Nitric oxide (NO) participates in the physiology and pathophysiology of every organ system. Of most importance, NO acts as an major mechanism of vasodilation, both under physiologic and toxic conditions. During the first 2.5 years of this project, we have identified several steps in the induction process of iNOS (microtubules, PTK, PKC, NF-kappaB), as well as mechanisms of NO-mediated control of soluble guanylate cyclase, the enzyme responsible for mediating most of the actions of NO. Recently, we also cloned 5.0 Kbases of the rat iNOS promoter and sequenced the 3.2Kbases apparently responsible for transcriptional control of the gene. In the proposed studies, we will investigate the molecular mechanisms involved in the promoter- dependent up-regulation of iNOS, and down-regulation of cNOS in vascular cells during immunological challenges. We hypothesize that NF-kB binding sites in the iNOS promoter are major elements in iNOS up- regulation in responding to immunomodulators; however, other factors (e.g., CRE) are also involved in iNOS expression; we further hypothesize that a similar NF-kappaB dependent signal transduction pathway is responsible for immunomodulator-induced endothelial cNOS down-regulation. Specifically, we will investigate 1) the essential element s of rat iNOS promoter in responding to cytokines and LPS stimulation, 2) the mechanism by which cAMP synergistically up-regulates the iNOS expression in rat ASMC in responding to cAMP and LPS, 3) the mechanism by which cytoskeleton disrupting agents decrease the iNOS expression and 4) differences between the rat and human iNOS promoter responsible for the weak iNOS up regulation in response to LPS and cytokines in human cells. Furthermore, 5) we will quantify LPS-, IL-1-, and TNF-induced oxidant stress and thiol depletion and determine the contribution of oxidants to reduced NO generation in LPS, IL-1 and TNF-treated EC, 6) we will investigate the kinetics and mechanisms of LPS, IL-1 and TNF-induced activation of protein kinases, NF- kappaB, cytoskeletal reorganization and determine the contribution of these mechanisms to cNOS down-regulation in cultured EC, 7) we will investigate the mechanisms of iNOS up-regulation in LPS- induced in rats, in vivo. Our findings should help clarify the role and mechanisms of iNOS activation and cNOS down-regulation under toxic conditions.
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