenaline (Patil 1986) and melanin concentrating hormone (Patil 1991) in Ca2+ deficient medium. The Ca2+ seems to be required for K+ stimulated responses and not for melanosome aggregation per se, since these agents induced normal responses in the absence of cation-Ca2+ (Fujii and Tagauchi 1969; Oshima et al 1985; Negishi 1986; Patil and Jain 1989; Jain and Patil 1990).

Such indispensability of cation-Ca2+ has also been reported for MSH action inXenopus (van de Veerdonk et al 1979) and the cyprinid fish Zacco (Iga andTakabatake 1982). Veseley and Hadley (1971) stated that in the lizard Anoliscarolinensis Ca2+ could regulate adenylate cyclase activity or inhibit cAMPphosphodiesterase activity leading to increased cAMP level which in turn dispersesthe melanosomes. Therefore this cation is not always required for melanosomedispersion per se, as it is not required for cAMP-stimulated dispersion ofmelanophores of the fish such as the guppy and the catfish (Miyashita et al 1984).

In the melanophores of Xenopus laevis α-MSH induced melanosome dispersion was inhibited by the prevention of Ca2+ influx through the Ca2+ antagonistsverapamil and lanthanum (van de Veerdonk et al 1979). In Oryzias, these

Role of calcium in melanophore regulation 89

Figure 4. The effect of increasing concentrations (0·5mM-l·mM) of Ca2+ on K+ -richsaline induced response of verapamil (10-5 M; 40min) pretreated melanophores. Themelanophores treated with CFK+ (—o—) represent the control.

antagonists caused reversal of light stimulated responses suggesting the possible role of Ca2+ to trigger the light stimulated dispersion of melanophores (Negishi 1986).

In Labeo the direction of pigment translocation due to CFK+ is different from that reported for Xenopus (van de Veerdonk et al 1979) and Oryzias (Negishi 1986) due to α-MSH and light stimulation respectively, but concentration dependent inhibition of CFK+ action by antagonists verapamil (figure 2) and lanthanum (figure 3) clearly suggests the indispensability of Ca 2+ for pigment aggregation due to K+.

In Oryzias, Obika and Negishi (1981) reported an abundant deposition of Ca2+ within the cytoplasm in pigment cells with aggregated melanosomes whereas in cells with dispersed pigment Ca2+ deposits were sparingly found within the cytoplasm, and these dispersed cells had extracellular deposition of Ca2+ around them. Other reports also demonstrate the aggregation of erythrosomes/melanosomes as a result of experimental elevation of intracellular Ca2+ (Luby-Phelps and Porter 1982; Negishi and Obika 1985; Oshima et al 1988). In the present study on melanophores treated with verapamil the increase in exogenous Ca2+ concentration was found to potentiate the aggregation effect due to K+. Thus in this fish too cytosolic concentration of Ca2+ appears to be responsible for aggregation of melanosomes within the melanophores.

90 Shashi Patil and Ajai Κ Jain

There are also reports that show the involvement of cAMP (Novales and Fujii 1970; Abramowitz and Chavin 1974; Fujii and Oshima 1986) in signal transduction in fish chromatophores. The most commonly observed effect of α1-receptoractivation is a rise in cytosolic calcium concentration. The mechanism by which the increase in calcium occurs is unclear. In certain smooth muscle cells, α1-receptor activation leads to an influx of Ca2+ across the membrane, probably through the opening of "receptor-operated" calcium channels. It has also been suggested that α1-receptor activation may cause breakdown of phosphatidylinositol in themembrane and thereby change intracellular calcium. A recent attempt in thisdirection comes from Fujii et al (1991). They were able to demonstrate that myo- inositol 1,4-biphosphate (IP2) and myoinositol 1-monophosphate (IP), both had no effect on motility of melanophores while exogenously applied myo-inositol 1,4,5- tr iphosphate (IP3) caused aggregation of melanosomes in the absence ofextracellular Ca2+. From their observation that adrenergic blocking agents wereineffective in blocking the action of IP3 , they concluded that IP3 does not act through interactions with α-adrenoceptors on the surface membrane, but through interactions with some intracellular compartments. Further they have concluded that intracellular messengers such as Ca2+, IP3 and cyclic nucleotides havedynamic interrelation in bringing about subtle and delicate changes of skin colour.

Thus from our study too it appears that Ca2+ might have a role in signal transduction of pigment aggregation within Labeo melanophores. Acknowledgements Research grant from Madhya Pradesh Council of Science and Technology, Bhopal (No. Z/122/87) to AKJ is acknowledged. We wish to thank late Prof. J Bahadur and to Prof. R Mathur for providing the laboratory facilities. References Abramowitz J and Chavin W 1974 In vitro response of goldfish (Carassius carassius L.) dermal

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