Abstract 1379: Angiotensin II Inhibits the Na+-K+ pump via PKC-dependent Activation of NAD(P)H Oxidase
Background: The sarcolemmal Na+-K+ pump, essentially the only export route for Na+ and pivotal for cardiac myocyte function, is inhibited by angiotensin II (Ang II). Since Ang II activates NAD(P)H oxidase we tested the hypothesis that NAD(P)H oxidase mediates Ang II-induced pump inhibition. This is important for our understanding of pathophysiology and treatment of heart failure, a condition with increases in circulating Ang II levels; intracellular myocyte Na+; NAD(PH oxidase activity; and myocardial oxidative stress. The raised myocyte Na+ is believed to contribute to the electro-mechanical phenotype of contractile abnormalities and cardiac arrhythmias.
Methods and results: Exposure to 100 nmol/L Ang II increased fluorescence of isolated rabbit ventricular myocytes loaded with a superoxide-sensitive dye. The increase was abolished by pegylated superoxide dismutase (SOD), by the NAD(P)H oxidase inhibitor apocynin and by myristolated inhibitory peptide to ϵ-protein kinase C (ϵPKC), previously implicated in Ang II-induced Na+-K+ pump inhibition. Exposure of voltage-clamped myocytes to Ang II decreased electrogenic Na+-K+ pump current, Ip, from 0.48 ± 0.02 pA/pF, N = 11 to 0.36 ± 0.03 pA/pF, N = 11 (P < 0.05). The decrease was abolished (P < 0.05) by inclusion in pipette solution of: SOD, the gp91ds inhibitory peptide that blocks assembly and activation of NAD(P)H oxidase and by ϵPKC inhibitory peptide. Since co-localization should facilitate NAD(P)H-oxidase dependent regulation of the Na+-K+ pump we examined if there is physical association between the pump and NAD(P)H oxidase. Cell lysate was immunoprecipitated with antibody to the α1 pump subunit. Immunoblotting demonstrated co-immunoprecipitation of the α1 subunit with caveolin 3 and with membrane-associated p22phox and cytosolic p47phox NAD(P)H oxidase subunits at baseline. Ang II had no effect on α1/caveolin3 or α1/p22phox interaction, but increased α1/p47phox co-immunoprecipitation.
Conclusion: Ang II is known to induce PKC-dependent phosphorylation and translocation of p47phox resulting in activation of NAD(P)H oxidase, and we propose this activation occurs in a caveolar microdomain and that it mediates the inhibition of the Na+-K+ pump by Ang II.