Chloride channel expression in cultured human fetal RPE cells

Response to oxidative stress

N. K. Wills, T. Weng, L. Mo, Helen Hellmich, A. Yu, T. Wang, S. Buchheit, B. F. Godley

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4247-4255
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume41
Issue number13
StatePublished - 2000

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Chloride Channels
Oxidative Stress
Chlorides
Hydrogen Peroxide
Cyclamates
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
1-Methyl-3-isobutylxanthine
Cell Line
Polymerase Chain Reaction
Membranes
Patch-Clamp Techniques
Colforsin
Gene Library
Anions
Organism Cloning
Permeability
Epithelium
Complementary DNA
Clone Cells
Immunohistochemistry

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Chloride channel expression in cultured human fetal RPE cells : Response to oxidative stress. / Wills, N. K.; Weng, T.; Mo, L.; Hellmich, Helen; Yu, A.; Wang, T.; Buchheit, S.; Godley, B. F.

In: Investigative Ophthalmology and Visual Science, Vol. 41, No. 13, 2000, p. 4247-4255.

Research output: Contribution to journalArticle

Wills, NK, Weng, T, Mo, L, Hellmich, H, Yu, A, Wang, T, Buchheit, S & Godley, BF 2000, 'Chloride channel expression in cultured human fetal RPE cells: Response to oxidative stress', Investigative Ophthalmology and Visual Science, vol. 41, no. 13, pp. 4247-4255.
Wills, N. K. ; Weng, T. ; Mo, L. ; Hellmich, Helen ; Yu, A. ; Wang, T. ; Buchheit, S. ; Godley, B. F. / Chloride channel expression in cultured human fetal RPE cells : Response to oxidative stress. In: Investigative Ophthalmology and Visual Science. 2000 ; Vol. 41, No. 13. pp. 4247-4255.
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abstract = "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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T2 - Response to oxidative stress

AU - Wills, N. K.

AU - Weng, T.

AU - Mo, L.

AU - Hellmich, Helen

AU - Yu, A.

AU - Wang, T.

AU - Buchheit, S.

AU - Godley, B. F.

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N2 - 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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