Nitric oxide (NO) is a small, reactive, and readily diffusible gas which has recently been shown to serve as a biosignaling role in mammalian cells. In blood vessels, NO is a potent relaxant which keeps blood pressure appropriately low in normal individuals. However, when produced in excess, NO causes profound vasodilatation and is a key mediator of septic shock, the leading cause of death in intensive care units in the U.S., afflicting an estimated 250-350 thousand individuals. Accordingly, the development of effective therapy necessitates that we attain a thorough understanding of cellular mechanisms that contribute to high- output NO production in the blood vessel. It is now appreciated that a major mechanism for NO overproduction in sepsis is increased expression of one of the three genes which encode isoforms of NO synthase (NOS), an enzyme which produces NO from the essential amino acid L-arginine. Induction of NOS gene expression is triggered by LPS, an immunostimulatory component of the cell wall of bacteria. While induction of NOS is necessary for NO overproduction, our experiments have shown that it is insufficient. Indeed, along with NO synthase, LPS triggers the synthesis of two important enzymes which endow cells with the capacity for high- output NO synthesis. These enzymes are (1) GTP cyclohydrolase (GTPCH), needed to produce the essential NOS cofactor, tetrahydrobiopterin, and, (2) argininosuccinate synthetase (AS), needed to regenerate L-arginine, the NOS substrate from, L-citrulline, the NOS product. The proposed research will analyze regulatory sequences of the genes which encode GTPCH and AS, to reveal the basis for their coordinate induction with NOS by immunostimulants. These studies take advantage of our novel cloning of the mammalian GTPCH gene. Additional experiments will study the relevance of post-transcriptional processes for regulating expression of GTPCH by immunostimulants. Pharmacological and biological studies will also be performed to assess the extent to which AS is required for sustained high- output NO synthesis; these experiments will utilize vascular cells from animals with an AS-gene targeted """"""""knockout"""""""" and vascular cells infected with viral AS-expressing vectors. The proposed investigations will shed light on pivotal processes involved in NO overproduction by vascular cells and may lead to novel approaches for therapeutic intervention.
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