Wiechmann AF et al. (1999), Melatonin receptor RNA expression in Xenopus re...

XB-ART-13717

Brain Res Mol Brain Res 1999 Jan 08;632:297-303. doi: 10.1016/s0169-328x(98)00292-7.

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Melatonin receptor RNA expression in Xenopus retina.

Wiechmann AF , Campbell LD , Defoe DM .

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Melatonin is an indolamine hormone presumably synthesized by retinal photoreceptors, and may act as a paracrine signal of darkness within the retina. Previous studies have suggested that melatonin, acting through specific receptors, may be involved in cyclic retinal functions such as photoreceptor outer segment disc shedding and phagocytosis, and modulation of neurotransmitter release in the inner retina. The goal of this study was to determine if melatonin receptor mRNA is expressed in the neural retina and retinal pigment epithelium (RPE) of Xenopus laevis. Sheets of RPE, devoid of contaminating cells, were obtained from Xenopus eyes, and epithelial cultures were subsequently established on microporous membrane filters in a defined medium. Total RNA was isolated from whole brain, neural retina, fresh RPE sheets, and cultured RPE cells. RNA expression of the three known Xenopus melatonin receptor subtypes (MEL1A, 1B, and 1C) was determined by reverse-transcription/polymerase chain reaction (RT/PCR) amplification, followed by Southern hybridization with RNA probes. PCR-amplified cDNA encoding melatonin receptor subtypes 1B and 1C, but not 1A, were detected in reverse-transcribed RNA obtained from brain, neural retina and RPE. RPE cells grown in culture for two weeks also demonstrated 1B and 1C receptor RNA expression. This study suggests that RNA encoding the 1B and 1C melatonin receptor subtypes is expressed in the neural retina and RPE of Xenopus retina, and the expression persists in RPE cells when grown in culture. The expression of melatonin receptor RNA in the RPE may reflect a regulatory role for melatonin in some diurnal events that occur in this tissue, such as phagocytosis of photoreceptor outer segment membranes, and intracellular migration of pigment granules.

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Species referenced: Xenopus laevis
Genes referenced: mtnr1a mtnr1b mtnr1c rpe

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	Fig. 1. Expression of melatonin receptor subtypes in Xenopus brain and neural retina. Agarose gel of RTrPCR products amplified from Xenopus brain and neural retina RNA using primers based on the melatonin receptor 1A, 1B, and 1C sequences. PCR bands of the predicted size appear in the receptor 1B 408 bp and 1C 250 bp lanes, but not in the Ž. Ž. receptor 1A lane 411 bp . MW Ž . smolecular weight markers.
	Fig. 2. Expression of melatonin receptor subtypes in wild-type WT and Ž . albino Xenopus neural retina and retinal pigment epithelium RPE . Ž . Agarose gel of RTrPCR products amplified from WT and albino Xenopus neural retina and RPE RNA using primers based on the melatonin receptor 1A, 1B, and 1C sequences. In both strains of frogs, PCR bands of the predicted size appear in the receptor 1B 408 bp and 1C 250 bp Ž. Ž. lanes, but not in the receptor 1A lane 411 bp . MW Ž . smolecular weight markers.
	Fig. 3. Expression of melatonin receptor subtypes in Xenopus RPE. Upper panel: Agarose gel of RTrPCR products amplified from Xenopus neural retina Ž. Ž . NR , fresh RPE, and two week RPE cultures CRPE RNA, using primers based on the melatonin receptor MEL1A, MEL1B, and MEL1C sequences. PCR bands of the predicted size appear in the receptor MEL1B and MEL1C lanes but not in the receptor MEL1A lane. The MEL1B PCR band in the cultured RPE sample has a 2.5-fold higher intensity than the fresh RPE sample. Lower panel: Southern blot of the agarose gel in the upper panel, probed with MEL1B and MEL1C digoxigenin-labeled riboprobes. The left side of the blot was probed with the MEL1B riboprobe, and the right side of the blot was probed with the MEL1C riboprobe. The MEL1B riboprobe hybridizes only with MEL1B PCR bands, and the MEL1C riboprobe hybridizes only with MEL1C PCR bands, indicating that the probes are highly specific for their respective receptor subtypes.
	Fig. 4. Expression of MEL1B and MEL1C receptor subtypes and b-actin in fresh vs. cultured Xenopus RPE. Agarose gel of RTrPCR products amplified from Xenopus fresh RPE F , and two week RPE cultures C Ž. Ž . RNA, using primers based on the melatonin receptor MEL1B, MEL1C, and b-actin sequences. When normalized to b-actin band density, the MEL1B, but not the MEL1C receptor subtype exhibits a twofold increase in expression after grown in culture for two weeks.