Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis

Felipe Simon, Diego Varela, Ana Luisa Eguiguren, Laín F. Díaz, Francisco Sala, Andrés Stutzin

Resultado de la investigación: Article

29 Citas (Scopus)

Resumen

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 Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ∼40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po 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 Po of both .OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. 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.

Idioma originalEnglish
PublicaciónAmerican Journal of Physiology - Cell Physiology
Volumen287
N.º4 56-4
DOI
EstadoPublished - oct 2004

Huella dactilar

Nitrobenzoates
Fenamates
Hydroxyl Radical
Cations
Necrosis
Epithelial Cells
Glutathione Disulfide
Oxidants
Cell Line
Monovalent Cations
Deferoxamine
Dithiothreitol
Liver
Reducing Agents
Patch-Clamp Techniques
Free Radicals
Cysteine
Reactive Oxygen Species
Adenosine Triphosphate
Membranes

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Citar esto

Simon, Felipe ; Varela, Diego ; Eguiguren, Ana Luisa ; Díaz, Laín F. ; Sala, Francisco ; Stutzin, Andrés. / Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis. En: American Journal of Physiology - Cell Physiology. 2004 ; Vol. 287, N.º 4 56-4.
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title = "Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis",
abstract = "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 Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ∼40{\%} without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po 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 Po of both .OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. 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.",
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Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis. / Simon, Felipe; Varela, Diego; Eguiguren, Ana Luisa; Díaz, Laín F.; Sala, Francisco; Stutzin, Andrés.

En: American Journal of Physiology - Cell Physiology, Vol. 287, N.º 4 56-4, 10.2004.

Resultado de la investigación: Article

TY - JOUR

T1 - Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis

AU - Simon, Felipe

AU - Varela, Diego

AU - Eguiguren, Ana Luisa

AU - Díaz, Laín F.

AU - Sala, Francisco

AU - Stutzin, Andrés

PY - 2004/10

Y1 - 2004/10

N2 - 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 Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ∼40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po 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 Po of both .OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. 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.

AB - 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 Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ∼40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po 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 Po of both .OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. 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.

KW - Ca-activated channels

KW - Oxidative stress

KW - Radical oxygen species

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U2 - 10.1152/ajpcell.00041.2004

DO - 10.1152/ajpcell.00041.2004

M3 - Article

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AN - SCOPUS:4544249174

VL - 287

JO - American Journal of Physiology - Cell Physiology

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