Skip to main content
PMC home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1996 Jul;118(6):1433–1440. doi: 10.1111/j.1476-5381.1996.tb15557.x

Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine.

S Pfeiffer 1, E Leopold 1, K Schmidt 1, F Brunner 1, B Mayer 1
PMCID: PMC1909689  PMID: 8832069

Abstract

1. The L-arginine derivatives NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2. A bolus of L-NAME and L-NOARG (0.25 micromol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +/- 0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3. Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 microM) than L-NOARG (IC50 = 1.4 microM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4. Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 +/- 11.2 min in buffer (pH 7.4), 207 +/- 1.7 min in human plasma, and 29 +/- 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5. These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.

Full text

PDF
1433

Selected References

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

  1. Baer H. P., Schmidt K., Mayer B., Kukovetz W. R. Pentamidine does not interfere with nitrite formation in activated RAW 264.7 macrophages but inhibits constitutive brain nitric oxide synthase. Life Sci. 1995;57(21):1973–1980. doi: 10.1016/0024-3205(95)02183-j. [DOI] [PubMed] [Google Scholar]
  2. Baydoun A. R., Mann G. E. Selective targeting of nitric oxide synthase inhibitors to system y+ in activated macrophages. Biochem Biophys Res Commun. 1994 Apr 29;200(2):726–731. doi: 10.1006/bbrc.1994.1511. [DOI] [PubMed] [Google Scholar]
  3. Bogle R. G., Moncada S., Pearson J. D., Mann G. E. Identification of inhibitors of nitric oxide synthase that do not interact with the endothelial cell L-arginine transporter. Br J Pharmacol. 1992 Apr;105(4):768–770. doi: 10.1111/j.1476-5381.1992.tb09053.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brouillet E., Roeda D., Valette H., Fuseau C., Guyot M. C., Crouzel C. In vivo metabolites of N omega-nitro-L-arginine methyl ester: methanol and N omega-nitro-L-arginine. Eur J Pharmacol. 1995 Dec 7;293(4):487–490. doi: 10.1016/0926-6917(95)90070-5. [DOI] [PubMed] [Google Scholar]
  5. Brunner F., Kühberger E., Schloos J., Kukovetz W. R. Characterization of muscarinic receptors of bovine coronary artery by functional and radioligand binding studies. Eur J Pharmacol. 1991 Apr 24;196(3):247–255. doi: 10.1016/0014-2999(91)90437-u. [DOI] [PubMed] [Google Scholar]
  6. Brunner F. Tissue endothelin-1 levels in perfused rat heart following stimulation with agonists and in ischaemia and reperfusion. J Mol Cell Cardiol. 1995 Sep;27(9):1953–1963. doi: 10.1016/0022-2828(95)90017-9. [DOI] [PubMed] [Google Scholar]
  7. Bryant C. E., Allcock G. H., Warner T. D. Comparison of effects of chronic and acute administration of NG-nitro-L-arginine methyl ester to the rat on inhibition of nitric oxide-mediated responses. Br J Pharmacol. 1995 Apr;114(8):1673–1679. doi: 10.1111/j.1476-5381.1995.tb14956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buxton I. L., Cheek D. J., Eckman D., Westfall D. P., Sanders K. M., Keef K. D. NG-nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists. Circ Res. 1993 Feb;72(2):387–395. doi: 10.1161/01.res.72.2.387. [DOI] [PubMed] [Google Scholar]
  9. Cheng D. Y., DeWitt B. J., McMahon T. J., Kadowitz P. J. Comparative effects of L-NNA and alkyl esters of L-NNA on pulmonary vasodilator responses to ACh, BK, and SP. Am J Physiol. 1994 Jun;266(6 Pt 2):H2416–H2422. doi: 10.1152/ajpheart.1994.266.6.H2416. [DOI] [PubMed] [Google Scholar]
  10. Collier J., Vallance P. Physiological importance of nitric oxide. BMJ. 1991 Jun 1;302(6788):1289–1290. doi: 10.1136/bmj.302.6788.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forray M. I., Angelo S., Boyd C. A., Devés R. Transport of nitric oxide synthase inhibitors through cationic amino acid carriers in human erythrocytes. Biochem Pharmacol. 1995 Dec 22;50(12):1963–1968. doi: 10.1016/0006-2952(95)02090-x. [DOI] [PubMed] [Google Scholar]
  12. Fukuto J. M., Chaudhuri G. Inhibition of constitutive and inducible nitric oxide synthase: potential selective inhibition. Annu Rev Pharmacol Toxicol. 1995;35:165–194. doi: 10.1146/annurev.pa.35.040195.001121. [DOI] [PubMed] [Google Scholar]
  13. Garthwaite J., Boulton C. L. Nitric oxide signaling in the central nervous system. Annu Rev Physiol. 1995;57:683–706. doi: 10.1146/annurev.ph.57.030195.003343. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Harteneck C., Klatt P., Schmidt K., Mayer B. Expression of rat brain nitric oxide synthase in baculovirus-infected insect cells and characterization of the purified enzyme. Biochem J. 1994 Dec 15;304(Pt 3):683–686. doi: 10.1042/bj3040683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heinzel B., John M., Klatt P., Böhme E., Mayer B. Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. Biochem J. 1992 Feb 1;281(Pt 3):627–630. doi: 10.1042/bj2810627. [DOI] [PMC free article] [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. Klatt P., Schmidt K., Brunner F., Mayer B. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. J Biol Chem. 1994 Jan 21;269(3):1674–1680. [PubMed] [Google Scholar]
  19. Krejcy K., Schwarzacher S., Raberger G. Distribution and metabolism of NG-nitro-L-arginine and NG-nitro-L-arginine methylester in canine blood in vitro. Naunyn Schmiedebergs Arch Pharmacol. 1993 Mar;347(3):342–345. doi: 10.1007/BF00167455. [DOI] [PubMed] [Google Scholar]
  20. Lambert L. E., Whitten J. P., Baron B. M., Cheng H. C., Doherty N. S., McDonald I. A. Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. Life Sci. 1991;48(1):69–75. doi: 10.1016/0024-3205(91)90426-c. [DOI] [PubMed] [Google Scholar]
  21. Lewis M. J. Nitric oxide and regulation of coronary vascular tone. Cardiovasc Res. 1992 May;26(5):555–556. doi: 10.1093/cvr/26.5.555. [DOI] [PubMed] [Google Scholar]
  22. Malenka R. C. Synaptic plasticity in the hippocampus: LTP and LTD. Cell. 1994 Aug 26;78(4):535–538. doi: 10.1016/0092-8674(94)90517-7. [DOI] [PubMed] [Google Scholar]
  23. Mayer B., Klatt P., Werner E. R., Schmidt K. Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole. Neuropharmacology. 1994 Nov;33(11):1253–1259. doi: 10.1016/0028-3908(94)90024-8. [DOI] [PubMed] [Google Scholar]
  24. Mayer B., Schmid M., Klatt P., Schmidt K. Reversible inactivation of endothelial nitric oxide synthase by NG-nitro-L-arginine. FEBS Lett. 1993 Oct 25;333(1-2):203–206. doi: 10.1016/0014-5793(93)80405-j. [DOI] [PubMed] [Google Scholar]
  25. Mayer B., Schmidt K., Humbert P., Böhme E. Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase. Biochem Biophys Res Commun. 1989 Oct 31;164(2):678–685. doi: 10.1016/0006-291x(89)91513-1. [DOI] [PubMed] [Google Scholar]
  26. Mayer B., Werner E. R. In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide. Naunyn Schmiedebergs Arch Pharmacol. 1995 May;351(5):453–463. doi: 10.1007/BF00171035. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Moore P. K., Oluyomi A. O., Babbedge R. C., Wallace P., Hart S. L. L-NG-nitro arginine methyl ester exhibits antinociceptive activity in the mouse. Br J Pharmacol. 1991 Jan;102(1):198–202. doi: 10.1111/j.1476-5381.1991.tb12153.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mülsch A., Busse R. NG-nitro-L-arginine (N5-[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedebergs Arch Pharmacol. 1990 Jan-Feb;341(1-2):143–147. doi: 10.1007/BF00195071. [DOI] [PubMed] [Google Scholar]
  30. Nathan C. F., Hibbs J. B., Jr Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol. 1991 Feb;3(1):65–70. doi: 10.1016/0952-7915(91)90079-g. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Palmer R. M., Rees D. D., Ashton D. S., Moncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun. 1988 Jun 30;153(3):1251–1256. doi: 10.1016/s0006-291x(88)81362-7. [DOI] [PubMed] [Google Scholar]
  33. Pou S., Pou W. S., Bredt D. S., Snyder S. H., Rosen G. M. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem. 1992 Dec 5;267(34):24173–24176. [PubMed] [Google Scholar]
  34. Rand M. J., Li C. G. Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission. Annu Rev Physiol. 1995;57:659–682. doi: 10.1146/annurev.ph.57.030195.003303. [DOI] [PubMed] [Google Scholar]
  35. Rees D. D., Palmer R. M., Hodson H. F., Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol. 1989 Feb;96(2):418–424. doi: 10.1111/j.1476-5381.1989.tb11833.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Rees D. D., Palmer R. M., Schulz R., Hodson H. F., Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol. 1990 Nov;101(3):746–752. doi: 10.1111/j.1476-5381.1990.tb14151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Santiago J. A., Garrison E. A., Kadowitz P. J. Comparative effects of N omega-nitro-L-arginine and N omega-nitro-L-arginine methyl ester on vasodilator responses to acetylcholine, bradykinin, and substance P. Eur J Pharmacol. 1994 Mar 21;254(3):207–212. doi: 10.1016/0014-2999(94)90456-1. [DOI] [PubMed] [Google Scholar]
  39. Schmidt K., Klatt P., Mayer B. Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors. Mol Pharmacol. 1993 Sep;44(3):615–621. [PubMed] [Google Scholar]
  40. Schmidt K., Klatt P., Mayer B. Uptake of nitric oxide synthase inhibitors by macrophage RAW 264.7 cells. Biochem J. 1994 Jul 15;301(Pt 2):313–316. doi: 10.1042/bj3010313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schmidt K., List B. M., Klatt P., Mayer B. Characterization of neuronal amino acid transporters: uptake of nitric oxide synthase inhibitors and implication for their biological effects. J Neurochem. 1995 Apr;64(4):1469–1475. doi: 10.1046/j.1471-4159.1995.64041469.x. [DOI] [PubMed] [Google Scholar]
  42. Takahashi N., Tanaka H., Abdullah N., Jing L., Inoue R., Ito Y. Regional difference in the distribution of L-NAME-sensitive and -insensitive NANC relaxations in cat airway. J Physiol. 1995 Nov 1;488(Pt 3):709–720. doi: 10.1113/jphysiol.1995.sp021002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. White D. G., Drew G. M., Gurden J. M., Penny D. M., Roach A. G., Watts I. S. The effect of NG-nitro-L-arginine methyl ester upon basal blood flow and endothelium-dependent vasodilatation in the dog hindlimb. Br J Pharmacol. 1993 Mar;108(3):763–768. doi: 10.1111/j.1476-5381.1993.tb12875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Whiting M. J., Rutten A. J., Williams P., Bersten A. D. Determination of NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester in plasma by high-performance liquid chromatography. J Chromatogr B Biomed Appl. 1994 Oct 3;660(1):170–175. doi: 10.1016/0378-4347(94)00257-6. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES