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.
