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 microM), and 4,4'-dithiodipyridine (DTDP; 30 microM) suppressed voltage-dependent K(+) current (I(K)) 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 I(K) caused by t-BHP. In cells dialyzed with ceramide (30 microM), neither t-BHP (1 mM) nor thimerosal (30 microM) had any effect on I(K), whereas DTDP (30 microM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 microM), and DTDP (30 microM) suppressed the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Dithiothreitol (10 microM) 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 microM), membrane potential remained unaltered in the presence of t-BHP.
Conclusions: The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress I(K) and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BK(Ca) channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BK(Ca) channels and indirectly from an increase in the intracellular production of ceramide.