Current evidence from this and other laboratories casts doubt on intracellular cyclic AMP acting as the sole intracellular signal involved in the regulation of melanogenesis in melanocytes and melanoma cells. Our data suggests that an intracellular inhibitory control can prevent the cell responding to an increase in cyclic AMP with pigmentation and that both calcium and calmodulin are involved in this inhibitory control of melanogenesis.

Our conclusions are based on several quite separate lines of evidence. Firstly, we have recently documented that the well known heterogeneity of pigment to MSH. Further investigation of this showed whereas all cell lines showed a significant cyclic AMP accumulation in response to MSH stimulation, only some B16 melanoma cells responded to this elevated cyclic AMP with an increase in tyrosinase activity and melanin production. In others, an equivalent, or indeed greater, rise in cyclic AMP did not lead to activation of tyrosinase. The possibility that the tyrosinase enzyme was defective in some way was excluded by the finding that all cell lines, when injected intradermally in mice, produced equally well pigmented tumours in vivo (Hill et al, 1989). Further B16 cells which failed to respond to agents elevating cyclic AMP could be made to pigment by the use of calmodulin antagonists. This latter observation was first made in 1984 (Mac Neil et al, 1984a) when cells incubated with the calmodulin antagonists prochlorperazine and W7   (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide) showed a significant increase in pigmentation, despite failing to pigment to MSH. These drugs inhibit the proliferation of melanoma cells (Mac Neil et al, 1984a). We have since confirmed this finding (Hill et al, 1989) with another calmodulin antagonist (N-(6-aminooctyl)-5-iodo-1-naphthalene-sulphonamide) which is significantly more potent and specific for the inhibition of calmodulin than the present drug W7 (Mac Neil et al, 1988). Our current studies with B16 melanoma lines include cells which show a gradation in their metastatic potential : highly metastatic cells in vivo have a poor pigmentary response in vitro, whereas cells with low metastatic potential respond to MSH in vitro with a predictable increase in melanogenesis (Hill et al, 1988a,b). As all of these cells lines pigment in vivo, it strongly suggests that the cells possess a similar potential for pigmentation but differ, either in respect to the pigmentary signals they are receiving (which cannot be the case in tissue culture ) or with respect to their interpretation of the pigmentary signals which they receive.

As with the concept that MSH is the sole intracellular mediator of melanogenesis acting through elevation of cAMP, the weight of increasing evidence makes it necessary to discard the simple hypothesis that the cell possesses a series of discrete intracellular signalling systems (Tomlinson et al, 1985). We now know, for example, that intracellular-free calcium and cyclic AMP signals can influence each other at several points within the cell. Thus, there are several opportunities for integration of signals which undoubtedly help the cell to handle a complex extracellular environment.

However, cancer cells achieve autonomy from their immediate environment by apparently becoming independent of some of these extracellular signals. We would hypothesise that what we are observing in the B16 melanoma cells is a gradation in the expression of an inhibitory signal for melanogenesis. That this is not an absolute block on melanogenesis can be demonstrated when the cells are reintroduced into mice. At present, we do not know what the adequate in vivo pigmentary stimuli for these B16 melanoma cells may be. Our experience with primary B16 melanoma cell lines leads us to suggest that these may not be so dissimilar to normal human melanocytes which also fail to pigment in response to MSH in vitro (Friedmann and Gilchrest, 1987) but will pigment to other stimuli such as UV. Indeed, in man, there does not appear to be a simple relationship between skin pigmentation and circulating levels of MSH peptide except when MSH levels are abnormaIly elevated in pathological conditions (Friedmann and Thody, 1986).

At present, despite the fact that calcium and calmodulin are intimately involved in both the production (Mac Neil etal, 1984b) and the degradation of (Walker et al, 1984) cyclic AMP, our evidence points to calmodulin antagonists initiating melanogenesis through some cyclic AMP independent mechanism. Indeed, in vitro calmodulin antagonists acutely inhibit cyclic AMP accumulation in intact cultured cells (Mac Neil et al, 1984a). Several other agents have also been reported to induce melanogenesis in melanoma cells and melanocytes by a presumably cyclic AMP independent mechanism vitamin D metabolites (Tomita et al, 1988), retinoids (Lotan and Lotan, 1981), Wirradiation (Friedmann and Gilchrest, 1987) and diacylglycerol analogues (Gordon et al, 1988). The intracellular mechanisms used have not been identified for any of these agents. Finally, recent very strong support for the idea of a calcium inhibitory control of melanogenesis has been provided by Fuller (1987). Fuller showed that the calcium ionophore A23187, which dramatically increases levels of intracellular free calcium within the cell, is inhibitory to basal and MSH-stimulated melanogenesis.

The majority of the actions of calcium within the cell are now known to be mediated by its binding to the ubiquitous calcium binding protein calmodulin. It is the calcium-caImodulin complex which then interacts with intracellular proteins, usually enzymes, to effect the biological response (Tomlinson et al, 1984). Thus, Fuller's data on A23187 is entirely consistent with ours using calmodulin antagonists, leading us to suggest that the inhibitory control we should be seeking in the control of melanogenesis is both calcium and calmodulin-dependent.

There is no evidence to either support or refute the possibility that one such inhibitory calcium/calmodulin control could explain the melanogenic response to such a diverse collection of pigmentary stimuli as Vitamin D metabolites, retinoids, UV irradiation and diacylglycerol analogues. In particular, the melanogenic response to diacylglycerol analogues, which mimic the natural substrate for the activation of protein kinase C, suggests that the immediate future in unravelling the intracellular mechanisms involved in melanogenesis, may be complicated. However, events have clearly overtaken the hypothesis and the concept of a dual cyclic AMP stimulatory control of melanogenesis working against an endogenous inhibitory control of melanogenesis, yet to be delineated, will perhaps be more useful for the next stage of the investigation of the regulation of melanogenesis.

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- Friedmann PS and Thody AJ (1986). Disorders in pigmentation. In : Scientific Basis of Dermatology (ed. AJ Thody and pS Friedmann), Churchill Livingstone, Edinburgh, pp. 244-261.
- Fuller BB (1987). Inhibition of tyrosinase activity and protein synthesis in melanoma cells by Ca2+ ionophore A23187. Pigment Cell Research 1:176-180.
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- Walker SW, Mac Neil S et al (1984). Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma . Biochem J 219:941-946.
- Tomita Y, Torinuki W and Tagami H (1988). Stimulation of human melanocytes by Vit D3 possibly mediates skin pigmentation after sun exposure. J Invest Dermatol 90:882-884.

Mac Neil S, Hill SE, Buffey J
Department of Medicine
Clinical Sciences Center
Northern General Hospital
UK - Sheffield