CALCIUM PLAYS A COMPLEX ROLE IN THE REGULATION OF MELANOGENESIS IN MURINE B16 MELANOMA-CELLS

To learn more of the role of calcium in the regulation of melanogenesis, we have used direct manipulation of medium calcium and pharmacological modulation of intracellular calcium to examine the consequences on unstimulated and cyclic AMP elevated tyrosinase activity and melanin synthesis and distribution in B16 melanoma cells. In unstimulated cells, calcium is clearly inhibitory to tyrosinase activity. However, in cells stimulated with cAMP-elevating agents the requirement for extracellular calcium was changed such that cells required a minimum of 0.4-0.6 mmol medium calcium for maximum tyrosinase response to these agents. Paradoxically, pharmacologically increasing intracellular calcium in cAMP-stimulated cells with ionophore inhibited tyrosinase activity, and the calcium-lowering agent TMB8 and the calcium channel blocker verapamil both stimulated tyrosinase activity. When melanin synthesis was measured in cAMP-stimulated cells, TMB8 was found to significantly increase the sensitivity and the maximum melanogenic response to alpha-MSH, suggesting the presence of at least one level of endogenous calcium inhibitory control operative in these cells. In addition, TMB8 changed the distribution of melanin between the cell and the medium such that, in the presence of (alpha-MSH and TMB8, significantly more melanin was secreted into the medium. These data suggest that calcium is required for several steps in melanogenesis, having an apparently inhibitory effect on pre-tyrosinase activity in unstimulated cells, but also showing evidence of a positive role in cyclic AMP-stimulated tyrosinase activity, as well as a further possible inhibitory role in melanin movement or secretion.