Nitric oxide (NO) is a potent endothelium-derived vasodilator that serves a physiological role in the regulation of blood pressure and vascular tone. Immunostimulants, such as bacterial lipopolysaccharide (LPS), act on many mammalian cell types to trigger transcription of the gene encoding the inducible form of NO synthase (iNOS). In the blood vessel, this results in NO over-production, hypotension and often vascular collapse and death. Induction of iNOS is considered to be the etiological basis for septic shock, a condition caused by systemic bacterial infection and which is the leading cause in intensive care units throughout the U.S.A. While iNOS gene expression is necessary for LPS-induced hypotension, we now know that is not sufficient. Indeed, immunostimulants also act on vascular cells to induce expression of GTP cyclohydrolase I (GTPCH), the rate limiting enzyme for the synthesis of the essential NOS co-factor, tetrahydrobiopterin (BH4). Immunostimulant- induced NO synthesis in vascular cells can be prevented by inhibitors of BH4 synthesis and accelerated by provision of exogenous BH4. Thus, BH4 availability limits iNOS activity. During the initial grant period, we cloned the GTPCH gene and found its transcription is upregulated by immunostimulants in vascular smooth muscle. However, additional important mechanisms have been uncovered that may have profound impact on intracellular levels of BH4. The overall goal of the proposed research is to elucidate how intracellular levels of BH4. The overall goal of the proposed research is to elucidate how intracellular levels of BH4 are regulated in vascular smooth muscle cells and how BH4 functions for iNOS catalysis. Toward this end, Specific Aims of our research are: 1) to specify post-translational modifications of GTPCH regulation by """"""""GFRP"""""""", a recently cloned GTPCH-binding protein that serves to balance BH4 with cellular needs; 3) to characterize processes that mediate cellular uptake and efflux of reduced pterins and the contribution of pterin transport to regulation of BH4 levels in vascular smooth muscle; 4) to elucidate structural requirements of pterin analogs for binding and function in iNOS catalysis. These studies will improve our understanding of BH4 regulation and function and may provide insights that lead to novel biopterin-based strategies for pharmacotherapy of septic shock and other conditions arising from NO excess.
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