Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite.

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- 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- 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-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.

Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase.

Peroxynitrite (ONOO(-)), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O(2)()) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817-826). Here we studied how ONOO(-) influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO(-) increased basal and agonist-stimulated Ser(1179) phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO(-) strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser(1179). Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation. In contrast to Akt, ONOO(-) significantly activated 5'-AMP-activated kinase (AMPK), as evidenced by its increased Thr(172) phosphorylation as well as increased Ser(92) phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O(2)(), ONOO(-) significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO(-) up-regulated eNOS-Ser(P)(1179). In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation as well as O(2)() release. We conclude that ONOO(-) inhibits Akt and increases AMPK-dependent Ser(1179) phosphorylation of eNOS resulting in enhanced O(2)() release.