The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre-and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (ϳ50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ϳ4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.Xanthine dehydrogenase is the rate-limiting step in the catabolism of purines, where it catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid. In this reaction, XDH 1 utilizes NAD ϩ preferentially as the electron acceptor. However, when XDH is converted to XO, the preferred electron acceptor becomes molecular oxygen resulting in the formation of superoxide and hydrogen peroxide. This generation of reactive oxygen species is thought to be the basis of XDH/XO involvement in various pathological conditions such as ischemia-reperfusion injury. Reversible conversion of XDH to XO can occur after the oxidation of eight cysteine residues in the molecule into four cystines by agents such as pyrimidines or oxidized glutathione (1, 2). This conversion may be reversed upon the addition of reducing agents such as dithiothreitol. XDH can also be converted into XO irreversibly through proteolysis (3). Experimental proteolysis by trypsin has allowed the identification of three different parts of the molecule: a 20-kDa N-terminal fragment, a 40-kDa flavin-binding fragment, and an 80-kDa molybdopterin-binding fragment, all of which remain attached after proteolysis (3). It is believed that both reversible and irreversible conversion of XDH to XO are due to conformational changes in the molecule that...

Left ventricular (LV) hypertrophy commonly develops in response to chronic hypertension and is a significant risk factor for heart failure and death. The serine-threonine phosphatase, calcineurin (CnA), plays a critical role in the development of pathologic hypertrophy. Previous experimental studies in murine models show that estrogen limits pressure overload-induced hypertrophy; our purpose was to explore further the mechanisms underlying this estrogen effect. Wild type, ovariectomized female mice were treated with placebo or 17β-estradiol (E2), followed by transverse aortic constriction (TAC) to induce pressure overload. At two weeks, mice underwent physiologic evaluation, immediate tissue harvest, or dispersion of cardiomyocytes. E2 replacement limited TAC-induced LV and cardiomyocyte hypertrophy while attenuating deterioration in LV systolic function and contractility. These E2 effects were associated with reduced abundance of CnA. The primary downstream targets of CnA are the nuclear factor of activated T-cell (NFAT) family of transcription factors. In transgenic mice expressing a NFAT-activated promoter-luciferase reporter gene, E2 limited TAC-induced activation of NFAT. Moreover, the inhibitory effects of E2 on LV hypertrophy were absent in CnA knockout mice supporting that CnA is an important target of E2-mediated inhibition. In cultured rat cardiac myocytes, E2 inhibited agonist-induced hypertrophy while also decreasing CnA abundance and NFAT activation. Agonist stimulation also reduced CnA ubiquitination and degradation that was prevented by E2; all in vitro effects of estrogen were reversed by an ER antagonist. These data support that E2 reduces pressure overload induced hypertrophy by an ER-dependent mechanism that increases CnA degradation, unveiling a novel mechanism by which E2 and ERs regulate pathologic LV and cardiomyocyte growth.