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Activation of cGMP phosphodiesterase(PDE) of frog rod outer segments (ROS) by purified green rod pigment (GRP) was analyzed. GRP activated PDE in a similar manner to purified rhodopsin. This activation required illumination of the pigment and presence of GTP.
The effect of calmodulin on the order of lipids in rhodopsin-free and rhodopsin-containing membranes has been studied using spin-label electron spin resonance methods. Calmodulin, up to 10(-6)M, did not change the measured order of lipids in bilayer membranes containing only rhodopsin. However, for bovine rod outer segment disc membranes, which contain rhodopsin and other proteins, calmodulin induced a significant concentration and temperature dependent increase in the order of the membrane lipids. This suggests that the site of calmodulin binding is remote from rhodopsin itself, and the nature of the binding appears to be a membrane surface phenomenon.
A previous study has shown that the long and very long chain FA (VLCFA) content of the rat retina responds to changes in dietary n-6/n-3 ratio of the fat fed (1). The present study tested whether similar changes in these FA are associated with alterations in rhodopsin content and rhodopsin phosphorylation after light treatment. Weanling rats were fed diets containing 20% (w/w, 40% energy) fat with either high (4.8%, w/w) or low (1.2%, w/w) n-3 FA. After 6 wk of feeding, half of the animals in each group were exposed to light for 48 h at 350 lx or were kept in complete darkness. In the rod outer segment, the high n-3 diet treatment increased the level of 20:5n-3 and 22:6n-3 and reduced the levels of 20:4n-6 and 24:4n-6 in PC, PE, and PS. After the feeding of a high n-3 FA diet, total n-3 pentaenoic VLCFA from C24 to C34 increased in PC, whereas the n-6 tetra- and pentaenoic VLCFA decreased. No changes occurred in n-3 hexaenoic VLCFA regardless of the level of 22:6n-3 in the diet. After light exposure, animals fed a high n-3 FA diet showed reduction in 22:6n-3 as well as in n-6 and n-3 VLCFA in PC. FFA and TG fractions contained increased levels of both 20:4n-6 and 22:6n-3 after light exposure. Dark-adapted rhodopsin content and rhodopsin phosphorylation in the rod outer segment of rats fed the low n-3 FA diet were higher than in animals fed a high n-3 FA diet. After light exposure, animals fed the low n-3 FA diet lost more rhodopsin compared to animals fed the high n-3 FA diet, resulting in less phosphorylation of rhodopsin. Results indicate that the FA composition, rhodopsin content, and phosphorylation in visual cells is influenced by the dietary n-3 FA fed as well as by light exposure. The results also imply that 22:6n-3 may not be the precursor for synthesis of hexaenoic VLCFA.
RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment.
The structural and functional properties of bovine rhodopsin from rod outer segment disk and plasma membranes were compared by high performance liquid chromatography (HPLC), mass spectrometric analyses, and in vitro rhodopsin phosphorylation assays. Disk and plasma membranes separated by a ricin gold-dextran affinity perturbation method were treated with trypsin or cyanogen bromide, and the N-terminal and C-terminal rhodopsin peptides were isolated by immunoaffinity chromatography using antirhodopsin monoclonal antibodies coupled to Sepharose. Reverse phase HPLC chromatograms of the C-terminal and N-terminal peptides from disk and plasma membrane rhodopsin were found to be similar. Mass spectrometric, PicoTag, and hexose analyses of the tryptic 1-16 N-terminal peptides further indicated that the post-translational glycosylation of plasma membrane rhodopsin is identical to that of disk membrane rhodopsin. HPLC analysis of soluble peptides obtained from cyanogen bromide and tryptic digestion of immunoaffinity purified rhodopsin also indicated that no significant differences exist between disk and plasma membrane rhodopsin. Light-induced phosphorylation of rhodopsin in disk and plasma membranes were also compared using in vitro phosphorylation assays. Plasma membrane rhodopsin was found to undergo light-dependent, rhodopsin kinase catalyzed phosphorylation to the same extent as disk membrane rhodopsin. These results indicate that the bulk rhodopsin in rod outer segment plasma membranes appears to be identical to rhodopsin in disk membranes in regard to primary structure, post-translational glycosylation and light-dependent phosphorylation. On this basis, it is unlikely that the sorting of rhodopsin between disk and plasma membranes occurs by a mechanism based on differences in structural properties of rhodopsin.
We have studied sterol distribution in the retinal pigment epithelial (RPE) microvillous and outer segment disc membranes of rats with inherited retinal degeneration (RCS; RCS-p/+) and of normal genetic controls (RCS-rdy+, RCS-rdy+-p/+) by using the polyene antibiotic filipin, which binds specifically to 3-B-hydroxy-sterols, and freeze-fracture techniques. Retinas were perfusion-fixed, incubated with filipin in the same fixative, and prepared routinely for freeze-fracture electron microscopy. In the normal retina, the distribution of filipin binding sites on both RPE microvillous and outer segment disc membranes changes during development. Prior to outer segment elongation and the onset of phagocytosis (10 days postnatal), filipin sterol complexes are homogeneously distributed in both microvillous and outer segment membranes. With the onset of phagocytosis (2 weeks postnatal and later) filipin binding in both tissues forms a proximal-to-distal gradient, and binding sites decrease as distance from the cell body increases. In the normal RPE microvillous membranes, binding sites are numerous proximally and sparse on the distal tips. In the normal outer segment disc membranes, binding sites are often present on the basal discs, but are sparse on the intact apical discs prior to shedding. As the discs are cast off and engulfed by the RPE, however, filipin binding increases on both disc and phagosome membranes. In the dystrophic retina, the distribution of filipin binding sites differs from the normal. First, in the microvillous membranes, the proximal-to-distal gradient in filipin binding is rarely present at 2 weeks postnatal and becomes prominent only after the buildup of membranous debris has begun (3-5 weeks postnatal). Second, as the photoreceptors degenerate and the membrane debris disappears (4 months postnatal), filipin binding on the microvillous membranes becomes relatively sparse and homogeneous. Third, filipin binding on the intact disc membranes does not change with outer segment elongation, and numerous filipin binding sites are present on both apical and basal outer segment disc membranes. Fourth, large aggregates of filipin binding sites occupy the vast expanses of particle-free areas of debris membranes which accumulate between the photoreceptors and the RPE. These changes in the amount and distribution of filipin binding sites in the dystrophic retina add to the evidence that the disease process involves outer segment as well as RPE membranes and suggest that alterations in cholesterol distribution could contribute to the phagocytic defect.
Retinal detachment with oral dialyses and tears of the nonpigmented epithelium of the ciliary body sometimes accompanies aqueous cells and high intraocular pressure with its marked fluctuation. The aqueous cells consist predominantly of photoreceptor outer segments which obstruct aqueous outflow at the trabecular meshwork and induce high intraocular pressure as ghost cells do in ghost cell glaucoma. A new clinical entity with the combination of these signs, namely, rhegmatogenous retinal detachment with "photoreceptor outer segment glaucoma" is designated as Schwartz-Matsuo syndrome in this report. The detection of photoreceptor outer segments in the anterior chamber indicates that a communication between subretinal space and aqueous humor has been established by some mechanism. The new syndrome should be included in differential diagnoses of iritis and glaucoma.
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.
Docosapentaenoic acid (22:5 omega 6), a minor constituent (less than 4%) of photoreceptor outer segment membranes in all vertebrate species examined to date, comprises 23% of the fatty acids in total lipids from rabbit outer segment membranes. This fatty acid is a significant constituent of each of the three major phospholipid classes in these membranes. The levels of docosahexaenoic acid (22:6 omega 3), the major polyunsaturated fatty acid of most other vertebrate outer segments, was 20%. The sum of 22:5 omega 6 and 22:6 omega 3 in rabbit outer segment membrane lipids is similar to the amount of 22:6 omega 3 usually found in membranes from other vertebrate species.
PURPOSE: Photoreceptor outer segments are in a dynamic state of membrane addition and disposal. This study was undertaken to determine how a standardized period of retinal detachment and varying periods of reattachment affect the renewal process. METHODS: To investigate the effects that retinal detachment and reattachment may have on this process, the neural retina from 12 adult rhesus monkeys (Macaca mulatta) was detached from the overlying retinal pigment epithelium (RPE) by subretinal injection of a balanced salt solution. After a standardized detachment period of 7 days, the two tissue layers were reapposed. Animals were labeled with 3H-fucose and killed at times ranging from 3-150 days after reattachment. RESULTS: During the 7 day detachment period, the majority of rod outer segments (ROS) and cone outer segments (COS) degenerated, but inner segments remained intact. During the first week after reattachment, a rapid increase in rod and cone outer segment length occurred in the absence of disc shedding. This was accompanied by re-establishment of a modified morphologic relationship between the apical processes of the RPE and the regenerating outer segments. ROS and COS regained approximately 40% of their control lengths after a 2 wk reattachment period. By 30 days of reattachment, ROS had regained 72% of their normal length and COS had regained approximately 48%. After 150 days of reattachment, photoreceptor outer segment mean length was not statistically different from control areas. Autoradiographic results confirmed that new disc membranes were synthesized after reattachment. The rate of ROS membrane assembly was subnormal at reattachment time points up to 30 days. CONCLUSIONS: Retinal detachment leads to a reduction in photoreceptor outer segment absolute length and membrane assembly rates. Increasing time of retinal reattachment is positively correlated with an increase in outer segment absolute length and a corresponding increase in membrane assembly rates. This recovery pattern in eyes without underlying pathology and after a relatively brief detachment interval may represent the upper limit of the recovery process.
Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)
The objective of this study was to determine whether addition of dietary 20:4n-6 and 22:6n-3 to a conventional infant formula fat blend influences membrane long-chain and very-long-chain fatty acid composition, rhodopsin content, and rhodopsin kinetics in developing rat photoreceptor cells. The dietary fats were formulated based on the fat composition of a conventional infant formula providing an 18:2n-6/18:3n-3 ratio of 7:1 (SMA, Wyeth Nutritionals), which served as the control fat blend. This dietary fat blend was modified to contain 20:4n-6 [arachidonic acid (AA)], 22:6n-3 [docosahexaenoic acid (DHA)], AA + DHA, or an 18:2n-6/18:3n-3 ratio of 4:1 (alpha-linolenic acid). Dams were fed diets from birth, and rat pups were fed the same diet after weaning. Retinas and rod outer segments were prepared in the dark from pups at 2, 3, and 6 wk of age for fatty acid analysis of individual phospholipids, rhodopsin content, and rhodopsin disappearance kinetics after light exposure. Feeding AA + DHA in the diet increased 22:6n-3 levels in phosphatidylcholine and phosphatidylethanolamine. In phosphatidylcholine, total n-6 tetraenoic very-long-chain fatty acids and total n-3 pentaenoic and n-3 hexaenoic very-long-chain fatty acids increased after feeding AA and DHA, respectively. Developmental changes were characterized by a decrease in 20:4n-6 in the major phospholipids, whereas 22:6n-3 increased with age in rod outer segments. The highest rhodopsin content occurred in the retina of rats fed diets containing AA and/or DHA. The kinetics of rhodopsin disappearance after light exposure was highest in rats fed DHA at 6 wk of age. This study demonstrates that small manipulations of the dietary level of 20:4n-6 and 22:6n-3 are important determinants of fatty acid composition of membrane lipid and visual pigment content and kinetics in the developing photoreceptor cell.
The retinal pigment epithelium (RPE) performs a critical role in maintaining the integrity and renewal of the retinal system by clearing the shed photoreceptor outer segment debris through the process of phagocytosis. If phagocytosis is impaired, accumulation of the debris occurs leading to the degeneration of the retina. Phagocytosis, measured by polyvinyl toluene bead uptake is a normal cell activity exhibited by the embryonic retinal pigment epithelium even before complete differentiation of the neuronal layers. It is significant that embryonic pigment epithelium from either normal or delayed amelanotic (DAM) strain is competent in the process of non-specific particle uptake. In normal chickens non-specific phagocytic activity is observable as early as embryonic day 9 and continues through the rest of the developmental period and throughout adult life. The retinal pigment epithelium in DAM chickens exhibits early phagocytic activity at embryonic day 9 and then through day 16, but by day 19 this function is lost. This loss of phagocytic capacity in RPE of the DAM strain precedes and may be instrumental in the gradual retinal degeneration exhibited by these animals. Specific phagocytosis of rod outer segments (ROS) was examined in 4-week-old post-hatch normal and DAM RPE. Phagocytosis of ROS was greatly reduced in DAM RPE implying that the defect in the DAM is expressed in non-specific and specific phagocytosis. This study indicates that the DAM retinal system can now serve as an alternative and comparative model for studying different aspects of retinal degeneration.
BETA2/NeuroD1 is a basic helix-loop-helix transcription factor that is expressed widely throughout the developing nervous system. Previous studies have shown that BETA2/NeuroD1 influences the fate of retinal cells in culture. To analyze the effect of BETA2/NeuroD1 on the structure and function of the retina, we examined a line of BETA2/NeuroD1 knock-out mice that survives until adulthood. At 2-3 months of age, homozygous null mice showed a 50% reduction in rod-driven electroretinograms (ERGs) and a 65% reduction in cone-driven ERGs. ERGs measured from knock-out mice that were >9 months of age were undetectable. At 2-3 months, the number of photoreceptors in the outer nuclear layer was reduced by 50%. In addition, electron microscopy showed that the surviving photoreceptors had shortened outer segments. The number of cones labeled by peanut agglutinin was decreased 50-60%. By 18 months, retinas from null mice were completely devoid of photoreceptors. There appeared to be few changes in the inner retina, although BETA2/NeuroD1 is expressed in this area. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining revealed a dramatic increase in cell death, peaking at approximately postnatal day 3 and continuing into adulthood. No defects in cell birth were detected using bromodeoxyuridine staining. Our results reveal that BETA2/NeuroD1 not only plays an important role in terminal differentiation of photoreceptors but also serves as a potential survival factor. Loss of BETA2/NeuroD1 results in an age-related degeneration of both rods and cones.
Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE.
Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methylation reaction in vitro, the peptide Leu-Ala-Arg-Tyr-Lys-Cys (LARYKC) and its S-isoprenylated and S-alkylated derivatives were synthesized and assayed as methyl-accepting substrates with subcellular fractions of rat tissues including liver microsomal membranes. While little or no peptide-specific methyltransferase activity was detected in the latter preparation using the unmodified hexapeptide, the C10, C15, and C20 isoprenylated derivatives were substrates with Km values of 389 microM for S-geranyl-LARYKC, 2.2 microM for S-farnesyl-LARYKC, and approximately 10.9 microM for S-geranylgeranyl-LARYKC. The methyl-acceptor activities of a variety of n-alkyl S-derivatives of LARYKC (C8, C10, C13, C15) were also tested; all of these compounds were poorer substrates than the S-geranyl derivative. This enzyme activity uses S-adenosyl-L-methionine as the methyl donor (Km = 2.1 microM) and can be inhibited by S-adenosylhomocysteine (Ki = 9.2 microM), a product of the methylation reaction. The S-farnesyl-LARYKC peptide can inhibit the carboxyl methylation of bovine retinal rod outer segment membrane proteins that was previously shown to occur at the alpha-carboxyl group of C-terminal cysteine residues, demonstrating that the same enzyme can methylate both peptides and proteins. These results suggest that the methyl esterification of proteins containing a C-terminal -CXXX sequence requires not only the removal of the 3 terminal amino acids, but the isoprenylation of the sulfhydryl group as well.
The protease inhibitor, leupeptin, has been previously shown to cause an accumulation of lysosomally associated intracytoplasmic dense bodies resembling lipofuscin when administered intraventricularly to the brains of young rats (Ivy et al., Science, 226:985, 1984). These findings support the idea that lipofuscin formation during normal aging involves perturbed proteolytic degradation. In the current study, we delineate more precisely the proteolytic mechanisms in question and examine the effects of protease inhibition on other tissues and species. Four sets of experiments were done. In the first, rats were administered leupeptin (L; an inhibitor of cysteine and some serine proteases), E-64C (a cysteine protease inhibitor), chloroquine (C; a lysosomal enzyme inhibitor), aprotinin (A; a serine protease inhibitor) or physiological saline (S) into the lateral ventricle of the brain at a constant rate for two weeks using an osmotic mini-pump. In the second set, beagle dogs were administered L or S intraventricularly in a similar fashion. In the third set, young rats received intraocular injections of L, E64C, C, A or S from 1 to 9 days at 24 hr intervals. In the fourth set, young rats and mice were administered L, E-64C or S intraperitoneally via an osmotic mini-pump for 2 or more weeks. The tissue of all animals was processed for light and electron microscopy as well as for fluorescence analysis. In rat brain cells (especially hippocampus and cerebellum), substances which by morphological, histochemical and fluorescence emission criteria resemble lipofuscin, accumulated following L, C or E64C, but not A or S treatment. A similar buildup of lipofuscin-like substance occurred in canine brain following L but not S infusion. In retinal pigment epithelial cells, undigested rod outer segment discs accumulated after L, C or E-64C and possibly A, but not after S injections. With increasing survival times, the accumulated substances became morphologically more similar to lipofuscin. Cells of liver (both hepatocytes and non-parenchymal cells) and kidney (mainly renal tubule cells) also accumulated substances resembling lipofuscin in response to L but not S infusion; other internal organs were differentially affected. These results demonstrate 1) that specific inhibition of cysteine proteases is sufficient to cause accumulation of lipofuscin-like substances in brain and retina and 2) that several species and organ systems are similarly affected by protease inhibition. Taken together, these findings are consistent with the idea that defective or decreased proteolysis by lysosomal cysteine proteases is responsible for lipofuscin accumulation during normal aging.
Ultrastructural features of the retinal pigment epithelium and photoreceptor cells were studied in vitiligo (C57BL/6 mivit/mivit) mice. Eyes from 12-day- to 56-week-old animals were analysed. Abnormal photoreceptors were seen in 12-day-old mice. By 3 weeks malformed outer segments were evident in the posterior and equatorial retina, but normal photoreceptors were present in the periphery. By 28 weeks, a marked gradient in cell loss was evident, with a progressive increase in cell viability along the posterior-peripheral axis. Viable intact photoreceptors were still present in the peripheral retina of 56-week-old mice. Melanosome content varied between adjacent pigment epithelium cells in both the posterior and peripheral retina. In the choroid, however, a steep posterior-peripheral gradient in melanosome content was evident with highest pigmentation in the periphery. In the optic nerve head region abnormal development of photoreceptors was correlated with proliferation of abnormal pigment epithelium cells. Accumulation of rod outer segment debris in the posterior and peripheral subretinal space preceded photoreceptor cell death. Short pigment epithelial microvilli without proper attachment to photoreceptors are suggestive of alterations in pigment-epithelium-photoreceptor interaction, which might affect photoreceptor differentiation and phagocytosis of rod outer segments by pigment epithelium cells.