Chloride channel expression in cultured human fetal RPE cells: Response tooxidative stress

PURPOSE. The human fetal cell line RPE 28 SV4 has been useful for studies o f oxidative stress and apoptosis in retinal pigmented epithelium. This cell model is now assessed in functional investigations of chloride channel act ivity. The study aims to determine the presence of specific chloride channe ls, including CFTR and ClC channels, to identify the properties of membrane chloride currents and to assess their modulation by hydrogen peroxide, cAM P, and other agents. METHODs. Channel expression was determined using RT-PCR and cDNA cloning an d biochemical and immunocytochemical methods. Membrane currents were analyz ed using whole-cell, patch-clamp techniques. RESULTS. RT-PCR results confined the presence of ClC-5 mRNA, and a full-len gth clone encoding ClC-3 was isolated from a cDNA Library for RPE 28 SV4 ce lls. Specific staining for CFTR and several CIC channels was detected by im munocytochemistry. Whole-cell chloride currents (under conditions of symmet rical chloride concentrations) averaged 16.9 +/- 3.4 pA/pF (at +100 mV; n = 8), showed outward rectification, and had an anion permeability sequence o f Cl- > I- > cyclamate. Currents were stimulated by cAMP cocktail (250 muM cAMP, 100 muM IBMX, and 25 muM forskolin) and were inhibited by 1 mM DIDS. The oxidative agent hydrogen peroxide (100 muM) decreased the current by 34 % +/- 10% (n = 4). CONCLUSIONS. These findings suggest that RPE 28 SV4 cells possess regulated chloride channels including CFTR and members of the ClC chloride channel f amily. The inhibition of chloride currents by H2O2 suggests that this cell line may be advantageous for studies of chloride channel modulation by oxid ative stress.