Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the beta(1)-Na+-K+ pump subunit, inhibiting activity of the catalytic alpha-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (I-p). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of I-p. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between beta(1)-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced beta(1)-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O-2(+)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease beta(1)-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in beta(1) pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo.