Nitrogen dioxide (NO/2), and environmental oxidant, is toxic to lung cells including pulmonary endothelial cells. Studies in progress reveal that NO/2 exposure results in specific increases in plasma membrane phosphatidylserine (PS) content, angiotensin-II (Ang-II) receptor binding, activation of phospholipase A1 (PLA1), and increase of lysophosphatidylethanolamine (lyso-PE) content of vascular endothelial cells. Since these responses appear to be causally related to increases in PS content and are directly involved in mediating pathophysiologic actions through the generation of second messengers and the catabolism of plasma membrane proteins and phospholipds, the specific aims of this study are: (I) identifying mechanisms of NO/2-induced increases of PS content of endothelial cell plasma membranes, (II) evaluating the effects of NO/2, PS, and lyso-PE on plasma membrane phospholipase C (PLC), phospholipase D (PLD), and redistribution of protein kinase C (PKC) from a predominantly cytosolic location to the plasma membrane fraction, (III) identifying the effects of NO/2 exposure, PS, and lyso- PE on Ang-II receptor binding and the production of second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG), that regulate Ca++ mobilization and PKC activity respectively, (IV) establishing the relationship between increased PS and/or lyso-PE as well as PKC translocation and its regulation of Ang-II binding site expression, and (V) identifying the functional response of increased Ang-II binding on cellular proliferation and hypertrophy. To achieve these aims, we will monitor PS synthesis, incorporation of exogenous PS and translocation of PS within the plasma membrane. To evaluate the effect of PLC, PLD, and translocation of PKC, we will use purified cytosolic and/or plasma membrane fractions from endothelial cells exposed to NO/2, PS or lyso- PE. To examine the stimulatory effect of Ang-II receptor binding on second messenger production, we will measure IP3 and DAG by HPLC and chromatographic analysis, Ca++ mobilization by fluorescent probe Fura-2, and PKC in purified cytosolic and plasma membranes of endothelial cells exposed to NO/2, PS or lyso-PE. Regulatory effects of PKC on Ang-II binding will be evaluated by modulating PKC activity by PS, lyso-PE, or phorbol ester and by PKC antagonists. Finally, stimulatory effects of increased Ang-II binding on cellular proliferation and hypertrophy will be evaluated by [3H] thymidine incorporation into DNA and [3H] leucine incorporation in protein, respectively.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES003989-05A1
Application #
3251788
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1987-09-01
Project End
1997-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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
Li, Y D; Patel, J M; Zhang, J et al. (1997) Overexpression of plasma membrane annexin II in NO2-exposed pulmonary artery endothelial cells. Free Radic Biol Med 23:120-6
Li, Y D; Patel, J M; Block, E R (1996) NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells. FEBS Lett 389:131-5
Li, Y D; Patel, J M; Block, E R (1994) Nitrogen dioxide-induced phosphatidylserine biosynthesis and subcellular translocation in cultured pulmonary artery endothelial cells. Toxicol Appl Pharmacol 129:114-20
Patel, J M; Block, E R (1993) Acrolein-induced injury to cultured pulmonary artery endothelial cells. Toxicol Appl Pharmacol 122:46-53
Patel, J M; Sekharam, K M; Block, E R (1992) Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells. J Biochem Toxicol 7:117-23
Patel, J M; Sekharam, M; Block, E R (1991) Vitamin E distribution and modulation of the physical state and function of pulmonary endothelial cell membranes. Exp Lung Res 17:707-23
Patel, J M; Sekharam, K M; Block, E R (1991) Angiotensin receptor-mediated stimulation of diacylglycerol production in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 5:321-7
Sekharam, K M; Patel, J M; Block, E R (1991) Plasma membrane-specific phospholipase A1 activation by nitrogen dioxide in pulmonary artery endothelial cells. Toxicol Appl Pharmacol 107:545-54
Bhat, G B; Patel, J M; Block, E R (1990) Exposure of pulmonary artery endothelial cells to nitrogen dioxide activates phospholipase A1. J Biochem Toxicol 5:67-9

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