Mechanism of inhibitory actions of oxidizing agents on calcium-activated potassium current in cultured pigment epithelial cells of the human retina

Shwu Jiuan Sheu, Sheng-Nan Wu

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24 Citations (Scopus)

Abstract

PURPOSE. To identify the mechanisms by which oxidative stress with oxidizing agents alters the activity of ion channels in human retinal pigment epithelial (RPE) cells. METHODS. The effects of oxidizing agents on ion currents were investigated in human RPE R-50 cells with the aid of the whole-cell, cell-attached, and inside-out configurations of the patch-clamp technique. RESULTS. In the whole-cell configuration, t-butyl hydroperoxide (t-BHP; 1 mM), thimerosal (30 μM), and 4,4′-dithiodipyridine (DTDP; 30 μM) suppressed voltage-dependent K+ current (IK) that was sensitive to inhibition by iberiotoxin or paxillin, yet not by apamin or 5-hydroxydecanoate sodium. Meclofenamic acid or Evans blue, but not diazoxide, reversed the decrease in IK caused by t-BHP. In cells dialyzed with ceramide (30 μM), neither t-BHP (1 mM) nor thimerosal (30 μM) had any effect on IK, whereas DTDP (30 μM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 μM), and DTDP (30 μM) suppressed the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Dithiothreitol (10 μM) reversed DTDP-induced decrease in channel activity. Under current-clamp conditions, cell exposure to oxidizing reagents caused membrane depolarization. In cells dialyzed with ceramide (30 μM), membrane potential remained unaltered in the presence of t-BHP. CONCLUSIONS. The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress IK and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BKCa, channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BKCa channels and indirectly from an increase in the intracellular production of ceramide.

Original languageEnglish
Pages (from-to)1237-1244
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number3
DOIs
Publication statusPublished - 2003 Mar 1

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Oxidants
Retina
Potassium
Epithelial Cells
Thimerosal
Calcium
Ceramides
Retinal Pigments
Meclofenamic Acid
Paxillin
Apamin
Diazoxide
tert-Butylhydroperoxide
Calcium-Activated Potassium Channels
Evans Blue
Membranes
Dithiothreitol
Patch-Clamp Techniques
Ion Channels
Membrane Potentials

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

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title = "Mechanism of inhibitory actions of oxidizing agents on calcium-activated potassium current in cultured pigment epithelial cells of the human retina",
abstract = "PURPOSE. To identify the mechanisms by which oxidative stress with oxidizing agents alters the activity of ion channels in human retinal pigment epithelial (RPE) cells. METHODS. The effects of oxidizing agents on ion currents were investigated in human RPE R-50 cells with the aid of the whole-cell, cell-attached, and inside-out configurations of the patch-clamp technique. RESULTS. In the whole-cell configuration, t-butyl hydroperoxide (t-BHP; 1 mM), thimerosal (30 μM), and 4,4′-dithiodipyridine (DTDP; 30 μM) suppressed voltage-dependent K+ current (IK) that was sensitive to inhibition by iberiotoxin or paxillin, yet not by apamin or 5-hydroxydecanoate sodium. Meclofenamic acid or Evans blue, but not diazoxide, reversed the decrease in IK caused by t-BHP. In cells dialyzed with ceramide (30 μM), neither t-BHP (1 mM) nor thimerosal (30 μM) had any effect on IK, whereas DTDP (30 μM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 μM), and DTDP (30 μM) suppressed the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Dithiothreitol (10 μM) reversed DTDP-induced decrease in channel activity. Under current-clamp conditions, cell exposure to oxidizing reagents caused membrane depolarization. In cells dialyzed with ceramide (30 μM), membrane potential remained unaltered in the presence of t-BHP. CONCLUSIONS. The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress IK and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BKCa, channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BKCa channels and indirectly from an increase in the intracellular production of ceramide.",
author = "Sheu, {Shwu Jiuan} and Sheng-Nan Wu",
year = "2003",
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doi = "10.1167/iovs.02-0330",
language = "English",
volume = "44",
pages = "1237--1244",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
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TY - JOUR

T1 - Mechanism of inhibitory actions of oxidizing agents on calcium-activated potassium current in cultured pigment epithelial cells of the human retina

AU - Sheu, Shwu Jiuan

AU - Wu, Sheng-Nan

PY - 2003/3/1

Y1 - 2003/3/1

N2 - PURPOSE. To identify the mechanisms by which oxidative stress with oxidizing agents alters the activity of ion channels in human retinal pigment epithelial (RPE) cells. METHODS. The effects of oxidizing agents on ion currents were investigated in human RPE R-50 cells with the aid of the whole-cell, cell-attached, and inside-out configurations of the patch-clamp technique. RESULTS. In the whole-cell configuration, t-butyl hydroperoxide (t-BHP; 1 mM), thimerosal (30 μM), and 4,4′-dithiodipyridine (DTDP; 30 μM) suppressed voltage-dependent K+ current (IK) that was sensitive to inhibition by iberiotoxin or paxillin, yet not by apamin or 5-hydroxydecanoate sodium. Meclofenamic acid or Evans blue, but not diazoxide, reversed the decrease in IK caused by t-BHP. In cells dialyzed with ceramide (30 μM), neither t-BHP (1 mM) nor thimerosal (30 μM) had any effect on IK, whereas DTDP (30 μM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 μM), and DTDP (30 μM) suppressed the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Dithiothreitol (10 μM) reversed DTDP-induced decrease in channel activity. Under current-clamp conditions, cell exposure to oxidizing reagents caused membrane depolarization. In cells dialyzed with ceramide (30 μM), membrane potential remained unaltered in the presence of t-BHP. CONCLUSIONS. The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress IK and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BKCa, channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BKCa channels and indirectly from an increase in the intracellular production of ceramide.

AB - PURPOSE. To identify the mechanisms by which oxidative stress with oxidizing agents alters the activity of ion channels in human retinal pigment epithelial (RPE) cells. METHODS. The effects of oxidizing agents on ion currents were investigated in human RPE R-50 cells with the aid of the whole-cell, cell-attached, and inside-out configurations of the patch-clamp technique. RESULTS. In the whole-cell configuration, t-butyl hydroperoxide (t-BHP; 1 mM), thimerosal (30 μM), and 4,4′-dithiodipyridine (DTDP; 30 μM) suppressed voltage-dependent K+ current (IK) that was sensitive to inhibition by iberiotoxin or paxillin, yet not by apamin or 5-hydroxydecanoate sodium. Meclofenamic acid or Evans blue, but not diazoxide, reversed the decrease in IK caused by t-BHP. In cells dialyzed with ceramide (30 μM), neither t-BHP (1 mM) nor thimerosal (30 μM) had any effect on IK, whereas DTDP (30 μM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 μM), and DTDP (30 μM) suppressed the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Dithiothreitol (10 μM) reversed DTDP-induced decrease in channel activity. Under current-clamp conditions, cell exposure to oxidizing reagents caused membrane depolarization. In cells dialyzed with ceramide (30 μM), membrane potential remained unaltered in the presence of t-BHP. CONCLUSIONS. The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress IK and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BKCa, channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BKCa channels and indirectly from an increase in the intracellular production of ceramide.

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