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The molecular mechanism of action for the pineal hormone melatonin was explored by testing melatonin interaction with the components of the hormone-sensitive adenylate cyclase complex in a Xenopus dermal melanophore bioassay. Forskolin was employed to stimulate melanosome dispersion. The ability of melatonin to reverse forskolin-stimulated pigment dispersion was assessed, as was the effect of pertussis toxin on the ability of melatonin to aggregate dispersed pigment. Forskolin elicited dispersal of melanosomes in a dose dependent manner (EC50 = 12 nM) in meninges from stage 52-56 tadpoles of Xenopus laevis. Maximal pigment dispersion was obtained with 100 nM forskolin. Melatonin reversed this effect of forskolin (EC50 = 1.5 nM), causing pigment aggregation. Pertussis toxin blocked the melatonin-induced aggregation (EC50 = 358 ng/ml). Prior treatment of the melanophore containing meningeal explants with pertussis toxin results in blockade of melatonin induced pigment aggregation. A 41 kDa pertussis toxin substrate is found in explant homogenates treated with 32P-NAD and pertussis toxin. The availability of this substrate is reduced by prior treatment of intact explants with pertussis toxin and depletion of melatonin responsiveness corresponds to depletion of the 41 kDa substrate. Together, these data suggest that melatonin action upon amphibian dermal melanosomes is mediated by a system requiring a protein similar to the regulatory protein Ni used by mammalian cells to mediate the action of hormones which inhibit adenylate cyclase through a cell surface receptor.
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