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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 microM methylene blue (IC50 = 5.3 and 9.2 microM in the absence and
presence of SOD, respectively), whereas guanylyl cyclase stimulated by S-nitrosoglutathione
was far less sensitive to the drug (50% inhibition at approximately 60 microM, 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.