Melatonin synthesis in the greenfrog retina in culture: I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis

Melatonin is synthesized in the pineal gland and the retina of vertebrates. Retinal serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels during the dark phase of the photocycle. In s ome vertebrates, these retinal NAT and melatonin rhythms are maintained in vitro. The aim of present work is to develop an eyecup culture system for t he greenfrog (Rana perezi), suitable to analyze the mechanisms of regulatio n of melatonin synthesis by simultaneous determination of NAT activity and melatonin release. The R. perezi eyecups released melatonin to the culture medium in a rhythmic manner at least over a 27-h period under photocycle co nditions. NAT activity and melatonin rhythms were similar to that observed in vivo under natural environmental conditions. Rana perezi retina exhibits a pronounced photosensitivity in vitro. Forskolin increased up to 2-fold t he NAT activity and 4-fold the melatonin production at any lighting conditi ons. The addition of the translation inhibitor, cycloheximide, to the mediu m reduced significantly both nocturnal NAT activity and melatonin release, suggesting that de novo protein synthesis is produced daily during darkness . Actinomycin D, a transcription inhibitor, needs a longer time of action, because pre-existing mRNA must be depleted before the inhibition of melaton in release can be observed. The eyecup culture system is highly sensitive t o light and chemical factors, which makes it particularly suitable as a mod el for the neurochemical analysis of melatonin biosynthesis in the retina o f Rana perezi.