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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL052136-01
Application #
2229322
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1994-04-01
Project End
1998-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
1
Fiscal Year
1994
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
Guo, Lu; Tian, Shuang; Chen, Yuguo et al. (2015) CAT-1 as a novel CAM stabilizes endothelial integrity and mediates the protective actions of L-Arg via a NO-independent mechanism. J Mol Cell Cardiol 87:180-91
Zharikov, Sergey; Krotova, Karina; Hu, Hanbo et al. (2008) Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells. Am J Physiol Cell Physiol 295:C1183-90
Su, Yunchao; Cui, Zhaoqiang; Li, Zhaozhong et al. (2006) Calpain-2 regulation of VEGF-mediated angiogenesis. FASEB J 20:1443-51
Qiu, Kai; Su, Yunchao; Block, Edward R (2006) Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation. Mol Cell Biochem 292:69-78
Krotova, Karina; Hu, Hanbo; Xia, Shen-Ling et al. (2006) Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation. Br J Pharmacol 148:732-40
Kondrikov, Dmitry; Han, Hye-Rim; Block, Edward R et al. (2006) Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 290:L41-50
Su, Yunchao; Kondrikov, Dmitry; Block, Edward R (2005) Cytoskeletal regulation of nitric oxide synthase. Cell Biochem Biophys 43:439-49
Cui, Zhaoqiang; Han, Zhaosheng; Li, Zhaozhong et al. (2005) Involvement of calpain-calpastatin in cigarette smoke-induced inhibition of lung endothelial nitric oxide synthase. Am J Respir Cell Mol Biol 33:513-20
Cui, Zhaoqiang; Zharikov, Sergey; Xia, Shen-Ling et al. (2005) Molecular cloning, characterization, and chromosomal assignment of porcine cationic amino acid transporter-1. Genomics 85:352-9
Zharikov, Sergey I; Krotova, Karina Y; Belayev, Leonid et al. (2004) Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity. Am J Physiol Lung Cell Mol Physiol 286:L974-83

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