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ADENOSINE AND ANTIOXIDANT ENZYMES

腺苷和抗氧化酶

基本信息

批准号：
2901240
负责人：
Vickram Ramkumar
金额：
$ 10.26万
依托单位：
SOUTHERN ILLINOIS UNIVERSITY SCH OF MED
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-04-01 至 2001-03-31
项目状态：
已结题

项目摘要

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.

氧自由基，如单线态氧、超氧阴离子和羟基自由基，是正常的副产品的氧利用的组织.减少这些自由基的细胞负荷是一个主要目标，自从自由基与阿尔茨海默病有关以来，帕金森病，癌症，缺血再灌注损伤，炎症也在老化过程中。抗氧化防御系统提供了保护细胞免受自由基的侵害，并包括酶，超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH.Px）和谷胱甘肽还原酶（GR）和非酶抗氧化剂，维生素A、C和E以及还原型谷胱甘肽。核苷腺苷， ATP的代谢产物，在心肌和脑缺血虽然细胞保护的机制尚不清楚，但我们最近证明腺苷和类似物R-苯基异丙基腺苷（R-PIA）促进快速激活（在30分钟内观察到）抗氧化酶2-3倍，通过激活AAR亚型偶联到磷脂酶C在大鼠嗜碱性克隆（RBL-2 H3细胞）中的表达。A23187（a Ca2+ 离子载体）和佛波醇酯（蛋白激酶C的活化剂）两者通过A3 AR模拟这些酶的激活。抑制蛋白质激酶C通过星形孢菌素减弱了这些酶的激活，被R-PIA和佛波醇酯所抑制。此外，纯化的活性抗氧化酶制剂可受蛋白激酶的调节 C-介导的磷酸化。这些数据表明， A3 AR导致抗氧化酶的激活，并且这种激活这一过程可能涉及蛋白激酶C的磷酸化作用。本研究的主要目的是： L.为了确定这种快速激活的机制，细胞表面的A3 AR导致抗氧化酶的活化。这研究将主要集中在蛋白激酶C上，确定抗氧化酶是否是蛋白激酶C的底物以及A3 AR是否促进这些蛋白的体内磷酸化，内切酶此外，潜在的定点诱变将进行磷酸化位点分析，以确定潜在位点蛋白激酶C的磷酸化。这些研究将涉及不同抗氧化酶的表位标记的cDNA的诱变。 2.为了确定是否通过A3 AR激活抗氧化酶在缺氧或氧化过程中保护细胞应力对RBL-2 H3细胞、牛主动脉内皮细胞的氧化应激和心肌细胞将诱导缺氧的补充氢过氧化物的混合物，或通过添加过氧化物的混合物，黄嘌呤/黄嘌呤氧化酶作用1-2小时。A3 AR介导保护将通过确定减少和氧化型谷胱甘肽、丙二醛和电子显微镜研究细胞器。 3.为了确定A3 AR激活对抗氧化剂的长期影响，内切酶细胞（RBL-2 H3和人内皮细胞）将用以下物质处理： 12-48 h的R-PIA和“稳态”活性以及各种抗氧化酶的水平分别通过分光光度法和蛋白质印迹法。北方印迹研究将确定A3 AR是否可以调节RNA 编码这些酶。总之，这些研究将探索一种新的机制，腺苷提供的细胞保护作用，可能有助于开发心肌和脑缺血的新治疗方法。