Nitric oxide (NO) is an endogenous product of lung endothelial cells as well as an exogenous gas known to play a critical role in the physiology and pathophysiology of the lung in animals and humans. Despite its regulatory function, NO is cytotoxic and causes S- nitrosylation and inhibition of enzyme activities including inhibition of the constitutively expressed endothelial cell NO synthase (ecNOS). NO also inhibits mRNA and protein expression of the redox signalling enzyme system thioredoxin/thioredoxin reductase (T/TR) in animal and human lung endothelial cells. T/TR plays a critical role in regulation of redox-sensitive enzymes essential for DNA synthesis and gene expression. As such, NO-induced inhibition of ecNOS and T/TR may be associated with impaired NO/cGMP and redox signalling functions of these cells. However, the molecular mechanisms of NO-induced endothelial cell dysfunction remain elusive. To identify these mechanisms, we hypothesize that: 1) NO's interaction with active site cysteines of ecNOS is causally linked with reduced catalytic activity and therefore impaired NO/cGMP signalling and 2) NO-induced reduction of T/TR expression is linked to inhibition of DNA synthesis and/or gene expression. To test these hypotheses, we will I) identify NO's interaction with active site cysteines of ecNOS and establish its implications for endothelial ell function by utilizing techniques of cell biology, biochemistry, and molecular biology, II) identify whether NO-induced reduction of DNA synthesis and gene expression are associated with reduced expression of T/TR by monitoring activity of redox-sensitive enzyme ribonucleotide reductase (RR) and DNA binding activity of redox-sensitive p50 submit of transcription factor NFKB, and III) determine whether endothelial cells supplemented with thiol-containing agents or genetically-engineered to hyperexpress T/TR are protected from NO-induced cell dysfunction. Understanding the molecular mechanisms of NO-induced endothelial cell dysfunction will result in the development of novel strategies for treatment of patients with pulmonary disorders that generate excessive NO or patients administered long term NO inhalation therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058679-04
Application #
6184048
Study Section
Special Emphasis Panel (ZRG4-ALTX-1 (04))
Project Start
1997-07-01
Project End
2001-10-31
Budget Start
2000-07-01
Budget End
2001-10-31
Support Year
4
Fiscal Year
2000
Total Cost
$237,631
Indirect Cost
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Zhang, Jianliang; Block, Edward R; Patel, Jawaharlal M (2002) Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev 123:1363-74
Zhang, Jianliang; Xia, Shen-Ling; Block, Edward R et al. (2002) NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells. Am J Physiol Cell Physiol 283:C1080-9
Li, Yong D; Block, Edward R; Patel, Jawaharlal M (2002) Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation. Am J Physiol Lung Cell Mol Physiol 283:L707-16
Zhang, Jianliang; Patel, Jawaharlal M; Block, Edward R (2002) Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev 123:613-25
Chen, S; Patel, J M; Block, E R (2000) Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release. Am J Physiol Lung Cell Mol Physiol 279:L849-56
Patel, J M; Li, Y D; Zhang, J et al. (1999) Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS. Am J Physiol 277:L794-801
Zhang, J; Velsor, L W; Patel, J M et al. (1999) Nitric oxide-induced reduction of lung cell and whole lung thioredoxin expression is regulated by NF-kappaB. Am J Physiol 277:L787-93
Patel, J M; Martens, J R; Li, Y D et al. (1998) Angiotensin IV receptor-mediated activation of lung endothelial NOS is associated with vasorelaxation. Am J Physiol 275:L1061-8
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