RESUMO
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.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Endotelinas/farmacologia , Opsinas de Bastonetes/metabolismo , Animais , Ritmo Circadiano/efeitos da radiação , Luz , Melaninas/metabolismo , Melanóforos/citologia , Melanóforos/efeitos dos fármacos , Melanóforos/metabolismo , Melanóforos/efeitos da radiação , Fotoperíodo , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo , Xenopus laevisRESUMO
The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 µM glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 µM glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli.