Protein Kinase C Activation Antagonizes Melatonin-induced Pigment Aggregation in Xenopus lae s Melanophores

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine Y,5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules, ot-Melanocyte stimulating hormone (oL-MSH), like PKC activators, overcame melatonininduced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after ct-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of RpcAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase. T RANSPORT of intracellular vesicles and organelles in eukaryotic cells is a ubiquitous phenomenon, essential for a variety of cellular processes including mitosis, exocytosis, endocytosis, fast axonal transport and the spatial organization of the Golgi apparatus, lysosomes and endoplasmic reticulurn. Pigment cells of vertebrates are an excellent experimental system for studying intracellular motility. In such cells the movement of pigment granules is rapid, reversible, readily quantitated and under cellular control. The development of methods for the isolation and culture of pigment cells (Ide, 1974a; Jackson et al., 1974) has allowed biochemical studies of the intracellular mechanisms regulating pigment granule transport (Rozdzial and Haimo, 1986a,b). The observation that pigment granule dispersion in goldfish xanthophores is accompanied by the phosphorylation of a 57-kD granule-associated protein by a cyclic AMP-dependent protein kinase (PKA) 1 has suggested a model mechanism by which phosphorylation/dephosphorylation of specific protein(s) can regulate organelle movement (Lynch et al., 1986a,b). Recent evidence indicates that the microtubule-dependent ATPase, kinesin, is the motor protein responsible for centrifugal movement (dispersion) of 1. Abbreviations used in this paper: PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C. pigment granules in melanophores (Rodionov et al., 1991), and it seems likely that a second micmtubule-dependent motor protein, dynein, is responsible for centripetal movement (aggregation) of pigment granules (Beckerle and Porter, 1982; Clark and Rosenbaum, 1982). In the melanophores of Xenopus/aev/s, pigment granules disperse within minutes after addition of low concentrations of the pituitary hormone, a-melanocyte stimulating hormone (ct-MSH) (-Bagnara et al., 1969). Pigment granule dispersion also occurs in response to agents which elevate the concentration of intracellular cyclic AMP such as theophylline (McGuire et al., 1972), forskolin (White et al., 1987) and cell-permeable cyclic AMP analogues (Ide, 1974b). ot-MSH has been shown to elevate the intracellular concentration of cyclic AMP in frog skin (Abe et al., 1969) and in isolated melanophores (Daniolos et al., 1990) suggesting that activation of the c~-MSH receptor stimulates adenylate cyclase in the melanophore cell membrane resulting in an increase in cyclic AMP, activation of PICA and phosphorylation of specific pigment cell proteins resulting in pigment granule dispersion. The role of protein kinases other than PKA in regulating pigment granule movement in Xenopus melanophores is not known. In Xenopus melanophores with dispersed pigment granules, addition of the pineal hormone, melatonin (5-methoxy 9 The Rockefeller University Press, 0021-9525/92/12/1515/7 $2.00 The Journal of Cell Biology, Volume 119, Number 6, December 1992 1515-1521 1515 on O cber 8, 2017 jcb.rress.org D ow nladed fom N-acetyltryptamine) induces a rapid aggregation of pigment granules around the nucleus. This change in pigment granule distribution is part of the physiological mechanism which adapts skin color to environmental illumination (Rollag, 1988). In melanophore-rich skin of Xenopus (Van de Veerdonk and Konijn, 1970) and in isolated cultured Xenopus melanophores (Daniolos et al., 1990), melatonin inhibits c~-MSH-induced changes in cyclic AMP and ot-MSH dispersion of pigment. Melatonin-induced aggregation of pigment is also blocked by prior incubation with pertussis toxin, which blocks inhibitory G-protein coupled receptors (Jakobs et al., 1985), both in Xenopus tissue explants (White et al., 1987) and in isolated Xenopus melanophores (Sugden, 1991). Together these observations suggest that the melanophore melatonin receptor is coupled to an inhibitory G-protein and that activation of the receptor reduces intracellular cyclic AMP resulting in pigment granule aggregation. The melanophore melatonin receptor appears to be pharmacologically identical to the high affinity melatonin binding sites described in the retina and brain of birds, mammals and a marsupial (Krause and Dubocovich, 1991; Sugden and Chong, 1991; Paterson et al., 1992). These binding sites are also G-protein coupled and, in some instances, their activation has been shown to reduce intracellular levels of cyclic AMP in a pertussis toxin-sensitive manner (Carlson et al., 1989; Vanecek and Vollrath, 1989; Morgan et al., 1989). However, melatonin receptors have also been reported to modulate other transduction systems. Melatonin reduced luteinizing hormone-releasing hormone stimulation of diacylglycerol production (after hydrolysis of phosphatidylinositol), the generation of arachidonic acid, the accumulation of cyclic GMP and the rise in intracellular free Ca 2+ ([Ca:+]i) in pituitary explants from immature rats (Vanecek and Vollrath, 1989, 1990; Vanecek and Klein, 1992). The present study was designed to examine the effect of protein kinase C (PKC) activation on melatonin-induced aggregation and ot-MSH-induced dispersion of pigment granules. The results indicate that PKC activatioa prevents and reverses melatonin-induced aggregation of pigment granules. PKC probably acts by phosphorylating intracellular proteins involved in granule translocation rather than at the melatonin receptor itself, c~-MSH-induced dispersion of pigment, which can also antagonize melatonin-induced aggregation, does not involve PKC activation. Materials and Methods Culture of Melanophores Xenopus laevis embryos were obtained from adult frogs induced to lay by injection of human chorionic gonadotrophin (Chorulon, Intervet Laboratories Ltd., UK., 400 IU/male, 600 IU/female). The embryos were reared in tap water until stage 20 assessed using the normal table of Xenopus development (Nieuwkoop and Faber, 1956). The neural plate and underlying notochord and somites from 20 embryos was dissected and dispersed into small aggregates as described previously (Messenger and Warner, 1977) onto collagen-coated (0.2 mg/ml) petri dishes. After 2-3 d, pigment ceils were readily visible among the many nerve, muscle and undifferentiated cells. Cells were initially cultured in medium containing (raM): NaCI 100, KCI 2.5, CaCI2 2, MgCI2 2, NaHCO3 5 plus 10% FCS, and penicillin 100 U/ml, and streptomycin 100 #g/ml. After •7 d the media was changed to Leibovitz Ld5 medium (Gibco Laboratories, Lawrence, MA) diluted (1:1) with deionized water containing 10% FCS, 200 iu/ml penicillin, 200 #g/rnl streptomycin, and 2.5 #g/ml amphotericin B (Daniolos et al., 1990). Figure L Photomicrographs of the same duster of Xenopus laevis melanophores before treatment (top), after melatonin treatment (center, 10 -s M, 15 min), after melatonin (10 -s M, 15 min) then 4flPMA (10 -7 M, 60 min) treatment (bottom). (Arrow) Melanophore showing increased dendricity more commonly seen after prolonged (>4 h) 4flPMA exposure. The horizontal bar represents 100