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Nancy K. Wills, Tianxiang Weng, Lijun Mo, Helen L. Hellmich, Alan Yu, Ting Wang, Sarah Buchheit, Bernard F. Godley; Chloride Channel Expression in Cultured Human Fetal RPE Cells: Response to Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4247-4255. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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 (100μ
M) decreased the current by 34% ± 10% (n =
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
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