Chloride channel expression in cultured human fetal RPE cells: response to oxidative stress.
OBJECTIVE
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 microM cAMP, 100 microM IBMX, and 25 microM forskolin) and were inhibited by 1 mM DIDS. The oxidative agent hydrogen peroxide (100 microM) 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 H(2)O(2) suggests that this cell line may be advantageous for studies of chloride channel modulation by oxidative stress.
Wills
N K
NK
Departments of Physiology, University of Texas Medical Branch, Galveston, Texas, USA. thieme@ukbf.fu-berlin.de
Weng
T
T
Mo
L
L
Hellmich
H L
HL
Yu
A
A
Wang
T
T
Buchheit
S
S
Godley
B F
BF
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis; D005333:Fetus; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D018408:Patch-Clamp Techniques; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C505032:CFTR protein, human; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D018076:DNA, Complementary; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D015056:1-Methyl-3-isobutylxanthine