Inhaled nitric oxide (NO) is a selective pulmonary vasodilator which ameliorates the hypoxemia and pulmonary hypertension associated with adult respiratory distress syndrome (ARDS). However, in approximately 30% of ARDS patients, NO fails to decrease pulmonary artery pressure or improve oxygenation or both. It is the long term objective of this research to characterize the cellular and molecular mechanisms regulating responsiveness to inhaled NO with the goal of improving the efficacy of this novel therapy. NO acts, at least in part, by stimulating soluble guanylate cyclase (sGC), a heterodimer, to produce the intracellular second messenger cGMP. cGMP activates cGMP-dependent protein kinases (cGDPK) leading to relaxation of vascular smooth muscle. Animal studies have demonstrated a critical role for sGC in the regulation of NO responsiveness. Our preliminary studies revealed that exposure of primary cultures of rat pulmonary artery smooth muscle cells (RPaSMC) to cytokines, NO-donor compounds, and cell-permeable cGMP analogues all decreased sGC subunit gene expression. Similarly, decreased pulmonary sGC subunit gene expression was found in animals breathing low concentrations of NO or exposed to lipopolysaccharide (LPS), which stimulates endogenous NO production. A unifying hypothesis for these observations is that LPS and inflammatory cytokines stimulate NO production and increase pulmonary cGMP levels leading to cGDPK activation and decreased sGC subunit gene expression. Decreased sGC in response to inflammatory conditions, such as acute pulmonary injury, may represent a homeostatic response to increase NO concentrations. The overall objective of the research described in this proposal is to characterize the cellular and molecular aspects of sGC regulation in pulmonary vascular smooth muscle. Specifically, mechanisms involved in the regulation of sGC by NO will be identified in RPaSMC and in rats exposed to LPS or NO. The effect of decreased sGC subunit gene expression associated with chronic NO inhalation on pulmonary vascular responsiveness to NO will be investigated using an isolated-perfused lung system. In addition, molecular mechanisms involved in NO-mediated regulation of sGC subunit gene transcription and/or mRNA stability will be characterized. Increased understanding of the mechanisms regulating pulmonary NO/cGMP signal transduction is expected to lead to the development of novel approaches to the treatment of ARDS.
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