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The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK coimmunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca 2+ -calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca 2+ -calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaMbinding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.z 1999 Federation of European Biochemical Societies.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

The presence of variable static hemin orientational disorder about the ␣-␥-meso axis in the substrate complexes of mammalian heme oxygenase, together with the incomplete averaging of a second, dynamic disorder, for each hemin orientation, has led to NMR spectra with severe spectral overlap and loss of key two-dimensional correlations that seriously interfere with structural characterization in solution. We demonstrate that the symmetric substrate, 2,4-dimethyldeuterohemin, yields a single solution species for which the dynamic disorder is sufficiently rapid to allow effective and informative Mammalian heme oxygenase (HO)1 is a ϳ300-residue, membrane-bound, non-heme enzyme that, using heme as cofactor and substrate, catalyzes the regiospecific conversion of heme to ␣-biliverdin, iron, and CO (1). The physiological roles of HO are heme catabolism (HO-1) (2-4) and the generation of CO as a putative neural messenger (HO-2) (5, 6). Detailed mechanistic (7-13) and spectroscopic (13-17) studies of the fully active recombinant, soluble 265-residue portion of HO-1 have shown that, in contrast to heme peroxidase and cytochrome P450, HO does not act through a ferryl intermediate. Recent crystal structures (18,19) of the substrate-bound, water-ligated complexes of a more truncated 233-residue human HO, hHO (20), and the complete rat HO (18), rHO, have revealed a largely helical enzyme that confirms the binding of heme by His-25 and locates a highly bent distal helix that is sufficiently close to the heme to sterically block all but the ␣-meso position (see Fig.

This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron--oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited.

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The action of heme oxygenase is graphically (but perhaps unsuspectingly) familiar to anyone who has observed the gradual discoloration of a bruise from "black and blue" to green and then yellow. The initial dark color is due to heme from the hemoglobin released into the damaged tissue by ruptured blood vessels. Oxidation of the heme to biliverdin by heme oxygenase provides the green tint, and subsequent reduction of biliverdin to bilirubin the yellow color (Figure 1). Heme and biliverdin are highly lipophilic and relatively difficult to eliminate, but bilirubin is readily excreted after conjugation with glucuronic acid.Heme oxygenase is highly unusual in that it uses heme as both its substrate and prosthetic group. It is also mechanistically distinct from the other classes of hemoproteins, including the cytochromes P450, peroxidases, catalases, nitric oxide synthases, prostaglandin synthases, thromboxane synthases, and prostacyclin synthases. Nevertheless, the reactions catalyzed by heme oxygenase are part of the same heme-dependent reaction manifold that underlies the catalytic action of all hemoproteins, and elucidation of its mechanism can be expected to shed considerable light on the function of all hemoproteins.

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