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

Abstract

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.

Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases

“…The research literature has identified the cofactor tetrahydrobiopterin (BH4) as a significant player in eNOS function [187,188]. BH4 shifts the heme iron in eNOS to a high spin state, as well as increasing arginine binding, thus catalyzing the synthesis of NO by eNOS [187].…”

“…BH4 shifts the heme iron in eNOS to a high spin state, as well as increasing arginine binding, thus catalyzing the synthesis of NO by eNOS [187]. The synthesis of BH4 from its substrate GTP is induced by IFN-γ, which, in turn, is induced by bacterial lipopolysaccharides (LPS) [189].…”

“…Thus, a bacterial infection will induce NO synthesis by eNOS. However, an excess of exogenous NO (as might be expected to occur through iNOS synthesis of NO during a bacterial infection) causes a decrease in NO synthesis by eNOS with a simultaneous increase in superoxide synthesis, an effect that can lead to severe hypertension in infants with congenital heart disease treated with inhaled NO [187]. Superoxide's reaction with NO to produce the highly toxic peroxynitrite (ONOO−), a potent bacteriocidal agent, is likely a critical factor.…”

Molecular mechanisms underlying the activation of eNOS

“…However, also in this case, the apparent uncoupling of eNOS does not seem to be linked to a measurable decrease in BH 4 levels. In fact, BH 4 levels and the expression of GTPCH were increased by NO donors via a mechanism linked to cyclic AMP and the cyclic AMP response element-binding protein [67]. Thus, it seems that mechanisms that regulate the BH 4 /BH 2 ratio independently of overall biopterin levels may play an important role in regulating eNOS.…”

GCH1, BH4 and Pain

“…(2). GCH1 expression is also under the control of estrogens [77], possibly through a NO-mediated activation of CREB [78, 79]. Interestingly, cAMP is a strong activator of GCH1 expression [80] and cAMP upregulation is also a central component of the axotomy response in dorsal root ganglion (DRG) neurons [81, 82].…”