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PubMed1985

Competition between retinal and 3-dehydroretinal for opsin in the regeneration of visual pigment.

Rhodopsin regenerated faster than porphyropsin in all preparations of bullfrog opsin, bullfrog rod outer segment membrane and cattle opsin. When opsin was incubated with excess amount of an equimolar mixture of 11-cis-retinal and 11-cis-3-dehydroretinal, the composition of the regenerated pigment was simply dependent on the ratio of regeneration rates of rhodopsin and porphyropsin. This result can provide a mechanism to account for the discrepancy in vitamin A1/A2 composition between the retina and the pigment epithelium. The property of opsin preferring retinal to 3-dehydroretinal may be one of the basic factors affecting vitamin A1/A2 visual pigment systems.

Animals↗

PubMed1984

Opsin-immunoreactive outer segments of photoreceptors in the eye and in the lumen of the optic nerve of the hagfish, Myxine glutinosa.

Opsin-immunoreactive sites in the eye and optic nerve of the hagfish, Myxine glutinosa, were studied by use of light-microscopic pre- and postembedding peroxidase-antiperoxidase or avidin-biotin-peroxidase techniques, and the immuno-electron-microscopic protein A-gold method. At the light-microscopic level, a strong opsin immunoreaction was obtained on the outer segments of the photoreceptor cells with sheep and rat antibodies against bovine (rhod)opsin. These outer segments were located in the marginal photoreceptor space and in follicles of the retina, as well as in the tubular lumen of the optic nerve. Ultrastructurally, two classes of outer segments can be distinguished; most of them exhibited a strong antiopsin reaction, while certain elements lacked immunoreactivity with the antisera employed. The protein A-gold particles marked opsin-immunoreactive sites on the photoreceptor membranes. The presence of opsin-immunoreactive material in the retina and optic nerve of the hagfish strengthens the view that this primitive eye lacking a cornea, lens and vitreous body is engaged in light perception. The morphological similarity between the eye and pineal tissue is discussed in connection with the absence of a pineal organ in this species.

Animals↗

PubMed1989

[The effect of anions on spectral properties of iodopsin and native cones in the frog retina (microspectrophotometric study)].

Absorption spectra of single outer segments of the frog Rana temporaria photoreceptors were registered. Effects of nitrate and chloride ions on spectral properties of cone and rod pigments were compared. These pigments proved to differ in structure of the native photoreceptor membrane and, therefore, in effect of hydrophile environment on the chromophore centrum. Substitution of chloride by nitrate ions led to the hypochromic shift of the cone absorption spectrum (20-25 nm) but does not affect the spectrum on case of rod pigment. The ionochromic behaviour of cone pigments resembles that of the light-sensitive halobacterium protein halorhodopsin, in native membrane. We suppose that the effect of anions on the chromophore centrum may be the cause of bathochromic shifts of absorption spectra of longwave-length retinal-containing pigments.

Animals↗

PubMed1990

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane.

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.

Amino Acid Sequence↗

PubMed1976

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

Interplay of phosphorylation and dephosphorylation in vision: protein phosphatases of bovine rod outer segments.

Two types of protein phosphatases were identified in carefully prepared bovine rod outer segments (ROS). Extraction of the ROS with a medium-salt buffer solubilized protein phosphatase activity that was mainly type 2A, since it was active toward phosphorylase a in the absence of divalent cations, was not retained by heparin-Sepharose, dephosphorylated the alpha-subunit of phosphorylase kinase faster that the beta-subunit, and was unaffected by inhibitor 2. Further extraction of the resulting membranes with a high-salt buffer solubilized additional phosphatase activity which was predominantly type 1, since it was retained by heparin-Sepharose and was blocked by inhibitor 2. The molecular mass of the type 2A phosphatase estimated by gel permeation chromatography on Superose 12 was 100 kDa, suggesting it may be the 2A2 form. Only the ROS type 2A phosphatase dephosphorylated opsin and rhodopsin efficiently. Concordant with this finding, the purified catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle dephosphorylated opsin efficiently, while the type 1 catalytic subunit isolated from this tissue was inactive. Together, the results suggest that the ROS type 2A protein phosphatase plays an important role in regenerating rhodopsin from the various phosphorylated species in vivo. The activity of the enzyme per retina (approximately 85 pmol of Pi released/min) is comparable to that of rhodopsin kinase (100 pmol of phosphate transferred/min).

Animals↗

PubMed1984

Opsin-induced experimental autoimmune retinitis in rats.

Experimental autoimmune retinitis has been induced in Lewis rats by injection of opsin in mycobacterial adjuvant and Hemophilus pertussis adjuvant. Clinical, histopathological and immunological parameters of the disease are reported. Two types of opsin were prepared from purified bovine retina outer segments, one type in Triton X-100 and the other in lithium dodecyl sulfate. Both preparations were free from S-antigen. Dodecyl sulfate-denaturated-opsin displayed lower antigenicity and pathogenicity than Triton-opsin. Triton-opsin (250 micrograms) induced moderate to severe non-granulomatous uveitis (predominantly retinitis) in 70% of the Lewis rats at the end of the second week after injection. The photoreceptor cell layer was destructed within a few days. This group displayed high responses to opsin in the lymphocyte transformation test. In view of observed histological features, the possible early involvement of vasoactive factors is discussed. Low opsin doses (50 or 100 micrograms) seldomly induced severe retinitis, while the incidence of mild pathology was low. Lewis rats appeared to be more susceptible for the development of experimental autoimmune retinitis than Wistar rats.

Animals↗

PubMed2005

Rod sensitivity of neonatal mouse and rat.

We have measured the sensitivity of rod photoreceptors isolated from overnight dark-adapted mice of age P12 (neonate) through P45 (adult) with suction-pipette recording. During this age period, the dark current increased roughly in direct proportion to the length of the rod outer segment. In the same period, the flash sensitivity of rods (reciprocal of the half-saturating flash intensity) increased by approximately 1.5-fold. This slight developmental change in sensitivity was not accentuated by dark adapting the animal for just 1 h or by increasing the ambient luminance by sixfold during the prior light exposure. The same small, age-dependent change in rod sensitivity was found with rat. After preincubation of the isolated retina with 9-cis-retinal, neonatal mouse rods showed the same sensitivity as adult rods, suggesting the presence of a small amount of free opsin being responsible for their lower sensitivity. The sensitivity of neonate rods could also be increased to the adult level by dark adapting the animal continuously for several days. By comparing the sensitivity of neonate rods in darkness to that of adult rods after light bleaches, we estimated that approximately 1% of rod opsin in neonatal mouse was devoid of chromophore even after overnight dark adaptation. Overall, we were unable to confirm a previous report that a 50-fold difference in rod sensitivity existed between neonatal and adult rats.

Aging↗

PubMed1988

A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Animals↗

PubMed1992

Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the isolated retina.

Hydroxylamine (NH2OH), a substance known to accelerate the decay of the metarhodopsin II bleaching intermediate of rhodopsin, was examined for its effect on the light-dependent phosphorylation of rhodopsin in the intact, isolated retina. Groups of ovine and bovine retinas that had been pre-incubated in darkness with 32P-inorganic phosphate were supplemented with NH2OH at final concentrations of up to 20 mM, then irradiated and further incubated in darkness. Rod outer segments isolated from the incubated retinas were subjected to SDS-PAGE; the gel was analysed for 32P (autoradiography) and protein (Coomassie staining), to determine the specific radioactivity (ratio of 32P and protein levels; '32P/opsin') of the opsin monomer band. Among retinas of a given experimental group, 32P/opsin declined with increasing concentration of added NH2OH. The relative value of 32P/opsin exhibited by controls (0 mM NH2OH) was halved in the presence of about 1-2 mM NH2OH, and was reduced by greater than or equal to 80% in the presence of 20 mM NH2OH. Supplementation of the retina with 20 mM NH2OH 1 min after irradiation caused relatively little reduction in 32P/opsin. The results indicate that the light-dependent phosphorylation of rhodopsin in situ is substantially inhibited by NH2OH at millimolar levels. The data are discussed in relation to previous electrophysiological studies that have examined rod dark adaptation in NH2OH-treated retinas.

Animals↗

PubMed1985

Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.

Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [14C]leucine was not significantly affected. In a companion single-label incubation, TM blocked the incorporation of [3H]leucine into rod outer segment (ROS) membrane Cl3CCOOH-precipitable material by 95% relative to controls. When retinas were labeled with [35S]methionine, fluorograms of NaDodSO4/polyacrylamide gels from control retinas and ROS membranes exhibited a heavily labeled component (apparent Mr approximately 37,000) which had the electrophoretic and antigenic properties of opsin, the rod visual pigment apoglycoprotein. TM-treated retinas exhibited a substantially reduced labeling of the Mr 37,000 component and incorporation of label into a component (apparent Mr approximately 32,000) not found in control retinas, which exhibited the electrophoretic and antigenic behavior of nonglycosylated opsin. ROS membranes isolated from TM-treated retinas contained neither the Mr 37,000 nor the Mr 32,000 radiolabeled species. Light-microscope autoradiograms of retinas incubated with [3H]leucine in the absence of TM exhibited bands of silver grains at the base of ROS, indicative of new membrane assembly. However, no such bands were observed in autoradiograms of TM-treated retinas. These results suggest that glycosylation of opsin is required for its incorporation into ROS membranes.

Animals↗

PubMed1996

Kinetics of rod outer segment phagocytosis by cultured retinal pigment epithelial cells. Relationship to cell morphology.

PURPOSE: To study phenotypic variation in primary cultures of rat retinal pigment epithelium (RPE) and to correlate cell morphology with rates of binding and ingestion of rod outer segments (ROS). METHOD: Replicate cultures were prepared using RPE cell sheets isolated with Dispase from Royal College of Surgeons normal (RCS rdy+ p+) and dystrophic (RCS p+) rats. Retinal pigment epithelial morphology was analyzed, and phagocytosis was assessed by fluorescence microscopy in cultures fixed at 2-hour intervals from 3 to 19 hours after continuous incubations with fluorescein isothiocyanate (FITC)-stained ROS. RESULTS: A wide range of RPE cell size, shape, and pigmentation was present at confluence; however, distinct morphologic subtypes were recognized, defined as types 1 to 3, and studied separately. In both normal and dystrophic cultures, the extent and rate of ROS binding varied with RPE phenotype. In normal cultures, highly spread pigmented binucleate cells (type 3) bound and rapidly ingested multiple ROS per cell starting at 3 hours and reached a peak at 9 hours. Lightly pigmented daughter cells (type 2) bound and ingested far fewer ROS per cell than did type 3 RPE, which had not divided. Patches of hexagonally packed cells with in vivo morphology (type 1) bound large numbers of ROS per cell only after prolonged (9- to 11-hour) incubations and ingested them synchronously. Comparison of normal versus dystrophic RPE subtypes 1 to 3 revealed the known ingestion defect in all three mutant phenotypes but indicated delayed ROS binding in type 2 and type 3 cells as well. CONCLUSIONS: Kinetics of ROS binding and ingestion differ markedly among phenotypic variants of RPE cells typically found in primary cultures at confluence. Thus, accurate quantitation requires comparison of equivalent microscopic fields or like RPE subtypes, and the heterogeneous responses of various RPE subtypes should be considered when interpreting phagocytic data obtained from entire cultures at a particular time.

Animals↗

PubMed1992

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.

Aging↗

PubMed1998

The role of taurine in osmotic, mechanical, and chemical protection of the retinal rod outer segments.

The ability of the photoreceptor cell to resist osmotic stress was examined by incubating isolated frog retina in medium of varying osmolality. An electron microscopic analysis of the rod outer segment following a severe hypoosmotic insult revealed connections between adjacent disks and between disk rims and the plasma membrane, which presumably provide mechanical stability to the rod outer segment. One surprising result was the extent of the damage incurred by the electrical signaling pathway of the photoreceptor cells subjected to a 50 mOsm insult; only the distal P111 component of the ERG remained unaffected. Thus, the rod outer segment is particularly resistant to osmotic-induced injury, presumably because of the effective osmoregulatory actions of taurine. Incubation of retina with tauret, retinylidentaurine, uncovered rose-like hexagonal structures on the surface of the rod outer segment. These structures purportedly consist of connections between disk rims and the plasma membrane of the rod outer segments. Based on the influence of tauret, it is likely that the calcium dependence of these channels is selective for retinoids. These data are discussed relative to taurine's role in the process of rhodopsin regeneration and in the protection of the rod outer segments against osmotic, mechanical and light induced damage.

Animals↗

PubMed1997

Porcine iris pigment epithelial cells can take up retinal outer segments.

This study investigates the ability of iris epithelial cells (IPE) to ingest rod outer segments (ROS) and compares the amount of phagocytosis of porcine RPE and IPE cells by the use of a pH sensitive fluorescent dye (carboxy SNAFL) at the light microscopic level. The dye allowed investigation of ingestion separately from binding of rod outer segments. In a second set of experiments, after exposing ferritin-labeled ROS to the cultured cells, phagosomes were also counted in electron microscopic sections. Additionally immunocytochemical staining was performed with IPE and RPE cells. Both cell types stained positive with polyclonal NaK-ATPase antibodies against the alpha 1 subunit from rat brain and kidney. The epithelial nature of the cultured cells was determined by monoclonal anti-human-cytokeratin antibodies. Moreover, the ultrastructure of the cells revealed high amounts of phagosomes smaller than 1 micron in diameter present in both RPE and IPE cells. The iron label of the phagosomes was determined by EELS spectra taken from individual phagosomes. Electron and light microscopic quantification shows that cultured IPE cells have 64% of the phagocytic capacity of the RPE with respect to phagosomes larger than 1 micron in diameter.

Animals↗

PubMed1994

RCS rat retinal rod outer segment membranes exhibit different cholesterol distributions than those of normal rats.

Royal College of Surgeons (RCS) rats exhibit an hereditary defect in phagocytosis of the tips of the photoreceptor cell rod outer segments (ROS) which leads to degeneration of the retinal visual cells. The lipid composition of outer segment membranes of these rats was analysed and compared to those of normal rats to determine whether there are differences between the normal and mutant rat ROS. The cholesterol distribution in ROS disk membranes from normal and RCS rats was investigated using a digitonin induced change in membrane density. Normal rat ROS disks varied in cholesterol to phospholipid mole ratio from 0.36 to 0.03. The disk membranes from RCS rats, however, do not exhibit the same marked cholesterol heterogeneity. The mean molar ratio of cholesterol to phospholipid in the disk membranes of normal rats is 0.11 while that found in the RCS rats is 0.14. The ROS plasma membrane of dystrophic rats also has a lower cholesterol to phospholipid ratio (0.20) than is found in the normal rat (0.40). The phospholipid headgroup composition of RCS disks and plasma membrane were determined. RCS disks were shown to differ from those of normal animals. The cholesterol content of ROS disks may be governed by the phospholipid composition.

Animals↗

PubMed1987

The influence of carbohydrates on the binding of rod outer-segment (ROS) disc membranes and intact ROS by the cells of the retinal pigment epithelium of the embryonic chick.

The role of carbohydrates in mediating the interaction of rhodopsin-containing membranes with retinal pigment epithelium (RPE) cells was investigated by studying the influence of various monosaccharides on their binding by RPE cells of the embryonic chick maintained in cell culture. Rod outer-segment (ROS) disc membranes were selected as a model rhodopsin-containing membrane system for these studies in view of their high concentration of rhodopsin and the relative purity with which they can be isolated. Disc membranes, frozen and thawed in order to expose the carbohydrate groups of rhodopsin which are oriented intraluminally in situ, were incubated with monolayers of RPE cells under various conditions, and the binding of the membranes by the cells was quantitated by radioimmunoassay for rhodopsin. Cell-membrane association was also verified by indirect immunofluorescence microscopy. The surface accessibility of the sugars in frozen-thawed discs was verified by succinyl concanavalin A-binding studies. From 15- to 20-fold increase in carbohydrate-reactive sites was obtained after freezing and thawing the discs. The RPE cell-membrane binding process was saturable, and time- and temperature-dependent. By means of competition studies carried out in the presence of high concentrations of various monosaccharides, and also by comparing the binding of disc membranes whose carbohydrate groups were either exposed (frozen-thawed) on the surface or inaccessible (native), it was concluded that the carbohydrates of rhodopsin, mannose and N-acetylglucosamine, were not involved in the interaction with the RPE. The possibility was also examined that enzymatically galactosylated rhodopsin might serve as a site for recognition by the RPE cell. The binding of ROS disc membranes modified in this manner was not enhanced, indicating that the presence of galactose groups on rhodopsin did not serve as a site for recognition by the RPE. The influence of monosaccharides on the binding of intact ROS by the RPE cells was also investigated. Similar to the results with the disc membranes, the process was not blocked by the presence in the incubation medium of high concentrations (up to 30,000-fold higher than that of rhodopsin) of mannose or GlcNAc, as with the disc membranes, or by glucose or galactose. Thus, from these studies it is concluded that a lectin-like carbohydrate-recognition process may not be involved in the interaction between rhodopsin-containing membranes and the RPE cells.

Animals↗

PubMed2006

Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: evidence of a light-dependent mechanism.

Photoreceptor cells contain rod outer segments (ROS) which are specialized light-sensitive organelles. The biological function of ROS is to generate a photoresponse, which occurs via the classic transducin-mediated pathway. Moreover, ROS undergo light-regulated membrane turnover and protein translocation whose mechanisms have not been fully elucidated to date. Phospholipase D (PLD) is a key enzyme involved in lipid signal transduction and membrane trafficking. We have previously reported that PLD activity is present in purified ROS (Salvador, G.A., Giusto, N.M., 1998. Characterization of phospholipase D activity in bovine photoreceptor membranes. Lipids 33, 853-860). We now demonstrate that ROS PLD activity is enhanced by phosphatidylinositol bisphosphate (PIP2) and cytosolic factors in a GTP dependent-manner. Western blot analysis demonstrates the presence of PLD1 isoform in purified ROS. In ROS obtained from dark-adapted retinas (DROS), PIP2-dependent PLD activity was higher than that observed in ROS obtained from light-adapted retinas (LROS). In addition, experiments carried out in the presence of C3 toxin inhibited PLD activity from DROS whereas pertussis toxin did not affect the enzyme activity. Western blot analysis demonstrates the presence of RhoA, a PLD upstream-regulator. Moreover, RhoA levels were higher in DROS with respect to those in LROS. The present study reports evidence of the involvement of the small G-protein, RhoA, in ROS PLD regulation. Our data strongly suggest that RhoA regulates ROS PLD activity under a light-dependent mechanism.

Adaptation, Ocular↗