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
Tetrahydrobiopterin (BH4) is one of the cofactors for nitric oxide synthase (NOS), and also has a protective effect against reactive oxygen species (ROS)- and/or nitric oxide (NO)-induced cytotoxicities. Lipopolysaccharide (LPS) induces NO production and BH4 synthesis, and also ROS release. This study examined whether hydrogen peroxide, one of the ROS, has a regulatory role in BH4 synthesis in vascular endothelial cells. Addition of LPS to the endothelial cells increased the BH4 content. Although the LPS-induced increase in BH4 content was not affected by the treatment with superoxide dismutase and catalase, co-treatment with hydrogen peroxide further stimulated the LPS-induced increase in BH4 content. Moreover, the hydrogen peroxide-induced increase in BH4 content in the presence of LPS was completely inhibited by the treatment with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP-cyclohydrolase I (GTPCH). These findings suggest that hydrogen peroxide may stimulate LPS-induced BH4 synthesis through the induction and/or activation of GTPCH. Under inflammation, hydrogen peroxide seems to stimulate BH4 synthesis.
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.
We investigate the incentive and welfare implications of a merger when heterogeneous oligopolists compete both in process R&D and on the product market. We examine how a merger affects the output, investment, and profits of firms. In addition, we examine whether firms have merger incentives, and, if so, whether such mergers are desirable from the viewpoint of social welfare. If R&D is not expensive and if large cost differences between efficient and inefficient firms exist, a merger between homogeneous firms tends to occur even though it harms welfare.
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.