Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice.

(6R)-5,6,7,8-Tetrahydro-biopterin (H(4)B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H(4)B synthesis. A relative shortage of H(4)B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H(4)B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BLxCBA) were compared. H(4)B levels were determined by high-performance liquid chromatography and NO synthase activity by [(3)H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H(4)B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with N(G)-nitro-L-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H(4)B, the differences between the 2 groups disappeared. Our findings demonstrate that H(4)B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H(4)B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.

Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse.

It has been suggested that decreased tetrahydrobiopterin (BH4) availability may be a useful tool for limiting excessive nitric oxide (NO) formation. In order to test this hypothesis we utilised cultured astrocytes derived from the brain of the hph-1 (BH4 deficient) mouse. In response to treatment with lipopolysaccharide and interferon-gamma (LPS/gammaIFN) levels of BH4 doubled in both wild type and hph-1 astrocytes. However, levels of BH4 in hph-1 astrocytes remained only 25% of the wild type astrocytes. Nitric oxide formation, measured with an NO-electrode, was 45% less from LPS/gammaIFN stimulated hph-1 astrocytes compared with wild type stimulated astrocytes. In contrast, iNOS specific activity and iNOS protein were enhanced in hph-1 stimulated astrocytes by 40 and 60%, respectively when compared with wild type. In conclusion it appears that whilst a decrease in BH4 may limit NO release per se, the possibility and consequences of long term 'over' induction of iNOS protein requires further consideration.

Tetrahydrobiopterin regulates cyclic GMP-dependent electrogenic Cl- secretion in mouse ileum in vitro.

1. Basal electrogenic Cl- secretion, measured as the short-circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)-deficient hph-1 mice and wild-type controls in vitro, although values were not significantly different. 2. The basal nitrite release and mucosal cyclic guanosine 3',5'-monophosphate (cyclic GMP) production were similar in control and BH4-deficient ileum. 3. Mucosally added Escherichia coli heat-stable toxin (STa, 55 ng ml-1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4-deficient ileum. 4. L-Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4-deficient ileum. 5. Serosal carbachol (1 mM) stimulated maximum short-circuit currents of similar magnitude in both control and BH4-deficient ileum, whilst nitrite release and cyclic GMP production were minimal. 6. E. coli STa and L-arginine increased electrogenic Cl- secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP. The inhibition of these processes in the hph-1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut.

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from the hph-1 mouse.

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes.

Nitric oxide (NO.) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO., as reflected by nitrite (NO2-) + nitrate (NO3-) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO2- + NO3- production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO2- + NO3- from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO. from nerve terminals. The ratios of antioxidant capacity to NO. production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.

Recessively inherited L-DOPA-responsive parkinsonism in infancy caused by a point mutation (L205P) in the tyrosine hydroxylase gene.

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of dopamine. This report describes a missense point mutation in the human TH (hTH) gene in a girl presenting parkinsonian symptoms in early infancy and a very low level of the dopamine metabolite homovanillic acid in the CSF. DNA sequencing revealed a T614-to-C transition in exon 5 (L205P). Both parents and the patient's brother are heterozygous for the mutation. Site-directed mutagenesis and expression in different systems revealed that the recombinant mutant enzyme had a low homospecific activity, i.e. approximately 1.5% of wt-hTH in E. coli and approximately 16% in a cell-free in vitro transcription-translation system. When transiently expressed in human embryonic kidney (A293) cells a very low specific activity (approximately 0.3% of wt-hTH) and immunoreactive hTH (< 2%) was obtained. The expression studies are compatible with the severe clinical phenotype of the L205P homozygous patient carrying this recessively inherited mutation. Treatment with L-DOPA resulted in normalisation of the CSF homovanillic acid concentration and a sustained improvement in parkinsonian symptoms.

Neurochemical effects following peripheral administration of tetrahydropterin derivatives to the hph-1 mouse.

The hph-1 mouse which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 mumol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N-benzyl-1, 4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1-4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 mumol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.

Inhibition of tetrahydrobiopterin synthesis reduces in vivo nitric oxide production in experimental endotoxic shock.

Nitric oxide synthesis requires the cofactor tetrahydrobiopterin. We have examined the effect on nitric oxide synthesis in experimental endotoxic shock of 2,4- diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, the first and rate limiting enzyme for tetrahydrobiopterin synthesis. Rats given lipopolysaccharide (LPS, 10 mg/kg) showed a large rise in plasma nitrate at 4 and 8 hours which was significantly reduced by DAHP (1 g/kg) given at the same time as LPS. There was a 40-50% reduction in the haem-NO signal detected in kidney by electron paramagnetic resonance spectroscopy. LPS produced hypotension at 3 hours and 6 hours and this was ameliorated at 6 hours in rats given DAHP. DAHP abolished the rise in kidney tetrahydrobiopterin levels seen 4 hours after LPS but no effect was seen on induction of inducible nitric oxide synthase (iNOS) as assessed by immunohistochemistry and reverse transcriptase PCR, consistent with the effect of DAPH being by reduction of tetrahydrobiopterin levels. The results show that inhibition of tetrahydrobiopterin synthesis is an effective strategy to reduce nitric oxide synthesis by iNOS in vivo.

Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse.

Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress.

[Psychiatric complications in patients under intensive care]. / Complications psychiatriques chez des patients de soins intensifs.

Ten adult patients with psychiatric disorders in the intensive care ward were examined. The length of stay varied from one week to four months and mechanical ventilation was necessary for all patients. Their experience of intensive care and their psychosensorial problems were as follows: temperospatial disorientation, perturbation of the sense of posture, hallucinations which could go as far as oneiric delirium, anguish and symptoms of depression. No psychotic syndrome, literraly speaking, was observed objectively. In the monthes that followed the stay under intensive care many patients presented important psychosomatic disorders. Organic factors are responsible for these complications, though the environment of the intensive care could induce a marked disafferentation. An effort by the attending staff, aimed at orientating or "reafferenting" these patients, could reduce these problems.