L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells. (1/830)

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.  (+info)

Biopterin derivatives in normal and phenylketonuric patients after oral loads of L-phenylalanine, L-tyrosine, and L-tryptophan. (2/830)

Plasma biopterin derivatives studied in 10 normal and 21 phenylketonuric children showed a significantly high concentration in the latter group. Biopterin derivatives correlated with plasma phenylalanine concentration, but in normal adults given an oral phenylalanine load the rate of increase with phenylalanine differed from that in phenylketonuric patients. A patient with hyperphenylalaninaemia, not due to phenylketonuria, had an abnormal biopterin derivatives response to phenylalanine distinct from that of patients with classical phenylketonuria. This may be a useful investigation to differentiate some variants of phenylketonuria.  (+info)

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice. (3/830)

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.  (+info)

Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs. (4/830)

Although hypertensive lungs of chronically hypoxic rats express increased levels of nitric oxide (NO) synthases (NOSs) and produce increased amounts of NO-containing compounds (NOx) during normoxic ventilation, the level of NO production during hypoxic exposure is unclear. Because hypoxia inhibits NO synthesis in normotensive lungs, we investigated whether hypoxic ventilation inhibited NO synthesis in isolated hypertensive lungs and chronically hypoxic rats. Measurement of perfusate NOx concentration in hypertensive lungs from male rats exposed to 4 wk of hypobaric hypoxia showed that basal NOx production was reduced during hypoxic (0% O2) vs. normoxic (21% O2) ventilation. Similarly, plasma NOx concentration was lower in chronically hypoxic rats breathing 10% O2 than in those breathing 21% O2. Hypoxic inhibition of lung NOx production was not prevented by supplementary L-arginine or tetrahydrobiopterin and was not mimicked by inhibition of Ca2+ influx. However, it was mimicked by inhibition of constitutive NOS with NG-monomethyl-L-arginine and chelation of intracellular Ca2+. The endothelin type B-receptor antagonist BQ-788 prevented the increases in NOx production associated with normoxic ventilation in both isolated hypertensive lungs and intact chronically hypoxic rats. These results suggest that a reduced supply of the cosubstrate molecular O2 to NOS counteracts an endothelin type B receptor-mediated stimulation of NO synthesis in hypertensive rat lungs. Thus, despite increased NOS protein in the lungs and pulmonary arteries of chronically hypoxic rats, direct hypoxic inhibition of NO production may contribute to the development of pulmonary hypertension.  (+info)

Enzymatic synthesis of biopterin from D-erythrodihydroneopterin triphosphate by extracts of kidneys from Syrian golden hamsters. (5/830)

An enzyme system was found in either crude homogenates of dialyzed extracts of liver, kidney, lung, and brain from Syrian golden hamsters that catalyzed the synthesis of radioactive 6(L-erythro-1',2'-dihydroxypropyl)pterin (biopterin) from [U-14C]6(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrolH2neopterin-PPP) preparation. The specific radioactivity of biopterin was found to be comparable to that of D-erythroH2neopterin-PPP. The enzyme system from hamster kidney was purified severalfold by fractionation with ammonium sulfate and with an Ultrogel AcA-34 column. It was demonstrated that (a) NADPH or NADAH was essential and that (b) Mg2+ was stimulatory for the enzymatic synthesis of biopterin from D-erythroH2-NEOPTERIN-PPP. Also GTP and nonphosphorylated neopterins were not converted to biopterin. Although 6-lactyl-7,8-dihydropterin (sepiapterin) was converted to biopterin in the presence of NADPH, sepiapterin was not detected from D-erythroH2neopterin-PPP in the absence of NADPH. A preliminary experiment was performed to identify dihydrobiopterin.  (+info)

Sepiapterin reductase producing L-threo-dihydrobiopterin from Chlorobium tepidum. (6/830)

A novel type of NADPH-dependent sepiapterin reductase, which catalysed uniquely the reduction of sepiapterin to l-threo-dihydrobiopterin, was purified 533-fold from the cytosolic fraction of Chlorobium tepidum, with an overall yield of 3%. The native enzyme had a molecular mass of 55 kDa and SDS/PAGE revealed that the enzyme consists of two subunits with a molecular mass of 26 kDa. The enzyme was optimally active at pH8.8 and 50 degrees C. Apparent Km values for sepiapterin and NADPH were 21 and 6.2 microM, respectively, and the kcat value was 5.0 s-1. Diacetyl could also serve as a substrate, with a Km of 4.0 mM. The inhibitory effects of N-acetylserotonin, N-acetyldopamine and melatonin were very weak. The Ki value of N-acetyldopamine was measured as 400 microM. The N-terminal amino acid sequence was revealed as Met-Lys-His-Ile-Leu-Leu-Ile-Thr-Gly-Ala-Xaa-Lys - Lys - Ile - Xaa - Arg - Ala - Ile - Ala - Leu - Glu - Xaa - Ala - Arg - Xaa-Xaa-Xaa-His-His-His-, which shared relatively high sequence similarity with other sepiapterin reductases.  (+info)

Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin. Functional evidence against reductive oxygen activation by the pterin cofactor. (7/830)

Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 microM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mM of the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.  (+info)

Functionally important residues tyrosine-171 and serine-158 in sepiapterin reductase. (8/830)

The active site of sepiapterin reductase (SPR), which is a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family and acts as the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor (BH4), was investigated by truncation and site-directed mutagenesis. The truncation mutants showed that N-terminal and C-terminal residues contribute to bind coenzyme and substrate, respectively. The mutant rSPRA29V showed decreased activity; however, the A-X-L-L-S sequence, which has been reported as a putative pterin binding site, was estimated to preferably work as a component in the region for binding coenzyme rather than substrate. Site-directed mutants of rSPRS158D, rSPRY171V, and rSPRK175I showed low, but significant, activity having similar Km values and kcat/Km values less than 25%, for both sepiapterin and NADPH. Both amino acids Tyr-171 and Ser-158 are located within a similar distance to the carbonyl group of the substrate in the crystal structure of mouse SPR, and the double point mutant rSPRY171V+S158D was indicated to be inactive. These results showed that Ser-158, Tyr-171, and Lys-175 contributed to the catalytic activity of SPR, and both Tyr-171 and Ser-158 are simultaneously necessary on proton transfer to the carbonyl functional groups of substrate.  (+info)