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.
