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(Investigative Ophthalmology and Visual Science. 2000;41:4247-4255.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Chloride Channel Expression in Cultured Human Fetal RPE Cells: Response to Oxidative Stress

Nancy K. Wills1, Tianxiang Weng1, Lijun Mo1, Helen L. Hellmich2, Alan Yu3, Ting Wang1, Sarah Buchheit1 and Bernard F. Godley4

1 From the Departments of Physiology and Biophysics, 2 Internal Medicine, and 3 Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas; and 4 Renal Division, Brigham and Women’s Hospital, Harvard Institutes of Medicine, Boston, Massachusetts.

PURPOSE. The human fetal cell line RPE 28 SV4 has been useful for studies of oxidative stress and apoptosis in retinal pigmented epithelium. This cell model is now assessed in functional investigations of chloride channel activity. The study aims to determine the presence of specific chloride channels, including CFTR and ClC channels, to identify the properties of membrane chloride currents and to assess their modulation by hydrogen peroxide, cAMP, and other agents.

METHODS. Channel expression was determined using RT-PCR and cDNA cloning and biochemical and immunocytochemical methods. Membrane currents were analyzed using whole-cell, patch-clamp techniques.

RESULTS. RT-PCR results confirmed the presence of ClC-5 mRNA, and a full-length clone encoding ClC-3 was isolated from a cDNA library for RPE 28 SV4 cells. Specific staining for CFTR and several ClC channels was detected by immunocytochemistry. Whole-cell chloride currents (under conditions of symmetrical chloride concentrations) averaged 16.9 ± 3.4 pA/pF (at +100 mV; n = 8), showed outward rectification, and had an anion permeability sequence of Cl- > I- > cyclamate. Currents were stimulated by cAMP cocktail (250 µM cAMP, 100 µM IBMX, and 25 µM forskolin) and were inhibited by 1 mM DIDS. The oxidative agent hydrogen peroxide (100 µM) 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 family. The inhibition of chloride currents by H2O2 suggests that this cell line may be advantageous for studies of chloride channel modulation by oxidative stress.




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