EFFECT OF LIGHT ON GANGLIOSIDES FROM CALF RETINA AND PHOTORECEPTORS

—The gangliosides of the whole calf retina and the rod outer segments have been analysed. This has been done in two functional states: before and after stimulation by light. After exposure to light no statistically significant change in the gangliosides of the whole retina was observed, but a 40 per cent increase in concentration was found in the rod outer segments. This difference was apparent only when using the same batch of rod outer segments. The major ganglioside in the whole calf retina is G_D3 which accounts for 46 per cent of the total. Three other gangliosides G_D1a, G_D1b and G_T1 are quantitatively important, each being between 12 and 16 per cent. G_Q1, G_M1, and G_M3 are minor constituents. In contrast to the chicken retina, G_M2 was not detected. The ganglioside N-acetylneuraminic acid of the rod outer segments accounts for only 1 per cent of the gangliosides of the whole retina. The composition of the gangliosides in the rod outer segments is essentially the same as that of the whole retina. No difference in the relative proportion of the gangliosides of either the rod outer segments or the whole retina was observed after exposure to light.     

Distribution of rhodopsin and retinochrome in the squid retina

The cephalopod retina contains two kinds of photopigments, rhodopsin and retinochrome. For many years retinochrome has been thought to be localized in the inner segments of the visual cells, whereas rhodopsin is in the outer segments. However, it is now clear that retinochrome can be extracted also from fragments of outer segments. In the dark-adapted retina of Loligo pealei retinochrome is distributed half-and-half in the inner and outer segments. Todarodes pacificus contains much more retinochrome than Loligo, and it is more abundant in the outer than in the inner segments. The outer segments of Loligo contain retinochrome and metarhodopsin in addition to rhodopsin, whether squids are kept in the dark or in the light. But there is extremely little metarhodopsin (about 3% of rhodopsin) even in light-adapted eyes. The inner segments contain only retinochrome, and much less in the light than in the dark. On the other hand, retinochrome in the outer segments increases markedly during light adaptation. These facts suggest the possibility that some retinochrome moves forward from the inner to the outer segments during light adaptation and there reacts with metarhodopsin to promote regeneration of rhodopsin.     

Whorl-like outer segments in the retina of the mole (Scalopus aquaticus)

The retina of the common mole, Scalopus aquaticus, has been studied with the transmission electron microscope. Structures examined include: pigment epithelium, outer and inner segments of the sensory cells, and synaptic ribbons of the outer plexiform layer. Rods and cones described in species by previous light microscopic studies are not seen with electron microscopic techniques. Instead, the mole retina contains peculiar outer segments consisting of whorls of membranes. These whorls have some similarities to dystrophic retinas of several vertebrates. The possibility of their being caused by extraordinary light exposure is discussed, also. The appearance of the sensory cells suggests that they are functional receptors.     

Carotenoids in the retina and lens: possible acute and chronic effects on human visual performance

(MP) that is composed of the hydroxy-carotenoids lutein and zeaxanthin. Although it appears that all humans have some quantity of these pigments within their retina, foveal concentrations tend to vary quite dramatically. This wide individual variability has prompted questions regarding possible functional consequences. At least two major nonexclusive hypotheses regarding the function of MP have been proposed. The "protection hypothesis" has received the most attention and is based on the possibility that MP could reduce the cumulative effects of damage due to light and oxygen and retard the development of age-related eye disease. The "acuity hypothesis" states that MP could improve visual resolution by absorbing short-wave light, which is easily scattered and poorly focused. In this article, we review evidence that lutein and zeaxanthin could improve human visual performance through both acute optical effects at the site of the retina and by maintaining health and functional integrity of the retina and crystalline lens.     

Metabolism of glucose and reduction of retinaldehyde in retinal photoreceptors

—The capacity of a variety of substrates to support reduction of retinaldehyde to retinol by preparations of outer segments from photoreceptor cells of bovine retina, the requirement for supplements, the effect of iodoacetate, and the stoichiometry of the metabolism of glucose and the reduction of retinaldehyde have been studied. Metabolism of glucose supported reduction of retinaldehyde by preparations of outer segments at approximately the rate observed for the intact retina. Throughout the physiological range of pH, the dehydrogenase reactions of the pentose cycle in outer segments provided virtually all of the reduced pyridine nucleotides that were utilized for reduction of retinaldehyde. It was concluded that reduction of retinaldehyde during light adaptation is exclusively dependent upon NADPH.     

Synthesis of nitric oxide in the bovine retina.

In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina.     

Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments

Phosphoinositides of chick and rat retina were labelled with [3H]inositol. Exposure of retinal preparations to light for 30 s caused loss of labelled phosphatidylinositol 4,5-bisphosphate and to a smaller extent of the other phosphoinositides. Similar light-induced changes were seen when rod outer segment preparations were used and, when these were illuminated in calcium-free media, phosphatidylinositol 4,5-bisphosphate was the only lipid affected. No inositol 1,4,5-trisphosphate was seen after either 30 s or 5 s of illumination of retina or 30 s illumination of rod outer segments. It is concluded that this compound plays no direct part in vertebrate photoreceptor light transduction, though phosphoinositide metabolism might relate to adaptation mechanisms.     

Superoxide dismutase of bovine and frog rod outer segments.

Bovine rod outer segments (ROS) contain soluble superoxide dismutase (SOD) which from cyanide sensitivity and electrophoretic mobility appears identical to CuZn SOD of erythrocytes. Enzyme activity of ROS extracts is 200–400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activate lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide.     

Evidence that light modulates protein nitration in rat retina

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

Rhythmic daily shedding of outer-segment membranes by visual cells in the goldfish

Goldfish were placed on a daily light cycle of 12 h light and 12 h darkness for 18 days or longer. The visual cells and pigment epithelium of the retina were then examined by microscopy at many intervals throughout the cycle. Goldfish rods and cones follow a rhythmic pattern in eliminating packets of photosensitive membranes from their outer segments. Rods shed membranes early in the light period. The detached membranes are ingested by pigment epithelial cells or by ameboid phagocytes, which degrade them during the remainder of the light period. Cones discard membranes from the ends of their outer segments early in the dark period. During the next several hours, this debris is digested by the pigment epithelium or by ameboid phagocytes. Thus, the disposal phase of the outer-segment renewal process is similar in rods and cones, but is displaced in time by about 12 h. There is evidence that this daily rhythm of membrane disposal in rods and cones is a general property of vertebrate visual cells.     

Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy

Retinochrome is readily reduced by sodium borohydride into an N-retinyl protein that emits visible fluorescence upon irradiation with near-ultraviolet light. Rhodopsin is also converted to a similar fluorescent product, but only when denatured with formaldehyde before reduction. Based upon this difference, retinochrome was discriminated from rhodopsin on frozen sections. The distribution of these two photopigments in various photosensitive tissues was examined by means of epifluorescence microscopy. In the octopus retina (Octopus vulgaris), the yellow-green fluorescence of reduced retinochrome was observed in both the basal regions of the outer segments and throughout the inner segments of the visual cells, while the fluorescence of reduced rhodopsin was restricted to within the rhabdomal layer of the outer segments. In the squid parolfactory vesicles (Todarodes pacificus), rhodopsin was present in the central lumen, which contains the distal processes of the photoreceptor cells, while retinochrome was detected in the myeloid bodies scattered within the vesicular wall. In the slug retina (Limax flavus), rhodopsin was found in the microvilli, and retinochrome appeared to be concentrated in the photic vesicles of the visual cells.     

A2E and blue light in the retina: the paradigm of age-related macular degeneration

The photoreceptors in the retina, designed to initiate the cascade of events which link the incoming light to the sensation of 'vision', are susceptible to damage by light, particularly blue light. The damage can lead to cell death and diseases. The turnover of retinal, an essential element of the visual process, is the basis of the events that lead to damage. Free retinal, absorbing in the blue region of the visible spectrum, is phototoxic, and is a precursor of the (photo)toxic compound A2E, which specifically targets cytochrome oxidase and thereby induces cell death by apoptosis. Cell death induced by A2E in the dark is prevented by negatively charged phospholipids. The blue light-filtering molecules lutein and zeaxanthin are tailor-made substances protecting the retina. In vitro, they protect cytochrome oxidase against the permanent damage caused by A2E in combination with light. These novel findings should enable us to prevent or cure the dry form of age-related macular degeneration, the leading cause of severe visual impairment in humans living in developed countries.     

Microchemical analysis of retina layers in pigmented and albino rats by Fourier transform infrared microspectroscopy

Fourier transform infrared (FT-IR) microspectroscopy is a powerful technique that can be used to collect infrared spectra from microscopic regions of tissue sections. The infrared spectra are evaluated to chemically characterize the absorbing molecules. This technique can be applied to normal or diseased tissues. In the latter case, FT-IR microspectroscopy can reveal chemical changes that are associated with discrete regions of lesion sites, which can provide insights into the chemical mechanisms of disease processes. In the present study, FT-IR microspectroscopy was used to analyze sections of retina from normal (pigmented) and albino rats. The outer segments of retinas from pigmented animals were found to have unusually strong absorption values for C&z.dbnd6;C-H unsaturation and carbonyl functional groups. Docosahexaenoic acid (DHA), a major constituent of lipids in the outer segments, also had particularly high absorption values for these functional groups, which suggests that it is responsible for those enhanced absorption values. Absorbance values for the unsaturation and carbonyl functional groups were substantially reduced in the outer segments of retinas from albino animals. This finding, together with data from other studies on light-induced oxidative events in the retina, indicates a loss of DHA by a light-induced mechanism in albino animals. The outer nuclear layer had strong absorbance values for H-C-OH and P&z. dbnd6;O functional groups, which is likely due to the sugar phosphate backbone of DNA. The outer and inner plexiform layers were found to contain greater concentrations of CH(2) and C&z.dbnd6;O functional groups than the outer and inner nuclear layers, which is due to the high concentration of synaptic connections in the former layers. In summary, FT-IR microspectroscopy revealed a unique chemical profile in the outer segments compared to other retinal layers, and this profile was altered in albino animals.     

Morphology of the retina in deep‐water fish Nezumia sclerorhynchus (Valenciennes, 1838) (Gadiformes: Macrouridae)

Using light microscopy, we examined the retina of benthopelagic fish Nezumia sclerorhynchus. Although the retina is typical of other vertebrates, having three nuclear and two synaptic layers, it presents some features associated with the animal's deep‐sea habitat. A stratum argenteum containing iridescent crystals is located in the choroid. The pigment cell layer shows bulky cells filled with melanin granules but without the typical apical processes. The visual cells, consisting of a big population of rods, are arranged in several banks. No cones were observed. The outer segments are very long and cylindrical, and the inner segments are constituted by a small ellipsoid at the proximal end. The outer nuclear layer contains several rows of oval nuclei, and the spherules in the outer plexiform layer have less regular outlines than nuclei. The inner retina is characterized by very large horizontal cells, and presumable bipolar and amacrine cells separated by large spaces that are occupied by neuronal processes. Finally, the low density of ganglion cells produces a thin nerve fibre layer. The results of this study suggest that the retina of Nezumia sclerorhyncus exhibits high visual sensitivity and that vision is a sense that plays an important role in its behaviour.     

Post-natal maturation of the retina in the albino rat. I. The pigment epithelium

The Authors studied the postnatal development of the retinal pigment epithelium in the albino rat, in order to elucidate its morphological and functional evolution, correlated to the numerous functional roles played in Vertebrates (Scheme 1). At birth, epithelial cells show few cytoplasmic organules and the apical surface provided of small depressions. From the third to the fifth postnatal day the first apical microfolds surround the depressions. From the seventh to the ninth day inner segments develop, whilst the apical surface of the epithelial cells is covered by many finger-like microfolds. During the eleventh postnatal day the buds of the outer segments and many lamellar microfolds can be demonstrated. During the sixteenth day the retina reaches its adult morphology. It is therefore well-evident that birth, similarly to many other Vertebrates, is not the last step, but only a moment, in the development of the retina: this process is completed only during postnatal life, when environmental light is able to stimulate every ocular structure.     

Morphological changes in the parr masou salmon Oncorhynchus masou retina in experimental change of geomagnetic field

Topography of photoreceptor cells in young salmon Oncorhynchus masou retina, their properties and morphology of cellular organelles in external and internal segments of photoceptors have been first described. Morphological changes of retina cells were analysed in day and night time, and also in the experiment for indemnification of geomagnetic field (GMF) in the body of the aquarium. A comparison of retina structure in fishes of night and day time controls with that in experimental fishes has shown that the external cone segments in the latter occupy, in relation to the external limiting membrane, an intermediate position, characteristic of retinae exposed to twilight lighting. It is supposed that GMF indemnification was equivalent to weak light pulse, which, however, could considerably change melatonin production by retina photoreceptor cells. Thus, at experimental indemnification of GMF, retina sensitive cells demonstrate typical retinomotor response. Some ultrastructural changes in retina cells were also detected, in particular, size changes in ribbon synapses in rod and cone terminations. In addition, nematosomes appeared in the internal nuclear layer, and in the spinules, i.e. digitiform invaginations of terminal dentrites of horizontal cells into cone nervous terminations, the quantity of an electron dense material was noticeably magnified in comparison with a night control. The noted changes testify, in our opinion, to essential modifications in metabolic processes of retina photoreceptors under effect of GMF variations, in particular, to changes in retinal melatonin synthesis.     

The photoreceptors of the west indian manatee

Using light and electron microscopy we found that the manatee retina has both rodlike and conelike photoreceptors in accord with its diurnal behavior pattern. Outer segment disks in both cell types appear to be enclosed along most of their length within the plasma membrane. The synaptic terminals are simple, with small, superficial postsynaptic contacts. The cones have long inner segments, short, conical outer segments and terminals with numerous synaptic ribbons and deeply embedded postsynaptic elements. There are two cone subclasses that may subserve color vision. Morphometry shows that there are more ganglion cells of small size ventrally, and that the thickness of the nerve fiber layer there is reduced, suggesting the presence of a specialized visual area in the retina. However, there were no pronounced differences in cone cell density in any of the regions examined.