Elsevier

Biochemical Pharmacology

Volume 45, Issue 2, 26 January 1993, Pages 367-374
Biochemical Pharmacology

Inhibition of nitric oxide synthesis by methylene blue

https://doi.org/10.1016/0006-2952(93)90072-5Get rights and content

Abstract

Methylene blue appears to inhibit nitric oxide-stimulated soluble guanylyl cyclase and has been widely used for inhibition of cGMP-mediated processes. We report here that endothelium-dependent relaxation of isolated blood vessels and NO synthase-dependent cGMP formation in cultured endothelial cells were both markedly more sensitive to inhibition by methylene blue than effects induced by direct activation of soluble guanylyl cyclase. These discrepancies were also observed when Superoxide dismutase (SOD) was present to protect NO from inactivation by Superoxide anion. Subsequent experiments showed that formation of l-citrulline by purified NO synthase was completely inhibited by 30 μM methylene blue (ic50 = 5.3 and 9.2 μM in the absence and presence of SOD, respectively), whereas guanylyl cyclase stimulated by 5-nitrosoglutathione was far less sensitive to the drug (50% inhibition at ~60μM, and maximal inhibition of 72% at 1 mM methylene blue). Experimental evidence indicated that oxidation of NADPH, tetrahydrobiopterin or reduced flavins does not account for the inhibitory effects of methylene blue. Our data suggest that methylene blue acts as a direct inhibitor of NO synthase and is a much less specific and potent inhibitor of guanylyl cyclase than hitherto assumed.

References (48)

  • B Mayer et al.

    Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase

    FEBS Lett

    (1991)
  • B Mayer et al.

    Purification of a Ca2+/ calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydro-biopterin

    FEBS Lett

    (1990)
  • P Klatt et al.

    Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase

    J Biol Chem

    (1992)
  • F Brunner et al.

    Characterization of muscarinic receptors mediating endothelium-dependent relaxation of bovine coronary artery

    Eur J Pharmacol

    (1991)
  • K Schmidt et al.

    Effect of calcium on endothelium-derived relaxing factor formation and cGMP levels in endothelial cells

    Eur J Pharmacol

    (1989)
  • PA Craven et al.

    Effects of thiol inhibitors on hepatic guanylate cyclase activity—evidence for involvement of vicinal dithiols in the expression of basal and agonist-stimulated activity

    Biochim Biophys Acta

    (1978)
  • RMJ Palmer et al.

    A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells

    Biochem Biophys Res Commun

    (1989)
  • LJ Ignarro et al.

    Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme

    Biochim Biophys Acta

    (1987)
  • M Feelisch et al.

    Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells

    Biochem Biophys Res Commun

    (1991)
  • D Servent et al.

    Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

    Biochem Biophys Res Commun

    (1989)
  • S Moncada et al.

    Nitric oxide physiology, pathophysiology, and pharmacology

    Pharmacol Rev

    (1991)
  • JS Pollock et al.

    Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells

  • EH Ohlstein et al.

    Purification and properties of heme-deficient hepatic soluble guanylate cyclase: effects of heme and other factors on enzyme activation by NO, NO-heme, and protoporphyrin IX

    Arch Biochem Biophys

    (1982)
  • SC Tsai et al.

    Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide

    Biochem J

    (1983)
  • Cited by (0)

    View full text