Nitric oxide (NO), a nitrogen-centered free radical that accounts for the biological properties of endothelium-derived relaxing factor, is generated by pulmonary vascular endothelial cells (PVEC). L-Arginine (L-arg) has been identified as the exclusive precursor of NO which is generated by the action of NO synthase on its substrate L-arg. The L-arg content of PVEC is derived primarily from plasma membrane-dependent transport of extracellular L-arg. The Principal Investigator has recently shown that physiological uptake of L-arg by PVEC in culture involves two distinct transport agencies: System y+, a saturable, sodium-independent transporter for cationic and neutral amino acids, and System BO,+, a saturable, sodium-dependent transporter that mediates uptake of both cationic and neutral amono acids. Hypoxia, a common accompaniment of a wide variety of lung disorders, has been shown to alter the structure and function of the plasma membrane of PVEC. The overall goal of this proposal is to determine whether and how hypoxia affects L-arg uptake by PVEC in culture and whether this has an impact on subsequent NO formation by these cells and reoxygenation injury to these cells.
Specific Aim #1 is to identify the effect of hypoxia on L-arg transport. In this aim, the time- and dose-dependent effects of low O2 tensions on the kinetics of each of the L-arg transport systems will be examined.
Specific Aim #2 is to define the mechanisms responsible for the hypoxic effects on L-arg transport observed in aim #1. Whole cells, isolated plasma membrane vesicles, and reconstituted proteoliposomes will be used to determine whether and how hypoxia affects the number or the activity of each of the L-arg transporters.
Specific Aim #3 is to evaluate the effect of hypoxia on intracellular L-arg content and NO formation. HPLC will be used to quantitate L-arg content and production of NO will be determined by measuring cyclic GMP content in RFL-6 cells and the NO products nitrite and nitrate. The final specific aim (#4) is to determine whether the effect of hypoxia on L-arg uptake and subsequent NO production by cultured PVEC affects reoxygenation injury to these cells. The results of the proposed studies will provide new insights into the effect of hypoxia on PVEC physiology and into the mechanisms responsible for hypoxia-mediated and reoxygenation-mediated pulmonary vascular injury.
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