池本 和久

<title>Abstract</title> Neopterin (NP), biopterin (BP) and monapterin (MP) exist in saliva. The physiological role of salivary NP as well as the pathophysiological role of increased NP in the immune-activated state has been unclear. Saliva is a characteristic specimen different from other body fluids. In this study, we analysed salivary NP and related pterin compounds, BP and MP and revealed some of its feature. High-performance liquid chromatography (HPLC) analysis of saliva and plasma obtained from 26 volunteers revealed that salivary NP existed mostly in its fully oxidized form. The results suggested that salivary NP as well as BP would mostly originate from the oral cavity, perhaps the salivary glands, and that salivary NP levels might not reflect those in the plasma. We also found that a gender difference existed in correlations between concentrations of salivary total concentrations of NP (tNP) and BP (tBP). HPLC analysis of saliva obtained from 5 volunteers revealed that the concentrations of salivary tNP as well as tBP fluctuated in an irregular fashion in various individuals. MP, a diastereomer of NP, might have come from oral cavity NP itself or its precursor. These results indicated that the nature of salivary NP might be different from that of NP in the blood or urine.

(6R)-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 α1 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 Parkinson's disease and spinal cord injury.

Aims: Cilostazol, a type. phosphodiesterase inhibitor, is utilized for the treatment of intermittent claudication and is considered to have the beneficial effects against the atherogenic process. In the present study, we examined the effects of cilostazol on BH4 biosynthesis in HUVEC treated with a mixture of the pro-inflammatory cytokines IFN-gamma and TNF-alpha. Methods: Isolated HUVECs were grown to confluence and treated with IFN-gamma (300 units/mL) and TNF-alpha (300 units/mL) for 16 h in order to stimulate BH4 biosynthesis. The BH4 levels were measured by HPLC. The mRNA expression of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 biosynthesis, and GTPCH feedback regulatory protein (GFRP) were quantified by real-time PCR. The GTPCH protein expression was assessed by western blot analysis. Results: Cilostazol significantly reduced the BH4 levels in cytokine-stimulated HUVEC. Cilostazol produced a concomitant increase in the cAMP levels in HUVEC. Cilostazol decreased the GTPCH activity as well as the expression of GTPCH mRNA and protein. 8-bromo-cAMP (8Br-cAMP), a cell-permeable cAMP analogue, did not reproduce the effects of cilostazol. Cilostazol did not affect the cytokine-induced inhibition of GFRP mRNA expression. Conclusions: We conclude that cilostazol inhibited cytokine-stimulated BH4 biosynthesis via a cAMP-independent mechanism in HUVEC. Our data indicate that cilostazol reduced GTPCH activity and did so by suppressing the GTPCH protein levels.

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.

5R-L-Erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) is an essential cofactor for tyrosine hydroxylase (TH). Recently, a type of dopa-responsive dystonia (DRD) (DYT5, Segawa&apos;s disease) was revealed to be caused by dominant mutations of the gene encoding GTP cyclohydrolase I (GCHI), which is the rate-limiting enzyme of BH(4) biosynthesis. In order to probe the role of BH(4) in vivo, we established BH(4)-depleted mice by disrupting the 6-pyruvoyltetrahydropterin synthase (PTS) gene (Pts(-/-)) and rescued them by introducing human PTS cDNA under the control of the human dopamine beta-hydroxylase (DBH) promoter (Pts(-/-)-DPS). The Pts(-/-)-DPS mice developed hyperphenylalaninemia. Interestingly, tyrosine hydroxylase protein was dramatically reduced in the dopaminergic nerve terminals of these mice, and they developed abnormal posture and motor disturbance. We propose that the biochemical and pathologic changes of Pts(-/-)-DPS mice are caused by mechanisms common to human DRD, and understanding these mechanisms could give us insight into other movement disorders.

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH4 biosynthesis and is widely used in order to elucidate the possible biological function of BH4 in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH4) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH4 might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH4 and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH4 level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH4 level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH4 production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH4-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappa B (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappa B level in the nucleus of HUVEC. (C) 2008 Elsevier B.V. All rights reserved.

Objective-Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results-Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions-In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.