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This Article Full Text Full Text (PDF) All Versions of this Article: 297/2/L309    most recent 90538.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Kumar, S. Articles by Black, S. M. PubMed PubMed Citation Articles by Kumar, S. Articles by Black, S. M.

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP

¹Vascular Biology Center, Medical College of Georgia, Augusta, Georgia; ²Cold Spring Harbor Laboratories, Cold Spring Harbor, New York; and ³Department of Pediatrics and ⁴Cardiovascular Research Institute, University of California, San Francisco, California 94143

Submitted 27 October 2008 ; accepted in final form 11 May 2009

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH₄) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH₄ could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH₄ levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH₄ levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH₄ levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH₄ levels.

tetrahydrobiopterin; adenosine 3',5'-cyclic monophosphate response element binding protein; cell signaling; rebound pulmonary hypertension