Isolated bovine retinal pigment epithelial cells express delayed rectifier type and M-type K+ currents

1997 ◽  
Vol 273 (3) ◽  
pp. C790-C803 ◽  
Author(s):  
M. Takahira ◽  
B. A. Hughes
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Type K ◽  
K Current ◽  
K Currents

Outwardly rectifying K+ currents in freshly isolated bovine retinal pigment epithelial (RPE) cells were characterized using the whole cell and perforated-patch configurations of the patch-clamp technique. All cells exhibited a delayed rectifier type K+ current. This current had an activation threshold voltage of approximately -40 mV, activated with a sigmoidal trajectory, and inactivated completely over a period of several seconds. External tetraethylammonium (TEA) was an effective blocker of the delayed rectifier current [apparent dissociation constant (Kd) = 5.1 mM], but external Ba2+ was relatively ineffective. Approximately 24% of the cells also exhibited a sustained outwardly rectifying K+ current that became activated at voltages positive to approximately -80 mV. This current resembled the neuronal M-current. External Ba2+ was a potent blocker of this current (apparent Kd = 1.1 mM), but external TEA and Cs+ were relatively ineffective. These results indicate that freshly isolated bovine RPE cells express K+ currents of both the delayed rectifier and M types. The latter may contribute to the resting K+ conductances of the apical and basolateral membranes.

An outwardly rectifying K+ current active near resting potential in human retinal pigment epithelial cells

1995 ◽  
Vol 269 (1) ◽  
pp. C179-C187 ◽  
Author(s):  
B. A. Hughes ◽  
M. Takahira ◽  
Y. Segawa
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Outward Current ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
K Current

Currents in freshly dissociated adult human retinal pigment epithelial (RPE) cells were studied using the perforated patch-clamp technique. The zero-current potential (V0) averaged -48.9 +/- 7.7 mV (n = 50). Depolarizing voltage pulses from -70 mV evoked an outward current that activated with first-order kinetics and that did not inactivate during prolonged depolarizations. Repolarizing the membrane potential produced tail currents that reversed near the K+ equilibrium potential, indicating that the sustained outward current was carried mainly by K+. The outwardly rectifying K+ conductance (gK) had an activation threshold voltage near -60 mV and was half-maximal at -37 mV. Approximately 25% of gK was active at the average V0. The K+ current was nearly completely blocked by 2 mM Ba2+ but was relatively insensitive to 20 mM tetraethylammonium. The kinetics, voltage dependence, and blocker sensitivity of this current clearly distinguish it from delayed rectifier K+ currents previously identified in RPE cells. We conclude that the sustained outward K+ current may help establish the resting potential of the apical and/or basolateral membranes and may also participate in K+ transport across the RPE.

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells

Planta Medica ◽  
2018 ◽  
Vol 84 (14) ◽  
pp. 1030-1037 ◽  
Author(s):  
Wayne Liu ◽  
Shorong-Shii Liou ◽  
Tang-Yao Hong ◽  
I-Min Liu
Keyword(s):  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Colorimetric Assay ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Antioxidant Enzyme Activities ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rpe Cells ◽  
Pigment Epithelial

AbstractThe present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility

2014 ◽  
Vol 31 (1) ◽  
pp. 1-10 ◽  
Author(s):  
CHRISTINA KING-SMITH ◽  
RONALD J. VAGNOZZI ◽  
NICOLE E. FISCHER ◽  
PATRICK GANNON ◽  
SATYA GUNNAM
Keyword(s):  
Epithelial Cells ◽  
Actin Filament ◽  
Concanavalin A ◽  
Actin Filaments ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

AbstractRetinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Carrier-dependent and carrier-independent uptake of myo-inositol in cultured retinal pigment epithelial cells: activation by heat and concentration

1988 ◽  
Vol 66 (9) ◽  
pp. 942-950 ◽  
Author(s):  
Mahin Khatami
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Retinal Pigment Epithelial Cells ◽  
Transport Systems ◽  
Rate Equations ◽  
Rpe Cells ◽  
Major Mechanism ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

Transport of myo-inositol (MI) was studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. At low external concentrations (0.01–1 mM), uptake appeared to follow saturation kinetics, although the reciprocal forms of the rate equations did not fit either Lineweaver–Burk or Eadie–Hofstee plots. Increasing external concentrations dramatically changed the pattern of MI entry. At two to three orders of magnitude higher than physiological concentrations, a second saturation occurred (pseudo saturation). Cells incubated with 20 μM [3H]MI for 60 min had a ratio of intracellular to extracellular radioactivity ≥ 8, indicating active transport.MI transport reduction by Na+ replacement or inhibitors (phlorizin, ouabain, amiloride, KSCN, iodoacetamide, MI analogues) was greater when RPE cells were incubated with low (20–400 μM) than with high (10–20 mM) MI concentrations. Cells incubated with 20 μM MI at 53 or 65 °C showed increased transport (up to 560%) compared with cells at 22 °C. The effect on MI uptake (20 μM) of Na+ replacement also was reduced at 53 °C. The uptake of MI involved at least two transport systems. The major mechanism at low external MI concentrations (physiological levels) was a carrier-mediated active process. At high external MI levels, uptake occurred by a diffusion process. A lipotropic effect of MI may be responsible for this increased rate of diffusion.

scholarly journals Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

2020 ◽  
Author(s):  
Janosch P Heller ◽  
Tristan G Heintz ◽  
Jessica CF Kwok ◽  
Keith R Martin ◽  
James W Fawcett
Keyword(s):  
Retinal Pigment Epithelial ◽  
Expression Profiles ◽  
Retinal Pigment ◽  
Cell Types ◽  
Human Cell Line ◽  
Retinal Pigment Epithelial Cells ◽  
Proper Function ◽  
Integrin Expression ◽  
Rpe Cells ◽  
Pigment Epithelial

AbstractRetinal pigment epithelial (RPE) cells have been used in disease modelling and transplantation studies in retinal diseases. Several types of RPE cells have been trialed, ranging from primary cells and immortalized cell lines to stem cell-derived RPE cells. During aging and in disease, the extracellular environment of the RPE cells changes, interfering with RPE cell adhesion. We hypothesize that this could be a key problem in transplantation studies that have reported lack of adhesion and survival of the transplanted RPE cells. Integrins are essential for the proper function of the RPE, mediating adhesion to Bruch’s membrane, and the binding and subsequent phagocytosis of photoreceptor outer segments. Variability that has been found in clinical trials might be due to the variability of cell types used and their expression profiles of surface molecules. Here, we set out to analyze integrin expression in primary rat RPE cells and in the human cell line ARPE-19 using immunochemistry. We found that both cell types express integrins to varying degrees. After long-term culturing, ARPE-19 cells resemble mature RPE cells, and increase integrin expression. We hence argue that it is important to test the properties of these cells prior to transplantation to avoid failure of adhesion and to facilitate correct function.

scholarly journals ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. C1372-C1383 ◽  
Author(s):  
Bret A. Hughes ◽  
Masayuki Takahira
Keyword(s):  
Retinal Pigment Epithelial ◽  
Atp Hydrolysis ◽  
Retinal Pigment ◽  
Underlying Mechanism ◽  
Retinal Pigment Epithelial Cells ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Pigment Epithelial ◽  
Cell Recording ◽  
Inwardly Rectifying

Inwardly rectifying K+ current ( I Kir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, I Kir ran down completely in <10 min [half time ( t 1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained I Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, I Kir ran down by ∼71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+( t 1/2 = 1.8 min). I Kir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5′-adenylylimidodiphosphate ( t 1/2 = 2.7 min) or 4 mM adenosine 5′- O-(3-thiotriphosphate) ( t 1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of I Kirin bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.

scholarly journals Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells

2019 ◽  
Vol 20 (6) ◽  
pp. 1387 ◽  
Author(s):  
Abdulwahab Alamri ◽  
Lincoln Biswas ◽  
David Watson ◽  
Xinhua Shu
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Nucleotide Metabolism ◽  
Retinal Pigment Epithelial Cells ◽  
Age Related Macular Degeneration ◽  
Clinical Feature ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Age Related ◽  
The Impact

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.

scholarly journals Depletion of Mitochondrial DNA in Differentiated Retinal Pigment Epithelial Cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xinqian Hu ◽  
Melissa A. Calton ◽  
Shibo Tang ◽  
Douglas Vollrath
Keyword(s):  
Mitochondrial Dna ◽  
Retinal Pigment Epithelial ◽  
Aerobic Glycolysis ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Response To Treatment ◽  
Flux Analysis ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Mitochondrial Dna Depletion

Abstract We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6–24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.

scholarly journals Gremlin in the Vitreous of Patients with Proliferative Diabetic Retinopathy and the Downregulation of Gremlin in Retinal Pigment Epithelial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-6 ◽  
Author(s):  
Dong Qin ◽  
Yan-rong Jiang ◽  
Zijun Meng
Keyword(s):  
Extracellular Matrix ◽  
Diabetic Retinopathy ◽  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Rpe Cells ◽  
Glucose Levels ◽  
Pigment Epithelial ◽  
Extracellular Matrix Molecules

Diabetic retinopathy (DR) is one of the most common causes of blindness globally. Proliferative DR (PDR), an advanced stage of DR, is characterized by the formation of fibrotic membranes at the vitreoretinal interface. The proliferation, migration, and secretion of extracellular matrix molecules in retinal pigment epithelial (RPE) cells contribute to the formation of fibrotic membranes in PDR. Gremlin has been reported to be upregulated in response to elevated glucose levels in the retina of diabetic rat and bovine pericytes. However, the role of gremlin in PDR remains unclear. In the present study, the vitreous concentrations of gremlin were significantly higher in the PDR (67.79±33.96) group than in the control (45.31±12.31) group, and high glucose levels induced the expression of gremlin in RPE cells. The elevated expression of extracellular matrix molecules, such as fibronectin and collagen IV, was significantly reduced by gremlin siRNA in human RPE cells under high-glucose conditions. Thus, gremlin may play a vital role in the development of PDR.

Protection of Photoreceptor Cells from Phototoxicity by Transplanted Retinal Pigment Epithelial Cells Expressing Different Neurotrophic Factors

2005 ◽  
Vol 14 (10) ◽  
pp. 799-808 ◽  
Author(s):  
Toshiaki Abe ◽  
Yoko Saigo ◽  
Masayoshi Hojo ◽  
Tetsuya Kano ◽  
Ryosuke Wakusawa ◽  
...  
Keyword(s):  
Neurotrophic Factors ◽  
Retinal Pigment Epithelial ◽  
Outer Nuclear Layer ◽  
Retinal Pigment ◽  
Green Fluorescence Protein ◽  
Retinal Pigment Epithelial Cells ◽  
Subretinal Space ◽  
Egfp Gene ◽  
Rpe Cells ◽  
Pigment Epithelial

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.

Sign in / Sign up

Export Citation Format

Share Document