Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25 degrees C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9 X 10(-7) M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled 'trans' and 'cis' isomers of vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.     

Comparison of retinal pigment epithelium cell preparations from the bovine eye

Summary Retinal pigment epithelium (RPE) cells were collected from, bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

Intercellular junctions of hyperplastic retinal pigment epithelium

Summary In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact, tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.Supported by a grant from the National Eye Institute to Dr R. Bellhorn, whose support is greatly appreciated, and an unrestricted grant and a Research Manpower Award from Research to Prevent Blindness, Inc.     

Carbonic anhydrase type II in regenerating retinal pigment epithelium. A histochemical study in the rabbit

Type II carbonic anhydrase (CAII) in the cytoplasm of the retinal pigment epithelium (RPE) may contribute to the transport of water and solutes across the RPE. The activity of this enzyme in RPE during its response to damage, e.g., during regeneration, is therefore of interest in understanding retinal disease. Immuno-histochemistry was used to compare CAII activity of normal RPE and RPE experimentally induced to regenerate. In normal rabbits, the RPE stained intensely with a peroxidase-linked antibody specific for human CAII. Regenerating RPE stained less intensely. Within the regenerating epithelium, staining appeared more intense in mature cells than in immature ones, suggesting that CAII activity gradually returns during RPE regeneration.     

Regulation of the retinal interphotoreceptor matrix Na by the retinal pigment epithelium during the light response

We examined the rabbit retinal pigment epithelium (RPE) for Na transport properties which would allow it to buffer undesirable changes in Na concentrations in the interphotoreceptor matrix (IPM) during light and dark cycles. The RPE is selectivity permeable to sodium. Open and short circuit transport studies with RPE indicate a circulating (choroid to retina and back) Na current which does not compromise the electrical integrity of the blood brain barrier but together with the Na permeselectivity is of sufficient magnitude to buffer both upwards and downwards movements of IPM [Na] during light or dark responses.     

Accumulation of oxidized lipid-protein complexes alters phagosome maturation in retinal pigment epithelium

Lipid peroxidation has been implicated in many age-associated disorders including macular degeneration of the retina. We sought to elucidate the mechanism by which accumulation of oxidized LDL (oxLDL) reduces the ability of retinal pigment epithelium (RPE) to process photoreceptor outer segments (OS) as a model of peroxidation-induced disruption of phagocytosis. OxLDL did not reduce the lysosomal hydrolytic capacity of the RPE, but efficiently inhibited processing of various internalized proteins. OxLDL caused a delay in the acquisition of late lysosomal markers by newly formed phagosomes. At the same time, an excessive accumulation of markers of early phagosomal compartments was also observed. The activity of phosphatidylinositol 3-kinase (PI3K) was reduced in phagosomes of the RPE treated with oxLDL. These results suggest that accumulation of oxidized lipid-protein complexes in the RPE impedes phagosome maturation by blocking PI3K recruitment to the phagosomal membrane, leading to delayed processing of internalized OS.Received 24 February 2004; received after revision 12 April 2004; accepted 4 May 2004     

Comparison of retinal pigment epithelium cell preparations from the bovine eye

Retinal pigment epithelium (RPE) cells were collected from bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.