2-[125I]-melatonin binding sites in the central nervous system and neural retina of the frog Rana perezi: regulation by light and temperature.
Melatonin is an output signal of an endogenous circadian clock of retinal photoreceptors, with highest levels occurring at night. Melatonin synthesized in the retina appears to act as a paracrine signal by binding to specific receptors in the eye. We have previously demonstrated that RNA encoding the Mel(1b) and Mel(1c) melatonin receptor subtypes is expressed in the Xenopus laevis retina. The goal of this study was to determine the distribution of the Mel(1b) and Mel(1c) receptor subtype RNA expression in the retina, and to determine if the level of expression of these receptors exhibits a diurnal rhythm. Sections of frog neural retina were analyzed by in situ hybridization with 35S-labeled Xenopus Mel(1c) and Mel(1b) riboprobes. Hybridization was present in cells of the inner nuclear layer and the ganglion cell layer. Moreover, there was hybridization in the photoreceptors, which has not been previously reported. To test the hypothesis that retinal melatonin receptor mRNA undergoes a diurnal rhythm of expression, total RNA was isolated from frog neural retinas obtained at 3-h intervals during a 24-h period. The total RNA was used in real-time PCR assays to quantify the differences in Mel(1b) and Mel(1c) receptor mRNA expression at various circadian times. Both the Mel(1b) and Mel(1c) receptor RNA demonstrated a diurnal rhythm of expression, with peak levels occurring late in the light period, and lowest levels late in the dark period. These results support the hypothesis that RNA encoding melatonin receptors undergo a diurnal rhythm of expression. To further investigate the possible expression of the Mel(1a) receptor subtype in Xenopus retina, we generated Mel(1a) PCR products in genomic DNA, and in reverse-transcribed neural retina and retinal pigment epithelium (RPE) RNA. The identity of the PCR product was confirmed by sequencing. Therefore, all three known Xenopus melatonin receptor subtypes appear to be expressed in the neural retina and RPE.