Rat Retinal Pigment Epithelium Research Articles

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mM KCl intracellular and 130 mM NaCl extracellular solutions, rat RPE cells possessed both inward and outward K+ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5'-O-(3-thiophosphate) (GTPgammaS, 0.1 mM), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs+-aspartate in the pipette, and was not affected by alterations in the extracellular Ca2+ or Cl- concentration. The GTPgammaS-activated current was found to be permeable to several monovalent cations (K+, Na+, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPgammaS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca2+ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K+ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K+.

Further Studies on the Identification of the Phagocytosis Receptor of Rat Retinal Pigment Epithelial Cells

We have previously produced a polyclonal antiserum (R 1S 5) against a plasma membrane-enriched fraction of rat retinal pigment epithelial (RPE) cells which inhibits the phagocytosis of photoreceptor outer segments (OS) by these cells. This antiserum has now been used to purify a subset of RPE membrane glycoproteins. Using a combination of lectin affinity chromatography, and chromatography on an affinity column made with R 1S 5-IgG, we have enriched an RPE membrane extract about 100-fold. This enriched extract contains only 12 components, all of which are glycoproteins, and retains the ability to adsorb out the inhibitory activity of antiserum R 1S 5. This shows that one or more of these glycoproteins recognizes an inhibitory IgG in R 1S 5and suggests that one or more of these glycoproteins may participate in the phagocytosis of OS by RPE cells, possibly as the phagocytosis receptor. We have performed N-terminal microsequencing of seven of these glycoproteins: four of the seven, with M rs of 34, 36, 51 and 55 kDa, show no sequence homology to any known proteins.

Role of LFA-1, ICAM-1, VLA-4 and VCAM-1 in lymphocyte migration across retinal pigment epithelial monolayers in vitro.

The blood-retinal barrier (BRB), which is composed of the retinal pigment epithelium (RPE) and retinal vascular endothelium, normally restricts the traffic of lymphocytes into the retina. During ocular inflammatory conditions such as posterior uveitis there is a large increase in lymphocyte migration across the BRB. The differential role played by the two barrier sites, however, remains unclear. To evaluate the role of the posterior BRB, the migration of CD4+ antigen-specific T-cell line through rat RPE cell monolayers was investigated in vitro using time-lapse videomicroscopy. The adhesion molecules involved in controlling transepithelial migration across normal and interferon-gamma (IFN-gamma)-activated RPE was assessed with monoclonal antibodies directed against cell adhesion molecules. Lymphocytes were treated with antibodies specific for CD11a (alpha L subunit of LFA-1), CD18 (beta 2 subnit of the leucam family) and CD49 d (alpha 4 subnit of very late activation antigen-4, VLA-4), and the RPE with antibodies specific for CD54 (intracellular adhesion molecule-1, ICAM-1) and CD 106 (vascular cell adhesion molecule-1, VCAM-1). Migration across unstimulated RPE was inhibited by antibodies to ICAM-1 (48.6 +/- 3.5% reduction), leucocyte functional antigen-1 (LFA-1) alpha (61 +/- 5.2%) and LFA-1 beta (63.2 +/- 4.7%), but not by antibodies to VLA-4. VCAM-1 was not expressed on untreated RPE. Following activation of the RPE monolayers for 72 hr with IFN-gamma, antibodies to LFA-1 alpha, LFA-1 beta and ICAM-1 inhibited migration by 49.9 +/- 9.4%, 63.6 +/- 5.5% and 47.7 +/- 4.2% respectively. Antibodies to VLA-4 and VCAM-1 blocked migration by 21.5 +/- 8.4% and 32.3 +/- 6.2%, respectively, which correlated with the induction of VCAM-1 expression on RPE and increased migration. Under these conditions blocking both VCAM-1 and ICAM-1 reduced migration by 70.9 +/- 2.3%, which was greater than the effect of blocking either of these molecules alone. These results demonstrate that the posterior barrier of the BRB utilizes the same principle receptor-ligand pairings in controlling lymphocyte traffic into the retina as the vascular endothelium of the anterior BRB.

Expression of transgenes in human and rat retinal pigment epithelium in vitro using an adenoviral vector.

Expression of transgenes in human and rat retinal pigment epithelium in vitro using an adenoviral vector.

Lymphocyte Migration across the Anterior and Posterior Blood–Retinal Barrierin Vitro

The migration of lymphocytes through monolayers of rat retinal pigment epithelium (RPE) and retinal vascular endothelium, which form the posterior and anterior blood–retinal barrier (BRB) respectively, was investigatedin vitro.After a 4-hr assay the migration of untreated peripheral lymph node (PLN) cells through RPE monolayers was negligible (<1%) with only a small increase found after activation of the PLN cells with concanavalin A or by cross-linking CD3. Activation of the RPE with IFN-γ augmented migration with maximal PLN cell migration being achieved with a combination of CD3 cross-linking and IFN-γ activation (17% migration). The highest level of lymphocyte migration was observed with three CD4+antigen-specific T cell lines specific for purified protein derivative (PPD; 33% migration), ovalbumin (OA; 31%), and S-antigen (S-Ag; 57%). Migration of both untreated and Con A-activated PLN cells through retinal endothelial cells (EC) from PVG rats was negligible, whereas the migration of the antigen-specific T cell lines was 23, 29, and 23% for PPD, OA, and S-Ag lines, respectively. Migration of these cell lines through retinal endothelium derived from Lewis rats was significantly greater (44% for PPD, 39% for OA, and 39% for S-Ag) which corresponded with a greater expression of ICAM-1 on the EC.

CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.

Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein. Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36. Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments. We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.

Cytokine effects on phagocytosis of rod outer segments by retinal pigment epithelial cells of normal and dystrophic rats.

Phagocytosis of rod outer segments (ROS) is an important function of retinal pigment epithelial (RPE) cells. Since the details of the process are not fully known, we studied effects of cytokines produced by RPE and photoreceptor cells on phagocytosis of ROS by rat RPE cells. RPE cells were isolated and cultivated from two strains of rats: Sprague-Dawley (SD) rats with normal phagocytosis and Royal College of Surgeons (RCS) rats, which have genetic deficiencies in ROS phagocytosis. A double immunofluorescence staining technique was used to study the effects in vitro of several cytokines on phagocytosis of ROS. We found that transforming growth factor beta-1 (TGF-beta 1) had dose-dependent effects on RPE cells of both strains of rat: at a concentration of 10 ng/ml, TGF-beta 1 significantly (p < 0.01) reduced total ROS (to 74% of control in SD rats and to 51% of control in RCS rats), reduced bound ROS (to 56% of control in SD rats and to 48% in RCS rats), and increased the ratio of ingested ROS to total ROS (to 140% in SD rats but not significantly in RCS rats). Treatment of medium with anti-TGF-beta 1 antibody before incubation of RPE cells of SD rats with TGF-beta 1 decreased the magnitude of these effects. The cytokine acidic fibroblast growth factor (aFGF, 10 ng/ml) affected RPE cells of SD rats only, decreasing ROS ingested to 56% of control and the ratio of ingested ROS to total ROS to 64% of control. We also examined effects of basic fibroblast growth factor and insulin-like growth factor. None of the cytokines tested increased ingestion of ROS by RPE cells of RCS rats. Our results suggest that TGF-beta 1 and aFGF have roles in regulating ROS phagocytosis by normal and dystrophic RPE cells in the rat.

Nitric oxide induces c- fos gene expression via cyclic AMP response element binding protein (CREB) phosphorylation in rat retinal pigment epithelium

We have examined whether nitric oxide (NO) induces the expression of c- fos proto-oncogene and the phosphorylation of cyclic AMP response element binding protein (CREB) in the rat retina. NO donor sodium nitroprusside (SNP) was injected into the vitreal cavity of the eye, and the retina and retinal pigment epithelium (RPE) was analysed by in situ hybridization using single-stranded RNA probes for c- fos transcripts and by immunocytochemistry using an anti-phospho Ser-133 antibody 20 min after the injection of SNP, phosphorylated-CREB immunoreactivity was found in RPE cells and weakly in some cells of the INL. Forty-five min after the SNP injection, the expression of c- fos mRNA was detected in the RPE. These results suggested that NO induced the c- fos expression via the phosphorylation of CREB in RPE cells, as it had been demonstrated in PC12 cells that the transcription of c- fos gene was activated by the CREB phosphorylation.

Retroviral gene transfer into retinal pigment epithelial cells followed by transplantation into rat retina.

In this preliminary report, we describe a technique for gene transfer into the retina using a retrovirus vector. We transferred the bacterial LacZ gene and the neomycin-resistance gene into pigmented wild-type rat retinal pigment epithelial (RPE) cells in culture. The RPE culture was exposed to retrovirus, and infected cells were selected with a neomycin analog (G418). The LacZ gene product was detected by X-Gal histochemistry in 95-100% of drug-resistant cells. These genetically labeled cells were transplanted into the subretinal space of two 15- to 25-day-old albino RCS rats, which have an inherited retinal degeneration syndrome. The retinas were fixed and stained with X-Gal at 3 and 6 weeks after transplantation. At both time points, pigmented, LacZ-containing cells were seen in the subretinal space. Further, there were several rows of photoreceptor nuclei in the transplant area of the approximately 2-month-old rats, while in the control contralateral eye the photoreceptor nuclei were virtually absent, as for untreated animals, suggesting that the transplanted LacZ-marked, wild-type RPE cells may have helped preserve photoreceptors. The technique for gene transfer into RPEs followed by transplantation thus provides a means for gene therapy in organisms with a genetic defect in RPE cells.

Identification of sodium channel subtypes induced in cultured retinal pigment epithelium cells.

Recent electrophysiological experiments have shown that retinal pigment epithelium (RPE) cells begin to produce neuronal-type voltage-dependent sodium currents when placed in dissociated cell culture. In this study, the sodium channel types induced in cultured rat RPE cells were identified. Sodium channel mRNAs encoding two distinct alpha subunits were detected in the cultured RPE cells, brain type II/IIA, and a novel rat mRNA which we have termed RET1. These two sodium channel types may correspond to the TTX-sensitive and TTX-insensitive components of sodium current reported previously in cultured rat RPE cells.

Cell-swelling induced amino acid efflux from human placental tissue

Cells of the retinal pigment epithelium (RPE) were isolated from neonatal rats. The perforated-patch clamp technique, using amphotericin-B, revealed a chloride current, which was detected as a 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)-sensitive component.A variety of chloride-channel inhibitors, other than DIDS, also blocked the chloride current, including 9-anthracenecarboxylic acid (9-AC), niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and N-phenylanthranilic acid (DPC). DIDS at 1 mmol l-1 and DPC at 2 mmol l-1 were both more potent than niflumic acid at 200 μmol l-1.The hyperosmotic condition (+ 50 mosmol) did not affect the chloride current. The hypo-osmotic condition (- 80 mosmol), however, led to an increase of total membrane conductance, during which a chloride conductance increase occurred. The membrane-conductance increase, evoked by the hypo-osmotic condition, ran up and then ran down by itself. As with the steady-state chloride conductance, 1 mmol l-1 DIDS and 2 mmol l-1 DPC were more potent blockers than 200 μmol l-1 niflumic acid. DIDS at 1 mmol l-1 was more effective in blocking the outward component than 2 mmol l-1 DPC, while the inward component was blocked oppositely.When the intracellular calcium concentration was presumed to have increased, using the calcium ionophore, ionomycin, membrane conductance abruptly increased, quickly ran up and then ran down by itself. This included an elevation of a DIDS-sensitive current, and was largely carried by chloride. Chloride currents of rat RPE cells did not appear to be regulated by cyclic AMP.

Acetyl- and butyrylcholinesterase activities in the rat retina and retinal pigment epithelium.

The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in the neural retina and retinal pigment epithelium (RPE) of adult rats were determined. The tissues were extracted with a saline buffer to release the soluble enzymes (S1) and the pellet re-extracted with Triton X-100 to detach the membrane-bound enzymes (S2). Less than 5% of the cholinesterase activity measured in retina and almost 30% of that assayed in RPE was due to BChE. About 20% and 10% of the AChE in retina and RPE was brought into solution with a saline buffer and the rest with a detergent-containing buffer. Main AChE molecular forms of 10.5S (hydrophilic G4H), 9.5S (amphiphilic G4A) and 3.0S (amphiphilic G1A) were identified in retina by subjecting the supernatant S1 to sedimentation analysis in sucrose gradients made with Brij 96. Amphiphilic G4 and G1 AChE were found in S2. Analysis of the soluble fractions obtained from RPE in the gradients made with Brij 96 revealed 16.0S (asymmetric A12), 10.5-10.0S (globular G4H + G4A), 4.5S (G2A), and 3.0S (G1A) AChE forms in S1, whereas G4A, G2A, and G1A enzyme molecules predominated in S2. Our results show that amphiphilic tetramers and monomers of AChE are abundant in neural retina, and enzyme tetramers, dimers, and monomers in RPE. The AChE in the neural retina might be involved in cholinergic actions. The enzyme function in the retinal pigment epithelium remains to be established.

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules.

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.

In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina

Divergence of neural retinal and retinal pigment epithelial (RPE) lineages from the optic vesicle neuroepithelium starts at a very early stage of eye development. Partially or even fully differentiated RPEs of some vertebrate species are capable of transforming into neural retina. In the present study, we have shown that mammalian RPE possesses the ability to transdifferentiate into neural retina at early embryonic stages. If cultured in serum-free medium, presumptive rat RPE became pigmented and expressed a molecular marker of mature RPE. In the presence of basic fibroblast growth factor (bFGF), cultured early embryonic rat RPE did not acquire pigment and grew to form retina-like multilayer structure containing neuronal cells and cells that express markers of retinal ganglion, amacrine and rod photoreceptor cells. The effects of bFGF occurred independently of effects on cell division and became irreversible after periods that varied with tissue age. This study has demonstrated that already differentiated embryonic rat RPE still retain the ability to become neural retina up to certain stage.

Müller cell survival and proliferation in response to medium conditioned by the retinal pigment epithelium

Müller cells have been implicated in the pathogenesis of proliferative vitreoretinopathy and subretinal scar formation; however, the source(s) and signal(s) responsible for their activation are unknown. This study was undertaken to determine if the retinal pigment epithelium (RPE) could be involved in this signaling process by studying its effects on Müller cell survival and division in vitro. A pure population of Müller cells isolated from 1–2 day Long-Evans rats was seeded at low density and treated with medium conditioned by neonatal rat RPE (RPE-CM) or a nonconditioned, defined medium. By day 3, Müller cells cultured in RPE-CM increased in number 2-fold. These cells survived up to 21 days, which was the longest time tested. In contrast, cell number decreased in control wells 75% by day 3, and 100 by day 4. The RPE-mediated survival and proliferation of the Müller cells occurred in a dose-dependent manner. The mitogenic response was specific for the RPE when compared with fibroblasts and non-retinal epithelial cells. Heat and trypsin treatment of the RPE-CM completely abolished its survival and mitogenic activity. These findings demonstrate the establishment of an in vitro model which can be used to investigate RPE-Müller cell interactions. This study also provides evidence for RPE involvement in Müller cell survival and proliferation.

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than −30 mV, a time-dependent outward current was activated. Inhibition by Ba 2+ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba 2+ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than — 70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.

Extracellular matrix proteins as substrate modulate the pattern of calcium channel expression in cultured rat retinal pigment epithelial cells.

We investigated the effect of different culture substrates on the expression of membrane conductances for calcium in cultured rat retinal pigment epithelial (RPE) cells using the perforated patch technique and barium as charge carrier. In younger cultures (up to 12 days old) the RPE cells expressed L-type calcium channels, in older cultures (more than 12 days old) LVA-type channels. The LVA-type channels have been characterized as a tetrodotoxin sensitive Ca2+ channels. Coating the culture substrate with laminin, shifted the culture age for expression of LVA-type channels to 7 days. When collagen type 4 was used as substrate LVA-type channels and L-type channels were expressed simultaneously in 7 days old cultures. We concluded that proteins of the extracellular matrix which are known to enhance cell differentiation in culture, enhance the expression of LVA-type channels in RPE cells.

Muscarinic receptors binding in retinal pigment epithelium during rat development

[3H]Quinuclidinyl benzylate (3H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of 3H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of 3H-QNB binding showed two high affinity sites to RPE, with KB = 2.6nM and 45 nM. Specific 3H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of 3H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.

Voltage-dependent sodium channels develop in rat retinal pigment epithelium cells in culture.

Whole-cell patch-clamp recordings were made from isolated cells of the retinal pigment epithelium (RPE) of neonatal rats. After 24 hr in cell culture, RPE cells developed a transient, voltage-activated inward current that was never observed in acutely isolated cells. The kinetics, voltage-dependence, and reversal potential of the current and its dependence on external sodium demonstrated that the current was due to the expression of voltage-activated Na+ channels in cultured RPE cells. The current was partly blocked by tetrodotoxin at low concentrations (< 100 nM), but a second component of Na+ current was unblocked by tetrodotoxin at concentrations up to 10 microM. Na+ channels were present in cultured RPE cells at sufficient density to support regenerative action potentials in voltage recordings. Both the epithelial cells of the RPE and the neurons of the retina derive embryonically from neural origin, and it is known that under certain circumstances, nonmammalian RPE cells retain the ability to take on neuronal characteristics. The development of voltage-activated Na+ channels and the presence of action potentials demonstrate that neonatal mammalian RPE cells are also capable of expressing neuronal characteristics in cell culture.

Chloride Currents in Freshly Isolated Rat Retinal Pigment Epithelial Cells

Cells of the retinal pigment epithelium (RPE) were isolated from neonatal rats. The perforated-patch clamp technique, using amphotericin-B, revealed a chloride current, which was detected as a 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)-sensitive component.A variety of chloride-channel inhibitors, other than DIDS, also blocked the chloride current, including 9-anthracenecarboxylic acid (9-AC), niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and N-phenylanthranilic acid (DPC). DIDS at 1 mmol l-1 and DPC at 2 mmol l-1 were both more potent than niflumic acid at 200 μmol l-1.The hyperosmotic condition (+ 50 mosmol) did not affect the chloride current. The hypo-osmotic condition (- 80 mosmol), however, led to an increase of total membrane conductance, during which a chloride conductance increase occurred. The membrane-conductance increase, evoked by the hypo-osmotic condition, ran up and then ran down by itself. As with the steady-state chloride conductance, 1 mmol l-1 DIDS and 2 mmol l-1 DPC were more potent blockers than 200 μmol l-1 niflumic acid. DIDS at 1 mmol l-1 was more effective in blocking the outward component than 2 mmol l-1 DPC, while the inward component was blocked oppositely.When the intracellular calcium concentration was presumed to have increased, using the calcium ionophore, ionomycin, membrane conductance abruptly increased, quickly ran up and then ran down by itself. This included an elevation of a DIDS-sensitive current, and was largely carried by chloride. Chloride currents of rat RPE cells did not appear to be regulated by cyclic AMP.