Induction of Tetrahydrobiopterin Synthesis in Cardiac Myocytes

Conference paper


Cardiac myocytes are now known to express the high-capacity inducible isoform of nitric oxide (NO) synthase (iNOS). Induction of iNOS by soluble inflammatory mediators, including cytokines, causes a marked depression in myocyte contractile responsiveness to b-adrenergic agonists and has been implicated as a contributor to the pathogenesis of heart failure. Since tetrahydrobiopterin (BH4) is an essential cofactor for NO formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultured rat cardiac myocytes. Activation of NO formation by cytokines in cardiac myocytes requires transcriptional induction of the genes that encode iNOS and GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in de novo BH4 synthesis. Given that nuclear factor κB (NF-κB) mediates the induction of iNOS gene expression in various cell types, the role of NF-κB in the induction of iNOS in cytokine-stimulated rat neonatal cardiac myocytes was assessed by examining the effects of pyrrolidine dithiocarbamate (PDTC). an inhibitor of NF-κB activation, on iNOS mRNA expression and subsequent NO production. The effects of PDTC on GTPCH mRNA expression and pterin synthesis were also examined. We here demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes. We show that PDTC inhibited in a dose-dependent manner both NO and BH4 synthesis induced by a combination of interleukin-1α (IL-1) and interferon-γ (IFN) and that PDTC also prevented the accumulation of iNOS and GTPCH mRNAs induced by IL-1 and IFN. The induction of both genes necessary for NO synthesis in cardiac myocytes appears to be regulated, at least in part, by a common mechanism: NF-κB activation. Our findings also suggest that regulation of pterin synthesis may be an important target for pharmacologic interventions for NO overproduction within the myocardium in cytokine-related cardiac dysfunction.

Key Words

nitric oxide (NO) nitric oxide synthase cardiac myocyte tetrahydrobiopterin (BH4) GTP cyclohydrolase I (GTPCH) 


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  1. 1.
    Barry WH (1994) Mechanisms of immune-mediated myocyte injury. Circulation 89: 2421–2432PubMedCrossRefGoogle Scholar
  2. 2.
    Lange LG, Schreiner GF (1994) Immune mechanisms of cardiac disease. N Engl J Med 174:493–496Google Scholar
  3. 3.
    Dallman MJ, Larsen CP, Morris PJ (1991) Cytokine gene transcription in vascularized organ grafts, analysis using semiquantitative polymerase chain reaction. J Exp Med 174:493–496PubMedCrossRefGoogle Scholar
  4. 4.
    Balligand J-L, Kelley RA, Marsden, PA, Smith TW, Mitchel T (1993) Control of cardiac muscle cell function by an endogenous nitric oxide signalling system. Proc Natl Acad Sci USA 90: 347–351PubMedCrossRefGoogle Scholar
  5. 5.
    Balligand J-L, Ungreanu D, Kelley RA, Kobzik L, Pimental D, Michel T, Smith TW (1993) Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest 91: 2314–2319PubMedCrossRefGoogle Scholar
  6. 6.
    Schulz R, Panas DL, Catena R, Moncada S., Olley PM, Lopaschuk GL (1995) The role of nitric oxide in cardiac depression induced by interleukin-lb and tumour necrosis factor-a. Brit J Pharmacol 114: 27–34CrossRefGoogle Scholar
  7. 7.
    Nathan C, Xie Q-W (1994) Regulation of biosynthesis of nitric oxide. J Biol Chem 269: 25722–25729Google Scholar
  8. 8.
    Werner-Felmayer G, Werner ER, Fuchs D, Hausen A., Reibnegger G & Wachter H (1990) Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J Exp Med 172: 1599–1607PubMedCrossRefGoogle Scholar
  9. 9.
    Gross SS, Jaffe EA, Levi R, Kilbourn RG (1991) Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent calmodullin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun 178: 823–829PubMedCrossRefGoogle Scholar
  10. 10.
    Gross SS, Levi R (1992) Tetrahydrobiopterin synthesis: An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 267: 25722–25729PubMedGoogle Scholar
  11. 11.
    Sakai N, Kaufmann S, Milstien S (1993) Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW264). Mol Pharmacol 43: 6–10PubMedGoogle Scholar
  12. 12.
    Sakai N, Kaufmann S, Milstien S (1995) Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glia cells. J Neurochem 65: 895–902PubMedCrossRefGoogle Scholar
  13. 13.
    Kasai K, Hattori Y, Nakanishi N, Manaka K, Banaba N, Motohashi S, Shimoda S (1995) Regulation of inducible nitric oxide production by cytokines in human thyrocytes in culture. Endocrinology 136: 4261–4270PubMedCrossRefGoogle Scholar
  14. 14.
    Werner-Felmayer G, Werner ER, Fuchs D, Hausen A., Reibnegger G Schmidt K, Weiss G, Wachter H (1993) Pterindine biosynthesis in human endothelial cells: Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem 268: 1842–1846PubMedGoogle Scholar
  15. 15.
    Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, Pober JS (1994) Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. J Clin Invest 93: 2236–2243PubMedCrossRefGoogle Scholar
  16. 16.
    Cosentino F, Katusic ZS. Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries. Circulation 1995;91:139–144PubMedCrossRefGoogle Scholar
  17. 17.
    Nathan C, Xie QW, 1994. Regulation of biosynthesis of nitric oxide. J Biol Chem 269: 25722–25729Google Scholar
  18. 18.
    Xie QW, Kashiwabara Y, Nathan C (1994) Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J Biol Chem 269: 4705–4708PubMedGoogle Scholar
  19. 19.
    Nunokawa Y, Oikawa S, Tanaka S (1996) Human inducible nitric oxide synthase gene is transcriptionally regulated by nuclear factor-kB dependent mechanism. Biochem Biophys Res Commun 223: 347–352PubMedCrossRefGoogle Scholar
  20. 20.
    Mulsch A, Schray-Utz B, Mordvibtcev PI, Hauschildt S, Busse R (1993) Diethyldithiocarbamate inhibits induction of macrophage NO synthase. FEBS lett 321: 215–218PubMedCrossRefGoogle Scholar
  21. 21.
    Sherman MP, Aeberhard EE, Wong VZ, Griscavage JM, Ignarro LJ, 1993. Pyrrolidine dithiocarbamate inhibits induction of nitric oxide synthase activity in rat alveolar macrophages, Biochem Biophys Res Commun 191: 1301–1308PubMedCrossRefGoogle Scholar
  22. 22.
    Eberhardt W, Kunz D, Pfeilschifer J (1994) Pyrrolidine dithiocarbamate differentially affects interleukin lb-and cAMP-induced nitric oxide synthase expression in rat mesangial cells. Biochem Biophys Res Commun 200: 163–170PubMedCrossRefGoogle Scholar
  23. 23.
    Nichol CA., Smith GK, Duch DS (1985) Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem 54:729–764PubMedCrossRefGoogle Scholar
  24. 24.
    Hattori Y, Gross SS (1993) GTP cyclohudrolase I mRNA is induced by LPS in vascular smooth muscle: charaterization, sequence and relationship to nitric oxide synthase. Biochem Biophys Res Commun 195: 435–441PubMedCrossRefGoogle Scholar
  25. 25.
    Gross SS, Levi R (1992) Tetrahydrobiopterin synthesis: an absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 267: 25722–25729PubMedGoogle Scholar
  26. 26.
    Hattori Y, So S, Hattori S, Kasai K, Shimoda S (1995) Vesnarinone inhibits induction of nitric oxide synthase in J774 macrophages and rat cardiac myocytes in culture. Cardiovasc Res 30: 187–192PubMedGoogle Scholar
  27. 27.
    Kamitani T, Ikeda U, Muto S, Kawakami K, Nagano K, Tsuruya Y, Oguchi A, Yamamoto K, Hara Y, Kojima T, Medford RM, Shimada K (1992) Regulation of Na. K-ATPase gene expression by thyroid hormone in rat cardiocytes. Circ Res 71: 1457–1464PubMedCrossRefGoogle Scholar
  28. 28.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 655: 55–63CrossRefGoogle Scholar
  29. 29.
    Fukushima T, Nixon JC (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 102: 176–188PubMedCrossRefGoogle Scholar
  30. 30.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275PubMedGoogle Scholar
  31. 31.
    Terada Y, Tomita K, Nonoguchi H, Marumo F (1992) Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments. J Clin Invest 90: 659–665PubMedCrossRefGoogle Scholar
  32. 32.
    Schreck R, Meier B, Mannel DN, Droge W, Baeueule PA (1992) Dithiocarbamates as potent inhibitors of nuclear factor kB activation in intact cells. J Exp Med 175: 1181–1194PubMedCrossRefGoogle Scholar
  33. 33.
    Chenn CC, Rosenbloom CL, Anderson DC, Manning AM (1995) Selective inhibition of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression by inhibitors of IkB-a phosphorylation. J Immunol 155: 3538–3545Google Scholar
  34. 34.
    Smith J, Gross SS (1996) Cloning and regulation of the rat GTP cyclohydrolase gene: co-ordinate transcriptional induction with iNOS by immunostimulants. In: Moncada S, Stamler J, Gross SS, Higgs EA (eds), The Biology of Nitric Oxide Part5, Portland Press, London, pp156Google Scholar

Copyright information

© Springer Japan 1999

Authors and Affiliations

  1. 1.Department of EndocrinologyDokkyo University School of MedicineMibu, TochigiJapan

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