The long-term goals of this project are to identify and elucidate the signal transduction mechanisms involved in the control of pulmonary vascular tone mediated by modulation of the production of the intracellular mediator of relaxation, cGMP, via regulation the soluble form of an integrated understanding of how various redox-related processes interact with the metabolism of reactive oxygen species and nitric oxide (NO) in the control of pulmonary vascular tone through mechanisms involving the regulation of sGC. The principal investigator proposes to systematically evaluate interactions that regulate sGC activity and its role in the control of pulmonary arterial smooth muscle force involving: (1) the potential role of endogenous vascular smooth muscle redox systems including cytosolic NAD and NADP, glutathione, microsomal and mitochondrial electron transport, with a focus on determining if H2O2, O2.- and the redox status of the heme on sGC are the central mechanisms through which these processes control the activity of sGC and vascular tone, (2) the relationships between these redox systems and the NO/O2.- interaction, with a focus on determining if thiol nitrosation is an important signaling process, and (3) to evaluate how interactions between the cellular redox systems examined, reactive O2 species and NO-derived species contribute to tone responses elicited by changes in PO2, with a focus on determining if the modulation of sGC activity is the primary signal transduction mechanism mediating responses that are observed. Much of the work in this proposal will also focus on developing an understanding of function and importance of NADH-cytochrome b558 O2.- producing electron transport chain, which the investigators have discovered during their recent work on this project in calf pulmonary arterial smooth muscle. This O2.- producing system seems to both be a major source of endogenous reactive O2 species and functions as a PO2 sensor that regulates sGC activity. The current proposal will employ isolated calf pulmonary arteries for tone studies with mechanistic probes, and measurements of endogenous reactive O2 species and cGMP production, and arterial smooth muscle subcellular fractions to identify the redox systems which generate activators and inhibitors of sGC. The processes examined in this proposal may contribute to further understanding aspects of oxygen-elicited regulation of the pulmonary circulation during normal physiology and when this circulation is exposed to pulmonary vascular pathophysiology associated with the formation of reactive oxygen and NO-derived species (e.g. acute lung injury/ARDS, ischemia reperfusion, hypertension, etc.).
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