PURPOSE:To measure outer segment phagocytosis in cultures of Royal College of Surgeons (RCS) rat retinal pigment epithelium (RPE) that have been treated with carbachol. Carbachol treatment of RCS RPE results in an increase in the second messenger inositol triphosphate, which mimics that observed in normal RPE after interaction with rod outer segments (ROS).METHODS:Cultures of RCS RPE were phagocytically challenged with isolated rat ROS for 2 hours. Carbachol (1 mM) was added to some cultures to stimulate inositol triphosphate synthesis, and incubation continued for 30 minutes at 37 degrees C. Inositol triphosphate concentration was measured by radioreceptor assay. Bound and ingested outer segments were quantified by double immunofluorescent staining. Ingestion of outer segment membranes was confirmed by electron microscopy and immunogold staining.RESULTS:Carbachol treatment was associated with a rapid and temporary increase in inositol triphosphate levels. Royal College of Surgeons rat RPE phagocytically challenged with outer segments and treated with carbachol showed significantly higher ingestion (34%) compared to untreated RCS RPE (9%) (P < 0.05).CONCLUSIONS:Exposure of cultured RCS RPE to carbachol increases the intracellular concentration of inositol triphosphate and enhances phagocytosis of bound ROS. These results support the hypothesis that the phagocytic defect in RCS RPE is related to an abnormality in the generation of inositol triphosphate as a second messenger after outer segment recognition and binding.

Purpose. To evaluate the generation of the second messenger molecule inositol triphosphate (IP3) in response to phagocytic challenge in cultures of retinal pigment epithelium (RPE) prepared from normal Long Evans or dystrophic Royal College of Surgeons (RCS) rats.Methods. RPE cultures were phagocytically challenged with isolated rat rod outer segments (ROS) or polystyrene latex spheres (PSL). Carbachol was added to some cultures as a positive control to stimulate the IP3 pathway. Inositol triphosphate levels in RPE cells were analyzed by high-pressure liquid chromatography and radioreceptor assay. Incorporation of H-3-myoinositol into phosphatidylinositol lipids was analyzed by thin layer chromatography and autoradiography.

Cultures of retinal pigment epithelium (RPE) from normal Long-Evans (LE) and dystrophic Royal College of Surgeons (RCS) rats were incubated with 32P-orthophosphate and then phagocytically challenged with isolated rod outer segments (ROS) or polystyrene latex spheres (PSL). The 32P incorporation into individual proteins was quantified by image analysis of two-dimensional gel autoradiograms, and changes in phosphorylation were identified by comparison with unchallenged control cultures. Phosphorylation changes that were similar in response to either ROS or PSL were classified as nonspecific and omitted from further analysis; those associated solely with ROS exposure were classified as ROS specific and compared between the two strains. None of the 30 ROS-specific changes in protein phosphorylation identified in normal LE RPE were the same as in RCS RPE. However, unique ROS-specific changes in phosphorylation were observed in 13 RCS RPE proteins. Three RCS proteins showed ROS-specific decreases; ten showed ROS-specific increases. Six of these ten RCS proteins with increased phosphorylation showed ROS-specific decreases in LE RPE. No other correspondence in ROS-specific changes was found among the remaining LE or RCS RPE proteins, but several RCS proteins were phosphorylated at abnormal levels under control conditions. Even though ROS-induced changes in phosphorylation were aberrant in RCS RPE, their presence indicated that a ROS-specific transmembrane signal was generated after interaction with ROS. The abnormal increases and decreases observed in ROS-specific phosphorylation in the RCS suggested that the defect in ROS phagocytosis was associated with the misregulation or malfunction of both protein kinases and phosphatases.

Changes in protein phosphorylation induced by phagocytic challenge were identified in cultured rat retinal pigment epithelium (RPE) following exposure to isolated rat rod outer segments (ROS) or to polystyrene latex microspheres (PSL). RPE phosphoproteins were characterized based on molecular weight and isoelectric point and 32P incorporation into phosphoproteins was quantified by digitized image analysis of two-dimensional gel autoradiograms. Changes in the phosphorylation of RPE proteins were determined by comparing 32P gel data from phagocytically challenged cultures with control cultures. ROS-specific changes were defined as those occurring only in response to ROS while nonspecific changes were those associated with either ROS or PSL phagocytosis. A parallel study was conducted to identify those proteins which also show increased phosphorylation following protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate. ROS-specific increases in the phosphorylation of 2 RPE proteins were found, 1 of which also showed an increase with PKC activation. Nonspecific increases included the phosphorylation of 11 RPE proteins, 10 of which were also phosphorylated with PKC activation. ROS-specific decreases were observed in 12 RPE phosphoproteins while 3 proteins showed nonspecific decreases in their phosphorylation. These findings demonstrate that phagocytic challenge of the RPE with either specific or nonspecific particles is linked to the activation of phosphatases and kinases and that activation of PKC may play a role in phagocytosis of both particle types. The identification of two distinct groups of changes in phosphorylation supports the hypothesis that different pathways exist for phagocytosis of ROS-specific and nonspecific particles by the RPE.

Interphotoreceptor matrix (IPM) proteins from a wide range of vertebrate species were examined by gel electrophoresis. Extensive similarities in the banding patterns of the proteins were found. S antigen, serum albumin and interphotoreceptor retinoid-binding protein (IRBP) were identified immunochemically. The latter two proteins dominate the IPM obtained from the apical surface of the retinal pigment epithelium, whereas IPM prepared from the retina washes contains IRBP plus outer-segment components including S antigen. IRBP is present in IPM from the all-cone lizard (Anolis) eye, as well as from rod- and cone-dominant animals. The ontogeny of IRBP in chick IPM is different from that of serum albumin in age of onset and rapidity of development. Comparison between Royal College of Surgeons rat IPM and normal rat IPM showed that several proteins are changed in amount. This study is a step toward a functional characterization of components common to the IPM of all vertebrates.

Incubation of confluent cultures of rat retinal pigment epithelium (RPE) with 32P-orthophosphate resulted in the incorporation of 32P into proteins, RNA and the nucleoside phosphates ADP, GDP, ATP and GTP. RPE cultures incubated with phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, did not significantly change the incorporation of 32P into total protein, RNA or the nucleoside phosphates ADP, GDP, ATP and GTP. However, PMA exposure specifically increased phosphorylation of five proteins with molecular weights of 80 kilodaltons (K), 56K, 35K, 33K, and 29K having isoelectric points between 4.3 and 6.5. PMA treated cultures also showed dephosphorylation of two proteins having molecular weights of about 33K. The observed increase in 80K phosphorylation suggests that protein kinase C is present and activated by PMA in the RPE.

Opsin, the alpha-subunit of transducin, S-antigen, interphotoreceptor retinoid-binding protein (IRBP) and cathepsin D were assessed in autopsy eyes from patients with retinitis pigmentosa (RP) and normal autopsy eyes. Immunochemical methods were used to determine the presence of these proteins on Western blots of retinal homogenates from five RP donors and on blots of interphotoreceptor matrix (IPM) preparations from six other RP eyes. The amounts of immunoreactive opsin, S-antigen, alpha-transducin, and IRBP appeared below normal in retinas from RP eyes. All six IPM samples from patients with advanced RP had reduced amounts of S-antigen and no detectable IRBP or transducin. Cathepsin D (an RPE protein) was present in IPM or RP eyes in amounts comparable to that in IPMs from normal eyes. Small amounts of cathepsin D were also detected in retinas from both normal and RP eyes. These studies show that proteins specific to the photoreceptor-pigment epithelium complex in normal eyes can be detected in autopsy eyes from patients with RP and suggest that the observed reductions in photoreceptor-specific proteins occur as a consequence of photoreceptor loss.

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.

A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.