Pigment Cell Research zyx 2:213-217 (1989) Ionic Requirements for Melanin Concentrating Hormone (MCH) Actions on Teleost zyxw PoeciZia reticuhta Melanophores MARIA zyxwvuts A. VISCONTI,' ANA MARIA DE L. CASTRUCCI,1.2 MAC E. HADLEY,2 AND VICTOR J. HRUBY' 'Departamento de Fisiologia Geral. Instituto de Biociencias. Universidade de Sao Paulo. CP 11176% CEP 05499, Sao Paulo. Brazil, Departments of 'Anatomy and 'Chemistry. Universitj of Arizona, Tucson. Arizona 85721 Melanin concentrating hormone (MCH)is a cyclic heptadecapeptide, Asp-Thr- zy Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hor- mone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a per- inuclear position within integumental melanophores. MCH and related fragment analogues, MCHSi7 and MCH,-,, were used to in- vestigate the ionic requirements for receptor activation by MCH on dermal me- lanophores of the fish Poecilia reticulntn. In calcium-free saline, the sensitivity of the melanophores to MCH and MCHi-14 increased, whereas the sensitivity of the cells to MCHS17 decreased. Verapamil diminished the sensitivity to MCHj17, but did not affect melanophore responses to MCH or MCHI-,,. The melanosome ag- gregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potas- sium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCHl-L4 also exhibit MSH-like melanosome disper- sion within melanophores, skin darkening activity on fish melanophores whereas MCHjl7 lacks this characteristic. Since the darkening activity of MCH and MCHI- 11 requires calcium, these analogues exhibited a diminished lightening (MCH- like) activity in the presence of the divalent cation. In the absence of the N- terminal tetrapeptide sequence (necessary for the expression of MSH-like activ- ity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements. Key words: MCH bioassay INTRODUCTION A bihumoral control of melanosome movements within melanophores for effecting color change was proposed as early as 1931 by Hogben and Slome (Hogben and Slome. 1931). However, it was not until 1955 that Ena- mi demonstrated in the teleost zyxwvutsrq Parasilurus asotus a second putative pituitary melanotropic hormone (in ad- dition to a-melanocyte-stimulating hormone. CX-MSH) with potent lightening (melanosome-aggregating) ac- tivity (Enami, 1955).Enami also suggested a hypotha- lamic origin for the so-called MCH (melanophore-con- trncting zyxwvutsrqp hormone, n~Clnll;ll-COIICC.II~I-~~;'l~ ~IWIIIVLIL.)~ confirmed yeara later by other investigators (Baker and Ball, 1975; Baker and Rance, 1983; Naito et al., 1985). In 1983, Kawauchi and colleagues purified and char- (-' 1989 Alan R. Liss, Inc. acterized the chemical structure of MCH from Oncor- hyrzch zyxwv us keta pituitaries, as a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg- Pro-Cys-Trp-Glu-Val, with a disulfide bridge between residues 5 and 14 (Kawauchi et al., 1983). MCH was subsequently synthesized. and its potent melanosome (melanin granulekaggregating activity was confirmed on a number of teleost fishes (Baker, 1988; Castrucci et al., 1987, 1988; Oshima et al., 1985, 1986; Wilkes et al., 1984 a,b). It has also been shown that the actions - ~~ ~ ilddtcnn wpi-in1 ri~~~ui~st~ lo l)r zyxwv M A Viwinti. I)r[iarlamrnLii de, E'I. siologia Geral. Instituto de Siocicncins da Univcrsidnde dc Sio I'aulo. CP 11176. CEP 05499. Sao Paulo. Brazil Received November 10. 1968: accepted December 20. 1988