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.
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