GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP.
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 (BH(4)) 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 BH(4) 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 BH(4) 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 BH(4) 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 BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.
Kumar
Sanjiv
S
Vascular Biology Center, Medical College of Georgia, Augusta, 30912, USA.
Sun
Xutong
X
Sharma
Shruti
S
Aggarwal
Saurabh
S
Ravi
Kandasamy
K
Fineman
Jeffery R
JR
Black
Stephen M
SM
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000097:blood; D019556:COS Cells; D050882:CREB-Binding Protein / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism*; D006136:GTP Cyclohydrolase / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D009569:Nitric Oxide / Q000493:pharmacokinetics*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology*; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection
D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D050882:CREB-Binding Protein; D006136:GTP Cyclohydrolase; C003402:sapropterin