Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1994 May;93(5):2236–2243. doi: 10.1172/JCI117221

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity.

P Rosenkranz-Weiss 1, W C Sessa 1, S Milstien 1, S Kaufman 1, C A Watson 1, J S Pober 1
PMCID: PMC294374  PMID: 7514193

Abstract

We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-gamma (200 U/ml) plus TNF (200 U/ml) or IL-1 beta (5 U/ml) increased NOS activity in HUVEC lysates, measured as conversion of [14C]L-arginine to [14C]L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steady-state mRNA levels of endothelial NOS fell by 94 +/- 2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 microM) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-gamma plus TNF or IL-1 beta increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme of tetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo.

Full text

PDF
2236

Images in this article

2239Image
on p.2239
2240Image
on p.2240
2241Image
on p.2241

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baek K. J., Thiel B. A., Lucas S., Stuehr D. J. Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J Biol Chem. 1993 Oct 5;268(28):21120–21129. [PubMed] [Google Scholar]
  2. Beutler B., Milsark I. W., Cerami A. C. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science. 1985 Aug 30;229(4716):869–871. doi: 10.1126/science.3895437. [DOI] [PubMed] [Google Scholar]
  3. Blau N., Niederwieser A. Guanosine triphosphate cyclohydrolase I assay in human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine nucleotides. Anal Biochem. 1983 Feb 1;128(2):446–452. doi: 10.1016/0003-2697(83)90399-8. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  6. Bredt D. S., Snyder S. H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990 Jan;87(2):682–685. doi: 10.1073/pnas.87.2.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Busse R., Mülsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 1990 Jun 4;265(1-2):133–136. doi: 10.1016/0014-5793(90)80902-u. [DOI] [PubMed] [Google Scholar]
  8. Busse R., Mülsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett. 1990 Nov 26;275(1-2):87–90. doi: 10.1016/0014-5793(90)81445-t. [DOI] [PubMed] [Google Scholar]
  9. Cho H. J., Xie Q. W., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med. 1992 Aug 1;176(2):599–604. doi: 10.1084/jem.176.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Denis M. Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates. J Leukoc Biol. 1991 Apr;49(4):380–387. doi: 10.1002/jlb.49.4.380. [DOI] [PubMed] [Google Scholar]
  12. Doherty G. M., Lange J. R., Langstein H. N., Alexander H. R., Buresh C. M., Norton J. A. Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha. J Immunol. 1992 Sep 1;149(5):1666–1670. [PubMed] [Google Scholar]
  13. Fuchs D., Weiss G., Reibnegger G., Wachter H. The role of neopterin as a monitor of cellular immune activation in transplantation, inflammatory, infectious, and malignant diseases. Crit Rev Clin Lab Sci. 1992;29(3-4):307–341. doi: 10.3109/10408369209114604. [DOI] [PubMed] [Google Scholar]
  14. Fukushima T., Nixon J. C. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem. 1980 Feb;102(1):176–188. doi: 10.1016/0003-2697(80)90336-x. [DOI] [PubMed] [Google Scholar]
  15. Geller D. A., Lowenstein C. J., Shapiro R. A., Nussler A. K., Di Silvio M., Wang S. C., Nakayama D. K., Simmons R. L., Snyder S. H., Billiar T. R. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3491–3495. doi: 10.1073/pnas.90.8.3491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geller D. A., Nussler A. K., Di Silvio M., Lowenstein C. J., Shapiro R. A., Wang S. C., Simmons R. L., Billiar T. R. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):522–526. doi: 10.1073/pnas.90.2.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gerritsen M. E. Functional heterogeneity of vascular endothelial cells. Biochem Pharmacol. 1987 Sep 1;36(17):2701–2711. doi: 10.1016/0006-2952(87)90252-8. [DOI] [PubMed] [Google Scholar]
  18. Gimbrone M. A., Jr Culture of vascular endothelium. Prog Hemost Thromb. 1976;3:1–28. [PubMed] [Google Scholar]
  19. Giovanelli J., Campos K. L., Kaufman S. Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7091–7095. doi: 10.1073/pnas.88.16.7091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gross S. S., Jaffe E. A., Levi R., Kilbourn R. G. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991 Aug 15;178(3):823–829. doi: 10.1016/0006-291x(91)90965-a. [DOI] [PubMed] [Google Scholar]
  21. Gross S. S., Jaffe E. A., Levi R., Kilbourn R. G. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991 Aug 15;178(3):823–829. doi: 10.1016/0006-291x(91)90965-a. [DOI] [PubMed] [Google Scholar]
  22. Gross S. S., Levi R. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992 Dec 25;267(36):25722–25729. [PubMed] [Google Scholar]
  23. Hatakeyama K., Inoue Y., Harada T., Kagamiyama H. Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway. J Biol Chem. 1991 Jan 15;266(2):765–769. [PubMed] [Google Scholar]
  24. Hattori Y., Gross S. S. GTP cyclohydrolase I mRNA is induced by LPS in vascular smooth muscle: characterization, sequence and relationship to nitric oxide synthase. Biochem Biophys Res Commun. 1993 Aug 31;195(1):435–441. doi: 10.1006/bbrc.1993.2062. [DOI] [PubMed] [Google Scholar]
  25. Hevel J. M., Marletta M. A. Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity. Biochemistry. 1992 Aug 11;31(31):7160–7165. doi: 10.1021/bi00146a019. [DOI] [PubMed] [Google Scholar]
  26. Ignarro L. J., Fukuto J. M., Griscavage J. M., Rogers N. E., Byrns R. E. Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8103–8107. doi: 10.1073/pnas.90.17.8103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Iida S., Ohshima H., Oguchi S., Hata T., Suzuki H., Kawasaki H., Esumi H. Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats. J Biol Chem. 1992 Dec 15;267(35):25385–25388. [PubMed] [Google Scholar]
  28. Kilbourn R. G., Belloni P. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J Natl Cancer Inst. 1990 May 2;82(9):772–776. doi: 10.1093/jnci/82.9.772. [DOI] [PubMed] [Google Scholar]
  29. Kwon N. S., Nathan C. F., Gilker C., Griffith O. W., Matthews D. E., Stuehr D. J. L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen. J Biol Chem. 1990 Aug 15;265(23):13442–13445. [PubMed] [Google Scholar]
  30. Kwon N. S., Nathan C. F., Stuehr D. J. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J Biol Chem. 1989 Dec 5;264(34):20496–20501. [PubMed] [Google Scholar]
  31. Lamas S., Michel T., Brenner B. M., Marsden P. A. Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha. Am J Physiol. 1991 Oct;261(4 Pt 1):C634–C641. doi: 10.1152/ajpcell.1991.261.4.C634. [DOI] [PubMed] [Google Scholar]
  32. Lamas S., Michel T., Collins T., Brenner B. M., Marsden P. A. Effects of interferon-gamma on nitric oxide synthase activity and endothelin-1 production by vascular endothelial cells. J Clin Invest. 1992 Sep;90(3):879–887. doi: 10.1172/JCI115963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lyons C. R., Orloff G. J., Cunningham J. M. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem. 1992 Mar 25;267(9):6370–6374. [PubMed] [Google Scholar]
  34. Marsden P. A., Schappert K. T., Chen H. S., Flowers M., Sundell C. L., Wilcox J. N., Lamas S., Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett. 1992 Aug 3;307(3):287–293. doi: 10.1016/0014-5793(92)80697-f. [DOI] [PubMed] [Google Scholar]
  35. Milstien S., Kaufman S., Summer G. K. Hyperphenylalaninemia due to dihydropteridine reductase deficiency: diagnosis by measurement of oxidized and reduced pterins in urine. Pediatrics. 1980 Apr;65(4):806–810. [PubMed] [Google Scholar]
  36. Nakane M., Schmidt H. H., Pollock J. S., Förstermann U., Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 1993 Jan 25;316(2):175–180. doi: 10.1016/0014-5793(93)81210-q. [DOI] [PubMed] [Google Scholar]
  37. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  38. Nichol C. A., Smith G. K., Duch D. S. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 1985;54:729–764. doi: 10.1146/annurev.bi.54.070185.003501. [DOI] [PubMed] [Google Scholar]
  39. Nunokawa Y., Ishida N., Tanaka S. Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem Biophys Res Commun. 1993 Feb 26;191(1):89–94. doi: 10.1006/bbrc.1993.1188. [DOI] [PubMed] [Google Scholar]
  40. Nussler A. K., Di Silvio M., Billiar T. R., Hoffman R. A., Geller D. A., Selby R., Madariaga J., Simmons R. L. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J Exp Med. 1992 Jul 1;176(1):261–264. doi: 10.1084/jem.176.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ochoa J. B., Udekwu A. O., Billiar T. R., Curran R. D., Cerra F. B., Simmons R. L., Peitzman A. B. Nitrogen oxide levels in patients after trauma and during sepsis. Ann Surg. 1991 Nov;214(5):621–626. doi: 10.1097/00000658-199111000-00013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ohlsson K., Björk P., Bergenfeldt M., Hageman R., Thompson R. C. Interleukin-1 receptor antagonist reduces mortality from endotoxin shock. Nature. 1990 Dec 6;348(6301):550–552. doi: 10.1038/348550a0. [DOI] [PubMed] [Google Scholar]
  43. Olson E. N. Regulation of muscle transcription by the MyoD family. The heart of the matter. Circ Res. 1993 Jan;72(1):1–6. doi: 10.1161/01.res.72.1.1. [DOI] [PubMed] [Google Scholar]
  44. Pacher R., Redl H., Frass M., Petzl D. H., Schuster E., Woloszczuk W. Relationship between neopterin and granulocyte elastase plasma levels and the severity of multiple organ failure. Crit Care Med. 1989 Mar;17(3):221–226. doi: 10.1097/00003246-198903000-00004. [DOI] [PubMed] [Google Scholar]
  45. Palmer R. M., Ferrige A. G., Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987 Jun 11;327(6122):524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  46. Petros A., Bennett D., Vallance P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet. 1991 Dec 21;338(8782-8783):1557–1558. doi: 10.1016/0140-6736(91)92376-d. [DOI] [PubMed] [Google Scholar]
  47. Pober J. S., Gimbrone M. A., Jr, Lapierre L. A., Mendrick D. L., Fiers W., Rothlein R., Springer T. A. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol. 1986 Sep 15;137(6):1893–1896. [PubMed] [Google Scholar]
  48. Pollock J. S., Förstermann U., Mitchell J. A., Warner T. D., Schmidt H. H., Nakane M., Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10480–10484. doi: 10.1073/pnas.88.23.10480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Radomski M. W., Palmer R. M., Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10043–10047. doi: 10.1073/pnas.87.24.10043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Radomski M. W., Vallance P., Whitley G., Foxwell N., Moncada S. Platelet adhesion to human vascular endothelium is modulated by constitutive and cytokine induced nitric oxide. Cardiovasc Res. 1993 Jul;27(7):1380–1382. doi: 10.1093/cvr/27.7.1380. [DOI] [PubMed] [Google Scholar]
  51. Sakai N., Kaufman S., Milstein S. Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). Mol Pharmacol. 1993 Jan;43(1):6–10. [PubMed] [Google Scholar]
  52. Schmidt K., Werner E. R., Mayer B., Wachter H., Kukovetz W. R. Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells. Biochem J. 1992 Jan 15;281(Pt 2):297–300. doi: 10.1042/bj2810297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sessa W. C., Harrison J. K., Barber C. M., Zeng D., Durieux M. E., D'Angelo D. D., Lynch K. R., Peach M. J. Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase. J Biol Chem. 1992 Aug 5;267(22):15274–15276. [PubMed] [Google Scholar]
  54. Stuehr D. J., Cho H. J., Kwon N. S., Weise M. F., Nathan C. F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7773–7777. doi: 10.1073/pnas.88.17.7773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Suschek C., Rothe H., Fehsel K., Enczmann J., Kolb-Bachofen V. Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma. J Immunol. 1993 Sep 15;151(6):3283–3291. [PubMed] [Google Scholar]
  56. Tayeh M. A., Marletta M. A. Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J Biol Chem. 1989 Nov 25;264(33):19654–19658. [PubMed] [Google Scholar]
  57. Thiemermann C., Vane J. Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol. 1990 Jul 17;182(3):591–595. doi: 10.1016/0014-2999(90)90062-b. [DOI] [PubMed] [Google Scholar]
  58. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  59. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Schmidt K., Weiss G., Wachter H. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem. 1993 Jan 25;268(3):1842–1846. [PubMed] [Google Scholar]
  60. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Wachter H. Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J Exp Med. 1990 Dec 1;172(6):1599–1607. doi: 10.1084/jem.172.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Werner E. R., Werner-Felmayer G., Fuchs D., Hausen A., Reibnegger G., Wachter H. Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-gamma. Biochem J. 1989 Sep 15;262(3):861–866. doi: 10.1042/bj2620861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Werner E. R., Werner-Felmayer G., Fuchs D., Hausen A., Reibnegger G., Yim J. J., Wachter H. Impact of tumour necrosis factor-alpha and interferon-gamma on tetrahydrobiopterin synthesis in murine fibroblasts and macrophages. Biochem J. 1991 Dec 15;280(Pt 3):709–714. doi: 10.1042/bj2800709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. White K. A., Marletta M. A. Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry. 1992 Jul 28;31(29):6627–6631. doi: 10.1021/bi00144a001. [DOI] [PubMed] [Google Scholar]
  64. Wood E. R., Berger H., Jr, Sherman P. A., Lapetina E. G. Hepatocytes and macrophages express an identical cytokine inducible nitric oxide synthase gene. Biochem Biophys Res Commun. 1993 Mar 31;191(3):767–774. doi: 10.1006/bbrc.1993.1283. [DOI] [PubMed] [Google Scholar]
  65. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]
  66. Yoshizumi M., Perrella M. A., Burnett J. C., Jr, Lee M. E. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993 Jul;73(1):205–209. doi: 10.1161/01.res.73.1.205. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES