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Results for “Rod Cell Outer Segment”

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PubMed1992

Retinal pigment epithelial cell transplantation in RCS rats: normal metabolism in rescued photoreceptors.

Photoreceptor cells in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy can be rescued by the transplantation of normal, wild-type retinal pigment epithelial (RPE) cells, if done before photoreceptor cell death. In the present study, we have examined several metabolic features of rescued photoreceptors and transplanted RPE cells at 2.3-3.3 months after transplantation. Rescued photoreceptors with a structurally normal RPE interface showed a rod outer segment renewal rate similar to that of normal control rats of 2.2 microns day-1, as measured in autoradiograms. Rod outer segment disc shedding had values indistinguishable from those in normal controls, as measured by the number of phagosomes in the transplanted RPE cells both during the burst of disc shedding soon after the onset of light in the morning and during the middle of the light cycle when disc shedding is low. The interphotoreceptor matrix, which is synthesized by both photoreceptors and the RPE, was distributed normally in the regions where normal-appearing photoreceptors were present underlying normal, transplanted RPE cells. Thus, the rescued photoreceptors show normal metabolic rates and normal interactions with the RPE in each of the parameters examined. These findings, combined with the previous demonstration of opsin and Na+,K(+)-ATPase expression by the rescued photoreceptors, support our interpretation that the surviving, normal-appearing photoreceptors may function normally. Moreover, transplantation of normal RPE cells reversed pathological changes in the photoreceptors that had already occurred by the time of transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The phagocytosis of ROS by RPE cells is not inhibited by mannose-containing ligands.

We have examined the ability of mannose and the mannose-rich ligands, mannan and mannosylated BSA, to inhibit the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. Mannose, at concentrations up to 0.25 M, had no effect on either the binding or the ingestion of ROS. At concentrations above 0.25 M, the cells were rounded and showed detachment from the substrate, and phagocytosis was markedly inhibited. Neither mannan (2 mg ml-1), nor mannosylated BSA(0.8 mg ml-1), affected the phagocytosis of ROS. These results suggest that the phagocytosis of ROS is probably not mediated by a mannose receptor on the surface of the RPE cells.

Animals↗
PubMed1999

Melanin granules of retinal pigment epithelium are connected with the lysosomal degradation pathway.

Melanosomes are closely related to lysosomes and lipofuscin granules. This paper indicates the potential involvement of lysosomal degradation processes in retinal pigment epithelial (RPE) cells. RPE cells cultured on Bruch's membrane and choroid were fed with indigestible latex beads. The RPE cells from this preparation were treated with chloroquine to investigate whether membrane swelling, typical for lysosomes under this condition, can be induced in melanosomes. To investigate the fate of indigestible material associated with rod outer segments (ROS), gold-labeled ROS were injected transsclerally into the subretinal space of Long Evans rats using a 32 gauge Hamilton syringe. The degradation of labeled ROS was observed after 5 and 12 days by electron microscopy. The following results were observed. Latex particles fuse with the melanin granules of the RPE. Following chloroquine treatment, the membranes of melanin granules fused, and formed large clusters and vacuoles. Gold granules were detected inside both early stage melanosomes and mature melanin granules of the RPE cells 5 and 12 days following subretinal injection of the labeled ROS. Higher numbers of gold granules were predominantly found in immature melanosomes containing still melanofilaments and in small fused mature melanin granules. In conclusion the effect of chloroquine clearly demonstrates that the melanosomes possess active proton pumps which is typical for lysosomes. In RPE cells stressed by overload with rod outer segments or by ingestion of undegradable material (latex beads, gold particles), fusion of these phagosomes with melanosomes of different maturity is more a general rule than an exception. Therefore, melanosomes are connected to lysosomal pathways in RPE cells.

Animals↗
PubMed1993

Molecular cloning, functional expression and mRNA analysis of human beta-adrenergic receptor kinase 2.

In the present study the cDNA of human beta ARK2 was cloned using both PCR and cDNA library screening, subcloned into an expression vector and transiently expressed in COS7 cells. The expressed kinase activity was approximately 40% as efficient as human beta ARK1 in phosphorylating bovine rod outer segments in vitro. Northern blot analysis of human and bovine mRNA revealed a species-specific pattern of multiple hybridization bands, with two major transcripts in human rather than one in bovine. High levels of mRNA expression were found in peripheral blood leukocytes.

Amino Acid Sequence↗
PubMed1991

ROS ingestion by RPE cells is turned off by increased protein kinase C activity and by increased calcium.

The activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) rapidly inhibits the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. PMA, at a concentration between 3.3 and 10 nM, blocks ROS ingestion by 50%, but does not inhibit the binding of ROS. The Ca2+ ionophore, A23187, also inhibits ROS phagocytosis, with an IC50 of about 0.5-1.0 microM and interferes with the ability of RPE cells to bind ROS. The effects of both of these drugs are reversible after drug washout. When PMA and A23187 are applied to cells consecutively, the effects are additive. These results suggest either that PMA and A23187, act upon the same proteins in the pathway which controls ROS ingestion, or that A23187 affects phagocytosis at the ROS binding level, while PKC affects steps further along the ingestion path. The effect of this process is to shut down the ingestion of ROS, as is seen during the prolonged feeding of ROS to RPE cells in culture.

Animals↗
PubMed1982

Vitamin A transport between retina and pigment epithelium--an interstitial protein carrying endogenous retinol (interstitial retinol-binding protein).

We have demonstrated and partially characterized an interstitial retinol-binding protein (IRBP) confined to bovine interphotoreceptor matrix (IPM). The native protein is a concanavalin A-binding glycoprotein with a mol. wt of 260 k as measured by gel-filtration and size-exclusion high-performance liquid chromatography. On SDS-gels, its mol. wt is 140-145 k. Since the protein is glycosylated, this value is probably too high. Hence, the native protein may be a dimer consisting of two identical subunits. The endogenous ligand has been analyzed by high-performance liquid chromatography--it consists mainly of all-trans retinol. Occasionally, retinal and 11-cis retinol are also associated with it. The amount of retinol bound to IRBP increases when the eyes are illuminated. The total binding capacity was estimated to represent 4-5% of the retinol released from a total rhodopsin bleach. We have established that, like serum retinol-binding protein, IRBP can be also bind retinoic acid, although it has not been established that retinoic acid is an endogenous ligand. The fluorescence emission lambda max for IRBP with its native ligand is at 470 nm and the excitation lambda max for this fluorescence is at 333 nm. Other retinoid carriers in the interphotoreceptor matrix have molecular weights of about 15 and 33 k. These probably correspond to cellular retinol- and retinal-binding proteins, respectively. Since both proteins have been identified in the pigment epithelium and retina cytosols, their presence in the IPM could be a result of cell damage. We conclude that interstitial retinol-binding protein is the best candidate for a transport protein carrying retinol between the rod outer segments and the pigment epithelium.

Animals↗
PubMed1978

Phagocytosis of light- and dark-adapted rod outer segments by cultured pigment epithelium.

Pigment epithelial cells in culture retain their ability to phagocytize rod outer segments. These cells phagocytize rod outer segments isolated from light-adapted rats, or from dark-adapted rats killed after the time at which the lights would normally be turned on. However, they phagocytize for fewer rod outer segments prepared in the dark from the retinas of rats killed before the onset of the normal light cycle. Phagocytosis of dark rod outer segments is variable, but that of light outer segments is reproducible. It is postulated that the effect of light is to synchronize the chemical events that occur at the surface of the rods to prepare them for phagocytosis. These processes also occur in the dark, but more slowly and irregularly than in the light.

Animals↗
PubMed1992

The phagocytosis of ROS by RPE cells is inhibited by an antiserum to rat RPE cell plasma membranes.

A polyclonal antiserum to a rat retinal pigment epithelium (RPE) plasma membrane-enriched fraction has been utilized to identify candidate receptor proteins which may be involved in the phagocytosis of rod outer segments (ROS) by the RPE. Immunoblots of RPE cell extracts show that the the antiserum recognizes a number of glycoproteins, including two with M(r)s of 174 and 75 kDa. The antiserum also recognizes their non-glycosylated counterparts, with M(r)s of 169 and 65 kDa, respectively, which are synthesized after treatment of the cells with tunicamycin B2. Immuno-precipitation of [35S]-methionine-labeled RPE cell extracts also demonstrates the presence of antibodies to these same glycoproteins as well as to other proteins. The antiserum inhibits the binding of ROS to the RPE, which subsequently results in a decrease in the ingestion of ROS. ROS phagocytosis by the RPE is inhibited by 97% in the presence of a 1:10 dilution of the IgG fraction of the antiserum. Phagocytosis recovers to normal levels after 4-6 hr of chase in the absence of antibodies. After sequential adsorption of the IgG fraction to monolayers of fixed RPE cells, which removes RPE surface-specific IgGs, the extent of inhibition of ROS phagocytosis produced by the IgG fraction is reduced. Using immunoblotting we have identified a number of surface-specific immunoreactive bands which are adsorbed out of the antiserum, including the 174 and 75 kDa bands. These data give further support to the hypothesis that ROS phagocytosis is a receptor-mediated process, which occurs via specific cell surface glycoprotein receptors.

Animals↗
PubMed1998

Phospholipase Cgamma1 in bovine rod outer segments: immunolocalization and light-dependent binding to membranes.

We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCbeta1, gamma1, and delta1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCgamma1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCgamma1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCgamma1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 microM. These results demonstrate the presence of PLCgamma1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.

Adaptation, Ocular↗
PubMed1994

Transforming growth factor-beta regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway.

BACKGROUND: Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of many ocular diseases, including proliferative vitreoretinopathy. We examined the effect of TGF-beta on the phagocytosis of rod outer segments by retinal pigment epithelium (RPE), which is a major function of RPE, and investigated the dependence of this effect on the protein kinase C (PKC) pathway. METHODS: Phagocytotic uptake of fluoresceinated bovine rod outer segments was determined by flow cytometry. RPE cells were treated with TGF-beta 1 or TGF-beta 2 and their effects on phagocytosis were examined. The effects of various PKC inhibitors (calphostin C, staurosporine, and extended exposure to phorbol 12-myristate 13-acetate, PMA) and a stimulator (brief exposure to PMA) on RPE phagocytosis was evaluated. RESULTS: Both TGF-beta 1 and TGF-beta 2 up-regulated RPE phagocytosis and PMA abolished the up-regulating effect of TGF-beta. In contrast, PKC inhibition by staurosporine and calphostin C resulted in increased phagocytosis. A combination of TGF-beta and PKC inhibitor treatment did not produced any additive effect on phagocytosis. CONCLUSION: We concluded that TGF-beta up-regulates human RPE phagocytosis, but that this effect is counteracted by PKC activation. It is possible that this TGF-beta-induced effect is due, in part, to a negative modulation of the PKC-dependent pathway.

Alkaloids↗
PubMed1984

Retinitis pigmentosa: a quantitative study of the apical membrane of normal and dystrophic human retinal pigment epithelial cells in tissue culture in relation to phagocytosis.

The phagocytic capabilities of both normal and dystrophic human retinal pigment epithelial cells were assessed by challenging cultures with both non-biological and biological particles. Cells from either source ingested both carbon particles and latex microspheres. Quantification of the phagocytic process in relation to microspheres showed that uptake was linear over an 8 h period and the rate was dose-dependent. All cultures phagocytosed rod outer segments from a variety of species, but excluded bacteria. By using bovine rod outer segments labelled with [H3] choline both normal and dystrophic cells were still accumulating label 24 h subsequent to challenge; however, this technique had significant problems. The introduction of any particulate matter into the culture medium resulted in changes in the apical membrane of the recipient cells. Such responses were quantified in terms of apical projections per unit area. Counts were always greater in cells challenged with biological material even if the material was not engulfed.

Aged↗
PubMed1989

Evidence that microtubules do not mediate opsin vesicle transport in photoreceptors.

The organization of the rod photoreceptor cytoskeleton suggests that microtubules (MTs) and F actin are important in outer segment (OS) membrane renewal. We studied the role of the cytoskeleton in this process by first quantifying OS membrane assembly in rods from explanted Xenopus eyecups with a video assay for disc morphogenesis and then determining if the rate of assembly was reduced after drug disassembly of either MTs or F actin. Membrane assembly was quantified by continuously labeling newly forming rod OS membranes with Lucifer Yellow VS (LY) and following the tagged membranes' distal displacement along the OS. LY band displacement displayed a linear increase over 16 h in culture. These cells possessed a longitudinally oriented network of ellipsoid MTs between the sites of OS protein synthesis and OS membrane assembly. Incubation of eyecups in nocodazole, colchicine, vinblastine, or podophyllotoxin disassembled the ellipsoid MTs. Despite their absence, photoreceptors maintained a normal rate of OS assembly. In contrast, photoreceptors displayed a reduced distal displacement of LY-labeled membranes in eyecups treated with cytochalasin D, showing that our technique can detect drug-induced changes in basal rod outer segment assembly. The reduction noted in the cytochalasin-treated cells was due to the abnormal lateral displacement of newly added OS disc membranes that occurs with this drug (Williams, D. S., K. A. Linberg, D. K. Vaughan, R. N. Fariss, and S. K. Fisher. 1988. J. Comp. Neurol. 272:161-176). Together, our results indicate that the vectorial transport of OS membrane constituents through the ellipsoid and their assembly into OS disc membranes are not dependent on elliposid MT integrity.

Animals↗
PubMed1990

Retinal detachment prevents normal assembly of disk membranes in vitro.

The effects of retinal detachment upon disk membrane assembly in rod outer segments were assessed in Xenopus laevis retinas that had been maintained in eyecup cultures for up to 4 days. In these cultures, assembly of disk membranes occurred at a normal rate in regions of the retina that remained attached to the retinal pigment epithelium. In regions of the retina that were detached from the pigment epithelium, the assembly of new disk membranes either was abnormal or was inhibited. This result cannot be attributed to reduced access of cells in the detached retina to oxygen and metabolites. The experiments described here suggest that the apposition of the retina with the pigment epithelium is a necessary condition for normal disk membrane assembly in Xenopus retinas. This effect may be mediated by contact between the rod outer segments and the pigment epithelium, or by trophic factors in the subretinal space.

Animals↗
PubMed2002

Tamoxifen induces changes in the lipid composition of the retinal pigment epithelium cell line D407.

Tamoxifen, the antioestrogenic drug prescribed for long-term, low-dose therapy of breast cancer, induces retinopathy. This study evaluates the effects of tamoxifen on the human retinal pigment epithelial cell line D407, attempting to identify the underlying mechanisms on tamoxifen-induced retinopathy and the involvement of cellular membranes in the cytotoxic action mechanism. We demonstrate that the tamoxifen-induced decrease in the cell growth of the D407 cell line results from pyknosis and cell cycle arrest rather than from necrosis. Furthermore, D407 cells influence the lipid composition of both plasma membrane and intracellular membranes in response to tamoxifen. Tamoxifen increases the physical order of the lipid bilayer. We observed a compensatory decrease in the cholesterol content of the plasma membrane which results in an increase of the plasma membrane fluidity. In intracellular membranes the phosphatidylcholine content is reduced to 50% of the controls. This reduction may be related to the formation of a second messenger via phospholipase pathway and sustained activation of protein kinase C. Since increased plasma membrane fluidity as well as sustained activation of protein kinase C influence the rod outer segments binding and/or ingestion by retinal pigment epithelial cells, our results suggest that membrane-mediated pathways contribute to the tamoxifen-induced retinopathy.

Antineoplastic Agents, Hormonal↗
PubMed2000

Retinal vascular changes induced by the oxidative stress of alpha-tocopherol deficiency contrasted with diabetic microangiopathy.

It has been proposed that oxidative tissue damage is involved in the development of diabetic angiopathies. To evaluate this hypothesis, experiments were conducted to identify the retinal vessel changes induced by the oxidative stress related to alpha-tocopherol deficiency and examine possible similarities with the lesions characteristic of diabetic retinopathy. Twenty-one-day-old male Fisher 344 albino rats were divided randomly to receive a basal, chemically defined diet either with (adequate group) or without (deficient group) alpha-tocopherol. After 6 and 8 months, some rats (n = 3 per group) were killed and the eyes removed. In order to evaluate cell integrity and localization of lipofuscin-specific autofluorescence by light and fluorescence microscopy, some of the retinas were prepared for cryostat-sections while others were digested by elastase to isolate intact retinal vasculatures. After 8 and 14 months, the central retina of one eye per rat (n = 6 to 8 per group) was examined by electron microscopy for retinal capillary basement membrane (RCBM) thickening and other ultrastructural changes. At 6 and 8 months, the deficient rats exhibited extensive shortening and disarray of rod outer segments (ROS), marked loss of photoreceptor cells, and pronounced increases in the numbers of granules with lipofuscin-specific autofluorescence in the retinal pigment epithelium (RPE) and retinal vessels. At 14 months, the ultrastructure revealed that the damage to ROS involved disruption of membranes and that the capillary lipofuscin was contained mainly within the endothelial cells. Membrane remnants were found in the lipofuscin granules of both the RPE and retinal vessels. In addition, there was an increase in RCBM thickness (98.7 +/- 2.6 nm vs. 86.9 +/- 2.9 nm). RCBM thickening was the only finding common with diabetic retinopathy, and the thickening was 13.6%, significantly less than that reported in diabetic rat models with 8 and 14 months durations (34% and 53.1%, respectively). Capillary lipofuscin accumulation, which was prominent in the deficient rats, is not notable in diabetes. Both the moderate RCBM thickening and marked lipofuscin accumulations seen in alpha-tocopherol-deficient rats were similar to changes occurring in the aging process, though more pronounced. The spectrum of microangiopathies characteristic of diabetic retinopathy did not develop in alpha-tocopherol-deficient rats. These findings suggest that oxidative damage, though probably involved, is unlikely to play a predominant role in the development of diabetic retinal microangiopathies.

Animals↗
PubMed1994

Superoxide inhibits proliferation and phagocytic internalization of photoreceptor outer segments by bovine retinal pigment epithelium in vitro.

Experiments were performed to investigate the effect of free radical damage on two aspects of retinal pigmented epithelium (RPE) metabolism, namely, proliferation and phagocytosis. Bovine RPE cells were maintained in monolayer cultures, either as passaged (for proliferation and lysosomal activity assays) or primary cultures (for phagocytosis measurements). Free radicals (superoxide anions) were generated by a xanthine oxidase (XO)-hypoxanthine (HX) reaction. Total phagocytosis (binding plus ingestion of rod outer segments (ROS)) was quantitated by radioimmunoassay using a specific anti-opsin antibody and iodinated secondary antibody. In some cases, agents with known or possible protective influences against oxidative damage, i.e., superoxide dismutase (SOD), vitamin E, and basic fibroblast growth factor (bFGF), were tested for their activity in this model system. RPE cell proliferation was inhibited in a HX-XO dose-dependent manner, in the absence of cell toxicity. Modifications of cell morphology were also noticed. Either simultaneous exposure of RPE cells to ROS membranes and HX-XO or pretreatment of ROS membranes with HX-XO prior to their addition to RPE monolayers led to a statistically significant 20-30% decrease in phagocytosis relative to control values. This decrease was essentially observed in the binding phase of phagocytosis, indicating damage to ROS surface molecules as the primary event. Addition of SOD or vitamin E prevented this loss of phagocytic activity, whereas bFGF had no effect. Superoxide radicals did not, however, affect phagocytosis when RPE cells were exposed to them alone, prior to incubation with ROS; nor did they alter a later stage in the phagocytic process, acid phosphatase activity. This tissue culture model represents a convenient system for analyzing free radical damage in different aspects of RPE-photoreceptor behavior and may be useful in studying this phenomenon in several retinal disorders.

Acid Phosphatase↗
PubMed1993

Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Adenosine Triphosphate↗
PubMed1989

Light exposure stimulates arachidonic acid metabolism in intact rat retina and isolated rod outer segments.

This paper presents evidence that short-term exposure to light increases synthesis of hydroxyeicosatetraenoic acid (HETE) and prostaglandin D2 (PGD2) and stimulates the uptake and metabolism of 20:4 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-14C-20:4 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 20:4 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 20:4 to leukotriene B4, two diHETEs, 5-HETE, 15-HETE, and PGD2. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 20:4 in isolated rod outer segments (ROS). There was more oxidation of 20:4 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 20:4 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs an PGD2 may be a consequence of the light-induced stimulation of the metabolism of 20:4 in membrane phospholipids in the retina.

Animals↗