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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES006219-02
Application #
2155083
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1994-06-01
Project End
1999-05-31
Budget Start
1995-06-01
Budget End
1996-05-31
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Zhang, J; Patel, J M; Block, E R (1998) Hypoxia-specific upregulation of calpain activity and gene expression in pulmonary artery endothelial cells. Am J Physiol 275:L461-8
Zhang, J; Li, Y D; Patel, J M et al. (1998) Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells. Am J Physiol 275:L288-93
Zhang, J; Patel, J M; Block, E R (1997) Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells. Comp Biochem Physiol B Biochem Mol Biol 116:485-91
Zhang, J; Patel, J M; Li, Y D et al. (1997) Proinflammatory cytokines downregulate gene expression and activity of constitutive nitric oxide synthase in porcine pulmonary artery endothelial cells. Res Commun Mol Pathol Pharmacol 96:71-87
Zhang, J L; Patel, J M; Li, Y D et al. (1996) Reductase domain cysteines 1048 and 1114 are critical for catalytic activity of human endothelial cell nitric oxide synthase as probed by site-directed mutagenesis. Biochem Biophys Res Commun 226:293-300
Patel, J M; Abeles, A J; Block, E R (1996) Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells. Free Radic Biol Med 20:629-37
Patel, J M; Zhang, J; Block, E R (1996) Nitric oxide-induced inhibition of lung endothelial cell nitric oxide synthase via interaction with allosteric thiols: role of thioredoxin in regulation of catalytic activity. Am J Respir Cell Mol Biol 15:410-9
Patel, J M; Block, E R (1995) Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 13:352-9