Activation of 5'-AMP-activated Kinase Is Mediated through c-Src and Phosphoinositide 3-Kinase Activity during Hypoxia-Reoxygenation of Bovine Aortic Endothelial Cells.

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO[sup -] acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO[sup -] formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO[sup -], both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO[sup -], significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO[sup -] favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO[sup -] activates AMPK in a c-Src-mediated, PI 3-kinase-dependent mannet, and suggest that ONOO[sup -]-induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC. [ABSTRACT FROM AUTHOR]