scholarly journals SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium

2020 ◽  
Vol 319 (4) ◽  
pp. C641-C656
Author(s):  
Xu Cao ◽  
Manoocher Soleimani ◽  
Bret A. Hughes
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Subretinal Fluid ◽  
Wild Type Mouse ◽  
Biologically Active ◽  
Rpe Cells ◽  
Anion Conductance

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl− between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN−), a property that does not correspond with any of the Cl− channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN− permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl− channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN− conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN−-selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium

1997 ◽  
Vol 273 (2) ◽  
pp. C456-C472 ◽  
Author(s):  
E. Kenyon ◽  
A. Maminishkis ◽  
D. P. Joseph ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Cytosolic Ph ◽  
Acid Recovery ◽  
Tightly Coupled ◽  
Intracellular Microelectrodes

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.

scholarly journals Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

2018 ◽  
Vol 115 (47) ◽  
pp. E11120-E11127 ◽  
Author(s):  
Tamara L. Lenis ◽  
Jane Hu ◽  
Sze Yin Ng ◽  
Zhichun Jiang ◽  
Shanta Sarfare ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Wild Type Mouse ◽  
Mouse Retina ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Rpe Cells ◽  
Photoreceptor Outer Segments ◽  
Quantitative Immunoblotting

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk−/− but not Abca4−/− mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4−/− background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4−/− mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

scholarly journals Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.

1991 ◽  
Vol 112 (5) ◽  
pp. 863-872 ◽  
Author(s):  
D Gundersen ◽  
J Orlowski ◽  
E Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Current Knowledge ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Opposite Polarity ◽  
Apical Surface ◽  
Rpe Cells ◽  
Membrane Cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

scholarly journals Spectral OCT-Angiography in En Face Mode for Detection of Morphological Changes in the Retinal Pigment Epithelium before and after Selective Micro-Pulse Laser Exposure in Patients with Central Serous Chorioretinopathy

2019 ◽  
Vol 16 (2) ◽  
pp. 192-201
Author(s):  
P. L. Volodin ◽  
E. V. Ivanova ◽  
E. Iu. Polyakova ◽  
A. V. Fomin
Keyword(s):  
Retinal Pigment Epithelium ◽  
Central Serous Chorioretinopathy ◽  
Morphological Changes ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Laser System ◽  
Subretinal Fluid ◽  
Micropulse Laser ◽  
En Face ◽  
Before And After

Purpose — to study the morphological changes of the retinal pigment epithelium (RPE) by optical coherence tomography-angiography (OCT-A) in En Face mode before and after selective micropulse laser irradiation in patients with central serous chorioretinopathy (CSC), determine the correspondence between the topographic location of RPE defects and detachment on the OCT-angiogram in En Face mode and points of leakage on the FAG.Patients and methods. There were 20 patients (21 eyes) with CSC before and after laser treatment under the observation. All patients underwent high-resolution FAG and OCT-A using Angio Retina 2×2 or 3×3 mm protocol and Angio Retina HD 6×6 mm. The treatment was carried out in a selective micropulse mode with individual selection of parameters using the Navilas 577s navigation laser system (OD-OS, Germany) or the IQ 577 laser system (IRIDEX, USA).Results were evaluated at 2 weeks and 1 month after treatment. Results. In all cases, the leaking points of the subretinal fluid on FAG corresponded to the topographic location of defects and detachments of RPE detachment on OKT-A En Face. According to OCT-A En Face, the following morphological changes were revealed: in 5 cases — single defects of RPE, in 7 cases — multiple defects of RPE, in 9 cases of slit-like detachment of RPE, in 3 patients a combination of slit-like detachment and defect RPE. The sizes of RPE defects varied in the range from 21 to 159 microns, while their rounded shape prevailed. 1 month after the selective micropulse laser effect on OCT-A in the En Face mode, the defects were closed and the RPE detachments fit in all patients, which resulted in resorption of the subretinal fluid and the neurosensory retina attachment.Findings. OCT-A in En Face mode is a highly informative diagnostic method that allows noninvasive detection of morphological changes in RPE with a clear topographic localization relative to the retinal vascular network, as well as evaluating the effectiveness of selective micropulse laser treatment in patients with CSC. 

scholarly journals Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium

2021 ◽  
Vol 22 (17) ◽  
pp. 9618
Author(s):  
Jérémie Canonica ◽  
Min Zhao ◽  
Tatiana Favez ◽  
Emmanuelle Gelizé ◽  
Laurent Jonet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Retinal Pigment Epithelium ◽  
Barrier Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Diseases ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Pathway Activation ◽  
And Migration

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood–retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone—an MR-specific agonist, or cortisol or cortisol + RU486—a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial–mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

Microfilament organization and wound repair in retinal pigment epithelium

1995 ◽  
Vol 73 (9-10) ◽  
pp. 709-722 ◽  
Author(s):  
Vitauts. I. Kalnins ◽  
Martin Sandig ◽  
Greg J. Hergott ◽  
Haruhiko Nagai
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
Retinal Pigment Epithelium ◽  
Wound Repair ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Wound Edge ◽  
Rpe Cells ◽  
Oriented Parallel ◽  
Experimentally Induced

Several systems of microfilaments (MF) associated with adherens-type junctions between adjacent retinal pigment epithelial (RPE) cells and between these cells and the substratum play an important role in maintaining the integrity and organization of the RPE. They include prominent, contractile circumferential MF bundles that are associated with the zonula adherens (ZA) junctions. In chick RPE, these junctions are assembled from smaller subunits thus giving greater structural flexibility to the junctional region. Because the separation of the junctions requires trypsin and low calcium, both calcium-dependent and -independent mechanisms are involved in keeping adjacent RPE cells attached to one another. Another system of MF bundles that crosses the cell at the level of ZA junctions can be induced to form by stretching the epithelium. The MF bundles forming this system are oriented in the direction in which the RPE is stretched, thereby preventing the overextension of the cell in any one direction. The system may be useful as an indicator of the direction in which tension is experienced by RPE during development of the eye, in animal models of disease and during repair of experimentally induced wounds. Numerous single-cell wounds resulting from death of RPE cells by apoptosis at various stages of repair are normally present in developing chick and adult mammalian RPE. These wounds are repaired by the spreading of adjacent RPE cells and by the contraction of MF bundles oriented parallel to the wound edge, which develop during this time. As a result of the spreading in the absence of cell proliferation, the RPE cells increase in diameter with age. Experimentally induced wounds made by removing 5–10 RPE cells are repaired by a similar mechanism within 24 h. In repair of larger wounds, over 125 μm in width, the MF bundles oriented parallel to the wound edge characteristic of spreading cells are later replaced by stress fibers (SFs) that run perpendicularly to the wound edge and interact with the substratum at focal contacts (FCs) as RPE cells start to migrate. Cell proliferation is induced in cells along the wound edge only when the wounds are wide enough to require cell migration. In the presence of antibodies to beta-1-integrins, a component of FCs, cell spreading is not prevented but both cell migration and cell proliferation are inhibited. Thus, only the organization of the cytoskeleton characteristic of migrating RPE cells that have SFs that interact with the substratum at FCs, is associated with the induction of cell proliferation.Key words: retinal pigment epithelium, microfilaments, wound repair.

JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

2017 ◽  
Vol 117 (04) ◽  
pp. 750-757
Author(s):  
Xin Jia ◽  
Chen Zhao ◽  
Qishan Chen ◽  
Yuxiang Du ◽  
Lijuan Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinal Pigment Epithelium ◽  
Cell Survival ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelium Cell ◽  
Photoreceptor Cells ◽  
Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

Experimental eye enlargement in mature animals changes the retinal pigment epithelium

1999 ◽  
Vol 16 (4) ◽  
pp. 619-628 ◽  
Author(s):  
ALISON M. HARMAN ◽  
ROBERT HOSKINS ◽  
LYN D. BEAZLEY
Keyword(s):  
Retinal Pigment Epithelium ◽  
Retinal Pigment Epithelial ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Expansion Process ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Multinucleated Cells ◽  
Eye Opening ◽  
Comparable Size

Form deprivation has been shown to result in myopia in a number of species such that the eye enlarges if one eye is permanently closed at the time of eye opening. In the quokka wallaby, the eye grows slowly throughout life. After form deprivation, the eye enlarges by 1–1.5 years of age to the size of that in a 4–6-year-old animal and the number of multinucleated retinal pigment epithelial (RPE) cells in the enlarged retina remains much lower than would be expected in eyes of comparable size. Here we have repeated the experiment but examined animals at 4 years of age. The sutured eye grew significantly larger than did its partner. Numbers of RPE cells were comparable between sutured and partner eyes but were lower than in normal animals of similar age. Reductions in RPE cell density were greater in nasal than in dorsal or ventral retina and were not seen in temporal retina. The distribution of multinucleated cells was quite different in the sutured and open eyes. As in normal eyes, partner eyes had most multinucleated cells in ventral retina, while in the sutured eyes such cells were located mainly in the far periphery. In conclusion, the RPE is significantly changed by the eye enlargement process. However, it is not known whether this change results from an active part played by the RPE in the retinal expansion process or whether the changes are simply a result of a passive increase in area of the RPE.

scholarly journals Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes

2020 ◽  
Vol 21 (11) ◽  
pp. 3830 ◽  
Author(s):  
Yan Levitsky ◽  
Sandra S. Hammer ◽  
Kiera P. Fisher ◽  
Chao Huang ◽  
Travan L. Gentles ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Mitochondrial Function ◽  
High Glucose ◽  
Citrate Synthase ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Intercellular Adhesion Molecule ◽  
Early Event ◽  
Rpe Cells

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

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