GTPCH mRNA Expression Research Articles

Cilostazol Inhibits Cytokine-Induced Tetrahydrobiopterin Biosynthesis in Human Umbilical Vein Endothelial Cells

Cilostazol, a type III 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 BH(4) biosynthesis in HUVEC treated with a mixture of the pro-inflammatory cytokines IFN-γ and TNF-α. Isolated HUVECs were grown to confluence and treated with IFN-γ (300 units/mL) and TNF-α (300 units/mL) for 16 h in order to stimulate BH(4) biosynthesis. The BH(4) levels were measured by HPLC. The mRNA expression of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) biosynthesis, and GTPCH feedback regulatory protein (GFRP) were quantified by real-time PCR. The GTPCH protein expression was assessed by western blot analysis. Cilostazol significantly reduced the BH(4) 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. We conclude that cilostazol inhibited cytokine-stimulated BH(4) 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.

Quantitative Regulation of Intracellular Endothelial Nitric-oxide Synthase (eNOS) Coupling by Both Tetrahydrobiopterin-eNOS Stoichiometry and Biopterin Redox Status: INSIGHTS FROM CELLS WITH TET-REGULATED GTP CYCLOHYDROLASE I EXPRESSION

Tetrahydrobiopterin (BH4) is a critical determinant of endothelial nitric-oxide synthase (eNOS) activity. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxide rather than NO. However, the stoichiometry of intracellular BH4/eNOS interactions is not well defined, and it is unclear whether intracellular BH4 deficiency alone is sufficient to induce eNOS uncoupling. To address these questions, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in BH4 synthesis, to selectively induce intracellular BH4 deficiency by incubation with doxycycline. These cells were stably co-transfected to express a human eNOS-green fluorescent protein fusion protein, selecting clones expressing either low (GCH/eNOS-LOW) or high (GCH/eNOS-HIGH) levels. Doxycycline abolished GTPCH mRNA expression and GTPCH protein, leading to markedly diminished total biopterin levels and a decreased ratio of BH4 to oxidized biopterins in cells expressing eNOS. Intracellular BH4 deficiency induced superoxide generation from eNOS, as assessed by N-nitro-L-arginine methyl ester inhibitable 2-hydroxyethidium generation, and attenuated NO production. Quantitative analysis of cellular BH4 versus superoxide production between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a striking linear relationship between eNOS protein and cellular BH4 stoichiometry, with eNOS uncoupling at eNOS:BH4 molar ratio >1. Furthermore, increasing the intracellular BH2 concentration in the presence of a constant eNOS:BH4 ratio was sufficient to induce eNOS-dependent superoxide production. This specific, reductionist approach in a cell-based system reveals that eNOS:BH4 reaction stoichiometry together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling, even in the absence of exogenous oxidative stress.

Sphingosine 1-Phosphate Inhibits Nitric Oxide Production Induced by Interleukin-1β in Rat Vascular Smooth Muscle Cells

Sphingosine 1-phosphate (S1P) is a lipid mediator that exerts potent and diverse biological effects on several cardiovascular cells. We investigated the effect of S1P on interleukin (IL)-1beta-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in rat vascular smooth muscle cells (VSMCs). S1P inhibited NO production at concentrations higher than 0.1 muM; this was associated with the inhibition of iNOS protein and mRNA expression. S1P also inhibited IL-1beta-induced GTP cyclohydrolase I (GTPCH) mRNA expression. Pertussis toxin (PTX) partially attenuated the inhibitory effects of S1P on NO production and iNOS protein induction, whereas it completely blocked the inhibitory effects on iNOS and GTPCH mRNA expression. S1P inhibited iNOS expression in Ca(2+)-depleted conditions; PTX did not modify this effect. The Rho kinase inhibitor Y 27632 partially but significantly attenuated the inhibitory effect of S1P on iNOS expression in Ca(2+)-depleted condition but did not affect it in the presence of Ca(2+). S1P significantly inhibited IL-1beta-induced persistent activation of extracellular signal-regulated kinase (ERK) but had no effect in Ca(2+)-depleted conditions. Thus, S1P inhibits IL-1beta induction of NO production and iNOS expression in rat VSMCs through multiple mechanisms involving both PTX-sensitive and -insensitive G proteins coupled to S1P receptors. Furthermore, Ca(2+)-dependent ERK inhibition and Ca(2+)-independent Rho kinase activation might be involved in the inhibitory mechanism of iNOS expression. Through its action on NO production by VSMCs, S1P may play an important role in the progression of local vascular injury associated with thrombosis, atherosclerosis, and hypertension.

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through activation of the Jak2 tyrosine kinase pathway in vascular endothelial cells

Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS). We previously described that hydrogen peroxide (H2O2) increases BH4 levels through the induction of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for the synthesis of BH4, in vascular endothelial cells. The aim of this study was to examine the underlying mechanism of H2O2-induced BH4 synthesis in vascular endothelial cells. The increases in BH4 levels induced by H2O2 were strongly reduced by a Janus kinase-2 (Jak2) inhibitor, AG490. The H2O2-induced increases in GTPCH mRNA expression and GTPCH activity were also blocked by treatment with AG490. H2O2 elicited an increase in the level of phosphorylated Jak2, suggesting that the induction of BH4 by H2O2 was mediated by the Jak2 pathway. Signal transducers and activators of transcription (Stats) are the best-known substrates for Jak2. The H2O2-induecd increases in BH4 levels were reduced by treatment with fludarabine, which is shown to cause a specific depletion of Stat1 protein but not of other Stats. Moreover, H2O2 caused the DNA binding of Stat1, and this was inhibited by AG490. Stat1 phosphorylation was enhanced by H2O2 treatment, and the phosphorylation was attenuated by AG490. These findings suggest that the stimulation of BH4 synthesis through the induction of GTPCH is mediated at least in-part by the Jak2–Stat1 pathway.

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells.

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.

Stimulation of Tetrahydrobiopterin Synthesis by Cyclosporin A during Lipopolysaccharide Treatment in Vascular Endothelial Cells

Abstract 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.

Interleukin-10 Inhibition of Nitric Oxide Biosynthesis Involves Suppression of CAT-2 Transcription

Interleukin-10 (IL-10) has been shown to attenuate lipopolysaccharide (LPS) stimulation of inducible nitric oxide synthase (iNOS) in various cell types. Guanosine triphosphate cyclohydrolase I (GTPCH) and type-2 cationic amino acid transporter (CAT-2) are enzymes that regulate iNOS activity. We therefore sought to assess the effects of IL-10 on the expression of these regulatory enzymes in LPS-stimulated macrophages that are known to express iNOS. Five minutes after adding LPS to these macrophage cultures, various doses of recombinant human IL-10 were also added. The samples were harvested for analysis 18 h after exposure to both LPS and IL-10. In LPS-stimulated macrophages, IL-10 attenuated the upregulation of nitric oxide and iNOS protein but not iNOS mRNA. IL-10 also attenuated the LPS-induced upregulation of CAT-2 mRNA. However, IL-10 and LPS had no effect on GTPCH mRNA expression. We therefore conclude that IL-10 inhibits nitric oxide formation in LPS-stimulated macrophages partly by decreasing iNOS protein expression. Moreover, our data suggests that transcriptional control of CAT-2 plays a role in IL-10 mediated influences upon nitric oxide biosynthesis.

Stimulation of Tetrahydrobiopterin Synthesis by 17β-Estradiol in Brain Microvascular Endothelial Cells

Abstract 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.

GTP cyclohydrolase I gene expression in the brains of male and female hph-1 mice.

The hph-1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double-label in situ hybridization technique was used to examine CNS GTPCH mRNA expression within serotonin, dopamine, and norepinephrine neurons of male and female wild-type and hph-1 mice. In wild-type male and female animals the highest levels of GTPCH mRNA expression were observed within serotonin neurons, followed by norepinephrine and then dopamine neurons. Wild-type female animals were found to express lower levels of GTPCH mRNA in each cell type when compared with levels seen in wild-type males. GTPCH mRNA abundance in all three cell types was lower in hph-1 male than in wild-type male mice, with the greatest reduction in serotonin neurons. GTPCH mRNA levels were also lower in hph-1 female than in wild-type female mice, again with the greatest reduction occurring in serotonin neurons. Comparison of hph-1 male and hph-1 female mice revealed that the sex-linked difference in GTPCH mRNA expression observed in wild-type neurons was only present within female dopamine neurons. Overall, these results indicate that not only are basal levels of GTPCH mRNA expression heterogeneous across wild-type murine monoamine cell types but that gene expression is also modified in a sex-linked and cell-specific fashion by the hph-1 gene locus. The hph-1 mutation does not lie within the GTPCH mRNA coding region. The 5' flanking region of the GTPCH gene was cloned and sequenced and shown to be identical for both wild-type and hph-1 genomic DNA. Transient transfection assays performed in PC12 cells demonstrated that this 5' flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph-1 mutation does not lie within the 5' flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression.

GTP Cyclohydrolase I mRNA Is Induced by LPS in Vascular Smooth Muscle: Characterization, Sequence and Relationship to Nitric Oxide Synthase

GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS). As the induction of NO synthesis by immunostimulants in vascular smooth muscle (VSM) cells requires de novo synthesis of BH4, we investigated whether immunostimulants enhance the expression of GTPCH mRNA. GTPCH mRNA and BH4 were measured in rat VSM cells after exposure to bacterial lipopolysaccharide (LPS) in combination with interferon-γ(IFN). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify a predicted 372 bp fragment of GTPCH mRNA, deduced from the known nucleotide sequence of rat liver GTPCH cDNA. Dideoxynucleotide sequencing of the PCR fragment revealed 100% identity between LPS/IFN-induced GTPCH mRNA of VSM and the constitutive GTPCH mRNA of liver. Although BH4 was below our limit of detection in untreated VSM, low levels of GTPCH mRNA were detectable. LPS/IFN treatment triggered the appearance of BH4 and markedly increased GTPCH mRNA. Induction of GTPCH mRNA was apparent by 2 h, peaked at 4 h, and was sustained at high levels for at least 24 h. Induction of GTPCH mRNA by LPS/IFN was substantially enhanced by cycloheximide,suggesting that mRNA levels are depressed by a labile protein. Measurement of LPS/IFN-induced NOS mRNA by RT-PCR, demonstrated a timecourse of induction which mirrors that of GTPCH. Similarly, the timecourse of appearance of cytosolic NOS activity following exposure of VSM to LPS/IFN paralleled that of the increase in BH4 content. Our studies demonstrate that immunostimulants co-induce NOS and GTPCH gene expression: both events are necessary for induction of NO synthesis by VSM.

Localization of GTP cyclohydrolase I (GTPCH) mRNA in the rat brain by in situ hybridization.

GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme in the tetrahydrobiopterin (BH4) biosynthetic pathway. BH4 is the essential cofactor for the pterin-dependent monooxygenases which include tyrosine and tryptophan hydroxylase, enzymes that are rate limiting in the biosynthesis of catecholamines and indolamines. BH4 is also required as a cofactor for the family of nitric oxide synthases1,2. We have obtained a clone for GTPCH based on the recently published full-length cDNA encoding GTPCH from rat liver3,4. The in situ hybridization technique was used to study the cellular localization and the relative levels of expression of GTPCH mRNA in the rat brain.KeywordsVentral Tegmental AreaLocus CoeruleusPineal GlandFourth VentricleDorsal RapheThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression of GTP cyclohydrolase I (GTPCH) mRNA in the rat: tissue distribution and effect of reserpine.

Disruption of vesicular monoamine storage by reserpine results in a marked depletion of monoamines in the central and peripheral nervous systems and a concomitant increase in tyrosine hydroxylase (TH) gene expression in response to the resulting acceleration of nerve impulse flow. This increase in TH mRNA appears to be catecholamine (CA) cell-type specific in that it is observed within NE-containing neurons of the locus coeruleus (LC), superior cervical ganglia (SCG) and adrenal gland or adrenal chromaffin cells but not in DA neurons of the substantia nigra (SN)1,2. Tetrahydrobiopterin (BH4) is synthesized by three or four enzymes from GTP3,4 and is the reduced pteridine cofactor required for CA and nitric oxide biosynthesis5,6. GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in this BH4 biosynthetic pathway3,4. Few studies have examined GTPCH gene expression in monoamine-containing neurons. A full-length cDNA encoding GTPCH has been isolated from rat liver7. We have used the reverse transcription-polymerase chain reaction (RT-PCR) to clone a coding portion of this sequence from rat adrenal gland. With this clone we have performed an analysis of GTPCH mRNA in rat tissues by Northern blot and nuclease protection assay and have examined the expression of GTPCH mRNA in CA neurons of the peripheral and central nervous systems following a single reserpine injection.