Modification of cysteine residues by nitric oxide changes the function of a broad spectrum of proteins. This protein modification represents a physiological signaling mechanism, termed S- nitrosylation, that has been proposed to be analogous to phosphorylation. It regulates many of the bioactivities of inflammatory cells and other cells relevant to pulmonary biology. However, unlike phosphorylation, the regulation of S-nitrosylation signaling is poorly understood. In our previous project, we studied the metabolic regulation in the airway of S-nitrosoglutathione (GSNO), an endogenous bronchodilator and S-nitrosylating agent. We have now accomplished most of the aims of this project. Indeed, GSNO is now entering clinical trials as an inhalational therapy. However, critical questions remain regarding human airway S-nitrosothiol metabolism as it relates to specific molecular targets in the lung. For example, we and others have shown that the airway epithelial expression of the enzyme, GSNO reductase, is an important determinant of airway hyper-responsiveness in animal models of asthma. Moreover, we have found evidence that the GSNO breakdown product, S-nitroso-L-cysteine, causes stereoselective, cyclic GMP-independent smooth muscle relaxation through S-nitrosylation of myosin. Further, several investigators have reported that GSNO can regulate the function and intracellular trafficking of proteins relevant to lung disease, including the cystic fibrosis transmembrane regulatory protein. We have developed novel techniques for measuring cellular S-nitrosothiols. In the current project, we will apply these novel techniques to study, in human airway tissue, 1) the regulation of GSNO reductase expression (Aim 1);2) the effect of GSNO reductase and other S-nitrosothiol regulatory enzymes on smooth muscle function (Aim 2);and 3) the effect of S-nitrosothiol regulatory enzymes on the metabolism and trafficking of S-nitrosothiol-modified proteins in epithelial cells, particularly in the context of GSNO therapy (Aim 3). We believe that our publication record and preliminary data suggest that we can accomplish these aims in the proposed time frame. Further, our data also suggest that these aims are innovative, scientifically sound and of potential relevance to the treatment of asthma and cystic fibrosis. Project Narrative: Our project is focused on the pulmonary metabolism of signaling molecules known as S-nitrosothiols. We have found evidence that abnormal S-nitrosothiol metabolism in the airway may help to explain airway narrowing in many patients with asthma. Further, it turns out that understanding this metabolism is likely to be important for the development of certain new therapies for asthma and cystic fibrosis.