ADENOSINE AND ANTIOXIDANT ENZYMES

氧自由基，如单线态氧、超氧阴离子和 羟基自由基，是人体利用氧气的正常副产品。 组织。减少这些自由基的细胞负荷是我们的主要目标 医学自从自由基与阿尔茨海默氏症有关以来， 帕金森病、癌症、缺血再灌注损伤、炎症 在老化过程中也是如此。抗氧化防御系统提供了 保护细胞免受自由基的伤害，并含有以下酶 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)和非酶类抗氧化剂 维生素A、C和E以及还原型谷胱甘肽。核苷腺苷，一种 三磷酸腺苷的代谢产物，为组织提供保护，在心肌和 脑缺血。虽然细胞保护的机制尚不清楚，但我们 最近证明了腺苷和类似的R-苯基异丙基 腺苷(R-PIA)促进快速激活(观察到30分钟内) 通过激活偶联的AAR亚型将抗氧化酶增加2-3倍 大鼠嗜碱性克隆(RBL-2H3细胞)中的磷脂酶C。A23187(钙离子 离子载体)和佛波醇酯(蛋白激酶C的激活剂) 通过A3AR模拟这些酶的激活。蛋白质的抑制作用 星状孢子菌素抑制这些酶的激活 R-PIA和佛波醇酯。此外，还测定了纯化产物的活性。 抗氧化酶的制备可受蛋白激酶的调节 C-介导的磷酸化。这些数据表明对大脑的刺激 A3AR导致抗氧化酶的激活，而这种激活 这一过程可能涉及蛋白激酶C的磷酸化。 这项研究的主要目的是： L。确定这种快速激活的机制(S) 细胞表面的A3AR导致抗氧化酶的激活。这 研究将主要集中在蛋白激酶C上，并将专门 确定抗氧化酶是否是蛋白激酶C的底物 以及A3AR是否促进这些蛋白在体内的磷酸化 酵素。此外，潜在的定点突变 将进行磷酸化位点以确定潜在的位点(S) 蛋白激酶C的磷酸化。这些后一项研究将涉及 不同抗氧化酶表位标记的cDNA的诱变作用。 2.确定抗氧化酶是否通过A3AR激活 在缺氧或氧化过程中为细胞提供保护 压力。氧化应激对RBL-2H3细胞、牛主动脉内皮细胞的影响 低氧加氢可诱导心肌细胞 过氧化氢添加到培养基中或通过添加 A3AR介导的1-2小时的黄嘌呤/黄嘌呤氧化酶 将通过确定减少的和减少的 氧化谷胱甘肽、丙二醛及电子显微镜研究 细胞器。 3.确定A3AR激活对抗氧化剂的长期影响 酵素。细胞(RBL-2H3和人内皮细胞)将被 12-48小时的R-PIA和“稳态”活动 并将测定各种抗氧化酶的水平。 分光光度法和Western blotting法。北方 印迹研究将确定A3AR是否可以调节RNA 编码这些酶。 综上所述，这些研究将探索一种新的 腺苷提供的细胞保护作用，可能有助于 开发治疗心肌和脑缺血的新方法。

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.