Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase

White CN, Figtree GA, Liu CC, Garcia A, Hamilton EJ, Chia KK, Rasmussen HH. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase. Am J Physiol Cell Physiol 296: C693-C700, 2009. First published February 4, 2009; doi:10.1152/ajpcell.00648.2008.-The sarcolemmal Na+-K+ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilon PKC), previously implicated in ANG II-induced Na+-K+ pump inhibition. A role for ePKC was also supported by an ANG II-induced increase in coimmunoprecipitation of ePKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na+-K+ pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by ePKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na+-K+ pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47phox NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na+-K+ pump via PKC-dependent NADPH oxidase activation.