Hydroxyl radical activation of a Ca(2+)-sensitive nonselective cation channel involved in epithelial cell necrosis.
In a previous work the involvement of a fenamate-sensitive Ca(2+)-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca(2+)-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe(2+), exposure of the intracellular side of NSCC to H(2)O(2) increased their open probability (P(o)) by approximately 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H(2)O(2), indicating that the increase in P(o) was mediated by.OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to.OH. The increase in P(o) induced by.OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P(o) of both.OH and DTNB. A similar increase in P(o) was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca(2+). The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.
Simon
Felipe
F
Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, 838-0453 Independencia, Santiago, Chile.
Varela
Diego
D
Eguiguren
Ana Luisa
AL
Díaz
Laín F
LF
Sala
Francisco
F
Stutzin
Andrés
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D051381:Rats; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002412:Cations; D007473:Ion Channels; D016877:Oxidants; D062367:ortho-Aminobenzoates; D017665:Hydroxyl Radical; C001209:fenamic acid; D006861:Hydrogen Peroxide; D003545:Cysteine; D002118:Calcium; D019803:Glutathione Disulfide