Distribution of 3':5'-cyclic AMP and 3':5'-cyclic GMP in rabbit retina in vivo: selective effects of dark and light adaptation and ischemia.

Abstract

By use of highly sensitive radioimmunoassays, 3':5'-cyclic AMP (cAMP) and 3':5'-cyclic GMP (cGMP) were measured in individual layers of light-and dark-adapted rabbit retinas, and the effects of ischemia were determined. In lightadapted retinas, cGMP levels ranged 50-fold, with over 90% of the total concentrated in the photoreceptor cells. The layer of outer segments contained 95 gmol/kg of dry weight, or three times the concentration present in the remainder of the photoreceptor cell layers. By contrast, levels of cAMP varied only 4-fold; the lowest level (6 gmol/kg of dry weight) was found in the outer segment layer and the highest level (22 ;mol/kg of dry weight) in the inner segment layer of the photoreceptor cells. Dark adaptation elevated cGMP levels only in retinal layers containing photoreceptor cells, and the greatest proportional increase was observed in the synaptic layer of photoreceptor cells. Dark adaptation also caused increases of cAMP that were restricted to the outer plexiform and outer nuclear layers. Ischemia lowered cGMP levels, but only in retinal layers containing photoreceptor cells, and elevated cAMP levels, primarily in the inner layers of the retina. emia was studied. The findings demonstrate that the cyclic nucleotides are discretely localized in retinas and that both light exposure and ischemia produce regionally selective changes in their levels. MATERIALS AND METHODS Tissue Preparation. Randomly bred, 6-month-old pigmented rabbits were obtained locally. Light adaptation consisted of 1 hr of exposure to two 100-watt tungsten bulbs about 25 cm above the rabbits. After the photopic stimulation, the rabbits were killed by decapitation. The eyes were removed, and after various ischemic periods in room light, were quickly frozen in liquid N2 cooled to its freezing point by partial evaporation under reduced pressure. Consequently, the liquid N2 does not boil when the eyes are immersed, and freezing is quicker. Dark adaptation consisted of an overnight period in cages wrapped with black cloth and placed in a completely dark room. After decapitation in this room, the dark-adapted eyes were dissected free with the aid of an infrared light source and an infrared image converter, and after various periods, were frozen in the dark as above. Under the most ideal conditions it required 1 min (46-69 sec) to remove and freeze either the light-or dark-adapted eyes. All eyes were stored at -70°until sectioned. Tangential sections (6 ,m) of the retinas were cut at -20°and dried at -400 under reduced pressure. Sections were taken from the paracentral regions of the retina, and care was taken to avoid that portion underlying the myelinated fiber bundle. Paracentral areas of rabbit retina are poorly vacularized (9). Samples (0.03-0.9 ,g dry weight) were dissected from the individual retinal layers and weighed on a quartz-fiber balance (10). Stained, undissected retinal sections were used to help identify individual layers.Assay of cGMP and cAMP. The procedures used for extraction and radioimmunoas...

Photic Regulation of ArylalkylamineN-Acetyltransferase Binding to 14-3-3 Proteins in Retinal Photoreceptor Cells

“…Photoreceptor cAMP is regulated by light exposure, which decreases levels of the second messenger (Orr et al, 1976;DeVries et al, 1978;Cohen, 1982). In photoreceptor-enriched chick retinal cell cultures, light treatment at night dramatically decreased the cAMP levels and AANAT activity, and the effects of light on AANAT activity were reversed by incubating the cells with 8Br-cAMP or lactacystin (Ivanova and Iuvone, 2003).…”

Cytochemical demonstration of guanylate cyclase activity in retinal photoreceptors with special reference to changes under light and dark adaptation.

“…In the dark, the cyclic GMP level in the rod outer segments is high, and illumination causes a decrease in the level of cyclic GMP in the whole retina (11,25,29,35), in photoreceptor cells (36), and in isolated outer segments (21,49,50), apparently through activation of the degradation enzyme, cyclic GMP-phosphodiesterase (9,24,33,48) . In view of the evidence of these studies, cyclic GMP is considered to be one of the possible visual transmitters that could transfer information from the site of photon absorption in the disc membranes to the permeability mechanism in the plasma membrane (16,27).…”

“…In this respect, information on histological distribution of cyclic GMP and measurement of cyclic GMP directly in photoreceptors in the retina after variation of the environment would be useful. Previous studies have been carried out concerning layer-by-layer analysis of quick-frozen eyes (2,19,36) or immunohistochemical localization of cyclic GMP (37). The ultrastructural localization of the enzymes required for cyclic nucleotide synthesis and degradation has been a matter of continuing interest.…”

Molecular genetics of retinitis pigmentosa

“…Cyclic GMP is localized mostly in rods,95 and the cells of the inner retina do not contribute substantially to cGMP levels. 96 …”