Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4442-4445, December 1976 Biochemistry 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 HARRY T. ORR*, OLIVER H. LOWRY, ADOLPH I. COHEN, AND JAMES A. FERRENDELLIt Department of Pharmacology, Department of Ophthalmology, and Department of Neurology and Neurological Surgery (Neurology), Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, Missouri 63110 Contributed by Oliver H. Lowry, September 22, 1976 ABSTRACT By use of highly sensitive radioimmunoassays, emia was studied. The findings demonstrate that the cyclic 3':5'-cyclic AMP (cAMP) and 3':5'-cyclic GMP (cGMP) were nucleotides are discretely localized in retinas and that both light measured in individual layers of light- and dark-adapted rabbit regionally selective changes retinas, and the effects of ischemia were determined. In light- exposure and ischemia produce adapted retinas, cGMP levels ranged 50-fold, with over 90% of in their levels. the total concentrated in the photoreceptor cells. The layer of outer segments contained 95 gmol/kg of dry weight, or three MATERIALS AND METHODS times the concentration present in the remainder of the photo- Tissue Preparation. Randomly bred, 6-month-old pig- receptor cell layers. By contrast, levels of cAMP varied only 4- mented rabbits were obtained locally. Light adaptation con- fold; the lowest level (6 gmol/kg of dry weight) was found in the sisted of 1 hr of exposure to two 100-watt tungsten bulbs about outer segment layer and the highest level (22 ;mol/kg of dry 25 cm above the rabbits. After the photopic stimulation, the weight) in the inner segment layer of the photoreceptor cells. were removed, Dark adaptation elevated cGMP levels only in retinal layers rabbits were killed by decapitation. The eyes containing photoreceptor cells, and the greatest proportional and after various ischemic periods in room light, were quickly increase was observed in the synaptic layer of photoreceptor frozen in liquid N2 cooled to its freezing point by partial cells. Dark adaptation also caused increases of cAMP that were evaporation under reduced pressure. Consequently, the liquid restricted to the outer plexiform and outer nuclear layers. N2 does not boil when the eyes are immersed, and freezing is Ischemia lowered cGMP levels, but only in retinal layers con- quicker. Dark adaptation consisted of an overnight period in taining photoreceptor cells, and elevated cAMP levels, primarily cages with black cloth and placed in a completely dark in the inner layers of the retina. The effects of ischemia were wrapped greater in dark-adapted than in light-adapted retinas. These room. After decapitation in this room, the dark-adapted eyes results indicate that cGMP and cAMP levels in retina are in- were dissected free with the aid of an infrared light source and fluenced by the light adaptational state, that ischemia markedly an infrared image converter, and after various periods, were modifies these processes, and that the effects of both light ex- frozen in the dark as above. Under the most ideal conditions it posure and ischemia are regionally selective. required 1 min (46-69 sec) to remove and freeze either the light- or dark-adapted eyes. All eyes were stored at -70° until Previous studies have detected the existence of adenosine 3': sectioned. Tangential sections (6 ,m) of the retinas were cut at 5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic -20° and dried at -400 under reduced pressure. Sections were monophosphate (cGMP) in mammalian retina and have taken from the paracentral regions of the retina, and care was demonstrated that this tissue is uniquely rich in cGMP (1, 2). taken to avoid that portion underlying the myelinated fiber Dark adaptation increases cGMP in outer segments of mam- bundle. Paracentral areas of rabbit retina are poorly vacularized malian photoreceptor cells in vitro (3-7), and Fletcher and (9). Samples (0.03-0.9 ,g dry weight) were dissected from the Chader (8) have shown that cGMP levels are elevated in outer individual retinal layers and weighed on a quartz-fiber balance segments isolated from frog retina that has been dark-adapted (10). Stained, undissected retinal sections were used to help in vivo. Recent preliminary studies in our laboratory have re- identify individual layers. vealed that dark adaptation, in vivo, increases cAMP as well Assay of cGMP and cAMP. The procedures used for ex- as cGMP in mouse retina and that ischemia affects the content traction and radioimmunoassay of the cyclic nucleotides are of both nucleotides. Although there is indirect evidence that described in detail elsewhere (J. A. Ferrendelli, E. H. Rubin, a substantial portion of the cyclic GMP of the retina is contained H. T. Orr, D. A. Kinscherf, and 0. H. Lowry, submitted to in the outer segments of the photoreceptor cells, its distribution Anal. Biochem.). Briefly, the samples were extracted at room in the remainder of the retina and the distribution of cAMP temperature in l-,Ml volumes of 10% trichloroacetic acid under throughout the retina have not been well defined. This infor- mineral oil. Aliquots (0.9 ,ul) were then removed from the oil mation is essential for the eventual understanding of the role wells, placed in 6 X 50 mm culture tubes, and dried on a Virtis of cyciic nucleotides in retinal function, as well as for evaluating Bio-Dryer to remove the acid. The residue was dissolved in 50 the physiological relations of cGMP and cAMP systems. ,ul of H20, and the cyclic nucleotides were acetylated by the Using highly sensitive radioimmunoassays and histochemical method of Harper and Brooker (11) with 1.0,l of triethylamine techniques, we have measured the content of both cAMP and and 0.5 ,ul of acetic anhydride. The tubes were dried again and cGMP in individual layers of rabbit retinas that were either the residues dissolved in 30 Ail of Na acetate buffer, 50 mM, pH light- or dark-adapted, in vivo. In addition, the effect of isch- 6.0. Both cyclic nucleotides were then assayed by the ra- dioimmunoassay described by Steiner et al. (12) scaled down Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; cGMP, to a final volume of 52 Ail. guanosine 3':5'-cyclic monophosphate. The results shown in Figs. 1 and 2 are the averages from three * Present address: The Biological Laboratories, Harvard University, Cambridge, Mass. 02138. rabbits at 1 min of ischemia and two rabbits at the other isch- t To whom reprint requests should be addressed: Department of emic times. Within each retina, each layer was assayed three Pharmacology, Washington University School of Medicine, 660 S. to seven times; values for SEM ranged from 5 to 20% of the Euclid Ave., St. Louis, Mo. 63110. mean for each case. 4442 Downloaded by guest on October 2, 2021 Biochemistry: Orr et al. Proc. Natl. Acad. Sci. USA 73 (1976) 4443 OS IS ONL OPL INL IPL GANG OS IS ONL OPL INL IPL GANG RETINAL LAYER FIG. 1. cGMP and cAMP in the layers of light- and dark-adapted rabbit retina. The eyes were removed and frozen after about 1 min of ischemia. This is the fastest that eyes could be removed from decapitated, unanesthetized rabbits. Each point is the average for three eyes +SEM, except for the dark-adapted OPL layer (cGMP and cAMP), where the average of two eyes is presented, in which case the bars represent ranges. Abbreviations: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GANG, ganglion cell layer. Lines connecting individual points are placed to assist visualization of the data, but do not imply smooth gradients of cyclic nucleotide content between layers. A schematic representation of the neural connections in the retina is illustrated under the left panel of the figure. RESULTS dry weight in the dark. A significant (P < 0.05) rise in cAMP was also found in the outer nuclear layer. The remaining layers Distribution of cGMP and cAMP in light-adapted of the retina did not show significant (P > 0.2) elevations in retina cAMP after dark adaptation. In the most rapidly frozen light-adapted retinas, cGMP levels ranged 50-fold. The layers containing the photoreceptor cell Effect of ischemia on cGMP levels had levels (30 ,umol/kg of dry weight) at least 10-fold higher In both light- and dark-adapted rabbit retinas, prolonged than any of the remaining layers of the retina (except the outer ischemia lowered cGMP concentrations only in retinal layers plexiform layer, which is partly composed of photoreceptor cell containing the photoreceptors (Fig. 2). The cGMP content of elements) (Fig. 1). Within the photoreceptor layer, cGMP levels dark-adapted retina changed more with ischemia than that of were highest at the outer segments, where the concentration light-adapted retina. In dark-adapted retina, 3 min of ischemia (95 Atmol/kg of dry weight) was three times greater than layers lowered cGMP levels in every photoreceptor cell layer to levels containing the remainder of the cell. By contrast, the cAMP equal to or somewhat lower than in light-adapted retinas. The distribution in these same light-adapted retinas varied only over cGMP in outer segments decreased 30%, while in the remaining a 4-fold range (Fig. 1). All but two of the retinal layers had photoreceptor cell layers, it fell 70-75%. In light-adapted retinas cAMP concentrations between 10 and 15 jzmol/kg of dry with lower cGMP to begin with, only levels in the outer nuclear weight. The two exceptional layers were the outer segments (5.5 and outer plexiform layers were altered by ischemia.