Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages.

Many groups of Gram-negative bacteria cause diseases that are harmful to sheep. Toll-like receptor 4 (TLR4), which is critical for detecting Gram-negative bacteria by the innate immune system, is activated by lipopolysaccharide (LPS) to initiate inflammatory responses and oxidative stress. Oxidation intermediates are essential activators of oxidative stress, as low levels of free radicals form a stressful oxidative environment that can clear invading pathogens. NO is an oxidation intermediate and its generation is regulated by nitric oxide synthase (iNOS). Guanosine triphosphate cyclohydrolase (GCHI) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for the production of inducible iNOS. Previously, we made vectors to overexpress the sheep TLR4 gene. Herein, first generation (G1) of transgenic sheep was stimulated with LPS in vivo and in vitro, and oxidative stress and GCHI expression were investigated. Oxidative injury caused by TLR4 overexpression was tightly regulated in tissues. However, the transgenic (Tg) group still secreted nitric oxide (NO) when an iNOS inhibitor was added. Furthermore, GCHI expression remained upregulated in both serum and monocytes/macrophages. Thus, overexpression of TLR4 in transgenic sheep might accelerate the clearance of invading microbes through NO generation following LPS stimulation. Additionally, TLR4 overexpression also enhances GCHI activation.

Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis.

BACKGROUND: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. METHODS AND RESULTS: In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. CONCLUSIONS: The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

A case of late-onset Segawa syndrome (autosomal dominant dopa-responsive dystonia) with a novel mutation of the GTP-cyclohydrase I (GCH1) gene.

We report a case of a 46-year-old Japanese woman with hereditary progressive dystonia with marked diurnal fluctuations and dopa-responsive dystonia (HPD/DRD). She developed difficulty in walking at the age of 44 years due to bradykinesia as well as hand tremors, muscle rigidity, increased tendon reflexes and mild dystonia in the lower extremities, all of which responded remarkably to low doses of levodopa (150 mg/day). Biopterin and neopterin concentrations in the cerebrospinal fluid (CSF) were decreased. Analysis of the guanosine 5'-triphosphate cyclohydrolase I (GCH1) gene revealed a novel mutation (W53X) in one allele. The GCH1 activity that was expressed in mononuclear blood cells was almost half the normal value (usually 2-20% of the normal value (39.0+/-9.2 pmol/ml) in patients with HPD/DRD). The relatively conserved GCH1 activity that is expressed in stimulated peripheral blood mononuclear cells may be related to the late clinical symptoms in this patient.

Functional tetrahydrobiopterin synthesis in human platelets.

BACKGROUND: Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. METHODS AND RESULTS: We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. CONCLUSIONS: Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric vasculature of cirrhotic rats.

In cirrhosis, arterial vasodilation and the associated hemodynamic disturbances are most prominent in the mesenteric circulation, and its severity has been linked to bacterial translocation (BT) and endotoxemia. Synthesis of nitric oxide (NO), the main vasodilator implicated, is dependent on the essential cofactor tetrahydrobiopterin (BH(4)). The key enzyme involved in BH(4) synthesis is GTP-cyclohydrolase I (GTPCH-I), which is stimulated by endotoxin. Therefore, we investigated GTPCH-I activity and BH(4) biosynthesis in the mesenteric vasculature of cirrhotic rats with ascites, as well as their relationship with BT and endotoxemia, serum NO, and mean arterial pressure (MAP). GTPCH-I activity and BH(4) content in mesenteric vasculature was determined by high-performance liquid chromatography. BT was assessed by standard bacteriologic culture of mesenteric lymph nodes (MLNs). Serum endotoxin was measured by a kinetic turbidimetric limulus amebocyte lysate assay, and serum NO metabolite (NOx) concentrations were assessed by chemiluminescence. BT was associated with local lymphatic and systemic appearance of endotoxin and was accompanied by increases in serum NOx levels. GTPCH-I activity and BH(4) content in mesenteric vasculature were both increased in animals with BT and correlated significantly (r = 0.69, P <.01). Both GTPCH-I activity and BH(4) levels significantly correlated with serum endotoxin and NOx levels (r = 0.69 and 0.54, 0.81 and 0.53, P <.05). MAP (a marker of systemic vasodilatation) correlated with endotoxemia (r = 0.58, P <.03) and with GTPCH-I activity (r = 0.69, P <.01). In conclusion, in cirrhotic animals BT appears to lead to endotoxemia, stimulation of GTPCH-I, increased BH(4) synthesis, and further enhancement of vascular NO production that leads to aggravation of vasodilatation.

Molecular mechanisms of hereditary progressive dystonia with marked diurnal fluctuation, Segawa's disease.

The causative gene for hereditary progressive dystonia with marked diurnal fluctuation/dopa-responsive dystonia (HPD/DRD) was discovered in 1994 to be guanosine triphosphate (GTP) cyclohydrolase I, an enzyme involved in tetrahydrobiopterin biosynthesis. To the present, more than 50 mutations have been found in this gene in HPD/DRD patients. Although it is clear that HPD/DRD is caused by partial deficiency of tetrahydrobiopterin in the brain, several important issues regarding the molecular etiology of HPD/DRD remain to be addressed. We review herein the recent progress in the molecular genetics of HPD/DRD and clarify the points to be answered.

[Segawa's disease].

Segawa's disease (dopa-responsive dystonia) is caused by a genetic mutation in the gene for GTP cyclohydrolase I, a rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin. Although the causative gene was identified, many issues remain to be addressed in this disease. In this short article, we have described and discussed the relevance of measurement of GTP cyclohydrolase I activity in phytohemagglutinin-stimulated mononuclear blood cells in patients with Segawa's disease. Although the enzyme activity in immune cells may not reflect the activity in the brain, we can evaluate the maximum ability in the induction of GTP cyclohydrolase I in the cell from this method. Under normal conditions without any stimulant, some compensatory mechanism(s) may attenuate the difference in the enzyme activity between patients and normal individuals. Since one allele is inactivated in patients with Segawa's disease, we can distinguish the patients from normal individuals without examining their gene-defect. Because we cannot identify a mutation in the coding region and the exon-intron junction of GTP cyclohydrolase I in about half of patients with Segawa's disease, this biochemical assay has great value in diagnosis of the disease.

Developmental changes of tetrahydrobiopterin in rat and human erythrocytes.

In the present study, we investigated the developmental changes of (1) plasma and erythrocyte tetrahydrobiopterin (BH4); (2) erythrocyte GTP cyclohydrolase (the rate-limiting enzyme of BH4 biosynthesis); (3) the permeability of erythrocyte membrane to BH4; and (4) plasma phenylalanine, both in healthy human subjects and Wistar rats. In vitro experiments demonstrated passive transport of BH4 into erythrocytes. In humans, BH4 levels as well as the other parameters were fairly consistent across all age groups. In contrast, Wistar rats showed significant developmental changes in erythrocyte BH4, which were not simply correlated to either GTP cyclohydrolase, permeability to BH4 or plasma phenylalanine levels. This may suggest the existence of other factors regulating the homeostasis of BH4, such as BH4-binding capacity in plasma and/or erythrocytes. These species/age differences in erythrocyte characteristics may influence the pharmacological behavior and clinical efficacy of BH4 in humans and experimental animals.

Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene.

Hereditary progressive dystonia with marked diurnal fluctuation (HPD) (also known as dopa responsive dystonia) is a dystonia with onset in childhood that shows a marked response without any side effects to levodopa. Recently the gene for dopa responsive dystonia (DRD) was mapped to chromosome 14q. Here we report that GTP cyclohydrolase I is mapped to 14q22.1-q22.2. The identification of four independent mutations of the gene for GTP cyclohydrolase I in patients with HPD, as well as a marked decrease in the enzyme's activity in mononuclear blood cells, confirms that the GTP cyclohydrolase I gene is a causative gene for HPD/DRD. This is the first report of a causative gene for the inherited dystonias.

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes.

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.

Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells.

The only known role for 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) is as the cofactor for the aromatic amino acid hydroxylases. However, BH4 has been shown to be synthesized by cells that do not contain any hydroxylase activity, suggesting that it may have still undiscovered functions. Our finding of much higher levels of BH4 and GTP cyclohydrolase, the first enzyme of de novo BH4 biosynthesis, in rat reticulocytes compared to mature erythrocytes raised the possibility that BH4 might play a role in erythrocyte maturation. We have now demonstrated, by using murine erythroleukemia (MEL) cells as a model for erythrogenesis, that BH4 synthesis is required for proliferation of these cells. Inhibition of BH4 biosynthesis in rapidly dividing MEL cells with N-acetylserotonin, a potent inhibitor of sepiapterin reductase, the terminal enzyme in the BH4 biosynthetic pathway, results in inhibition of DNA synthesis and mitogenesis without induction of hemoglobin synthesis. The inhibition of DNA synthesis is reversed by repletion of cellular BH4 levels with sepiapterin, a pterin that is readily taken up by the cells and converted to BH4 by the sequential reductions of sepiapterin reductase and dihydrofolate reductase. Treatment of MEL cells with hexamethylene bisacetamide, an inducer of differentiation, results in a decrease in BH4 synthesis accompanied by a cessation of growth and concomitant hemoglobin synthesis. The inhibition of proliferation induced by hexamethylene bisacetamide can be reversed by maintaining high intracellular levels of BH4, which also decreases the amount of hemoglobin. The mechanism of the BH4 effect has not yet been elucidated, but it appears as though BH4 synthesis is more intimately linked with cell proliferation than with the differentiation process.

Localization of GTP cyclohydrolase I in human peripheral blood smears using a specific monoclonal antibody and an immune-alkaline phosphatase labeling technique.

GTP cyclohydrolase I, the enzyme catalyzing the first step in the cofactor biosynthesis for the aromatic amino acid hydroxylases, has been localized in situ. By the use of a monoclonal antibody specific to human GTP cyclohydrolase I, the enzyme has been visualized immuno-enzymatically by alkaline phosphatase monoclonal anti-alkaline phosphatase labeling. In routine blood smears lymphocytes, monocytes/macrophages, and granulocytes show strong intraplasmatic staining. Premature erythrocytes show clear staining of the reticulated cytoplasmatic structure, while mature erythrocytes are completely negative. Neither is there any staining for GTP cyclohydrolase I in the blast cells of a case of T-cell acute lymphoblastic leukemia. These results closely confirm the prior finding that mature erythrocytes as well as most malignant mononuclear cells lack GTP cyclohydrolase I activity, and they indicate that in these cells the enzyme protein may be absent.

Increase of GTP cyclohydrolase I activity in mononuclear blood cells by stimulation: detection of heterozygotes of GTP cyclohydrolase I deficiency.

An assay is described for GTP cyclohydrolase I activity in human mononuclear cells isolated from 20 ml of heparinized blood. The activity of this enzyme was low in unstimulated cells and increased 5-10 times after stimulation by phytohemagglutinin (formation of 0.8-1.3 pmol dihydroneopterin triphosphate/min per mg protein at 37 degrees C, n = 15) or mixed lymphocyte culture. No activity was detected in phytohemagglutinin-stimulated mononuclear cells of a patient with proven GTP cyclohydrolase I deficiency in liver; the samples from the father and mother of the patient showed 30 and 46%, respectively, of the mean of 15 healthy controls. In unstimulated cells, neopterin was the main component of the total intracellular pterins (after oxidation). After stimulation, dihydroneopterin triphosphate, measured as neopterin triphosphate by high performance liquid chromatography, was increased 10-30 times; neopterin and pterin were increased only 2- to 6-fold. Since the immunoreactive cells from this patient were unable to produce pterins and all immunological tests on the patient were normal, it is concluded that neither dihydroneopterin triphosphate, nor one of its metabolites are of primary importance for an immune reaction. The assay described can be used for the detection of heterozygotes of GTP cyclohydrolase I deficiency.