Nitric oxide synthase (NOS) activation can result in formation of relevent nitrogen oxide diseases, other than nitric oxide radicals. One of these is nitrite. Additionally, covalent cysteine modifications form Snitrosothiol bonds. When this cysteine modification signals a change in protein function, it is often termed Snitrosylation. These reactions are increasingly recognized to represent metabolically regulated cell signaling processes. Disorders of ainway epithelial S-nitrosylation signaling have been observed in a range of diseases, including asthma and cystic fibrosis (CF). However, the formation and location of S-nitrosothiolmodified proteins is poorly understood in airway epithelial cell cultures in general, and has not been studied in primary, pseudostratified columnar ainway epithelium. In order to begin to understand NOS-dependent Snitrosothiol formafion in the ainways as it relates to disease, we will test the three hypotheses that 1) specific proteins in normal human airway epithelial cells are S-nitrosylated by NOS (Aim 1);2) protein Snitrosylation occurs in specific subcellular locations in human airway epithelial cells (Aim 2);and 3) S-nitrosylation signaling is disordered in the human CF airway epithelium (Aim 3). We chose to study S-nitrosylation by the inducible and endothelial NOS (iNOS and eNOS) isoforms because each is expressed and active in normal human ainway epithelial cells;and because decreased INOS expression in the CF ainway epithelium may have important disease implications. Hsp70/Hsp90 organizing protein was chosen to study the paradigm of S-nitrosothiol signaling downstream from iNOS and eNOS activity because 1) it is S-nitrosylated at baseline;2) its S-nitrosylation increases with Snitrosoglutathione treatment;and 3) its S-nitrosylation appears to be important to ainway epithelial cell biology in general, and to the pathobiology and treatment of CF in particular. We have chosen to perform our studies primarily in human airway pseudostratified columnar epithelial cultures because these most closely to recapitulate the human ainway in vivo. We will do additional in vivo studies in a mouse model. This project will make extensive use of interactions with investigators on the other projects in this program, and each aim will make use of one or more cores in the program. At the conclusion of this project, we anticipate that we will have 1) a functional model of mechanisms by which NOS activation leads to Snitrosylation of specific proteins in specific cellular locations in human pseudostratified columnar epithelium;and 2) the relevance of disorders of S-nitrosothiol formation to the development of new therapies for CF.

Public Health Relevance

The mechanisms by which NOS signals S-nitrosylation in the human ainway epithelium are pooriy understood. Our preliminary data suggest that disorders of S-nitrosylation signaling are relevant to the development of new corrector therapies for CF patients. Additionally, this mechanism?and the targets of Snitrosylation we will study?may be relevant to the underiying pathophysiology not only of CF, but of other pulmonary diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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University of Virginia
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