Hph-1 Mouse Research Articles

1710-P: Maternal Supplementation of Tetrahydrobiopterin Regulates Differentiation of Fetal Brown Adipose Tissue and Contributes to Offspring Metabolic Health

Brown adipose tissue (BAT) is a key organ that produces heat and dissipates energy, and may be clinically relevant to the treatment of obesity and diabetes. Tetrahydrobiopterin (BH4) is an essential co-factor of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis including that of norepinephrine (NA) and nitric oxide (NO) synthase. Although both NA and NO are well-known factors in BAT differentiation, the role of BH4 in the differentiation of BAT is poorly understood. We investigated the role of BH4 in differentiation of BAT using a mouse model of BH4 deficiency, the Hph-1 mouse. Hph-1 neonatal mice exhibit dysplasia of BAT as well as attenuated thermogenesis-related gene expressions (UCP1, Dio2, Pgc1a) compared with control mice. As differentiation capacity is considered to be at maximum during late gestation, we administered BH4 to fetal Hph-1 mice via placental transfer using intraperitoneal injection to pregnant Hph-1 mice for 6 days just before birth. Maternal BH4 supplementation ameliorated dysplasia of neonatal BAT and restored thermogenesis-related genes expression. Intriguingly, offspring of the maternal BH4-supplementation group showed less weight gain, ameliorated glucose intolerance, and improved cold tolerance after growth under high fat chow diet. We then evaluated the direct effect of BH4 on brown adipocyte differentiation using brown pre-adipocytes isolated from control and Hph-1 mice. Compared with control mice, brown pre-adipocytes from Hph-1 mice showed reduced differentiation capacity, while BH4 supplementation in brown pre-adipocytes from Hph-1 mice ameliorated differentiation capacity nitric oxide-dependently. Taking these findings together, BH4 plays an integral role in the differentiation of BAT, especially in the fetal period, and may represent a novel target for prevention of metabolic disorders such as obesity and diabetes. Disclosure H. Minamino: None. Y. Fujita: None. Y. Oguri: None. T. Goto: None. A. Ohashi: None. F. Furuya: None. N. Isomura: None. T. Kohei: None. Y. Li: None. S. Kawarasaki: None. T. Kawada: None. H. Hasegawa: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Drawbridge Health, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Life Scan Japan, Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Novo Nordisk Pharma Ltd.,, Sanofi K.K., Sanwa Kagaku Kenkyusho, Terumo Medical Corporation. Speaker’s Bureau; Self; Astellas Pharma Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Intestinal microbiota as a tetrahydrobiopterin exogenous source in hph-1 mice

Tetrahydrobiopterin (BH4) is a cofactor of a number of regulatory enzymes. Although there are no known BH4 exogenous sources, the tissue content of this biopterin increases with age in GTP cyclohydrolase 1-deficient hyperphenylalaninemia-1 (hph-1) mice. Since certain bacteria are known to generate BH4, we hypothesize that generation of this biopterin by the intestinal microbiota contributes to its tissue increase in hph-1 adult mice. The goal of this study was to comparatively evaluate hph-1 mice and wild-type C57Bl/6 controls for the presence of intestinal BH4-producing bacteria. Newborn and adult mice fecal material was screened for 6-pyruvoyltetrahydropterin synthase (PTPS-2) an enzyme only present in BH4-generating bacteria. Adult, but not newborn, wild-type control and hph-1 mouse fecal material contained PTPS-2 mRNA indicative of the presence of BH4-generating bacteria. Utilizing chemostat-cultured human fecal bacteria, we identified the PTPS-2-producing bacteria as belonging to the Actinobacteria phylum. We further confirmed that at least two PTPS-2-producing species, Adlercreutzia equolifaciens and Microbacterium schleiferi, generate BH4 and are present in hph-1 fecal material. In conclusion, intestinal Actinobacteria generate BH4. This finding has important translational significance, since manipulation of the intestinal flora in individuals with congenital biopterin deficiency may allow for an increase in total body BH4 content.

Erythropoietin prevents endothelial dysfunction in GTP-cyclohydrolase I-deficient hph1 mice.

: In this study, we used the mutant hph1 mouse model, which has deficiency in GTP-cyclohydrolase I (GTPCH I) activity, to test the hypothesis that erythropoietin (EPO) protects aortic wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and tetrahydrobiopterin (BH4) levels were reduced in hph1 mice, whereas 7,8-dihydrobiopterin (7,8-BH2) levels were significantly increased. Furthermore, BH4 deficiency caused increased production of superoxide anion and hydrogen peroxide in the aorta thus resulting in impairment of endothelium-dependent relaxations to acetylcholine. Treatment of hph1 mice with recombinant human EPO (1000 U/kg, subcutaneously for 3 days) significantly decreased superoxide anion production by eNOS and improved BH4 to 7,8-BH2 ratio in aortas. EPO also significantly decreased production of hydrogen peroxide and improved endothelium-dependent relaxations in aortas of hph1 mice. In addition, EPO treatment increased protein expressions of copper-/zinc-superoxide dismutase, manganese-superoxide dismutase, and catalase in the aorta of hph1 mice. Our findings demonstrate that treatment with EPO prevented oxidative stress and endothelial dysfunction caused by eNOS uncoupling. Increased vascular expressions of antioxidants seem to be an important molecular mechanism underlying vascular protection by EPO during chronic BH4 deficiency.

Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin

Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depression-like behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depression-like behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression.

Impaired Behavioural Pain Responses in hph-1 Mice with Inherited Deficiency in GTP Cyclohydrolase 1 in Models of Inflammatory Pain

BackgroundGTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a “pain-protective” (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain.ResultsIn the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms.ConclusionsIn this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to study the consequence of congenital deficiency of GCH1 in painful conditions.

Regulation of β-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin.

Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency. In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. β1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation. Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced β-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.

Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

Pulmonary hypertension in the newborn GTP cyclohydrolase I-deficient mouse

Tetrahydrobiopterin (BH4) is a regulator of endothelial nitric oxide synthase (eNOS) activity. Deficient levels result in eNOS uncoupling, with a shift from nitric oxide to superoxide generation. The hph-1 mutant mouse has deficient GTP cyclohydrolase I (GTPCH1) activity, resulting in low BH4 tissue content. The adult hph-1 mouse has pulmonary hypertension, but whether such condition is present from birth is not known. Thus, we evaluated newborn animals’ pulmonary arterial medial thickness, biopterin content (BH4+BH2), H2O2 and eNOS, right ventricle-to-left ventricle+septum (RV/LV+septum) ratio, near-resistance pulmonary artery agonist-induced force, and endothelium-dependent and -independent relaxation. The lung biopterin content was inversely related to age for both types, but significantly lower in hph-1 mice, compared to wild-type animals. As judged by the RV/LV+septum ratio, newborn hph-1 mice have pulmonary hypertension and, after a 2-week 13% oxygen exposure, the ratios were similar in both types. The pulmonary arterial agonist-induced force was reduced (P<0.01) in hph-1 animals and no type-dependent difference in endothelium-dependent or -independent vasorelaxation was observed. Compared to wild-type mice, the lung H2O2 content was increased, whereas the eNOS expression was decreased (P<0.01) in hph-1 animals. The pulmonary arterial medial thickness, a surrogate marker of vascular remodeling, was increased (P<0.01) in hph-1 compared to wild-type mice. In conclusion, our data suggest that pulmonary hypertension is present from birth in the GTPCH1-deficient mice, not as a result of impaired vasodilation, but secondary to vascular remodeling.

Phosphorylation of endothelial nitric oxide synthase is increased in mouse model of GTP‐cyclohydrolase I deficiency

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS). Suboptimal level of BH4 reduces formation of nitric oxide leading to uncoupling of eNOS. However, the exact molecular mechanisms underlying the effects of eNOS uncoupling on vascular endothelium in‐vivo are not completely understood. In the present study, we used the hph‐1 mouse, which displays GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that BH4 levels were significantly reduced in the aortas of hph‐1 mice (3.9±0.5 pmol/mg; P&lt;0.05 vs. wild‐type mice: 5.9±0.6 pmol/mg), while oxidative products of BH4, 7,8‐dihydrobiopterin (7,8‐BH2), were unchanged. Consequently, the BH4 to 7,8‐BH2 ratio was significantly reduced in the aorta of hph‐1 mice (P&lt;0.05). Expression of eNOS protein was not different between wild‐type and hph‐1 mice. Unexpectedly, protein expressions of phosphorylated Ser1177‐eNOS and Ser473‐Akt1 were significantly increased in hph‐1 mice aortas (P&lt;0.05). Consistent with uncoupling of eNOS, L‐NAME sensitive superoxide anion production and 3‐nitrotyrosine formation were significantly increased in aortas of hph‐1 mice (P&lt;0.05). Protein expressions of CuZnSOD, MnSOD, and EC‐SOD were unaltered in the aortas of hph‐1 mice as compared with wild‐type mice. Our results suggest that uncoupling eNOS increases phosphorylation of Akt1 and eNOS. This effect is most likely mediated by increased formation of eNOS‐derived reactive oxygen species including superoxide anion and hydrogen peroxide.

In vivo regulation of phenylalanine hydroxylase in the genetic mutant hph-1 mouse model

The hph-1 mouse has low liver activity of GTP cyclohydrolase 1, the rate limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH 4). BH 4 is the cofactor for phenylalanine hydroxylase (PAH) and in the early stages of life the hph-1 mouse is hyperphenylalaninemic. At approximately 15 days after birth the blood phenylalanine levels normalize. During this period the animals provide an in vivo model which can be used to study the regulatory effects of phenylalanine on PAH, and for related pediatric metabolic disease in humans; from birth to youth. We therefore, examined; liver PAH activity using BH 4 and 6-methyltetrahydropterin (6MPH 4) as cofactor; PAH total enzyme concentration by Western blotting using the PH8 antibody, and PAH state of phosphorylation using the PH7 antibody from 4 to 18 days after birth. The findings were compared to the wild type animals that are not hyperphenylalaninemic during this period. PAH (6MPH 4) activity and total protein (PH8 antibody) rose steadily in the hph-1 mice. In control mice, both activity and total protein fluctuated. The degree of phosphorylation of PAH in the mutants and the state of activation (as measured by the 6MPH 4/BH 4 activity ratio) increased as phenylalanine levels rose, and decreased when they fell. Similar patterns were not seen in the control animals. These studies provide in vivo evidence that phenylalanine concentration regulates the activity of PAH in the hph-1 mouse and that this acts via a mechanism that includes phosphorylation of the PAH molecule. The kinetic values ( K m and V max) for mouse PAH are also reported.

GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

Tetrahydrobiopterin availability, nitric oxide metabolism and glutathione status in the hph‐1 mouse; implications for the pathogenesis and treatment of tetrahydrobiopterin deficiency states

Tetrahydrobiopterin (BH4) is an essential cofactor for all isoforms of nitric oxide synthase. While it is well established that BH4 deficiency states are associated with impairment of dopamine, serotonin and phenylalanine metabolism, less is known with regard to the effects of deficiency of the cofactor upon nitric oxide (NO) metabolism. In this study, we have evaluated the effects of partial BH4 deficiency upon (a) tissue availability of the antioxidant glutathione, (b) basal NO production and (c) NO generation following exposure to lipopolysaccharide (LPS), which is known to increase expression of the inducible form of nitric oxide synthase. Using the hph-1 mouse, which displays a partial BH4 deficiency owing to impaired activity of GTP cyclohydrolase, we report decreased levels of glutathione in brain and kidney and evidence for decreased basal generation of nitric oxide in the periphery (as judged by the plasma nitrate plus nitrite concentration). Following LPS administration, peripheral NO generation increases. However, the concentration of plasma nitrate plus nitrite achieved was significantly decreased in the hph-1 mouse. Furthermore, LPS administration caused loss of glutathione in both wild-type and hph-1 liver and kidney. It is concluded that cofactor replacement, sufficient to fully correct a cellular BH4 deficiency, may be of benefit to patients with inborn errors of BH4 metabolism.

Pivotal Role for Endothelial Tetrahydrobiopterin in Pulmonary Hypertension

Pulmonary hypertension is a fatal disease characterized by vasoconstriction and vascular remodeling. Loss of endothelial nitric oxide bioavailability is implicated in pulmonary hypertension pathogenesis. Recent evidence suggests that the cofactor tetrahydrobiopterin (BH4) is an important regulator of nitric oxide synthase enzymatic function. In the hph-1 mouse with deficient BH4 biosynthesis, BH4 deficiency caused pulmonary hypertension, even in normoxic conditions, and greatly increased susceptibility to hypoxia-induced pulmonary hypertension. In contrast, augmented BH4 synthesis in the endothelium, by targeted transgenic overexpression of GTP-cyclohydrolase I (GCH), prevented hypoxia-induced pulmonary hypertension. Furthermore, specific augmentation of endothelial BH4 in hph-1 mice by crossing with GCH transgenic mice rescued pulmonary hypertension induced by systemic BH4 deficiency. Lung BH4 availability controlled pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling in a dose-dependent manner in both normoxia and hypoxia. Furthermore, BH4 availability had striking effects on the immediate vasoconstriction response to acute hypoxia. These effects of BH4 were mediated through the regulation of nitric oxide compared with superoxide synthesis by endothelial nitric oxide synthase. Endothelial BH4 availability is essential for maintaining pulmonary vascular homeostasis, is a critical mediator in the pathogenesis of pulmonary hypertension, and is a novel therapeutic target.

Pulmonary Hypertension in a GTP-Cyclohydrolase 1–Deficient Mouse

GTP-cyclohydrolase 1 (GTP-CH1) catalyzes the first step for the de novo production of tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthase (NOS). The hyperphenylalaninemic mutant mouse (hph-1) displays a 90% reduction in GTP-CH1 activity. Reduced BH4 decreases NOS activity and may lead to endothelial dysfunction, and there is increasing evidence that a dysfunction of the NOS pathway may be implicated in pulmonary hypertension. The aim of the study was to investigate whether reduced BH4 in the hph-1 mouse results in a pulmonary hypertensive phenotype. Morphological characterization of the heart, lung, and kidney and measurements of systemic and right ventricular blood pressures were performed in both hph-1 and wild-type mice. BH4 and NO(x) levels were also measured. Hph-1 mice had significantly lower NO(x) and BH4 levels, consistent with previous findings. Both morphological and in vivo data were indicative of a pulmonary but not systemic hypertensive phenotype. We observed increased right ventricle-left ventricle plus septum ratios attributable only to an increase in right ventricular mass, increased smooth muscle medial area in pulmonary resistance vessels, and significantly higher right ventricular pressures in vivo. There were no significant differences between left ventricular masses and systemic pressures, and there was no observed evidence of systemic hypertension in kidney sections between wild-type and hph-1. This study demonstrates that mice deficient in GTP-CH1/BH4 display a pulmonary hypertensive but not systemic hypertensive phenotype.

Alterations in expression of dopamine receptors and neuropeptides in the striatum of GTP cyclohydrolase-deficient mice

The hph-1 mice have defective tetrahydrobiopterin biosynthesis and share many neurochemical similarities with l-dopa-responsive dystonia (DRD) in humans. In both, there are deficiencies in GTP cyclohydrolase I and low brain levels of dopamine (DA). Striatal tyrosine hydroxylase (TH) levels are decreased while the number of DA neurones in substantia nigra (SN) appears normal. The hph-1 mouse is therefore a useful model in which to investigate the biochemical mechanisms underlying dystonia in DRD. In the present study, the density of striatal DA terminals and DA receptors and the expression of D-1, D-2, and D-3 receptors, preproenkephalin (PPE-A), preprotachykinin (PPT), and nitric oxide synthase (NOS) mRNAs in the striatum and nucleus accumbens and nigral TH mRNA expression were examined. Striatal DA terminal density as judged by specific [3H]mazindol binding was not altered while the levels of TH mRNA were elevated in the SN of hph-1 mice compared to control (C57BL) mice. Total and subregional analysis of the striatum and nucleus accumbens showed that D-2 receptor ([3H]spiperone) binding density was increased while D-1 receptor ([3H]SCH 23390) and D-3 receptor ([3H]7-OH-DPAT) binding density was not altered. In the striatum and nucleus accumbens, expression of PPT mRNA was elevated but PPE-A mRNA, D-1, D-2 receptor, and nNOS mRNA were not changed in hph-1 mice compared to controls. These findings suggest that an imbalance between the direct strionigral and indirect striopallidal output pathways may be relevant to the genesis of DRD. However, the pattern of changes observed is not that expected as a result of striatal dopamine deficiency and suggests that other effects of GTP cyclohydrolase I deficiency may be involved.

Congenic mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse

The hph-1 ENU-mutant mouse provides a model of tetrahydrobiopterin deficiency for studying hyperphenylalaninaemia, dopa–response dystonia, and vascular dysfunction. We have successively localized the hph-1 mutation to a congenic interval of 1.6–2.8 Mb, containing the GCH gene encoding GTP cyclohydrolase I (GTP-CH I). We used these data to establish a PCR method for genotyping wild type, hph-1 and heterozygote mice, and found that heterozygote animals have partial tetrahydrobiopterin deficiency. These new findings will extend the utility of the hph-1 mouse in studies of GTP-CH I deficiency.

The hph-1 mouse: a model for dominantly inherited GTP-cyclohydrolase deficiency.

Dominantly inherited guanosine triphosphate (GTP)-cyclohydrolase deficiency, otherwise known as Segawa's disease or dopa-responsive dystonia, has a wide spectrum of phenotypic expression ranging from asymptomatic to very severe. Penetrance is more frequent in women as compared with men, and there is a variable occurrence of diurnal variation in symptom intensity. Biochemical characterization of the disease has demonstrated lower cerebrospinal fluid levels of tetrahydrobiopterin (BH4), neopterin, and homovanillic acid and low levels of tyrosine hydroxylase protein in the striatum. To investigate the pathophysiology, we have begun to characterize biogenic amine and BH4 metabolism in the GTP cyclohydrolase deficient hph-1 mouse. The data show low brain levels of BH4, catecholamines, serotonin, and their metabolites together with low levels of tyrosine hydroxylase protein within the striatum. The hph-1 mouse therefore provides a good model system in which to study the human disease.

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.

Studies on the genotype-phenotype relation in the hph-1 mouse mutant deficient in guanosine triphosphate (GTP) cyclohydrolase I activity.

The guanosine triphosphate (GTP) cyclohydrolase I (GTP-CHI) catalyses the rate-limiting step in the de novo synthesis of tetrahydrobiopterin, a cofactor of three aromatic amino acid hydroxylases, one of which is phenylalanine hydroxylase. The hph-1 mouse mutant deficient in GTP-CHI activity exhibits hyperphenylalaninemia which peculiarly disappears at 3 weeks of age, thus corresponding to the increase in liver GTP-CHI activity. The present gas chromatographic-mass spectrometric analysis of the phenylalanine and catecholamine metabolisms demonstrated the former metabolism to remain disturbed even in adult hph-1, which demonstrated a metabolic basis for sensitivity to the phenylalanine challenge in adult hph-1. A Northern blot analysis showed the hepatic GTP-CHI RNA expression in hph-1 at 2, 3 and 4 weeks of age to parallel the peculiar time course of the enzyme activity previously reported. No mutation was detected in either the coding region or the 5' flanking region (nt.-1 to -746) of the GTP-CHI gene of the hph-1. Further molecular genetic analyses are therefore required to elucidate the mechanism of the peculiar phenotype of hph-1.

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse.

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.