Oxygen free radicals, such as singlet oxygen, superoxide anion and hydroxyl radicals, are normal byproducts of oxygen utilization by the tissue. Reducing the cellular load of these radicals is a major goal in medicine since free radicals have been implicated in Alzheimer's disease, Parkinson's disease, cancers, ischemia-reperfusion injuries, inflammation and also in the aging process. An antioxidant defense system provides protection of the cell from free radicals and comprises enzymes such as superoxide dismutase (SOD), catalase CAT), glutathione peroxidase (GSH.Px) and glutathione reductase (GR) and nonenzymatic antioxidants such as vitamins A, C and E and reduced glutathione. The nucleoside adenosine, a metabolite of ATP, provides protection to tissues during myocardial and cerebral ischemia. While the mechanism of cytoprotection is not clear, we have recently demonstrated that adenosine and an analog R-phenylisopropyl adenosine (R-PIA) promote rapid activation (observed within 30 min) of antioxidant enzymes by 2-3 fold by activating an AAR subtype coupled to phospholipase C in a rat basophilic clone (RBL-2H3 cells). A23187 (a Ca2+ ionophore) and phorbol esters (activators of protein kinase C) both mimicked activation of these enzymes via the A3AR. Inhibition of protein kinase C by staurosporine attenuated activation of these enzymes elicited by both R-PIA and phorbol esters. Furthermore, the activities of purified preparations of antioxidant enzymes could be regulated by protein kinase C-mediated phosphorylation. These data suggest that stimulation of the A3AR leads to activation of antioxidant enzymes, and that this activation process likely involves phosphorylation by protein kinase C. The major aims of this study are: l. To determine the mechanism(s) by which this rapid activation of the A3AR on the cell surface leads to activation of antioxidant enzymes. This study will focus mainly on protein kinase C and would specifically determine whether antioxidant enzymes are substrates of protein kinase C in vitro and whether the A3AR promotes in vivo phosphorylation of these enzymes. In addition, site-directed mutagenesis of potential phosphorylation sites will be performed to determine the potential site(s) of phosphorylation by protein kinase C. These latter studies will involve mutagenesis of epitope tagged cDNAs of the different antioxidant enzymes. 2. To determine whether activation of antioxidant enzymes via the A3AR provides protection to cells during hypoxia or undergoing oxidative stress. Oxidative stress to RBL-2H3 cells, bovine aortic endothelial cells and cardiac myocytes will be induced by hypoxia the addition of hydrogen peroxide to the culture medium or by the addition of a mixture of xanthine/xanthine oxidase for periods ranging from 1-2 h. A3AR-mediated protection will be assessed by determining the levels of reduced and oxidized glutathione, malondialdehyde and by electron microscopic studies of cellular organelles. 3. To determine the long term effect of A3AR activation on antioxidant enzymes. Cells (RBL-2H3 and human endothelial cells) will be treated with R-PIA for periods ranging from 12-48 h and the """"""""steady state"""""""" activities and levels of various antioxidant enzymes will be determined spectrophometrically and by Western blotting, respectively. Northern blotting studies will determine whether the A3AR can regulate the RNA encoding these enzymes. Taken together, these studies will explore a novel mechanism of cytoprotection provided by adenosine and might contribute to the development of new treatments for myocardial and cerebral ischemia.
