Reactive oxygen species and the regulation of renal Na⁺-K⁺-ATPase in opossum kidney cells

Abstract

Culture of opossum kidney (OK) cells over 40 passages resulted in marked overexpression of alpha(1)-subunit and beta(1)-subunit Na(+)-K(+)-ATPase, which paralleled with increases in Na(+)-K(+)-ATPase activity, and enhanced availability in hydrogen peroxide (H(2)O(2)) (J Membr Biol 212: 163-175, 2006). The present study evaluated the mechanisms involved in increased H(2)O(2) production in OK cells cultured over 40 passages (up to passage 80) and whether H(2)O(2) plays a role in the regulation of Na(+)-K(+)-ATPase expression and activity. The accumulation of H(2)O(2) in the extracellular medium over 24 h (236.5 +/- 2.7 vs. 319.3 +/- 2.9 nM) and the rate of H(2)O(2) production (5.21 +/- 0.02 vs. 7.37 +/- 0.02 nM/min) in cells with 40 passages was lower (P < 0.05) than in cells with 80 passages. The increase in H(2)O(2) production in cells with 80 passages was accompanied by overexpression of NOX1, but not of NOX2, and overexpression of SOD1, SOD2, and SOD3. Apocynin, a NADPH inhibitor, markedly attenuated the increase in H(2)O(2) production in cells with 80 passages and significantly decreased Na(+)-K(+)-ATPase activity (26% reduction) and alpha(1)-subunit Na(+)-K(+)-ATPase overexpression. Increases in Na(+)-K(+)-ATPase alpha(1)-subunit mRNA abundance were also observed in OK cells with increased number of cell passages, but apocynin did not affect transcript abundance. In conclusion, the increased availability of H(2)O(2) in the intracellular milieu plays an important role in the long-term regulation of Na(+)-K(+)-ATPase expression and activity. These cells may constitute an interesting model to study events related to oxidative stress, in which adaptation to increases in H(2)O(2) had progressively adapted in a more natural manner.