Regulation of melanopsins and Per1 by α -MSH and melatonin in photosensitive Xenopus laevis melanophores.

α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins' expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

Endothelin modulates the circadian expression of non-visual opsins.

The non-visual opsin, melanopsin, expressed in the mammalian retina, is considered a circadian photopigment because it is responsible to entrain the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Pigment dispersion can also be triggered by endothelin-3 (ET-3). Here we show that melanin translocation is greater when a blue light pulse was applied in the presence of ET-3. In addition, we demonstrated that mRNA levels of the melanopsins Opn4x and Opn4m exhibit temporal variation in melanophores under light/dark (LD) cycles or constant darkness, suggesting that this variation is clock-driven. Moreover, under LD cycles the oscillations of both melanopsins show a circadian profile suggesting a role for these opsins in the photoentrainment mechanism. Blue-light pulse decreased Opn4x expression, but had no effect on Opn4m. ET-3 abolishes the circadian rhythm of expression of both opsins; in addition the hormone increases Opn4x expression in a dose-, circadian time- and light-dependent way. ET-3 also increases the expression of its own receptor, in a dose-dependent manner. The variation of melanopsin levels may represent an adaptive mechanism to ensure greater melanophore sensitivity in response to environmental light conditions with ideal magnitude in terms of melanin granule dispersion, and consequently color change.