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 language | English |
|---|---|
| Pages (from-to) | 1237-1244 |
| Number of pages | 8 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 44 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2003 Mar 1 |
Fingerprint
All Science Journal Classification (ASJC) codes
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience
Cite this
}
Mechanism of inhibitory actions of oxidizing agents on calcium-activated potassium current in cultured pigment epithelial cells of the human retina. / Sheu, Shwu Jiuan; Wu, Sheng-Nan.
In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 3, 01.03.2003, p. 1237-1244.Research output: Contribution to journal › Article
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.
UR - http://www.scopus.com/inward/record.url?scp=0037336141&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037336141&partnerID=8YFLogxK
U2 - 10.1167/iovs.02-0330
DO - 10.1167/iovs.02-0330
M3 - Article
C2 - 12601054
AN - SCOPUS:0037336141
VL - 44
SP - 1237
EP - 1244
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 3
ER -