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. 1993 Dec 1;90(23):11419–11423. doi: 10.1073/pnas.90.23.11419

Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.

I G Charles 1, R M Palmer 1, M S Hickery 1, M T Bayliss 1, A P Chubb 1, V S Hall 1, D W Moss 1, S Moncada 1
PMCID: PMC47994  PMID: 7504305

Abstract

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.

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Selected References

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

  1. Aisaka K., Gross S. S., Griffith O. W., Levi R. NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo? Biochem Biophys Res Commun. 1989 Apr 28;160(2):881–886. doi: 10.1016/0006-291x(89)92517-5. [DOI] [PubMed] [Google Scholar]
  2. Alexander J., Snoke K., Ruppert J., Sidney J., Wall M., Southwood S., Oseroff C., Arrhenius T., Gaeta F. C., Colón S. M. Functional consequences of engagement of the T cell receptor by low affinity ligands. J Immunol. 1993 Jan 1;150(1):1–7. [PubMed] [Google Scholar]
  3. Arend W. P., Dayer J. M. Cytokines and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum. 1990 Mar;33(3):305–315. doi: 10.1002/art.1780330302. [DOI] [PubMed] [Google Scholar]
  4. Beckman J. S., Beckman T. W., Chen J., Marshall P. A., Freeman B. A. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1620–1624. doi: 10.1073/pnas.87.4.1620. [DOI] [PMC free article] [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. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989 Nov;86(22):9030–9033. doi: 10.1073/pnas.86.22.9030. [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. Charles I. G., Keyte J. W., Brammar W. J., Smith M., Hawkins A. R. The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans. Nucleic Acids Res. 1986 Mar 11;14(5):2201–2213. doi: 10.1093/nar/14.5.2201. [DOI] [PMC free article] [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. Di Rosa M., Radomski M., Carnuccio R., Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1246–1252. doi: 10.1016/0006-291x(90)91583-e. [DOI] [PubMed] [Google Scholar]
  11. Furchgott R. F., Zawadzki J. V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980 Nov 27;288(5789):373–376. doi: 10.1038/288373a0. [DOI] [PubMed] [Google Scholar]
  12. Garthwaite J., Charles S. L., Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature. 1988 Nov 24;336(6197):385–388. doi: 10.1038/336385a0. [DOI] [PubMed] [Google Scholar]
  13. Garthwaite J. Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 1991 Feb;14(2):60–67. doi: 10.1016/0166-2236(91)90022-m. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gillespie J. S., Liu X. R., Martin W. The effects of L-arginine and NG-monomethyl L-arginine on the response of the rat anococcygeus muscle to NANC nerve stimulation. Br J Pharmacol. 1989 Dec;98(4):1080–1082. doi: 10.1111/j.1476-5381.1989.tb12650.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  17. Hibbs J. B., Jr, Vavrin Z., Taintor R. R. L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol. 1987 Jan 15;138(2):550–565. [PubMed] [Google Scholar]
  18. Ignarro L. J., Buga G. M., Wood K. S., Byrns R. E., Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9265–9269. doi: 10.1073/pnas.84.24.9265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. James S. L., Hibbs J. B., Jr The role of nitrogen oxides as effector molecules of parasite killing. Parasitol Today. 1990 Sep;6(9):303–305. doi: 10.1016/0169-4758(90)90261-2. [DOI] [PubMed] [Google Scholar]
  20. Janssens S. P., Shimouchi A., Quertermous T., Bloch D. B., Bloch K. D. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem. 1992 Jul 25;267(21):14519–14522. [PubMed] [Google Scholar]
  21. Knowles R. G., Palacios M., Palmer R. M., Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5159–5162. doi: 10.1073/pnas.86.13.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lamas S., Marsden P. A., Li G. K., Tempst P., Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6348–6352. doi: 10.1073/pnas.89.14.6348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liew F. Y., Millott S., Parkinson C., Palmer R. M., Moncada S. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol. 1990 Jun 15;144(12):4794–4797. [PubMed] [Google Scholar]
  25. Lowenstein C. J., Glatt C. S., Bredt D. S., Snyder S. H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6711–6715. doi: 10.1073/pnas.89.15.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moncada S., Palmer R. M., Higgs E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991 Jun;43(2):109–142. [PubMed] [Google Scholar]
  30. 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]
  31. Nguyen Q., Mort J. S., Roughley P. J. Preferential mRNA expression of prostromelysin relative to procollagenase and in situ localization in human articular cartilage. J Clin Invest. 1992 Apr;89(4):1189–1197. doi: 10.1172/JCI115702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Palmer R. M., Andrews T., Foxwell N. A., Moncada S. Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes. Biochem Biophys Res Commun. 1992 Oct 15;188(1):209–215. doi: 10.1016/0006-291x(92)92371-4. [DOI] [PubMed] [Google Scholar]
  34. Palmer R. M., Ashton D. S., Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988 Jun 16;333(6174):664–666. doi: 10.1038/333664a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Palmer R. M., Hickery M. S., Charles I. G., Moncada S., Bayliss M. T. Induction of nitric oxide synthase in human chondrocytes. Biochem Biophys Res Commun. 1993 May 28;193(1):398–405. doi: 10.1006/bbrc.1993.1637. [DOI] [PubMed] [Google Scholar]
  37. Palmer R. M., Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun. 1989 Jan 16;158(1):348–352. doi: 10.1016/s0006-291x(89)80219-0. [DOI] [PubMed] [Google Scholar]
  38. Palmer R. M., Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun. 1989 Jan 16;158(1):348–352. doi: 10.1016/s0006-291x(89)80219-0. [DOI] [PubMed] [Google Scholar]
  39. Radomski M. W., Palmer R. M., Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5193–5197. doi: 10.1073/pnas.87.13.5193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Rees D. D., Palmer R. M., Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A. 1989 May;86(9):3375–3378. doi: 10.1073/pnas.86.9.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Salter M., Knowles R. G., Moncada S. Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases. FEBS Lett. 1991 Oct 7;291(1):145–149. doi: 10.1016/0014-5793(91)81123-p. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schulz R., Smith J. A., Lewis M. J., Moncada S. Nitric oxide synthase in cultured endocardial cells of the pig. Br J Pharmacol. 1991 Sep;104(1):21–24. doi: 10.1111/j.1476-5381.1991.tb12378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Stadler J., Stefanovic-Racic M., Billiar T. R., Curran R. D., McIntyre L. A., Georgescu H. I., Simmons R. L., Evans C. H. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J Immunol. 1991 Dec 1;147(11):3915–3920. [PubMed] [Google Scholar]
  47. Stuehr D. J., Griffith O. W. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Mol Biol. 1992;65:287–346. doi: 10.1002/9780470123119.ch8. [DOI] [PubMed] [Google Scholar]
  48. Stuehr D. J., Ikeda-Saito M. Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical. J Biol Chem. 1992 Oct 15;267(29):20547–20550. [PubMed] [Google Scholar]
  49. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]

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