scholarly journals Mouse retinal pigment epithelial cells exhibit a thiocyanate-selective conductance

2018 ◽  
Vol 315 (4) ◽  
pp. C457-C473 ◽  
Author(s):  
Xu Cao ◽  
Bikash R. Pattnaik ◽  
Bret A. Hughes
Keyword(s):  
Ion Selectivity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Potential Gradient ◽  
Retinal Pigment Epithelial Cells ◽  
Transport Pathway ◽  
Intact Cells ◽  
Anion Conductance ◽  
Apical Membranes

The basolateral membrane anion conductance of the retinal pigment epithelium (RPE) is a key component of the transepithelial Cl− transport pathway. Although multiple Cl− channels have been found to be expressed in the RPE, the components of the resting Cl− conductance have not been identified. In this study, we used the patch-clamp method to characterize the ion selectivity of the anion conductance in isolated mouse RPE cells and in excised patches of RPE basolateral and apical membranes. Relative permeabilities ( PA/ PCl) calculated from reversal potentials measured in intact cells under bi-ionic conditions were as follows: SCN− >> ClO4− > [Formula: see text] > I− > Br− > Cl− >> gluconate. Relative conductances ( GA/ GCl) followed a similar trend of SCN− >> ClO4− > [Formula: see text] > I− > Br− ≈Cl− >> gluconate. Whole cell currents were highly time-dependent in 10 mM external SCN−, reflecting collapse of the electrochemical potential gradient due to SCN− accumulation or depletion intracellularly. When the membrane potential was held at −120 mV to minimize SCN− accumulation in cells exposed to 10 mM SCN−, the instantaneous current reversed at −90 mV, revealing that PSCN/ PCl is approximately 500. Macroscopic current recordings from outside-out patches demonstrated that both the basolateral and apical membranes exhibit SCN− conductances, with the basolateral membrane having a larger SCN− current density and higher relative permeability for SCN−. Our results suggest that the RPE basolateral and apical membranes contain previously unappreciated anion channels or electrogenic transporters that may mediate the transmembrane fluxes of SCN− and Cl−.

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.

Pigment Epithelium-Derived Factor: A Novel Therapeutic Target for Cardiometabolic Diseases and Related Complications

2018 ◽  
Vol 25 (13) ◽  
pp. 1480-1500 ◽  
Author(s):  
Sho-ichi Yamagishi ◽  
Takanori Matsui
Keyword(s):  
Cardiovascular Disease ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Visceral Obesity ◽  
Retinal Pigment Epithelial Cells ◽  
Atherosclerotic Cardiovascular Disease ◽  
Cardiometabolic Diseases ◽  
The Metabolic Syndrome ◽  
Pigment Epithelium Derived Factor ◽  
Cardiometabolic Disorders

Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors, serpins. It was first identified as a neuronal differentiating factor secreted by human retinal pigment epithelial cells, and then found to be the most potent inhibitor of pathological angiogenesis in mammalian eyes. Recently, PEDF has been shown not only to suppress oxidative stress and inflammatory reactions in vascular wall cells, T cells and macrophages, and adipocytes, but also to exert antithrombotic and anti-fibrotic properties, thereby protecting against the development and progression of various cardiometabolic diseases and related complications. Furthermore, accumulating evidence has suggested that circulating PEDF levels may be a biomarker of severity and prognosis of these devastating disorders. Number of subjects with visceral obesity and insulin resistance is increasing, and the metabolic syndrome and its related complications, such as diabetes, nonalcoholic fatty liver disease/non-alcoholic steatohepatits, and atherosclerotic cardiovascular disease are a growing health challenge. Therefore, in this study, we review the pathophysiological role of PEDF in obesity and metabolic disorders, cardiovascular disease, diabetic eye and kidney complications, liver diseases, and reproductive system disorders, and discuss the potential clinical utility of modulating the expression and actions of PEDF for preventing these cardiometabolic disorders. We also refer to the clinical value of PEDF as a biomarker in cardiometabolic complications.

scholarly journals A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 179
Author(s):  
Laurence Klipfel ◽  
Marie Cordonnier ◽  
Léa Thiébault ◽  
Emmanuelle Clérin ◽  
Frédéric Blond ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Age Related Macular Degeneration ◽  
Lactate Transport ◽  
Ips Cell ◽  
Risk Alleles ◽  
Age Related ◽  
A Genome

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

scholarly journals Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances.

1988 ◽  
Vol 92 (3) ◽  
pp. 281-306 ◽  
Author(s):  
D Chang ◽  
D C Dawson
Keyword(s):  
Ion Selectivity ◽  
Basolateral Membrane ◽  
Permeability Barrier ◽  
Buffer System ◽  
Bathing Solution ◽  
Marker Enzyme ◽  
Cell Layers ◽  
Mucosal Side ◽  
Apical Membranes ◽  
Conductive Properties

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.

A double-membrane model for urinary bicarbonate secretion

1985 ◽  
Vol 249 (4) ◽  
pp. F546-F552 ◽  
Author(s):  
D. L. Stetson ◽  
R. Beauwens ◽  
J. Palmisano ◽  
P. P. Mitchell ◽  
P. R. Steinmetz
Keyword(s):  
Channel Blocker ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ultrastructural Examination ◽  
Transport Pathway ◽  
Anion Conductance ◽  
Luminal Membrane ◽  
Disulfonic Stilbene ◽  
Ph Stat

To define the transport pathway for HCO-3 secretion (JHCO3) across the apical and basolateral membranes of turtle bladder, we examined the effects of cAMP, isobutylmethylxanthine (IBMX), the Cl- channel blocker 9-anthroic acid (9-AA), and the disulfonic stilbene DIDS (4,4'-diisothiocyanostilbene-2,2'-sulfonic acid) on the electroneutral and electrogenic components of JHCO3. Total JHCO3 was measured by pH stat titration of the mucosal compartment after Na+ absorption and H+ secretion were abolished by ouabain and a delta pH, respectively. Addition of cAMP or IBMX increased total JHCO3 and induced a short-circuit current (ISC), accounting for a large part of JHCO3; net Cl- absorption was reduced. Mucosal 9-AA inhibited the IBMX-induced electrogenic component of JHCO3, whereas mucosal DIDS inhibited the electroneutral component and acetazolamide reduced both. We suggest that HCO-3 is generated within the cell by a Na-independent primary active acid-base transport at the basolateral membrane (H+ extrusion into the serosal compartment). Cellular HCO-3 accumulation drives JHCO3 via a Cl-HCO3 exchanger at the luminal membrane. IBMX and cAMP activate a 9-AA-sensitive anion conductance parallel to the exchanger. The apparent reversal of the transport elements between the two cell membranes (compared with H+-secreting cells) led to an ultrastructural examination of the carbonic anhydrase-rich cells.

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 Characterization of artificially re-pigmented ARPE-19 retinal pigment epithelial cell model

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Hellinen ◽  
Marja Hagström ◽  
Heidi Knuutila ◽  
Marika Ruponen ◽  
Arto Urtti ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Epithelial Cell ◽  
Cell Line ◽  
Retinal Pigment Epithelial ◽  
Cell Model ◽  
Retinal Pigment Epithelial Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Pigment Epithelial

Abstract Melanin pigment has a significant role in ocular pharmacokinetics, because many drugs bind at high extent to melanin in the retinal pigment epithelial cells. Most retinal pigment epithelial cell lines lack pigmentation and, therefore, we re-pigmented human ARPE-19 cells to generate a pigmented cell model. Melanosomes from porcine retinal pigment epithelium were isolated and co-incubated with ARPE-19 cells that spontaneously phagocytosed the melanosomes. Internalized melanosomes were functionally integrated to the cellular system as evidenced by correct translocation of cellular Rab27a protein to the melanosomal membranes. The pigmentation was retained during cell cultivation and the level of pigmentation can be controlled by altering the amount of administered melanosomes. We used these cells to study melanosomal uptake of six drugs. The uptake was negligible with low melanin-binders (methotrexate, diclofenac) whereas most of the high melanin-binders (propranolol, chloroquine) were extensively taken up by the melanosomes. This cell line can be used to model pigmentation of the retinal pigment epithelium, while maintaining the beneficial cell line characteristics, such as fast generation of cultures, low cost, long-term maintenance and good reproducibility. The model enables studies at normal and decreased levels of pigmentation to model different retinal conditions.

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