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.

氧气自由基，例如单线氧，超氧化阴离子和羟基自由基是正常的氧气副产品组织。减少这些自由基的细胞负荷是一个主要目标由于自由基已与阿尔茨海默氏病有关帕金森氏病，癌症，缺血 - 再灌注损伤，炎症以及在衰老过程中。抗氧化剂防御系统提供保护细胞免受自由基的保护，包括酶，例如超氧化物歧化酶（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，例如维生素A，C和E并减少谷胱甘肽。核苷腺苷，A ATP的代谢产物，为心肌和脑缺血。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物R-苯基丙二醇胺腺苷（R-PIA）促进快速激活（在30分钟内观察）抗氧化剂酶通过激活AAR亚型耦合到2-3倍大鼠嗜碱性克隆（RBL-2H3细胞）中的磷脂酶C。 A23187（Ca2+ 离子载体）和佛波酯（蛋白激酶的活化剂C）均为通过A3AR模仿这些酶的激活。抑制蛋白质通过星形孢菌素减弱了这些酶的激活，激活了这些酶的激活由R-PIA和Phorbol酯都通过。此外，纯化的活动抗氧化剂的制剂可以由蛋白激酶调节 C介导的磷酸化。这些数据表明刺激 A3AR导致抗氧化酶的激活，并且这种激活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细胞表面上的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，并专门确定抗氧化剂是否是蛋白激酶C的底物体外以及A3AR是否促进这些体内磷酸化酶。另外，电位定向诱变将进行磷酸化位点以确定潜在位点蛋白激酶C的磷酸化。这些后一种研究将涉及表位的诱变标记了不同抗氧化剂酶的cDNA。 2。确定是否通过A3AR激活抗氧化酶在缺氧期间为细胞提供保护或经历氧化的保护压力。对RBL-2H3细胞，牛主动脉内皮细胞的氧化应激缺氧将诱导心肌细胞和心肌细胞的添加过氧化培养基或通过添加混合物黄嘌呤/黄嘌呤氧化酶的周期范围为1-2小时。 A3AR介导的通过确定降低的水平和氧化的谷胱甘肽，丙二醛和电子显微镜研究细胞细胞器。 3。确定A3AR激活对抗氧化剂的长期影响酶。细胞（RBL-2H3和人内皮细胞）将用 R-PIA的期间为12-48 h，“稳态”活动将确定各种抗氧化剂的水平分光图和通过蛋白质印迹。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疗法。