MOLECULAR MECHANISMS OF ACROLEIN LUNG CELL INJURY

丙烯醛是一种环境污染物，也是一种反应性代谢物， 环磷酰胺，多胺和环境化学品，是有毒的 肺细胞导致通透性增加和肺水肿。 因为肺内皮细胞质膜的损伤可能是 这些细胞的总体功能障碍，包括血浆的扰动 膜结构和功能，我们假设丙烯醛诱导的 调节质膜特异性蛋白巯基（P-SH）， 细胞溶质和线粒体谷胱甘肽（GSH），和钙（Ca 2+） 体内平衡是导致血浆中 膜依赖性功能，导致调节和/或 内皮细胞的分解代谢过程。 为了验证这一假设， 具体目标是：1）评估剂量和时间依赖性效应 丙烯醛对完整细胞和/或 分离的质膜、胞质溶胶和线粒体，II）建立 质膜P-SH丢失与质膜透性的关系 - 3、建立相互关系; Ca 2+稳态的改变和对Ca 2+依赖性调节的影响 和分解代谢酶，和IV）评估巯基- 含有试剂和恢复丧失的细胞功能。 实现 这些目标，我们将确定丙烯醛诱导的影响， 质膜ATP酶和血管紧张素II（Ang II）亚型I（TA 1） 受体以及细胞质和线粒体GSH含量对45 Ca 2 + 内流、外排和Ca 2+动员（使用荧光探针） fura-2对脂质过氧化和GSH氧化还原循环酶活性的影响。 为了评估对质膜依赖性功能的影响，我们将 监测Na+/K+ ATP酶活性，转运所需的氨基酸， GSH、蛋白质和内皮衍生舒张因子（EDRF）的合成， Ca ~（2+）-ATP酶活性和~（45）Ca ~（2+）内流及血管紧张素Ⅱ AT_1受体 介导的信号转导级联刺激。 评价 改变的Ca 2+稳态对调节和分解代谢过程的影响， 我们将监测NO合酶的Ca 2+依赖性活性和 EDRF、蛋白激酶C（PKC）的活化和/或易位，以及 膜蛋白的磷酸化和磷脂酶A2、C和D 介导的膜磷脂水解。 检查保护性的 含巯基试剂的效果，我们将使用N-乙酰基-L-半胱氨酸， 二硫苏糖醇和2-巯基乙磺酸（美司钠）之前，期间， 和丙烯醛暴露后。

Acrolein, an environmental pollutant as well as a reactive metabolite of cyclophosphamide, polyamines, and environmental chemicals, is toxic to lung cells resulting in increased permeability and pulmonary edema. Because injury to lung endothelial cell plasma membranes may account for overall dysfunction of these cells including perturbation of the plasma membrane structure and function, we hypothesize that acrolein-induCed modulations of plasma membrane specific protein sulfhydryls (P-SH), of cytosolic and mitochondrial glutathione (GSH), and of calcium (Ca2+) homeostasis are responsible for alterations in the plasma membrane.dependent functions leading to modulation of regulatory and/or catabolic processes in endothelial cells. To test this hypothesis the specific aims are: I) evaluating the dose - and time - dependent effects of acrolein on various biochemical parameters in intact cells and/or in isolated plasma membranes, cytosol, and mitochondria, Il) establishing the relationship between the loss of plasma membrane P-SH and plasma membrane - dependent functions, III) establishing the relationship between alterations in Ca2+ homeostasis and effect on Ca2+-dependent regulatory and catabolic enzymes, and IV) evaluating the protective effect of thiol- containing agents and recovery of loss of cellular functions. To achieve these aims, we will identify acrolein-induced effects on SH containing plasma membrane ATPase and angiotensin II (Ang II) subtype I (TA1) receptors as well as on cytosolic and mitochondrial GSH contents on 45Ca2+ influx, on efflux and Ca2+ mobilization by using the fluorescent probe fura-2, on lipid peroxidation, and on GSH redox cycling enzyme activities. To evaluate the effects on plasma membrane-dependent functions, we will monitor Na+/K+ ATPase activity, transport of amino acids required for synthesis of GSH, protein, and endothelium derived relaxing factor (EDRF), Ca2+-ATPase activity and influx of 45Ca2+, and Ang-lI AT1 receptor mediated stimulation of the signal transduction cascade. To evaluate the effect of altered Ca2+ homeostasis on regulatory and catabolic processes, we will monitor Ca2+-dependent activities of NO synthase and production of EDRF, activation and/or translocation of protein kinase C (PKC) and phosphorylation of membrane proteins, and phospholipases A2, C, and D mediated hydrolysis of membrane phospholipids. To examine the protective effect of thiol- containing agents, we will use N-acetyl -L-cysteine, dithiothreitol, and 2-mercaptoethanesulfonic acid (mesna) before, during, and after acrolein exposure.