Function Of BH4 Research Articles

Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation

Abstract Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Accordingly, administration of BH4 is considered as a promising therapy of cardiovascular diseases associated with endothelial dysfunction and oxidative stress. In a recent study we identified a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. As demonstrated with cultured porcine aortic endothelial cells, oxidative inactivation of soluble guanylate cyclase with nitroglycerin or the heme-site inhibitor ODQ (1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one) resulted in a decrease in NO-induced cGMP accumulation that was insensitive to scavengers of reactive oxygen species but prevented upon supplementation of the cells with BH4. Tetrahydroneopterin had the same effect and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of soluble guanylate cyclase against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Tetrahydrobiopterin Protects Soluble Guanylate Cyclase against Oxidative Inactivation

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Specific interaction of the diastereomers 7(R)‐ and 7(S)‐tetrahydrobiopterin with phenylalanine hydroxylase: implications for understanding primapterinuria and vitiligo

Pterin-4a-carbinolamine dehydratase (PCD) is an essential component of the phenylalanine hydroxylase (PAH) system, catalyzing the regeneration of the essential cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin [6(R)BH4]. Mutations in PCD or its deactivation by hydrogen peroxide result in the generation of 7(R,S)BH4, which is a potent inhibitor of PAH that has been implicated in primapterinuria, a variant form of phenylketonuria, and in the skin depigmentation disorder vitiligo. We have synthesized and separated the 7(R) and 7(S) diastereomers confirming their structure by NMR. Both 7(R)- and 7(S)BH4 function as poor cofactors for PAH, whereas only 7(S)BH4 acts as a potent competitive inhibitor vs. 6(R)BH4 (Ki=2.3-4.9 microM). Kinetic and binding studies, as well as characterization of the pterin-enzyme complexes by fluorescence spectroscopy, revealed that the inhibitory effects of 7(R,S)BH4 on PAH are in fact specifically based on 7(S)BH4 binding. The molecular dynamics simulated structures of the pterin-PAH complexes indicate that 7(S)BH4 inhibition is due to its interaction with the polar region at the pterin binding site close to Ser-251, whereas its low efficiency as cofactor is related to a suboptimal positioning toward the catalytic iron. 7(S)BH4 is not an inhibitor for tyrosine hydroxylase (TH) in the physiological range, presumably due to the replacement of Ser-251 by the corresponding Ala297. Taken together, our results identified structural determinants for the specific regulation of PAH and TH by 7(S)BH4, which in turn aid in the understanding of primapterinuria and acute vitiligo.

Modulation of Inducible Nitric Oxide Synthase mRNA Stability by Tetrahydrobiopterin in Vascular Smooth Muscle Cells

Tetrahydrobiopterin (BH4) regulates inducible nitric oxide synthase (iNOS) as cofactor and allosteric effector. The present paper describes a novel function of BH4 in vascular smooth muscle cells (SMC). By varying BH4 levels with dicumarol (an inhibitor of BH4 synthesis) and sepiapterin (an exogenous source of cofactor), we investigated iNOS expression in activated rat aortic SMC. In sepiapterin-supplemented cells, iNOS protein levels were increased while in dicumarol-treated cells, iNOS levels were diminished. Time-kinetic experiments revealed that inhibition or supplementation of BH4 synthesis had no effects on iNOS induction or transcription rate. However, iNOS mRNA was present over a prolonged time in sepiapterin-supplemented SMC. Analysis of iNOS mRNA levels showed stable iNOS mRNA in sepiapterin-treated cells 8 hours after transcription inhibition, while in dicumarol-treated cells iNOS mRNA disappeared. The decrease of iNOS mRNA by dicumarol was abolished by sepiapterin. These data indicate that BH4 post-transcriptionally stabilizes iNOS mRNA in SMC. By this way BH4 modulates iNOS expression in the vascular system.

Regulation of the Expression of the Inducible Isoform of Nitric Oxide Synthase

All forms of NOS contain four prosthetic groups: flavin-adenine dinucleotide; flavin mononucleotide, tetrahydrobiopterin (BH4), and a heme complex, iron protoporphyrin IX. All NOS isoforms are dependent on calmodutin; in the inducible isoform calmodulin is already present in a tightly bound form. The role of calmodulin is to control the electron transfer from NADPH to the flavins, possibly by causing a reorientation of the reductase and oxygenase domains of NOS, perhaps moving them into a favorable position for electron transfer among them. The function of BH4 is unclear, but may substitute for peroxy-heme to carry out nucleophilic attack on hydroxyarginine or perhaps may act as a stabilizer of the active structure of NOS—that is, assembling dimeric forms of NOS in the case of bNOS and iNOS. The cellular and molecular mechanisms of iNOS induction, or its inhibition by pharmacological agents, are not fully understood. They clearly involve transcription of mRNA and novel protein biosynthesis and take at least several hours. The sequencing of the DNA regions upstream of the iNOS gene (that is, the promoter regions) revealed separate promoter regions for the induction of iNOS by endotoxin and interferon-γ (IFN-γ).