Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport

2005 ◽  
Vol 288 (1) ◽  
pp. C132-C140 ◽  
Author(s):  
David Reigada ◽  
Claire H. Mitchell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Vesicular Transport ◽  
Atp Release ◽  
Retinal Pigment Epithelial Cells ◽  
Subretinal Space ◽  
Transport Pathways ◽  
Rpe Cells

The retinal pigment epithelium (RPE) faces the photoreceptor outer segments and regulates the composition of the interstitial subretinal space. ATP enhances fluid movement from the subretinal space across the RPE. RPE cells can themselves release ATP, but the mechanisms and polarity of this release are unknown. The RPE expresses the cystic fibrosis transmembrane conductance regulator (CFTR), and CFTR is associated with ATP release in other epithelial cells. However, an increasing number of reports have suggested that the exocytotic pathway contributes to release. In the present study, we examined the involvement of CFTR and the vesicular pathway in ATP release from RPE cells. Release from cultured human ARPE-19 cells and across the apical membrane of fresh bovine RPE cells in an eyecup was studied. A cAMP cocktail to activate CFTR triggered ATP release from fresh and cultured RPE cells. Release from both RPE preparations was largely prevented by the broad-acting blocker glibenclamide and the specific thiazolidinone CFTR inhibitor CFTR-172. The block by CFTR-172 was enhanced by preincubation and prevented ATP release with 3.5 μM IC50. The rise in intracellular Ca2+ accompanying hypotonic challenge was prevented by CFTR-172. The vesicular transport inhibitor brefeldin A prevented ATP release after stimulation with both hypotonic and cAMP conditions, suggesting vesicular insertion was also involved. These results show an intimate involvement of CFTR in ATP release from RPE cells which can autostimulate receptors on the apical membrane to modify Ca2+ signaling. The requirement for both CFTR and vesicular transport pathways suggests vesicular insertion of CFTR may underlie the release of ATP.

scholarly journals Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Antonio E. Paniagua ◽  
Alicia Segurado ◽  
Jorge F. Dolón ◽  
Julián Esteve-Rudd ◽  
Almudena Velasco ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Cell Junction ◽  
Subretinal Space ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Apicobasal Polarity

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

Degradation of extracellular ATP by the retinal pigment epithelium

2005 ◽  
Vol 289 (3) ◽  
pp. C617-C624 ◽  
Author(s):  
David Reigada ◽  
Wennan Lu ◽  
Xiulan Zhang ◽  
Constantin Friedman ◽  
Klara Pendrak ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Adenosine Diphosphate ◽  
Cell Types ◽  
P2y Receptors ◽  
Extracellular Atp ◽  
Pcr Analysis ◽  
Subretinal Space ◽  
Rpe Cells ◽  
Mrs 2179

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and βγ-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

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.

scholarly journals Isolation of Bovine Retinal Pigment Epithelial Cells Using Adhesion to Agarose: Demonstration of Cellular and Regional Heterogeneity

2003 ◽  
Vol 51 (1) ◽  
pp. 121-124 ◽  
Author(s):  
Eleonore Fröhlich ◽  
Elke Maier ◽  
Christian Klessen
Keyword(s):  
Enzymatic Activity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Histochemical Staining ◽  
Retinal Pigment Epithelial Cells ◽  
Isolated Cells ◽  
Regional Heterogeneity ◽  
Rpe Cells ◽  
A New Technique

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

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.

scholarly journals Changes in apical [K+] produce delayed basal membrane responses of the retinal pigment epithelium in the gecko.

1984 ◽  
Vol 83 (2) ◽  
pp. 193-211 ◽  
Author(s):  
E R Griff ◽  
R H Steinberg
Keyword(s):  
Membrane Potential ◽  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basal Membrane ◽  
Subretinal Space ◽  
Bathing Solution ◽  
Subsequent Decrease ◽  
Opposite Sequence

We describe here a new retinal pigment epithelium (RPE) response, a delayed hyperpolarization of the RPE basal membrane, which is initiated by the light-evoked decrease of [K+]o in the subretinal space. This occurs in addition to an apical hyperpolarization previously described in cat (Steinberg et al., 1970; Schmidt and Steinberg, 1971) and in bullfrog (Oakley et al., 1977; Oakley, 1977). Intracellular and extracellular potentials and measurements of subretinal [K+]o were recorded from an in vitro preparation of neural retina-RPE-choroid from the lizard Gekko gekko in response to light. Extracellularly, the potential across the RPE, the transepithelial potential (TEP), first increased and then decreased during illumination. Whereas the light-evoked decrease in [K+]o predicted the increase in TEP, the subsequent decrease in TEP was greater than predicted by the reaccumulation of [K+]o. Intracellular RPE recordings showed that a delayed hyperpolarization generated at the RPE basal membrane produced the extra TEP decrease. At light offset, the opposite sequence of membrane potential changes occurred. RPE responses to changes in [K+]o were studied directly in the isolated gecko RPE-choroid. Decreasing [K+]o in the apical bathing solution produced first a hyperpolarization of the apical membrane, followed by a delayed hyperpolarization of the basal membrane, a sequence of membrane potential changes identical to those evoked by light. Increasing [K+]o produced the opposite sequence of membrane potential changes. In both preparations, the delayed basal membrane potentials were accompanied by changes in basal membrane conductance. The mechanism by which a change in extracellular [K+] outside the apical membrane leads to a polarization of the basal membrane remains to be determined.

Stress-induced ATP release from and growth modulation of human lens and retinal pigment epithelial cells

2003 ◽  
Vol 31 (6) ◽  
pp. 1213-1215 ◽  
Author(s):  
J.A. Eldred ◽  
J. Sanderson ◽  
M. Wormstone ◽  
J.R. Reddan ◽  
G. Duncan
Keyword(s):  
Epithelial Cells ◽  
Retinal Pigment ◽  
Atp Release ◽  
Cell Types ◽  
Cellular Systems ◽  
Retinal Pigment Epithelial Cells ◽  
Gene Profiling ◽  
Human Lens ◽  
Growth Modulation ◽  
Pigment Epithelial Cells

ATP release has been shown to occur following stimulation in several cellular systems. This study was undertaken to determine if lens and retinal epithelial cells release ATP in response to physiological stresses and to elucidate a possible role for ATP. Analysis of human aqueous humour samples showed a mean ATP level of 37.8±7.7 nM. Hyper-osmotic stress induced a dose- and time-dependent release of ATP. Both cell types were found to proliferate in serum-free medium, and the addition of ATP and adenosine at concentrations as low as 0.1 nM inhibited growth. Gene profiling also demonstrated the presence of the ectonucleotidases CD39 and CD73 and the A1 adenosine receptor on both cell types.

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