cyclic-gmp and 2-4-diaminohypoxanthine

Tetrahydrobiopterin is an essential cofactor for NOS enzymes to synthesize NO. It has been suggested that reduced intrahepatic tetrahydrobiopterin decreases intrahepatic NO and contributes to increase hepatic vascular resistance and portal pressure in cirrhosis. The main aim of the study was to evaluate the effect of tetrahydrobiopterin supplementation in portal pressure in CCl4 cirrhotic rats.. Cirrhotic rats received vehicle or tetrahydrobiopterin (10mg/kg/day i.p.) for 3 days. Hepatic and systemic hemodynamics and hepatic tetrahydrobiopterin, NOS activity and cGMP levels were measured. In addition, hepatic and systemic hemodynamics were evaluated in normal rats in which tetrahydrobiopterin deficiency was induced by administrating 2,4-diamino-6-hydroxy-pyrimidine (DAHP) for 8h.. In cirrhotic rats, tetrahydrobiopterin administration increased liver NOS activity and cGMP levels and markedly and significantly reduced portal pressure. Amelioration of portal hypertension was associated with a normalization of arterial pressure. In normal rats DAHP decreased hepatic tetrahydrobiopterin and NOS activity and increased hepatic vascular tone. These effects of DAHP administration were corrected by tetrahydrobiopterin supplementation.. The present study shows that tetrahydrobiopterin markedly reduces portal hypertension and improves systemic hemodynamics in cirrhotic rats. These data support the concept that tetrahydrobiopterin supplementation may represent a new therapeutic strategy for portal hypertension.

Topics: Animals; Biopterins; Carbon Tetrachloride; Cyclic GMP; Enzyme Inhibitors; Hypertension, Portal; Hypoxanthines; Liver; Liver Cirrhosis; Male; Nitric Oxide Synthase; Rats; Rats, Wistar; Splanchnic Circulation

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre-incubating neurones with 5 mm 2,4-diamino-6-hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia-induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4-deficient neurones. All these effects were counteracted when neuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia-induced damage in BH4-deficient neurones was prevented when Nomega-nitro-l-arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability to hypoxia-induced mitochondrial damage and necrosis.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Biopterins; Cell Death; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; GTP Cyclohydrolase; Hypoxanthines; Hypoxia, Brain; Mitochondria; Neurons; Nitric Oxide Synthase; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

Topics: Ascorbic Acid; Biopterins; Cells, Cultured; Citrulline; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; GTP Cyclohydrolase; Humans; Hypoxanthines; Nitric Oxide; Nitric Oxide Synthase; Phosphorus-Oxygen Lyases; Pteridines; Pterins; RNA, Messenger; Solutions; Umbilical Cord

The L-arginine/nitric oxide pathway plays a key role in the regulation of arterial tone. Biosynthesis of nitric oxide requires activation of nitric oxide synthase in the presence of tetrahydrobiopterin as a cofactor. Biochemical studies demonstrated that activation of purified nitric oxide synthase at suboptimal concentrations of tetrahydrobiopterin leads to production of hydrogen peroxide. The present experiments were designed to determine whether in coronary arteries inhibition of tetrahydrobiopterin synthesis may favor nitric oxide synthase-catalyzed production of hydrogen peroxide.. Primary branches of canine left anterior descending artery were incubated for 6 hours in minimum essential medium in the presence or in the absence of the tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP; 10(-2) mol/L). Arterial rings were suspended for isometric tension recording. Production of cGMP was measured by radioimmunoassay. Experiments were performed in the presence of indomethacin (10(-5) mol/L). During contractions to the thromboxane A2/prostaglandin H2 receptor agonist U46619 (10(-7) mol/L), calcium ionophore A23187 (10(-9) to 10(-6) mol/L) caused endothelium-dependent relaxations. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (3 x 10(-4) mol/L), significantly inhibited these relaxations. In DAHP-treated arteries, relaxations to A23187 and its stimulating effect on cGMP production were significantly reduced in the presence of catalase (1200 U/mL). By contrast, catalase did not exert any effect in rings incubated in the absence of DAHP. Furthermore, the inhibitory effect of catalase on A23187-induced relaxations was abolished when coronary arteries were incubated in the presence of DAHP plus a liposoluble analogue of tetrahydrobiopterin, 6-methyltetrahydropterin (10(-4) mol/L).. The present study suggests that hydrogen peroxide may be a mediator of endothelium-dependent relaxations in coronary arteries depleted of tetrahydrobiopterin. This initially compensatory response, triggered by a dysfunctional nitric oxide synthase, may represent an important mechanism underlying oxidative vascular injury.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Calcimycin; Coronary Vessels; Cyclic GMP; Dogs; Endothelium, Vascular; Hydrogen Peroxide; Hypoxanthines; Muscle Relaxation; Nitric Oxide Synthase

Stimulation of nitric oxide (NO) synthase in endothelial cells by Ca2+ influx leads to increased intracellular levels of cGMP. NO synthase from various sources is known to use tetrahydrobiopterin, flavins, and NADPH as cofactors. We studied the effect of interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide on tetrahydrobiopterin biosynthetic activities in human umbilical vein endothelial cells (HUVEC). These stimuli led to an up to 40-fold increase of GTP cyclohydrolase I (EC 3.5.4.16) activity and to increased accumulation of neopterin and tetrahydrobiopterin in HUVEC. Further enzyme activities of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), remained unchanged. NO synthase activity in protein fractions from homogenates of cells treated with interferon-gamma plus tumor necrosis factor-alpha was not influenced as compared with untreated controls. However, interferon-gamma alone or in combination with tumor necrosis factor-alpha significantly increased intracellular cGMP formation in intact HUVEC by 50 and 80%, respectively. These stimuli increased intracellular tetrahydrobiopterin concentrations up to 14-fold. NO-triggered cGMP formation was similarly increased by incubation of otherwise untreated cells with sepiapterin, leading to elevated intracellular tetrahydrobiopterin levels. Thus, cytokines indirectly stimulate the activity of constitutive NO synthase in HUVEC by upregulating production of the cofactor tetrahydrobiopterin.

Topics: Amino Acid Oxidoreductases; Biopterins; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; GTP Cyclohydrolase; Humans; Hypoxanthines; Interferon-gamma; Lipopolysaccharides; Neopterin; Nitric Oxide; Nitric Oxide Synthase; Pteridines; Pterins; Tumor Necrosis Factor-alpha; Umbilical Veins