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Basic Science
Cardiac and Vascular Signaling

Abstract 5263: Hydrogen Peroxide Activates AMP-Activated Protein Kinase in Endothelial Cells by Stimulating Ca2+/Calmodulin-Dependent Protein Kinase Kinase β, and Reveals a Critical Role for eNOS in AMPK Modulation by H2O2

Originally publishedCirculation. 2008;118:S_516

    Hydrogen peroxide (H2O2) is intimately involved in endothelial cell signaling, but the underlying molecular mechanisms are incompletely understood. The pathways that control AMP-activated protein kinase (AMPK) activity in endothelial cells remain to be completely defined. We used a combination of RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAEC). H2O2 treatment of BAEC rapidly (within minutes) and significantly increases phosphorylation of AMPK at Thr172 (3.0±0.6-fold increase in AMPK phosphorylation relative to vehicle-treated cells, n=4, p<0.01), as analyzed in immunoblots of BAEC probed with phosphorylation state-specific antibodies. The EC50 for H2O2-promoted phosphorylation of AMPK or of the AMPK substrate acetyl-coA carboxylase is 50±15 μM (n=5). Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) by the inhibitor STO-609 (10μg/ml) completely abolishes H2O2-dependent AMPK activation. siRNA-mediated “knockdown” of CaMKKβ using a specific duplex siRNA completely abrogates AMPK activation. Thus, CaMKKβ serves as the key upstream modulator of H2O2-modulated AMPK activity. Treatment of BAEC with eNOS inhibitors L-NAME (100μM) or NNA (10μM) significantly enhances AMPK phosphorylation (2.0±0.5-fold increase, p<0.02, n=6). siRNA-mediated knockdown of eNOS leads to marked enhancement of AMPK phosphorylation (1.9±0.2-fold increase compared to control siRNA-transfected cells, p<0.01, n=4). We next analyzed AMPK expression and phosphorylation in tissues isolated from eNOSnull mice. Compared to wild-type mice, eNOSnull mice show a striking increase in AMPK phosphorylation in liver (3.8±0.3-fold increase, p<0.001, n=3), and lung (4.9±0.3-fold increase, p<0.001, n=3), with no change in total AMPK expression. Endothelial cells from lungs of eNOSnull mice show a 1.5±0.2-fold increase in AMPK phosphorylation compared to wild-type mice (n=3, p<0.01). Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation.

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