Melatonin-binding in the frog retina: autoradiographic and biochemical analysis

• Published in: Investigative ophthalmology & visual science Vol. 27; no. 2; pp. 153 - 163
• Main Authors: Wiechmann, AF, Bok, D, Horwitz, J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD ARVO 01.02.1986; Association for Research in Vision and Ophtalmology
• Subjects: Animals; Autoradiography; Binding Sites - drug effects; Biological and medical sciences; Choroid - analysis; Choroid - metabolism; Circadian Rhythm; Darkness; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; Indoles - pharmacology; Light; Melanocytes - analysis; Melanocytes - metabolism; Melatonin - analogs & derivatives; Melatonin - analysis; Melatonin - metabolism; Melatonin - pharmacology; Pigment Epithelium of Eye - analysis; Pigment Epithelium of Eye - metabolism; Radioimmunoassay; Rana pipiens; Retina - analysis; Retina - metabolism; Retinal Ganglion Cells - analysis; Retinal Ganglion Cells - metabolism; Vertebrates: nervous system and sense organs; Frog; Amphibia; Rana pipiens; Retina; Salientia; Biological rhythm; Binding site; Circadian rhythm; Metabolism; Melatonine; Eye; Autoradiography; Vertebrata; Transmission electron microscopy; Pigments
• ISSN: 0146-0404; 1552-5783

Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.