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Background: Ascorbic acid (AsA) therapy for sepsis is thought to have a protective effect on vascular endothelial cells. However, the effect of AsA therapy on endothelial cell dysfunction over time and the appropriate timing for AsA administration to demonstrate efficacy is unclear.Methods: Septic mice were induced by cecal ligation and puncture (CLP). The effect of AsA administration (200 mg/kg) on vascular endothelial cell dysfunction in sepsis was examined using septic mice at two administration timings: early group (AsA was administered immediately after CLP) and late group (AsA was administered 12 h after CLP). First, survival rates were compared between the early and late administration groups. Next, the dihydrobiopterin/tetrahydrobiopterin (BH2/BH4) ratio, serum syndecan-1, and endothelial nitric oxide synthase (eNOS) were measured as indicators of vascular endothelial cell damage, and liver tissue was examined for organ damage to verify the effect of early AsA administration in septic mice.Results: In survival experiments, the early group showed significantly improved survival compared to the non-treatment group (p < 0.05), while the late group showed no improved survival compared to the non-treatment group. The BH2/BH4 ratio increased after 6 hours following CLP, and the increase was suppressed by early AsA administration (0.12 ± 0.02 vs. 0.07 ± 0.016, p < 0.05). Serum syndecan-1 levels were significantly elevated at 12 h after CLP, and early AsA administration prevented the increase (25.4 ± 12.1 vs. 9.9 ± 2.7 mg/ml, p < 0.05). The expression of eNOS at 12 h after CLP was decreased in the non-treatment group, but the expression was protected by early AsA administration. Moreover, hematoxylin and eosin staining of hepatocytes showed reduced liver damage in the early group compared to the non-treatment group.Conclusions: In septic mice, early AsA administration immediately after CLP may have protective effects on vascular endothelial cells by decreasing the BH2/BH4 ratio, resulting in reduced organ dysfunction and improved survival. To achieve this effect, early administration of AsA is required before BH4 is oxidized.

Because treatment for postsurgical pain (PSP) remains a major unmet medical need, the emergence of safe and innovative nonopioid drugs has been strongly coveted. Tetrahydrobiopterin (BH4) is an interesting molecule for gaining a better understanding the pathological mechanism of neuropathic pain. However, whether BH4 and its pathway are involved in the pathogenesis of PSP remains unclear. In this study, we found that early in a rat paw incision model, the gene expression of GTP cyclohydrolase 1 (GTPCH) and sepiapterin reductase (SPR), BH4-producing enzymes in the de novo pathway, were significantly increased in incised compared with naive paw skin. Although a significant increase in GTPCH protein levels was observed in incised paw skin until only 1 day after incision, a significant increase in BH4 levels was observed until 7 days after incision. In vivo, Spr-knockout mice showed an antinociceptive phenotype in the hind paw incision compared with the wild-type and Spr heterozygote groups. Furthermore, QM385, the SPR inhibitor, showed a significant dose-dependent, antinociceptive effect, which was supported by a reduction in BH4 levels in incised skin tissues, with no apparent adverse effects. Immunohistochemical analysis demonstrated that macrophages expressing GTPCH protein were increased around the injury site in the rat paw incision model. These results indicate that BH4 is involved in the pathogenesis of PSP, and that inhibition of the BH4 pathway could provide a new strategy for the treatment of acute PSP.

Purpose Acrylamide is known to induce disorders in the central nervous system in humans and experimental animals. The present study investigated effects of exposure to acrylamide on adult neurogenesis, noradrenergic axons and the level of norepinephrine in the brain of male rats. Method Four groups of 12 male Wistar rats each were exposed to acrylamide at 0, 0.2, 2 and 20 mg/kg body weight by gavage for 5 weeks. Six rats of each groups were injected with 5-bromo-2′-deoxy-uridine (BrdU) after five-week exposure to acrylamide to examine proliferative cells in the dentate gyrus using immunostaining. Density of noradrenergic and serotonergic axons in the prefrontal cortex, hippocampus and cortex behind the bregma was quantified. Remaining 6 rats were decapitated after the last exposure and brains were dissected out to measure monoamine level in the hippocampus and prefrontal cortex using high performance liquid chromatography. Result Exposure to acrylamide dose-dependently decreased the density of noradrenergic axons in the prefrontal cortex with a significant change at 20 mg/kg. Norepinephrine level decreased in the hippocampus at 20 mg/kg. Exposure to acrylamide at 20 mg/kg or less did not change the number of BrdU positive cells, but the result should be considered preliminary. Conclusion The results show that oral exposure to acrylamide induces decrease in noradrenergic axons and norepinephrine level in the brain of rats. Given the similar effects are observed in 1-bromopropane-exposed rats, there may be the common mechanism in the toxicity of soft electrophiles to the central nervous system.

Parkinson's disease (PD) is the second common neurodegenerative disorder. Deficit of the nigro-striatal dopaminergic neurons causes the motor symptoms of PD. While the oxidative stress is thought to be deeply involved in the etiology of PD, molecular targets for the oxidative insults has not been fully elucidated. 6R-5,6,7,8-Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase (TH), the rate-limiting enzyme for production of dopamine, and easily oxidized to its dihydro-form. In this study, we examined the alteration in the metabolism of BH4 caused by a parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP reduced the dopamine content and the in vivo activity of TH in the striatum prior to degeneration of the dopaminergic neurons. We found that administration of BH4 could restore the dopamine content and in vivo TH activity in the striatum of MPTP-treated mice. Unexpectedly, when BH4 was administered with MPTP, BH4 contents in the brain were far higher than those injected without MPTP even at 23 h after the last injection. Because MPTP has been shown to increase ROS production in the dopaminergic neurons, we assumed that the increased ROS oxidizes BH4 into its dihydro-form, excreted from the dopaminergic neurons, taken-up by the neighboring cells, reduced back to BH4, and then accumulated in the brain. We also investigated the action of MPTP in mice lacking quinonoid-dihydropteridine reductase (Qdpr), an enzyme catalyzing regeneration of BH4 from quinonoid dihydrobiopterin. The dopamine depletion induced by MPTP was severer in Qdpr-deficient mice than in wild-type mice. The present data suggest that perturbation of the BH4 metabolism would be the cause of early and persistent dopamine depletion in the striatum.

6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI–TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2. S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.

Epinephrine is a hormone secreted primarily by medullary cells of the adrenal glands which regulates permeability of blood–brain barrier (BBB). Recent studies showed signaling by epinephrine/epinephrine receptor in T cells is involved in autoimmune diseases. Nevertheless, the production of epinephrine by T cells and its pathogenic function in T cells are not well investigated. Our results show that phenylethanol N-methyltransferase (PNMT), a rate-limiting enzyme of epinephrine synthesis, is specifically expressed in vitro in differentiated TH17 cells and in tissue-resident TH17 cells. Indeed, expression levels of enzymes involved in epinephrine production are higher in TH17 cells from animals after EAE induction. The induction of PNMT was not observed in other effector T cell subsets or regulatory T cells. Epinephrine producing TH17 cells exhibit co-expression of GM-CSF, suggesting they are pathogenic TH17 cells. To delineate the function of epinephrine-production in TH17 cells, we generated a TH17-specific knockout of tyrosine hydroxylase (Th) by breeding a Th-flox and a ROR-gt-CRE mouse (Th-CKO). Th-CKO mice are developmentally normal with an equivalent T lymphocyte number in peripheral lymphoid organs. Th-CKO mice also show an equivalent number of TH17 cells in vivo and following in vitro differentiation. To test whether epinephrine-producing TH17 cells are key for breaching the BBB, migration of T cells through mouse brain endothelial cells was investigated in vitro. Both epi+ wild-type and epi- TH17 cells migrate through an endothelial cell barrier. Mice were immunized with MOG peptide to induce experimental autoimmune encephalitis (EAE) and disease progression was monitored. Although there is a reduced infiltration of CD4+ T cells in Th-CKO mice, no difference in clinical score was observed between Th-CKO and wild-type control mice. Increased neutrophils were observed in the central nervous system of Th-CKO mice, suggesting an alternative pathway to EAE progression in the absence of TH17 derived epinephrine.

Background Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. Objectives The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. Methods To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FceRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. Results Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FceRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FceRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FceRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FceRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. Conclusions Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

OBJECTIVE The hemodialysis (HD) diet, which is a high-calorie and high-fat regimen, may inadvertently lead to an inadequate dietary intake, resulting in undernutrition among elderly HD patients. Therefore, an attempt was made to improve the dietary intake by implementing a modified diet regimen in eligible elderly HD patients. SUBJECTS Elderly HD patients who had ingested < 50% of the meals provided and were diagnosed with undernutrition among all elderly patients institutionalized at the special elderly nursing home annexed to Nagasaki Kidney Hospital between June and November 2012. RESULTS Of the elderly HD patients in the nursing home (n = 27), the study included a total of 7 consecutive patients (male/female, 1/6; mean age, 84.1±6.4 years old; duration of HD, 4.3±3.8 years; geriatric nutritional index [GNRI], 83.5±8.3; normalized protein catabolic ratio [nPCR], 0.78±0.14). The modified diet regimen, which involved reducing food portion sizes and incorporating a liquid diet, led to a significant increase in their dietary intake from 48.1% at baseline to 97.1% of the meals provided 3 months after the start of the modified HD diet regimen. Their GNRI also significantly increased from 83.5±8.3 to 86.1±10.2, and their serum albumin levels significantly increased from 3.2±0.2 g/dL to 3.4±0.4 g/dL, suggesting improvements in their nutritional status. CONCLUSIONS The attempted dietary approach for elderly HD patients was shown to potentially increase their dietary intake and improve their nutritional status without affecting the efficiency of HD being implemented.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase and tryptophan hydroxylase, which are essential enzymes for the biosynthesis of dopamine, norepinephrine, and serotonin. It has been known that BH4 is a labile molecule and easily oxidized. As ascorbic acid (AsA) is an antioxidant that is rich in the brain, alteration in the AsA concentration in the brain may affect the proper metabolism of BH4. Here, we examined the effect of AsA deficiency on the concentration of BH4 using ODS rats, which are defective in the gene for AsA synthesis. Intake of an AsA-deficient diet for 2 weeks in ODS rats resulted in great reductions in the AsA levels up to 7 % in the liver and up to 55 % in the brain compared to animals fed a basal diet containing an adequate amount of AsA. The BH4 concentrations in ODS rats fed an AsA-free diet were decreased to 71 % in the liver and 88 % in the brain of those fed a basal diet. We found that the levels of dopamine, norepinephrine, and serotonin were also decreased compared with the ODS rats fed a basal diet. Our data showed that AsA deficiency can affect the BH4 concentrations in the liver and brain, resulting in decreases in the monoamine levels in the brain. These results suggest the importance of AsA in the pathophysiology of neuropsychiatric and cardiovascular disorders through alteration in the BH4 metabolism.

The intra-renal dopamine (DA) system is highly expressed in the proximal tubule and contributes to Na+ and blood pressure homeostasis, as well as to the development of nephropathy. In the kidney, the enzyme DOPA Decarboxylase (DDC) originating from the circulation. We used a twin/family study design, followed by polymorphism association analysis at DDC locus to elucidate heritable influences on renal DA production. Dense single nucleotide polymorphism (SNP) genotyping across the DDC locus on chromosome 7p12 was analyzed by re-sequencing guided by trait-associated genetic markers to discover the responsible genetic variation. We also characterized kinetics of the expressed DDC mutant enzyme. Systematic polymorphism screening across the 15-Exon DDC locus revealed a single coding variant in Exon-14 that was associated with DA excretion and multiple other renal traits indicating pleiotropy. When expressed and characterized in eukaryotic cells, the 462Gln variant displayed lower Vmax (maximal rate of product formation by an enzyme) (21.3 versus 44.9 nmol/min/mg) and lower Km (substrate concentration at which half-maximal product formation is achieved by an enzyme.)(36.2 versus 46.8 μM) than the wild-type (Arg462) allele. The highly heritable DA excretion trait is substantially influenced by a previously uncharacterized common coding variant (Arg462Gln) at the DDC gene that affects multiple renal tubular and glomerular traits, and predicts accelerated functional decline in chronic kidney disease.

Some bipolar cells in the human retina are known to express voltage-gated Na+ channels. However, it is unclear which types of channels are expressed, and whether Na+ channel expression is limited to specific types of bipolar cells. In the present study, we examined the types of voltage-gated Na+ channels expressed in human bipolar cells and the morphology of bipolar cells with voltage-gated Na+ currents. To investigate the expression of voltage-gated Na+ channels in human bipolar cells, we examined whether Na+ channel transcripts could be detected in single bipolar cells using the reverse transcription polymerase chain reaction (RT-PCR) technique. The voltage-gated Na+ current was recorded from isolated bipolar cells using the patch-clamp recording technique. Types of bipolar cells that have the Na+ currents were investigated by analyzing their morphology after staining with Lucifer yellow. Using RT-PCR, the SCN2A Na+ channel was detected in 5 of 6 isolated bipolar cells. This suggests that a subset of human bipolar cells expresses the SCN2A Na+ channel. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current in cone bipolar cells with axon terminal boutons that stratified at the ON layer, which includes the stratum 3, 4, and 5 of the inner plexiform layer (IPL, n = 2/11 cells). The fast transient inward current of isolated bipolar cells was blocked by 1 μM of tetrodotoxin (TTX), a voltage-gated Na+ channel blocker. No fast transient inward current was recorded with axon terminals that stratify at the OFF layer, which includes stratum 1 and 2 of the IPL (n = 4). Thus, a subset of ON cone bipolar cells at least expresses the putative voltage-gated Na+ channel SCN2A in the human retina. The Na+ channels in the bipolar cells may serve to amplify the release of neurotransmitter, glutamate, when membrane potential is rapidly depolarized and thereby selectively accelerating light responses.

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.

Parkinson's disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD; (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene; and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. Proper function of DCs is crucial to elicit an effective immune response against pathogens and to induce antitumor immunity. Different members of the nuclear receptor (NR) family of transcription factors have been reported to affect proper function of immune cells. Nur77 is a member of the NR4A subfamily of orphan NRs that is expressed and has a function within the immune system. We now show that Nur77 is expressed in different murine DCs subsets in vitro and ex vivo, in human monocyte-derived DCs (moDCs) and in freshly isolated human BDCA1+ DCs, but its expression is dispensable for DC development in the spleen and lymph nodes. We show, by siRNA-mediated knockdown of Nur77 in human moDCs and by using Nur77−/− murine DCs, that Nur77-deficient DCs have enhanced inflammatory responses leading to increased T cell proliferation. Treatment of human moDCs with 6-mercaptopurine, an activator of Nur77, leads to diminished DC activation resulting in an impaired capacity to induce IFNγ production by allogeneic T cells. Altogether, our data show a yet unexplored role for Nur77 in modifying the activation status of murine and human DCs. Ultimately, targeting Nur77 may prove to be efficacious in boosting or diminishing the activation status of DCs and may lead to the development of improved DC-based immunotherapies in, respectively, cancer treatment or treatment of autoimmune diseases.

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.

(6 R )‐l‐ erythro ‐5,6,7,8‐Tetrahydrobiopterin (BH4) is an essential cofactor for monoamine and nitric oxide (NO) production. Sepiapterin reductase (SPR) catalyzes the final step in BH4 biosynthesis. We analyzed the cardiovascular function of adult Spr gene‐disrupted ( Spr −/− ) mice for the first time. After weaning, Spr −/− mice suffered from hypertension with fluctuation and bradycardia, while the monoamine contents in these mice were less than 10% of those in the wild‐type mice as a result of BH4 depletion. Heart rate variability analysis indicated the sympathetic dominant state in Spr −/− mice. The endothelium‐dependent vascular relaxation in response to acetylcholine was significantly impaired in Spr −/− mice after sexual maturation (above 4 months old). Protein amounts of α 1 adrenergic receptor and eNOS in the aorta were not altered. Spr −/− mice exhibited hypoglycemia and elevation of plasma renin activity. Our results suggest that the hypertension with fluctuation and bradycardia of Spr −/− mice would be caused by an imbalance of sympathetic and parasympathetic input and impaired nitric oxide production in endothelial cells. We suggest an important role of BH4 and SPR in age‐related hypertension and a possible relationship with the cardiovascular instabilities in autonomic diseases, including Parkinson9s disease and spinal cord injury.

Microglia cells fulfill key homeostatic functions and essentially contribute to host defense within the CNS. Altered activation of microglia, in turn, has been implicated in neuroinflammatory and neurodegenerative diseases. In this study, we identify the nuclear receptor (NR) Nr4a1 as key rheostat controlling the activation threshold and polarization of microglia. In steady-state microglia, ubiquitous neuronal-derived stress signals such as ATP induced expression of this NR, which contributed to the maintenance of a resting and noninflammatory microglia phenotype. Global and microglia-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia and resulted in increased cytokine and NO production as well as in an accelerated and exacerbated form of experimental autoimmune encephalomyelitis. Ligand-induced activation of Nr4a1 accordingly ameliorated the course of this disease. Our current data thus identify Nr4a1 as regulator of microglia activation and potentially new target for the treatment of inflammatory CNS diseases such as multiple sclerosis.

Aims Progressive cognitive decline has been an important issue in the treatment and care of patients with schizophrenia. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of catecholamine, including dopamine and noradrenaline. In this report, we examined a possible association of a genetic variant in the TH promoter region. Methods Association of a genetic variant in the TH promoter region, C-824T (rs10770141), with intellectual ability in 132 patients with schizophrenia and 282 healthy subjects was examined. The transcriptional activity of the plasmids harboring the TH promoter region with either C or T nucleotide at −824 was assayed using a luciferase gene as a reporter. Results We found significant effects of the genotype on the full-scale IQ, verbal IQ, and performance IQ, in patients with schizophrenia. IQ was lower in individuals with the C/C genotype than those with T carriers. The plasmid with the T allele at −824 showed higher transcriptional activity than that with the C allele in a transient transfection experiment using a luciferase gene as a reporter, implying that the T carriers may have higher TH activities and retain higher levels of catecholamines in the brain. Conclusions The present data suggest that the biosynthesis of catecholamine by the action of TH should be deeply involved in decreased intellectual ability in patients with schizophrenia. This is the first report, as far as we know, showing a correlation between TH expression and IQ in humans.

Guanosine triphosphate cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. As monoamine neurotransmitters are synthesized by the reactions catalyzed by tyrosine hydroxylase and tryptophan hydroxylase, alterations in the content of tetrahydrobiopterin affect the monoamine levels in the brain. Here, we examined the possible association of a functional single-nucleotide polymorphism (SNP) of the GCH1 gene, rs841 (C+243T), with attentional function as assessed by the Continuous Performance Test-Identical Pairs version (CPT-IP) in healthy individuals. We found that homozygous T/T genotype carriers of rs841 scored lower performance on the CPT-IP test. Our data suggest that alterations in GCH1 activity affect attentional function, especially sustained attention and vigilance.

Nurr1 is an orphan nuclear transcription factor expressed in the brain. While Nurr1 is assumed to be an immediate early gene, it is not fully understood how Nurr1 expression is regulated in an activity-dependent manner in the central nervous system. Here, we investigated the molecular mechanisms underlying the regulation of Nurr1 expression in cultured hippocampal and cortical neurons. We found that upregulation of neural activity by high KCl and bicuculline enhances Nurr1 levels, while blockade of its activity by tetrodotoxin reduces Nurr1 levels. The induction of Nurr1 expression was mediated by voltage-dependent calcium channels (VDCCs), as shown by cadmium and VDCC-specific inhibitors. Furthermore, calcineurin, but not calcium/calmodulin-dependent protein kinase (CaMK) was critical for the induction. Thus, Nurr1 expression is regulated by VDCC and calcineurin in a cell-autonomous, neural activity-dependent manner.

Dopamine (DA) is a neurotransmitter in the brain, playing a central role in several disease conditions, including tetrahydrobiopterin (BH4) metabolism disorders and Parkinson's disease (PD). BH4 metabolism disorders present a variety of clinical manifestations including motor disturbance via altered DA metabolism, since BH4 is a cofactor for tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis. Genetically, BH4 metabolism disorders are, in an autosomal recessive pattern, caused by a variant in genes encoding enzymes for BH4 synthesis or recycling, including 6-pyruvoyltetrahydropterin synthase (PTPS) or dihydropteridine reductase (DHPR), respectively. Although BH4 metabolism disorders and its metabolisms have been studied, it is unclear how gene variants cause aberrant DA synthesis in patient neurons. Here, we generated induced pluripotent stem cells (iPSCs) from BH4 metabolism disorder patients with PTPS or DHPR variants, corrected the gene variant in the iPSCs using the CRISPR/Cas9 system, and differentiated the BH4 metabolism disorder patient- and isogenic control iPSCs into midbrain DA neurons. We found that by the gene correction, the BH4 amount, TH protein level and extracellular DA level were restored in DA neuronal culture using PTPS deficiency iPSCs. Furthermore, the pharmacological correction by BH4 precursor sepiapterin treatment also improved the phenotypes of PTPS deficiency. These results suggest that patient iPSCs with BH4 metabolism disorders provide an opportunity for screening substances for treating aberrant DA synthesis-related disorders., 患者由来iPS細胞とゲノム編集技術を用いて, BH4代謝病のドパミン合成異常の疾患モデル系構築に成功 : iPS細胞利用による代謝改善生理活性物質の同定と疾患再定義. 京都大学プレスリリース. 2016-10-28.

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.

In the autumn two patients with general malaise were consecutively admitted to the department of internal medicine of our hospital in the northern region of Nagasaki Prefecture. Since both patients were engaged in rice farm work and showed conjunctival suffusion and pain of the gastrocnemius muscle, leptospirosis was suspected. The first patient required temporary hemodialysis for renal dysfunction, whereas liver dysfunction developed in the second patient. The disease was remitted by antimicrobial agents. A diagnosis of leptospiro sis was made serologically in both patients. Leptospirosis should be considered as a differential diagnosis when a patient engaged in farm work in the autumn has typical symptoms, and an early initiation of treatment after onset is important., Internal Medicine, 49(12), pp.1143-1147; 2010

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson's disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin-proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-(40)Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin-proteasome system in PC12D cells.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.

Background Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing α-satellite DNA with CENP-B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthase. Results: We constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co-transfection with the α-satellite DNA-containing BAC to a human fibroblast cell line. Two cell lines carrying a HAC with GCH1 genes were obtained. Both HACs were composed of multiple copies of precursor BACs and were maintained stably in human and mouse cell lines. The GCH1 activities of the HAC-carrying human fibroblast cell lines were elevated but still highly sensitive to IFN-γ induction, mimicking the response of the gene expression from the authentic chromosomal genes. Conclusion: These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.

Blood pressure is regulated by extrinsic factors including noradrenaline, the sympathetic neurotransmitter that controls cardiovascular functions through adrenergic receptors. However, the fine-tuning system of noradrenaline signaling is relatively unknown. We here show that l-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of catecholamines, sensitizes the vascular adrenergic receptor alpha1 (ADRA1) through activation of L-DOPA receptor GPR143. In WT mice, intravenous infusion of the ADRA1 agonist phenylephrine induced a transient elevation of blood pressure. This response was attenuated in Gpr143 gene-deficient (Gpr143-/y) mice. Specific knockout of Gpr143 in vascular smooth muscle cells (VSMCs) also showed a similar phenotype, indicating that L-DOPA directly modulates ADRA1 signaling in the VSMCs. L-DOPA at nanomolar concentrations alone produced no effect on the VSMCs, but it enhanced phenylephrine-induced vasoconstriction and intracellular Ca2+ responses. Phenylephrine also augmented the phosphorylation of extracellular signal-regulated kinases in cultured VSMCs from WT but not Gpr143-/y mice. In WT mice, blood pressure increased during the transition from light-rest to dark-active phases. This elevation was not observed in Gpr143-/y mice. Taken together, our findings provide evidence for L-DOPA/GPR143 signaling that exerts precursor control of sympathetic neurotransmission through sensitizing vascular ADRA1.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

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.

β Both neopterin and biopterin concentrations in cerebrospinal fluid from patients with Parkinson's disease, in which the nigrostriatal dopamine neurons degenerate, were lower than those from age-matched older control subjects. However, the decrease in biopterin was more marked than that in neopterin, resulting in the increase in the neopterin/ biopterin ratio in Parkinson's disease. These results suggests that neopterin in cerebrospinal fluid in Parkinson's disease may partly be derived from immunoactivated glial cells, besides catecholamine or serotonin n eurons including nigrostriatal dopamine neurons. In accordance to this hypothesis, cytokines (TNF-α, IL-1, IL-2 , IL-6, EGF, TGF-α, TGF-β1) were found to be increased in the striatum and/or in cerebrospinal fluid. The increment of cytokines in the brain in Parkinson's disease may be related to the mechanism of neurodegeneration of dopaminergic neurons in Parkinson's disease . In contrast to Parkinson's disease, in hereditary progressive dystonia/ dopa-responsive dystonia, which is a dopamine deficiency caused by mutations in GTP cyclohydrolase I without neuronal cell death (Segawa's disease), neopterin and biopterin in cerebrospinal fluid decreases in parallel owing to the decreased activity in GTP cyclohydrolase I .

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels. 2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4. 3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)]. 4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.

Objectives. AicardiGoutieres syndrome (AGS) is a rare, genetically determined, early onset progressive encephalopathy associated with autoimmune manifestations. AGS is usually inherited in an autosomal recessive manner. The disease is rare, therefore the clinical manifestations and genotypephenotype correlations, particularly with regard to autoimmune diseases, are still unclear. Here we performed a nationwide survey of AGS patients in Japan and analysed the genetic and clinical data. Methods. Patients were recruited via questionnaires sent to paediatric or adult neurologists in Japanese hospitals and institutions. Genetic analysis was performed and clinical data were collected. Results. Fourteen AGS patients were identified from 13 families; 10 harboured genetic mutations. Three patients harboured dominant-type TREX1 mutations. These included two de novo cases: one caused by a novel heterozygous p.His195Tyr mutation and the other by a novel somatic mosaicism resulting in a p.Asp200Asn mutation. Chilblain lesions were observed in all patients harbouring dominant-type TREX1 mutations. All three patients harbouring SAMHD1 mutations were diagnosed with autoimmune diseases, two with SLE and one with SS. The latter is the first reported case. Conclusion. This study is the first to report a nationwide AGS survey, which identified more patients with sporadic AGS carrying de novo dominant-type TREX1 mutations than expected. There was a strong association between the dominant-type TREX1 mutations and chilblain lesions, and between SAMHD1 mutations and autoimmunity. These findings suggest that rheumatologists should pay attention to possible sporadic AGS cases presenting with neurological disorders and autoimmune manifestations.

Aromatic l-amino acid decarboxylase (AADC) is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the syntheses of dopamine and serotonin. Defects in the AADC gene result in neurotransmitter deficiencies. Patients with AADC deficiency have severe motor and autonomic dysfunctions. A mouse model of AADC deficiency was recently established. These mice grow poorly and move awkwardly during infancy. They also show high anxiety when they grow up. Because drug therapy provides little or no benefit for many patients with AADC deficiency, a gene therapy has been attempted. The gene therapy employed an adeno-associated virus viral vector that can express the human AADC protein. The vector was injected to the brain of several children with AADC deficiency. The therapy was well tolerated, and all treated patients showed improvement. In the future, the mouse model will also help the development of treatments for AADC deficiency.

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning ∼30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5′-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

The “nutcracker” phenomenon results from compression of the left renal vein between the superior mesenteric artery and the aorta. The main features of this phenomenon are non-glomerular haematuria on urinalysis and stenosis of the left renal vein with dilatation of the vein distal to the stenosis. The characteristics of two patients with the “nutcracker” phenomenon complicated by immunoglobulin A nephropathy were compared with those of 10 patients showing only the “nutcracker” phenomenon. Patients with the “nutcracker” phenomenon complicated by immunoglobulin A nephropathy showed aggravation of haematuria after upper respiratory infections, urinary red cell morphology indicating haematuria of glomerular origin, elevation of serum IgA, persistence of proteinuria, and granular casts in the urine. The coexistence of immunoglobulin A nephropathy was suspected when these characteristics were observed in patients with the “nutcracker” phenomenon. In such cases, a renal biopsy is needed for a final diagnosis.

Human tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.