Induction of tetrahydrobiopterin synthesis in rat cardiac myocytes: impact on cytokine-induced NO generation

1997 ◽  
Vol 273 (2) ◽  
pp. H665-H672 ◽  
Author(s):  
K. Kasai ◽  
Y. Hattori ◽  
N. Banba ◽  
S. Hattori ◽  
S. Motohashi ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
De Novo ◽  
Limit Of Detection ◽  
Interleukin 1 ◽  
Significant Rise ◽  
No Production ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Ifn Gamma ◽  
Rat Cardiac Myocytes

Because tetra-hydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultured rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below our limit of detection. However, treatment with interleukin-1 alpha (IL-1 alpha) + interferon-gamma (IFN-gamma) caused a significant rise in biopterin levels and induced NO synthesis. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I (the rate-limiting enzyme for de novo BH4 synthesis), completely abolished the elevation in biopterin levels induced by IL-1 alpha + IFN-gamma. DAHP also caused a concentration-dependent inhibition of (IL-1 alpha + IFN-gamma)-induced NO synthesis. Similarly, N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, blocked increases in biopterin levels as well as NO synthesis induced by IL-1 alpha + IFN-gamma. Sepiapterin, substrate for BH4 synthesis via the pterin salvage pathway, prevented this inhibition by DAHP or N-acetylserotonin, and this effect was blocked by methotrexate. Sepiapterin and, to a lesser extent, BH4 dose dependently enhanced (IL-1 alpha + IFN-gamma)-induced NO synthesis, suggesting that the concentration of BH4 limits the rate of NO production. Inducible NO synthase mRNA and GTP cyclohydrolase I mRNA were induced by IL-1 alpha + IFN-gamma in parallel. We thus demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.

Tetrahydrobiopterin Synthesis in Rat Cardiac Myocytes; An Event Required for Cytokine-induced NO Generation

Pteridines ◽  
1998 ◽  
Vol 9 (1) ◽  
pp. 8-12
Author(s):  
Yoshiyuki Hattori ◽  
Nobuo Nakanishi ◽  
Steven S. Gross ◽  
Kikuo Kasai
Keyword(s):  
Cardiac Myocytes ◽  
Limit Of Detection ◽  
High Capacity ◽  
Interferon Γ ◽  
Significant Rise ◽  
Inos Mrna ◽  
No Production ◽  
No Formation ◽  
Absolute Requirement ◽  
Rat Cardiac Myocytes

SummaryCardiac myocytes are known to express the high-capacity inducible isoform of nitric oxide (NO) synthase (iNOS). Since tetrahydrobiopterin (BH4) is an essential cofactor for NO formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultutred rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below our limit of detection. However, treatment with inter-leukin-1α. and interferon-γ (IL-1/IFN) caused a significant rise in biopterin levels and induced NO synthesis. iNOS mRNA and GTP cyclohydrolase I (GTPCH) mRNA were induced by IL-1/IFN in parallel. 2,4-Diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTPCH, inhibited both the increase in cellular levels of BH4 as well as the concomitant formation of NO caused by IL-1/IFN. This inhibition by DAHP was reversed by coaddition of sepiapterin which is a substrate for BH4 synthesis. Thus BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency

2000 ◽  
Vol 349 (1) ◽  
pp. 353-356 ◽  
Author(s):  
Cynthia J. MEININGER ◽  
Rebecca S. MARINOS ◽  
Kazuyuki HATAKEYAMA ◽  
Raul MARTINEZ-ZAGUILAN ◽  
Jose D. ROJAS ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Diabetic Rats ◽  
Bb Rat ◽  
No Production ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Bb Rats ◽  
Metabolic Basis ◽  
Rate Limiting

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH4), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH4 levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH4 levels with sepiapterin increased NO production, suggesting that BH4 deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH4. GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

NF-kappa B and GTP cyclohydrolase regulate cytokine-induced nitric oxide production by cardiac myocytes

1996 ◽  
Vol 270 (5) ◽  
pp. H1864-H1868 ◽  
Author(s):  
C. V. Oddis ◽  
M. S. Finkel
Keyword(s):  
Nitric Oxide ◽  
Cardiac Myocytes ◽  
Nuclear Translocation ◽  
Interleukin 1 ◽  
No Synthase ◽  
Neonatal Rat ◽  
Inos Mrna ◽  
No Production ◽  
Gtp Cyclohydrolase ◽  
Nf Kappa B

We previously reported that interleukin-1 beta (IL-1) alone stimulated nitric oxide (NO) production by neonatal rat cardiac myocytes (CM) in culture. The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. Translocation from the cytosol to the nucleus of nuclear factor-kappa B (NF-kappa B) and activation of guanosine 5'-triphosphate (GTP) cyclohydrolase [rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis] have been implicated in IL-1 signaling. Accordingly, the effects of the NF-kappa B inhibitor pyrolidine dithiocarbamate (PDTC) and the GTP cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) on IL-1-induced NO production by CM were studied. PDTC and DAHP inhibited IL-1-induced NO2-production by CM (6.7 +/- 0.6 vs. 0.9 +/- 0.3 and 0.3 +/- 0.1 nmol. 1.25 x 10(5) cells(-1).48 h-1, respectively, P < 0.01, n = 12 for each). Immunohistochemical staining revealed that PDTC blocked IL-1-stimulated nuclear translocation of NF-kappa B. The membrane-permeable analogue of the NO synthase cofactor BH4, methyl-BH4 (mBH4), only partially reversed DAHP inhibition of NO2- formation (6.7 +/- 0.6 vs. 2.4 +/- 0.3 nmol. 1.25 x 10(5) cells-1.48 h-1, P < 0.01, n = 12). Semiquantitative reverse transcription polymerase chain reaction revealed no inducible NO synthase (iNOS) mRNA production in cells treated with IL-1 + PDTC.CM treated with IL-1 + DAHP did express iNOS mRNA. We report for the first time that nuclear translocation of NF-kappa B is essential for II-1-induced iNOS mRNA expression and GTP cyclohydrolase activity is required in addition in addition to BH4 for optimal NO production by CM.

scholarly journals Tetrahydrobiopterin biosynthesis, regeneration and functions

2000 ◽  
Vol 347 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Beat THÖNY ◽  
Günter AUERBACH ◽  
Nenad BLAU
Keyword(s):  
De Novo ◽  
Recombinant Expression ◽  
Regulatory Protein ◽  
Cellular Level ◽  
No Synthase ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Nmr Studies ◽  
Sepiapterin Reductase ◽  
Mammalian Cell Lines

Tetrahydrobiopterin (BH4) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH4 is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH4 from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH4, but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH4 are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH4 has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH4 is a self-protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH4 deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be a consequence of restricted cofactor availability.

scholarly journals Nitric oxide formation in acutely rejecting cardiac allografts correlates with GTP cyclohydrolase I activity

2005 ◽  
Vol 391 (3) ◽  
pp. 541-547 ◽  
Author(s):  
Galen M. Pieper ◽  
Vani Nilakantan ◽  
Nadine L. N. Halligan ◽  
Ashwani K. Khanna ◽  
Gail Hilton ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Graft Rejection ◽  
Ex Vivo ◽  
No Production ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Protein Levels ◽  
Acute Graft Rejection ◽  
Cardiac Allografts ◽  
Acute Graft

Inducible nitric oxide synthase (iNOS) is a prominent component of the complex array of mediators in acute graft rejection. While NO production is determined by iNOS expression, BH4 (tetrahydrobiopterin), a cofactor of iNOS synthesized by GTP cyclohydrolase I, has been considered critical in sustaining NO production. In the present study, we examined time-dependent changes in iNOS and GTP cyclohydrolase I in rat cardiac allografts. The increase in iNOS protein and mRNA in allografts was similar at POD4 (post-operative day 4) and POD6. However, the peak increase in intragraft NO level at POD4 was not sustained at POD6. This disparity could not be explained by any decrease in iNOS enzyme activity measured ex vivo with optimal amounts of substrate and cofactors. Lower iNOS activity could be explained by changes in total biopterin levels in allografts at POD4 that was decreased to baseline at POD6. Changes in biopterin production correlated with lower GTP cyclohydrolase I protein levels but not by any change in GTP cyclohydrolase I mRNA. Functionally, allografts displayed bradycardia and distended diastolic and systolic dimensions at POD6 but not at POD4. Likewise, histological rejection scores were increased at POD4 but with a secondary increased stage at POD6. It is hypothesized that the dissimilar amounts of NO at early and later stages of rejection is due to uncoupling of iNOS arising from disproportionate synthesis of BH4. These findings provide insight into a potential pathway regulating NO bioactivity in graft rejection. Such knowledge may potentially assist in the design of newer strategies to prevent acute graft rejection.

Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle

1994 ◽  
Vol 266 (4) ◽  
pp. L455-L460 ◽  
Author(s):  
D. K. Nakayama ◽  
D. A. Geller ◽  
M. Di Silvio ◽  
G. Bloomgarden ◽  
P. Davies ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
De Novo ◽  
Interleukin 1 ◽  
No Production ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pulmonary Artery Smooth Muscle ◽  
Inducible Nitric Oxide

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes

1996 ◽  
Vol 271 (1) ◽  
pp. C429-C434 ◽  
Author(s):  
C. V. Oddis ◽  
R. L. Simmons ◽  
B. G. Hattler ◽  
M. S. Finkel
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cardiac Myocytes ◽  
Interleukin 1 ◽  
Neonatal Rat ◽  
Inos Mrna ◽  
No Production ◽  
Neonatal Rat Cardiac Myocytes ◽  
Kinase A

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.

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