Bovine Retinal Pigment Epithelium Research Articles

Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells.

Regulation of intracellular pH (pHi) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pHi was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pHi after induction of an acid load by removal of NH4Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive 22Na+-uptake into Na+-loaded cells. Both results suggest the presence of a Na+/H+ antiport. (2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO3(-)-Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl-/HCO3- exchange mechanism. (3) We found no evidence for a Na+/HCO3(-)-cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pHi in RPE: A Na+/H+ antiport responsible for recovery of pHi from acid load, and a DIDS-sensitive Cl-/HCO3- exchange mechanism responsible for recovery of pHi after alkalinization.

Ascorbate peroxidase in bovine retinal pigment epithelium and choroid.

Peroxidase, catalyzing hydrogen peroxide reduction concurrent with ascorbate oxidation, was demonstrated in the extract of retinal pigment epithelium and choroid. The peroxidase in the choroid, RPE, and retina are 236.1, 25.1, and 0.5 units/mg protein respectively. Ammonium sulfate fractionation and high pressure liquid chromatography showed that the peroxidase in the RPE-choroid is associated with a group of heme proteins with absorption maxima at 410 nm, and optimal activity at pH 4.5. The high peroxidase activity in the RPE-choroid explains the observation of dehydroascorbate in these tissues and indicates a possible role of this enzyme in the removal of H2O2.

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.

Vitamin A uptake from retinol-binding protein in a cell-free system from pigment epithelial cells of bovine retina. Retinol transfer from plasma retinol-binding protein to cytoplasmic retinol-binding protein with retinyl-ester formation as the intermediate step.

We have investigated the steps by which retinol, released from plasma retinol-binding protein (RBP), enters the cells and is accumulated for the most part as a retinyl-ester, only a small fraction of it being present as a complex with cytoplasmic retinol-binding protein (CRBP). For this purpose, we have developed a cell-free system composed of plasma membrane-enriched fractions from bovine retinal pigment epithelium which selectively incorporates exogenous vitamin A when presented as a retinol-RBP complex. Upon incubation in the presence of [3H]retinol-RBP, isolated plasma membrane fractions take up and esterify retinol. A 4-fold reduction of total vitamin A incorporation is observed in conditions which specifically inhibit retinyl-ester formation, thus indicating that the two processes of retinol uptake and esterification are functionally coupled. Evidence is presented that retinol bound to a plasma membrane receptor sharing functional and structural similarities with CRBP is the actual substrate for esterification. Vitamin A accumulation seems to require retinol esterification to allow the recycling of a limited number of free, plasma membrane-associated, retinol receptors. Mobilization of retinol stored as a membrane-bound retinyl-ester is mediated by a membrane-associated hydrolase activity selectively controlled by the level of apo-CRBP which acts as a carrier for the released retinol. Up to 90% of membrane-bound vitamin A is released upon incubation in the presence of apo-CRBP (11 microM) with concomitant formation of retinol-CRBP. The overall process, in which retinol never needs to leave its binding proteins, allows the accumulation of vitamin A in the form of a membrane-bound retinyl-ester and its regulated mobilization as a retinol-CRBP complex.

Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells

Cultured cells derived from bovine retinal pigment epithelium (RPE) express the basic fibroblast growth factor (bFGF) gene and they contain 3.7 and 7.0 kb bFGF gene transcripts which are translated into immunoreactive bFGF of M r 17,500 and 18,000, respectively. The RPE cell-derived bFGF is bioactive, i.e., it can stimulate the proliferation of capillary endothelial cells and the stimulation of cell proliferation is blocked by anti-bFGF antibodies. We suggest that RPE cell-derived bFGF may be involved in the repair mechanisms following retinal injury and in the intravitreal pseudoneoplastic proliferation of the injured RPE.

Short-term organ culture of the retinal pigment epithelium in microtitration plates: ultrastructural studies.

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2-3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. (3)H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol.mg protein(?1).min(?1) when (3)H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of (3)H-labeled all-trans retinol and (3)H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of (3)H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 +/- 3 ng CRBP.?g cytosol protein(?1). In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of (3)H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific (3)H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.

A serum-free defined medium for retinal pigment epithelial cells

Human and bovine RPE cells underwent changes in morphology and culture doubling times when passaged in serum-supplemented medium (CM). Furthermore, late passage human RPE cells subcultured in CM medium increased synthesis of three acidic, 43 000–63 000 D proteins. In order to provide a controlled environment for the study of RPE cells in vitro, we have devloped a method for growing human and bovine RPE in a serum-free defined medium (DM). RPE cells grown in DM required a 24 h pretreatment with CM to allow the cells to attach and spread on the substrate. Cells grown in DM retained an epithelioid morphology, a stable culture doubling time, and similar 2-D PAGE patterns through several subculturings.

The enzymatic cleavage of rhodopsin by the retinal pigment epithelium. I. Enzyme preparation, properties and kinetics: Characterization of the glycopeptide product

An enzyme system has been detected in the bovine retinal pigmented epithelium (RPE) which carries out the degradation of rhodopsin. The substrate for these studies was bovine rhodopsin labeled by in vitro techniques with [ 3H]-glucosamine or [ 3H]-mannose, the former being the derivative most extensively examined. Labeled rhodopsin was purified by adsorption and affinity chromatography followed by preparative isoelectric focusing. Cell-free preparations from bovine RPE cleaved rhodopsin to a single glycopeptide. The pH optimum for the reaction was about 3·5. The apparent K m for rhodopsin was about 5 μm. Rhodopsin was cleaved about 55% under saturating conditions, the limited extent of reaction due primarily to its partial inactivation for use as a substrate during the course of the reaction. Inhibition by cleavage products was not observed. Among several tissues which were examined, the RPE was the most active in rhodopsin-cleaving activity. Although stable as a crude extract, the activity of the rhodopsin-cleaving enzyme, as well as the bovine serum albumin (BSA)-cleaving enzyme (cathepsin-D) were rapidly lost upon purification by DEAE-Sephacel and pepstatin-Sepharose. Differences in relative specific activity among tissues as well as differential patterns of purification suggest that the rhodopsin-cleaving enzyme and the BSA-cleaving enzyme of the RPE may not be the same. Using N-retinylopsin as a substrate for the rhodopsin-cleaving enzyme, it was shown that the regions of the cleavage products containing vitamin A were not associated with the glycopeptide. Some of the properties of the product were examined. Its response to ion exchange resins, susceptibility to further cleavage by pronase, amino acid analysis, solubility properties, isoelectric point, and binding by concanavalin A (conA)-Sepharose, were consistent with the product being described as an acidic glycopeptide. The glycopeptide was reactive with the antibody prepared against bovine rhodopsin, and retained the full antigenicity of the intact molecule. This enzyme system may be one of the means whereby the RPE participates in maintaining the physiological concentration of rhodopsin which is synthesized in the retinal and catabolized in the RPE.

Effect of temperature and pH on release of enzymes from lysosomes of the bovine retinal pigment epithelium in vitro

The effects of buffer concentrations, pH, and temperatures on the release of enzymes from lysosomes of the bovine retinal pigment epithelium were studied in vitro. Cathepsin D, arylsulfatase, and acid phosphatase were used as lysosomal marker enzymes. Elevation of temperature caused a marked increase in the release of cathepsin D and arylsulfatase from lysosomes, but little changes in the release of acid phosphatase. Acidic conditions accelerated the release of arylsulfatase and acid phosphatase. Different buffer concentrations had little effect on the release of these enzymes from lysosomes.

The relative proportions of lysosomal enzyme activities in bovine retinal pigment epithelium

The lysosome fractions from bovine retina, liver and retinal pigment epithelium were isolated by subcellular fractionation and compared with regard to the relative proportions of several hydrolytic enzyme activities. It was found that the lysosome fraction of the retinal pigment epithelium is more than three times as active as the lysosome fractions from other tissues in degrading the rhodopsin of photoreceptor (rod) cell outer segments. This proteolytic activity is attributable to a cathepsin D-like proteinase, and the possible biochemical bases for its increased activity in the pigment epithelium are discussed, including interaction with phospholipase A. It is suggested that the lysosomes of the retinal pigment epithelium are specialized in their content of hydrolytic enzymes for the degradation of photoreceptor cell outer segments.

Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.

The endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium have been identified by their spectral characteristics and their co-migration with authentic retinoids on high performance liquid chromatography. All-trans-retinol is the only retinoid associated with the cellular retinol-binding protein from retina and retinal pigment epithelium. The saturation of the binding site with native ligand is 0.95 +/- 0.05 mol of retinoid/mol of protein for cellular retinol-binding protein purified from frozen and fresh retina and retinal pigment epithelium. All-trans-retinoic acid has been identified as the endogenous ligand associated with the cellular retinoic acid-binding protein of cattle retina. The saturation of the binding site of cellular retinoic acid-binding protein from frozen and fresh retina (the protein is absent in extracts of retinal pigment epithelium) is about 0.2 mol of retinoid/mol of protein, probably a minimum value due to losses of the ligand. Retinoic acid has not been detected previously in retina. Cellular retinaldehyde-binding protein from retina purifies with two endogenous ligands which cochromatograph on high performance liquid chromatography with 11-cis-retinaldehyde and 11-cis-retinol and occur in a ratio of approximately 3:1, respectively. The binding site of cellular retinaldehyde-binding protein from frozen retina is nearly fully occupied with these two ligands (saturation greater than 0.9 mol of retinoids/mol of protein). In contrast, purified cellular retinaldehyde-binding protein from retinal pigment epithelium carries only 11-cis-retinaldehyde as an endogenous ligand. The saturation of the binding site with this ligand is greater than 0.95.

Mercapturic acid pathway enzymes in bovine ocular lens, cornea, retina and retinal pigmented epithelium

Analogous to the liver, ocular tissues contain large concentrations of glutathione and are exposed to potentially damaging chemical compounds. Since glutathione has been shown to have a detoxification function, via mercapturic acid production in the liver, we investigated whether glutathione has a similar function in ocular tissues. We have demonstrated the presence of all of the enzymes involved in the mercapturic acid pathway i.e. glutathione S-transferase, gamma-glutamyl transpeptidase, cysteinylglycinase, and N-acetyl transferase, in the ocular tissues of bovine lens, cornea, retina, and retinal pigmented epithelium. Therefore glutathione may have another function in ocular tissues, that of the detoxification of xenobiotics.

α-Fucosidase, α-mannosidase and β-N-acetylglucosaminidase of the bovine retinal pigment epithelium

α-Fueosidase, α-mannosidase and β-N-acetylglucosaminidase in the bovine retinal pigment epithelium were studied biochemically. p-Nitrophenyl-α- l-fucoside, p-nitrophenyl-α- d-mannoside and p-nitrophenyl-N-acetyl-β- d-glucosaminide were used as substrate. The optimal pH of the glycosidases were acidic. The K m values of α-fucosidase, α-mannosidase and β-N-acetylglucosaminidase were 0·37, 0·52 and 1·37 m m, respectively.

Retinol uptake by isolated retinal pigment epithelium plasma membranes

The uptake of retinol has been investigated by incubating suspensions of isolated bovine retinal pigment epithelium plasma membranes in the presence of [ 3 H]retinol-retinol-binding protein complex. After SDS gel electrophoresis of the labelled membranes, [ 3 H]retinol is found to be associated with a protein band of molecular weight 16000. The binding process is saturable, specific and shows a linear dependence on the membrane concentration in the incubation mixture. A dissociation constant of the order of 10 −9 has been calculated for the retinol-receptor interaction. These observations confirm previous data obtained with isolated pigment epithelium cells and suggest the existence of an intermediate step, at the membrane level, in the transfer of retinol from its plasma carrier to the cell cytoplasm.

Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues

The fatty acid composition of retinal tissues was measured in rats maintained for 26–32 weeks on each of the following diets: a purified basal diet deficient in α-tocopherol and selenium, an identical control diet supplemented with α-tocopherol and selenium, and a commercial laboratory rat chow. Dietary deficiencies of antioxidant nutrients were found to cause a large decrease in total polyunsaturated fatty acids in the retinal pigment epithelium, a small decrease in the retinal rod outer segments, but no change in the whole retina or liver when compared to tissues from animals fed the vitamin E- and selenium-supplemented control diet. The polyunsaturated fatty acid content which we have observed for the retinal pigment epithelium from rats fed commercial lab chow is similar to that which we observed for bovine retinal pigment epithelium. Our results indicate that changes in fatty acid composition are not generalized to all tissues in severely antioxidant-deficient animals, but that changes do occur in some tissues, such as the retinal pigment epithelium, which appears to be particularly sensitive to in vivo lipid peroxidation.

Subcellular distribution of free and esterified forms of vitamin a in the pigment epithelium of the retina and in liver

1. 1. The distribution of vitamin A was examined in various subcellular fractions of rat liver and bovine retinal pigment epithelium. In rat liver, the major portion of the vitamin is in the cytosol, whereas in pigment epithelium, it is concentrated mainly in the microsomes. The microsomal vitamin A of pigment epithelium is tightly bound to membranes, as shown by the inability to release it except by organic solvent extraction or incubation with Triton X-100. 2. 2. In both tissues, two different forms of cytosol vitamin A could be distinguished by ultracentrifugation. The major portion in liver is in the flotating lipid phase and consists mainly of retinyl ester. The remainder (less than 10% of the total) is in the underlying infranatant; about 90% of the vitamin A in this fraction is esterified. By contrast, two-thirds of the vitamin A of pigment epithelial cell cytosol is in the infranatant; it consists of both esterified and unesterified retinol. The floating layer in the pigment epithelial cytosol consists entirely of retinyl ester. 3. 3. These two forms of cytosol vitamin A in the pigment epithelium could also be separated by gel filtration on Sephadex G-100 which yielded two distinct fluorescent peaks. The first, which appeared in the void volume and corresponded in all probability to the floating layer obtained by ultracentrifugation, consisted only of retinyl ester. The second peak, which was eluted in approximately the same position as myoglobin, contained only unesterified retinol. It was abolished completely by preincubation with pronase. These findings support the view that the second peak represents the endogenous retinol-retinol binding protein complex of pigment epithelial cytosol. The fluorescent enhancement of the retinol bound to protein in this peak was about 4–5-fold compared to retinol in organic solvents.

Partial purification and properties of acid lipase in the bovine retinal pigment epithelium

Lysosomal acid lipase in the bovine retinal pigment epithelium was studied biochemically using triglyceride as substrate. Acid lipase was purified about 21-fold from a crude extract of the retinal pigment epithelium by ammonium sulfate fractionation, Sepharose 6B and DEAE-Sephadex column chromatography. The enzyme activity in the partially purified preparation was proportional to both the enzyme concentration and incubation time. When the enzyme was heated, the enzyme activity was lost completely. The optimal pH of the enzyme was 4·0 to 5·0. The enzyme was activated by Triton X-100, cysteine and β-mercaptoethanol, and inhibited by iodoacetic acid and p-chloromercuribenzoate. The partially purified enzyme exhibited a maximal activity with glycerol tridecanoate as substrate.

In vitro effect of prostaglandins and cyclic nucleotides on differential release of enzymes from lysosomes of the bovine retinal pigment epithelium

The effect of prostaglandins and cyclic nucleotides on lysosomes of bovine retinal pigment epithelial cells was studied in vitro using acid phosphatase and cathepsin D as lysosomal marker enzymes. Prostaglandin E 1 (PGE 1), cyclic AMP and cyclic GMP had no effect on the release of the two marker enzymes from lysosomes. Prostaglandin F 2α (PGF 2α) increased considerably the cathepsin D release from lysosomes, while it had little effect on the acid phosphatase release. However, PGE 1, PGF 2α, cyclic AMP and cyclic GMP did not affect the enzymatic activity of acid phosphatase or cathepsin D.

Lipids of bovine retinal pigment epithelium

Bovine retinal pigment epithelial cells have been prepared in high purity and their lipid class composition determined. The two major phospholipids are phosphatidyl ethanolamine and phosphatidyl choline, with phosphatidyl serine, phosphatidyl inositol and sphingomyelin being minor components. This composition is similar to that reported for bovine rod photoreceptor membranes. However, the fatty acids of the phospholipids of the retina pigment epithelium differ substantially from those of rod outer segments. The former contains high levels of saturated and low levels of long chain polyunsaturated fatty acids in all phospholipid classes.