Abstract
Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2-4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.
Highlights
By oxidants such as aldehydes [6, 7] and hydrogen peroxide [7, 8] and under conditions associated with oxidative stress such as myocardial ischemia [9], heart failure [10], vascular inflammation [11], and alcoholic liver disease [12]; and that inhibition of Aldose reductase (AR) increases aldehyde toxicity in rat vascular smooth muscle cell lines [7] and Chinese hamster fibroblast cell lines [8]
The major findings of this study are that AR is activated in the ischemic heart and that the increase in AR activity could be attributed to the oxidation of the cysteine residues of the enzyme to sulfenic acids
Many investigators over several years have reported that activated and nonactivated forms of AR are present in purified enzyme preparations or in diseased tissue [13, 22,23,24,25,26], the present study provides clear evidence for the first time that AR is intrinsically activated in the ischemic heart by a ROSsensitive mechanism
Summary
Separation of proteins from hearts subjected to coronary occlusion by twodimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303 These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids. Because of its ability to catalyze the reduction of glucose, AR regulates the flux of glucose in the heart and inhibition of the enzyme has been shown to prevent myocardial ischemia-reperfusion injury by increasing glycolysis and preserving NADH/NADϩ levels [13,14,15]. AR is subject to post-translational modifications and protein kinase C-dependent phosphorylation of the enzyme has been described [21] It remains unclear whether such mechanisms regulate AR activity under conditions of oxidative stress or ischemia. Preliminary findings of this study have been published as an abstract [33]
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