β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade

Dysregulated nitric oxide (NO)-and superoxide (O-2(center dot-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of beta(3)-adrenergic receptors (beta(3)-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na+-K+ pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na+-K+ pump beta(1)-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47phox to p22phox with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na+-K+ pump beta(1)-subunit and decrease in pump current. In vivo treatment with the beta(3)-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22phox with p47phox, abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na+-K+ pump beta(1)-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na+-K+ pump beta(1)-subunit, which may reflect facilitation of deglutathionylation. In vivo beta(3)-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes.