Exogenous nitric oxide (NO) has been increasingly used in inhalation therapy in newborns, children, and adult patients with diverse pulmonary disorders. In vitro and in vivo studies show that exogenous NO inhibits basal NO release from vascular endothelium. This NO-induced negative- feedback regulation is associated with inhibition of constitutively expressed endothelial NO synthase (eNOS) at a post-transcriptional level involving S-nitrosylation of active site thiol and intramolecular disulfide formation in eNOS. The loss of eNOS activity is reversible by the redox regulatory proteins thioredoxin/thioredoxin - reductase (Thx/ThxR) whose gene expression is mediated through the Rel family transcription factor NFkB. NO is also known to inhibit mRNA and protein expression of Thx/ThxR and of NFkB subunit proteins as well as NFkB/DNA binding activity in lung endothelial cells and in intact lung. Because NFkB regulates expression of multiple genes including genes of its own subunits and of Thx/ThxR, and because Thx/ThxR levels are critical for regulation of redox-sensitive processes and endothelium-dependent lung function, understanding NO-induced regulation of Thx/ThxR expression is important. Our preliminary data lead us to hypothesize that NO- induced inhibition of the upstream redox-sensitive effector kinase, protein kinase C isoforms (PKC-zeta and lambda) in transcriptional signaling, and p70 ribosomal S6 kinase (p70S6k) in translational signaling pathways, serves as a molecular switching mechanism for reduced synthesis of NFkB subunit proteins and of Thx/ThxR, and that Thx/ThxR gene expression can restore NO-mediated endothelial cell dysfunction. To test this hypothesis, we will: 1) identify the role of Thx/ThxR in NO-induced modulations of PKC isoforms and p70S6k expression and activities, 2) identify the transcriptional and translational signaling pathways involved in NO-induced inhibition of NFkB- associated proteins and of Thx/ThxR protein synthesis, and 3) verify that Thx/ThxR gene transfection restores endothelium-dependent NO/cGMP- mediated vasoregulatory function in isolated pulmonary artery. Understanding the molecular mechanisms of NO-induced redox modulation and endothelial cell dysfunction will result in the development of novel treatments for patients with pulmonary disorders that generate excessive NO or patients administered long term NO inhalation therapy.
