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Studies on the endoplasmic reticulum. V. Its form and differentiation in pigment epithelial cells of the frog retina.

Pigment epithelial cells of the frog's retina have been examined by methods of electron microscopy with special attention focused on the fine structure of the endoplasmic reticulum and the myeloid bodies. These cells, as reported previously, send apical prolongations into the spaces between the rod outer segments, and within these extensions, pigment migrates in response to light stimulation. The cytoplasm of these cells is filled with a compact lattice of membrane-limited tubules, the surfaces of which are smooth or particle-free. In this respect, the endoplasmic reticulum here resembles that encountered in cells which produce lipid-rich secretions. The myeloid bodies comprise paired membranes arranged in stacks shaped like biconvex lenses. At their margins the membranes are continuous with elements of the ER and in consequence of this the myeloid body is referred to as a differentiation of the reticulum. The paired membranes resemble in their thickness and spacings those which make up the outer segments; they are therefore regarded as intracellular photoreceptors of possible significance in the activation of pigment migration and other physiologic functions of these cells. The fuscin granules are enclosed in membranes which are also continuous with those of the ER. The granules seem to move independently of the prolongations in which they are contained. The report also describes the fine structure of the terminal bar apparatus, the fibrous layer intervening between the epithelium and the choroid blood vessels, and comments on the functions of the organelles depicted.

Animals↗

Cloning of a major human retinal pigment epithelial lysosomal aspartic protease and mapping its transcriptional start sites.

PURPOSE: It has been proposed that the major proteolytic enzyme responsible for the proteolysis of photoreceptor outer segments is an aspartic protease similar or identical to cathepsin D. The aim of this study was to determine if the major retinal pigment epithelial (RPE) aspartic protease was cathepsin D, to study its distribution and investigate its transcription start sites (TSS). METHODS: An RPE cDNA library was screened at low stringency for the presence of aspartic proteases. DNA sequencing was used to analyze the identified clones. The expression of cathepsin D mRNA was analyzed by Northern blot while immunohistochemistry was used to demonstrate cathepsin D related immunoreactivity in an eye section. RNase protection assay was used to map the Cathepsin D TSS in RPE cells. RESULTS: The aspartic protease identified in the RPE cDNA library was identical to the DNA sequence of cathepsin D found in other tissues. Northern blot analysis of a wide range of cell types showed that the highest level of cathepsin D mRNA expression was found in RPE cells which was similar only to cathepsin D expression in MCF7 breast cancer cells. Immunohistochemical staining of a human retina confirmed the inherent nature of high cathepsin D expression in RPE cells. An RNase protection assay demonstrated two major cathepsin D TSS in RPE cells. One of them was identical to the TSS responsible for the constitutive expression of cathepsin D and the other, a TATA box-controlled TSS, was identical to a TSS found in MCF7 cells. Cathepsin D expression was not enhanced by the phagocytosis and digestion of rod outer segments (ROS) and ROS phagocytosis did not induce the use of other cathepsin D TSS in the RPE cells. CONCLUSION: The major aspartic protease identified in RPE cells was cathepsin D. In addition, it was demonstrated that the high level of cathepsin D expression in RPE cells is the intrinsic nature of these cells and that it is linked to a TATA box-controlled TSS. This TSS has previously been described as an estrogen regulated TSS and further studies are required to identify the RPE-specific inducer or indirect factors that may be responsible for the enhanced expression of cathepsin D in the RPE cells.

Blotting, Northern↗

Nitric oxide as a second messenger in phagocytosis by cultured retinal pigment epithelial cells.

PURPOSE: To investigate a possible role of the nitric oxide (NO)-cGMP signal transduction system in phagocytosis of rod outer segments (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Primary cultures of RPE cells from 10-day-old Brown Norway rats were used to study the phagocytosis of ROS by these cells. Phagocytosis of ROS was evaluated with or without an inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine (L-NNA), and the reverse effects of L-NNA by L-arginine and 8-bromo-cGMP on phagocytosis were also studied. NO-associated cGMP production by RPE cells was monitored during phagocytosis using L-NNA. NOS activity was assayed in RPE cells and ROS to locate the source of NO. RESULTS: Phagocytosis of ROS was inhibited by L-NNA but not by D-NNA. L-NNA inhibited the ingestion in a dose-dependent manner, but not the binding of ROS. The inhibition was reversed by L-arginine and also by an NO donor, SIN-1. RPE cells challenged with ROS showed increased cGMP activity, which was significantly reduced by L-NNA and again restored by an overdose of L-arginine. NOS activity was found in RPE cells but not in ROS. CONCLUSIONS: Our data show that cGMP plays a role in the ingestion phase of ROS phagocytosis by RPE cells via a cGMP second-messenger system.

Animals↗

Modulation of early response gene expression by prostaglandins in cultured rat retinal pigment epithelium cells.

PURPOSE: To explore the role of prostaglandins (PGs) as modulators of retinal pigment epithelium (RPE) rod outer segment (ROS)-phagocytosis and ROS-phagocytosis-induced gene expression. METHODS: RPE cells in primary cell culture were pre-incubated with PGE( 2), PGD(2), PGF(2)alpha, PGJ(2), 15-deoxy-Delta( 12,14)-PGJ(2) or U-46619 (stable analog of thromboxane A(2)), and fed with a suspension of ROS. Expression of zif-268 and tis-1 mRNA was determined by Northern blotting. DNA-binding activity of TIS-1 protein was assessed by electrophoretic mobility shift assay. Concentration of PGE (2) and PGD (2) in the tissue culture medium was measured by enzyme immuno-assay. Phagocytis-tosis was quantified by counting of double-immunostained bound and ingested ROS. RESULTS: PGE 2, the most potent of PGs, strongly elevated both basal and ROS-phagocytosis-induced levels of tis-1 mRNA, while significantly inhibiting both basal and phagocytosis-induced expression of zif-268 mRNA. PGD(2), PGJ(2) and 15-deoxy-Delta(12,14)-PGJ( 2) elevated ROS-phagocytosis-induced, but not basal, expression of tis-1 mRNA expression. PGF(2alpha) super-induced both phagocytosis-induced and basal tis-1 mRNA expression. U-46619 and carbaprostacyclin had no effect on expression of tis-1 mRNA. PGE(2) was the only PG to affect zif-268 expression. Exogenous PGE(2), PGD( 2) and PGF(2alpha), when added to the medium at 1-microM concentrations, significantly inhibited ingestion of ROS, with PGE(2) being the most potent PG affecting ROS-phagocytosis. CONCLUSIONS: PGs act as selective regulators of phagocytosis-induced transcription factor gene expression in RPE cells, as well as of ROS-phagocytosis itself. This modulation may help to ensure specificity in the differential activation of target genes by ROS-phagocytosis receptor-mediated signal transduction in RPE cells.

Animals↗

Photoreceptor-specific degeneration caused by tunicamycin.

The antibiotic tunicamycin inhibits the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyisoprenol, a key intermediate in the formation of the asparagine-linked oligosaccharides of glycoproteins. The effects of tunicamycin have been studied in various biological systems, primarily with the aim of elucidating the role of the carbohydrate moieties in the cellular function of glycoproteins. Rhodopsin, the visual pigment of retinal rod photoreceptor cells, is a membrane glycoprotein which consists of a single polypeptide chain (opsin) to which a chromophoric prosthetic group (II-cis-retinaldehyde) and two asparagine-linked oligosaccharide chains are covalently attached. The glycosylation of opsin can be blocked with tunicamycin in vitro in conditions where polypeptide synthesis is only slightly decreased. We have reported that tunicamycin can disrupt the normal assembly of rod outer segment membranes in vitro without significantly inhibiting the biosynthesis or intracellular transport of opsin. Here we report that intraocular injection of tunicamycin produces a photoreceptor-specific degeneration characterized by progressive shortening of rod outer segment, decreased membrane assembly, and eventual photoreceptor cell death.

Animals↗

Nitric oxide decreases in vitro phagocytosis of photoreceptor outer segments by bovine retinal pigmented epithelial cells.

The presence of nitric oxide synthase (NOS) in the retina, the constitutive isoform in photoreceptor outer segments and the inducible form in retinal pigmented epithelial (RPE) cells, has been demonstrated, but the role of the free radical NO produced, remains unknown. We have investigated the effect of NO on the process of rod outer segment (ROS) phagocytosis. Using an in vitro assay for phagocytosis in primary cultures of bovine RPE cells, we demonstrate that NO released by SIN-1 (3-morpholinosydnonimine) in the culture medium inhibits the phagocytosis of ROS. Furthermore, endogenous NO, produced by RPE cells cotreated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), is also able to decrease RPE cell phagocytic activity. This effect depends upon the continuous presence of NO during the assay and is abolished by the scavenging of NO by hemoglobin or by the inhibition of NO synthase activity by L-arginine analog, NG-monomethyl-L-arginine. Pretreatment of ROS with SIN-1 failed to impair subsequent phagocytosis, demonstrating that NO directly affects the RPE cells ability to phagocytose ROS. The inhibitory effect of NO is cGMP independent, since 8-bromo-cGMP does not modify this process. This decrease of ROS phagocytosis by RPE cells caused by NO may occur as a result of retinal inflammation, and could lead to photoreceptor degeneration.

Animals↗

Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex.

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.

Animals↗

Double fluorescent vital assay of phagocytosis by cultured retinal pigment epithelial cells.

PURPOSE: The goal of this study was to develop the first vital assay system for in vitro analysis of phagocytosis of rod outer segments (ROS) by normal retinal pigment epithelial (RPE) cells and for study of the phagocytic defect in RPE of the Royal College of Surgeons (RCS) rat with inherited retinal degeneration. Required features included ability to directly visualize and quantitate the phagocytic process in living RPE cultures, and capability for subsequent quantitative analysis after fixation of the cells at any chosen time after incubation with ROS. METHODS: A double fluorescent method was designed, based on the process of phagosome-lysosome fusion. For vital staining of lysosomes, confluent cultures of rat RPE cells were incubated with sulforhodamine (SR), a red fluorescent lysosomotropic dye. SR-stained cultures were challenged with isolated rat ROS tagged with fluorescein isothiocyanate (FITC), a green fluorescent probe. RESULTS: This method was used to observe all phases of the phagocytic process in the living cells and the kinetics of ROS binding, ingestion, and phagosome-lysosome fusion were determined. Control studies showed no differences in binding, ingestion, or digestion of unstained versus FITC-stained ROS. Additionally, the phagocytic defect in dystrophic RCS rat RPE cells was confirmed using this technique. CONCLUSIONS: This relatively simple new method is useful in that it uses inexpensive, readily available reagents, it enables real-time analysis of phagocytosis experiments, and it does not require termination of the cultures for analysis of phagocytic ability.

Animals↗

Morphological recovery in the reattached retina of the toad Bufo marinus: a new experimental model of retinal detachment.

BACKGROUND: The retinal pigment epithelium (RPE) of the toad (Bufo marinus) has been used in many studies as a model for understanding its role and interaction with the neural retina. The toad's retina has been used to establish a new in vitro model of experimental retinal detachment (RD) and replacement . It has been shown that the electrophysiological measures of retinal function recovered following complete RD. The toad was chosen because its RPE is similar to the mammalian RPE . In this report, light microscopy was used to characterize the morphologic changes that occur in the RPE and neural retina following RD/replacement and to correlate these findings with recovery of electrophysiologic function. METHODS: Retinas from Bufo marinus were studied in vitro. The neural retina completely detached from the RPE and then replaced. At various times after replacement, neural retina-RPE tissues were processed for light microscopy. RESULTS: At 30 min after replacement, the subretinal space was greatly expanded, and the apical processes that normally ensheath the rod outer segments were short and no longer contacted the rod outer segments. The RPE was swollen, contained many vacuoles and the apical surface was rounded. By 2 h after replacement, the subretinal space was significantly resorbed and contained many shredded rod outer segments; RPE cells were still swollen, although less. During the next 5-10 h, the number of phagosomes in the RPE cytoplasm increased and the number of shredded rod outer segments in the subretinal space decreased. RPE cells regained their normal size and interdigitation of apical processes and rod outer segments were observed. CONCLUSIONS: These results demonstrate the re-establishment of morphological interactions between the RPE and neural retina within hours following RD/replacement. Morphological recovery coincides with recovery of electrophysiologic parameters. This is a good model to investigate the retinal pigment epithelium (RPE) and neural retina mechanisms involved in retinal adhesion and recovery from retinal detachment.

Animals↗

RPE cells from normal rats do not secrete a factor which enhances the phagocytosis of ROS by dystrophic rat RPE cells.

Retinal pigment epithelial (RPE) cells from normal and dystrophic rats were grown separately and in mixed culture for 7 days, without a change of growth medium. Isolated rod outer segments (ROS) were suspended in the conditioned medium from these cells, and were fed to the mixed or pure RPE cell cultures. No increase or decrease in the phagocytosis of ROS by dystrophic or normal RPE cells, respectively, was observed. These results suggest that normal RPE cells do not secrete a diffusible factor(s) which enhances the phagocytosis of ROS by dystrophic RPE cells.

Animals↗

Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure.

Visible light-induced photoreceptor cell damage resulting from exposure to multiple intermittent light-dark periods was compared with damage resulting from continuous light in albino rats maintained in a weak cyclic-light environment or in darkness before light treatment. The time course of retinal damage was determined by correlative measurements of rhodopsin and visual cell DNA at various times after light exposure, and by histopathological evaluation. The effect of intense light exposures on rhodopsin regeneration and on the level of rod outer segment docosahexaenoic acid was also determined. For rats previously maintained in weak cyclic light, 50% visual cell loss was measured 2 weeks after 12 1 hr light/2 hr dark periods, or following 24 hr of continuous light. A comparable 50% loss of visual cells was found in dark-reared rats after only 5 hr of continuous illumination or 2-3 hr of intermittent light. As judged by histology, cyclic-light-reared rats incurred less retinal pigment epithelial cell damage than dark-reared animals. In both experimental rat models intermittent light exposure resulted in greater visual cell damage than continuous exposure. Visual cell damage from intermittent light was found to depend on the duration of light exposure and on the number of light doses administered. Measurements of rhodopsin and DNA 2 hr and 2 weeks after light exposure of up to 8 hr duration revealed that visual cell loss occurs largely during the 2 week dark period following light treatment. The loss of docosahexaenoic acid from rod outer segments was also greater in rats exposed to intermittent light than in animals treated with continuous light. It is concluded that intermittent light exposure exacerbates Type I light damage in rats (involving the retina and retinal pigment epithelium) and the schedule of intense light exposure is a determinant of visual cell death.

Animals↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗