Inhibition by methylene blue of the L-arginine metabolism to L-citrulline coupled with nitric oxide synthesis in cultured endothelial cells.
The effect of methylene blue on the metabolism of L-arginine to L-citrulline coupled with nitric oxide synthesis in cultured endothelial cells was investigated. The addition of the calcium ionophore ionomycin (10(-8)-10(-5) M) to endothelial cells stimulated L-citrulline formation from L-arginine. Ionomycin-stimulated L-citrulline formation was inhibited by NG-nitro-L-arginine (10(-6)-10(-4) M), a potent inhibitor of nitric oxide synthase. These results indicate that ionomycin-stimulated L-citrulline formation was coupled to nitric oxide synthesis. Therefore, L-citrulline formation from L-arginine was used as a marker for nitric oxide synthesis. Methylene blue (10(-4)-10(-3) M) inhibited ionomycin-stimulated L-citrulline formation in bovine aortic endothelial cells. Moreover, the formation of L-citrulline by the particulate fraction obtained from bovine cultured endothelial cells was also inhibited by methylene blue concentrations in excess of 10(-5) M. Although the addition of methylene blue to various tissues has been reported to release superoxide anions (Wolin et al., 1990; Marczin et al., 1992), inhibition of L-citrulline formation by methylene blue was not affected by the presence of superoxide dismutase (100 micrograms/mL) in either cultured endothelial cells or the particulate fraction. These findings suggest that methylene blue is a direct inhibitor of nitric oxide synthase in bovine aortic endothelial cells.
Shimizu
S
S
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Yamamoto
T
T
Momose
K
K
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors; D000818:Animals; D001013:Aorta, Thoracic; D001120:Arginine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015759:Ionomycin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008751:Methylene Blue / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D013482:Superoxide Dismutase / Q000494:pharmacology
D019335:Nitroarginine; D002956:Citrulline; D009569:Nitric Oxide; D015759:Ionomycin; D001120:Arginine; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D000594:Amino Acid Oxidoreductases; D008751:Methylene Blue