Involvement of the mitochondrial KATP+ channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures

Soybean suspension cell cultures were treated by H₂O₂ or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H₂O₂, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H₂O₂-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (ΔΨ) weakly decreased. The addition of KCl to the ΔΨ formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced ΔΨ formation, due to a K⁺ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H₂O₂-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, ΔΨ generation significantly inhibited, and CsA-dependent ΔΨ formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a \(\mathrm{K}_{\mathrm{ATP}}^{{+}}\) channel during the manifestation of PCD induced by H₂O₂ or NO in plants.