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Tetrahydrobiopterin (BH4) content in the pancreas from GK (Goto-Kakizaki) rats as a type II diabetic model was markedly decreased compared to Wistar rats as a healthy control. Moreover, insulin stimulated BH4 synthesis in a hamster pancreatic β-cell line, HIT-T15 cells. Therefore, in diabetic condition, the reduction of insulin action is likely to lead to the low levels of BH4 content in pancreatic β-cells. In this study, we examined the effects of the decrease in BH4 content on insulin secretion by L-arginine (L-Arg) or glucose using HIT-T15 cells. Cytosolic calcium concentration ([Ca2+]i) in HIT-T15 cells was increased by L-Arg. Under the low BH4 content using 2,4- diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of BH4 synthesis, L-Arg-induced both Ca2+ response and insulin secretion were stimulated. These stimulating effects were recovered by BH4 content recovering, using sepiapterin, a precursor of BH4 biosynthesis. By contrast, glucose-induced Ca2+ response was not affected by the BH4 content. These results suggest that the decrease of BH4 content in β-cells may stimulate insulin secretion by L-Arg, but not by glucose, under diabetic conditions.

We examined the role of endogenous tetrahydrobiopterin (BH4) on L-tyrosine-induced increase in cytosolic Ca: concentration ([Ca2+],) in the ß-cell line, HIT-T15. Addition of L-tyrosine to the cells increased [Ca2+], in a conccntration- depcndent manner. Moreover, the L-tyrosine-induced [Ca2+], elevation was blocked by L-3,4-dihydroxyphenylalanine (L-DOPA). Under low levels of BH4 by treatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 biosynthesis, elevation of [Ca2+], by L-tyrosine was stimulated. Moreover, co-prctreatment with sepiapterin, a precursor of BH4 biosynthesis, reduced the decrease of BH4 content by DAHP, and suppressed L-tyrosine-induccd [Ca2+ ]¡ elevation in DAHP-pretreated cells. These findings suggest that BH4 negatively regulates L-tyrosine-induced [Ca2+] elevation via the production of L-DOPA in ß-cells.

5,6,7,8-Tetrahydrobiopterin (BH4) is an csscntial cofactor for production of nitric oxide (NO) by N O synthase (NOS). Nitric oxide (NO) is an important signaling mo!ecu!e for the regulation o f vita! functions such as vascular tone, neurotransmission and immune surveiüance. Accumu!ated evidences show that although NOS mainly re!eases N O under nonnal conditions, NOS also produces Superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of reactive oxygen species (ROS) under pathological conditions. In fact, supplementation of BH4 restores N O production and availability in various diseases including hyperlipidemia, diabetes, hypertension and ischemia-reperfusion. Moreover, recent studies show that administration o f BH4 protects tissues against ischemia-reperfusion injuiy in heart, stomach and liver. Oxidative stress is a common mechanism underlying the development of vascular diseases and reperfusion injury. Regulation of BH4 levels under oxidative stress is important to understand the role of BH4 on the development of vascular diseases and reperfusion injury. We recently described that oxidative stress transiently decreased BH4 levels, and then markedly increased its levels in vascular endothelial cells. Supplementation of BH4 appears to be important therapeutic strategies in vascular diseases and reperfusion injury, and induction of BH4 synthesis may endogenous defense system against oxidative stress-mediated cardiovascular diseases.

Abstract.: Intracellular ADP-ribose is an activator of TRPM2, which is a Ca2+-permeable channel and mediates H2O2-induced cell death, in the TRPM2-expressing rat β-cell line RIN-5F. We examined the effect of extracellular-added ADP-ribose on intracellular Ca2+ concentration in RIN-5F cells. ADP-ribose induced Ca2+ release from the thapsigargin-sensitive Ca2+ store, but not Ca2+ entry across the plasma membrane. A phospholipase C (PLC) inhibitor and a non-specific IP3 receptor inhibitor blocked its Ca2+ release. H2O2-induced Ca2+ entry through TRPM2 was not affected by extracellular ADP-ribose. These findings suggest that extracellular-added ADP-ribose induces Ca2+ release through the PLC-IP3 pathway and does not act as a TRPM2 activator. Keywords:: ADP-ribose, hydrogen peroxide, TRPM2

The purpose of this study was to examine whether basic fibroblast growth factor (bFGF) stimulates tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells. BH4 content was determined by oxidation under acidic conditions as biopterin and analysed with reversed-phase high Performance liquid chromatography. Measurement of the mRNA level of QTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of the de novo pathway of BH4 synthesis. The addition of bFGF to endothelial cells increased the BH4 content and GTPCH mRNA levels in an incubation period- and a concentration-dependent manner. 2,4-Diamino-6- hydroxypyrimidine, an inhibitor of GTPCH, strongly reduced the bFGF-induced increase in BH4 content. These findings suggest that bFGF stimulates BH4 synthesis via a de novo pathway with the induction of GTPCH.

We examined the effect of immunosuppressant cyclosporin A (CsA) on the synthesis of tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase (NOS), during treatment with lipopolysaccharide (LPS) in mouse brain microvascular endothelial cells. Addition of LPS to the endothelial cells increased the BH4 content and the mRNA level of GTP-cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis, and the LPSínduced increases in both the BH4 content and expression of GTPCH mRNA were further stimulated by the cotreatment with CsA. 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, blocked the increase in BH4 content induced by CsA during the LPS treatment. Moreover. CsA stimulated the expression of inducible NOS (iNOS) mRNA during the LPS treatment. These findings suggest that CsA stimulates LPS-induced BH4 synthesis through the induction of GTPCH, and iNOS expression. CsA may increase NO production during LPS treatment in brain microvascular endothelial cells.

Vascular endothelial cells are one of the major biological targets of reactive oxygen species (ROS) in ischemiareperfusion, diabetes, hypercholesterolemia and inflammation. Accumulated evidence has shown that tetrahydrobiopterin (BH4), which is an essential cefactor for nitric oxide synthase (NOS) activity, protected the cells against ROS and nitric oxide (NO) toxicities. Although NOS releases NO, which regulates vascular tone and immune surveillance under normal conditions, NOS also produces superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of ROS under pathological conditions. Since BH4 is easily oxidized by ROS, oxidative stress may induce ROS release from NOS by the decomposition of BH4. BH4 also has an antioxidative activity and scavenging activity for ROS. In this review, the r o l e of BH4 in the function of' NOS, and the mechanisms underlying the protective effect of BH4 against ROS- and/or NO-induced endothelial cell injury will be discussed.

We examined the role ofprotein kinase C (PKC) in the insulin-induced biosynthesis oftetrahydrobiopterin (BH4)' which is one of the co factors of nitric oxide (NO) synthase (NOS), in vascular endothelial cells. The BH. level was determined as biopterin by reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level ofrnRNA for GTP cyclohydrolase I (GTPCH), which is the fust and the rate-limiting enzyme for the novo pathway for BH4 synthesis, was performed by reverse transcription-polymerase chain reaction (RT-PCR). Treatment with insulin increased the BH4 level and the GTPCH rnRNA level in endothelial cells. The insulin-induced increases of both GTPCH rnRNA and BH4 levels were enhanced by co-treatment with bisindolylmaleimide I or Ro 31-8220, inhibitors of PKC. On the other hand, the insulin-induced increases of BH4 synthesis and expression of GTPCH rnRNA were also enhanced by co-incubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA) for 24 h. Long-term treatment with PMA is known to down-regulate PKC. Addition of insulin after pretreatment with PMA for 24 h markedly enhanced BH4 synthesis and expression of .:JTPCH rnRNA. These findings suggest that PKC negatively regulates the induction of BH4 synthesis by insulin -:ndothelial cells. Insulin-induced BH4 synthesis may be suppressed in diabetes mellitus, since PKC has been shown to be up-regulated in diabetes mellitus.

The purpose of this study was to examine whether 17β-estradiol stimulates the synthesis of tetrahydrobiopterin : BH4), which is one of the cofactors of nitric oxide (NO) synthase, in mouse brain microvascular endothelial cells. Addition of 17()-estradiol to endothelial cells time- and concentration-dependently increased intracellular BH4 level. 17β-Estradiol also stimulated the mRNA level of GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme of the de novo BH4 synthetic pathway. In addition, the 17β-estradiol-induced expression of GTPCH mRNA was strongly attenuated by treatment with an inhibitor of 17β-estradiol receptor 4-hydroxy-tamoxlfen. These results suggest that 17β-estradiol stimulates BH4 synthesis through the induction of GTPCH by tamoxifensensitive receptor in vascular endothelial cells. The 17β-estradiol-induced increase in BH4 level might be implicated in not only NO production, but also protective effects of 17β-estradiol against ischemic brain damage and atherosclerosis, since BH4 is an intracellular antioxidant.

We investigated the effects of troglitazone, which is a ligand of nuclear transcription factor peroxisome proliferator- activated receptor-γ (PPAR-γ), on insulin-induced tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). BH4 content was determined as biopterin, by reversed-phase high performance liquid chromatography using fluorometric detection. Measurement of mRNA level of GTP cyclohydrolase I (GTPCH), which is a rate-limiting enzyme for de novo BH4 synthesis, was performed by reverse transcription- polymerase chain reaction (RT -PCR). Addition of insulin (10µg/ml) to endothelial cells increased intracellular BH4 content and GTPCH mRNA level, and the insulin-induced increase in BH4 content and GTPCH mRNA level was further stimulated by co-treatment with troglitazone (0.3-3 µM). However, the expression of PPAR-γ mRNA in untreated and insulin-treated endothelial cells was not detected. Moreover, bezafibrate (1-10 µM), a ligand of PPAR-δ, did not affect BH4 synthesis in insulin-treated endothehal cells. These findings suggest that troglitazone stimulates insulin-induced BH4 synthesis in endothelial cells with the induction of GTPCH through PPAR-γ- and PPAR-δ-independent mechanism

We examined the effects of insulin on tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). Treatment of MBMECs with insulin increased the intracellular BH4 content in a time- and concentration-dependent manner. The insulin-induced increase in BH4 content was inhibited by treatment with 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTP cyclohydrolase I, and Nacetylserotonin, a selective inhibitor of sepiapterin reductase. These findings indicate that insulin stimulates BH4 synthesis in MBMECs through a de novo synthetic pathway of BH4.

The purpose of this study was to characterize the protective effect of tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, against NO-induced endothelial cell injury. The addition of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, to endothelial cells induced the release of lactate dehydrogenase (LDH), a marker for cell injury. The SNAP-induced endothelial cell injury was markedly reduced by pretreatment with sepiapterin, a precursor of BH4 synthesis. On the other hand, exogenous BH4 had little effect on the SNAP-induced endothelial cell injury. We recently found that NO-induced endothelial cell injury involves a part of H202 production, since the injury was blocked by the treatment with catalase. Although BH4 released reactive oxygen species (ROS) in cell-free conditions, the increase in intracellular BH4 by pretreatment with sepiapterin strongly reduced H202-induced intracellular oxidative stress. These findings suggest that the increase in intracellular BH4 content but not extracellular BH4, strongly attenuates NO-induced endothelial cell injury by at least one of the mechanisms by which BH4 reduces H202-induced oxidative stress. Intracellular BH4 seems mainly to play a role as an antioxidant or as a ROS-scavenger.

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane [Ca.sup.2+]-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide ([H.sub.2][O.sub.2]) evokes [Ca.sup.2+] influx through TRPM2 to activate [Ca.sup.2+]-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-[kappa]B essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, [H.sub.2][O.sub.2]-induced [Ca.sup.2+] influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 [Ca.sup.2+] influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases., The biological purpose of inflammation is to bring fluids, proteins and inflammatory cells such as neutrophils and monocytes from the blood into the damaged tissues to eliminate the injuring agents [...]

Ets-1, which stimulates metalloproteinase gene transcription, has a key role in angiogenesis. We first examined whether activated polymorphonuclear leukocytes (PMNs) enhanced angiogenesis through the induction of Ets-1. Addition of activated PMNs to endothelial cells stimulated both in vitro angiogenesis in collagen gel and Ets-1 expression. Both angiogenesis and Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide, suggesting that Ets-1 is an important factor in PMN-induced angiogenesis. Although intercellular adhesion molecule (ICAM)-I and E-selectin are involved in PMN-induced angiogenesis, the mechanisms underlying their roles in angiogenesis have yet to be elucidated. PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. Moreover, the stimulation of angiogenesis by [H.sub.2][O.sub.2] without PMNs was inhibited by a monoclonal antibody to E-selectin but not ICAM-i. These findings suggested that ICAM-1 in endothelial cells may act as a signaling receptor to induce Ets-1 expression, whereas E-selectin seems to function in the formation of tubelike structures in vascular endothelial cell cultures. endothelial cell; intercellular adhesion molecule-1; Ets-1

The gene ets-1 codes for an activating factor for the expression of matrix proteinases necessary for the remodelling of the extracellular substance during repair following injuries. The regulation of ets-1 is investigated by treating cultured rat smooth muscle cells with factors known to promote proliferation and migration. Results reveal that the platelet-derived growth factor BB and endothelin-1 are potent activators of ets-1. Calcium and protein kinase C appear to be important secondary messengers in signal transduction.

Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture. Am J Physiol Cell Physiol 281: C24-C32, 2001.--Hypoxia (95% [N.sub.2]-5% [CO.sub.2]) elicits an endothelium-independent relaxation (45-80%) in freshly dissected porcine coronary arteries. Paired artery rings cultured at 37 [degrees] C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 [micro]M U-46619. However, relaxation in response to hypoxia was sharply attenuated compared with control (fresh arteries or those-stored at 4 [degrees] C for 24 h). Hypoxic vasorelaxation in organ cultured vessels was reduced at both high and low stimulation, indicating that both [Ca.sup.2+]-independent and [Ca.sup.2+]-dependent components are altered. In contrast, relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolin and isoproterenol) activation was not significantly affected by organ culture. Additionally, there was no difference in relaxation after washout of the stimulus, indicating that the inhibition is specific to acute hypoxia-induced relaxation. Simultaneous force and intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) measurements indicate the reduction in [[[Ca.sup.2+]].sub.i] concomitant with hypoxia at low stimulus levels in these tissue is abolished by culture. Our results indicate that organ culture at 37 [degrees] C specifically attenuates hypoxic relaxation in vascular smooth muscle by altering dynamics of [[[Ca.sup.2+]].sub.i] handling and decreasing a [Ca.sup.2+]-independent component of relaxation. Thus organ culture can be a novel tool for investigating the mechanisms of hypoxia-induced vasodilation. hypoxia