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Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs 1
Author(s) -
Stuehr Dennis J.,
Fasehun Olufunmilayo A.,
Kwon Nyoun Soo,
Gross Steven S.,
Gonzalez Jose A.,
Levi Roberto,
Nathan Carl F.
Publication year - 1991
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.5.1.1703974
Subject(s) - nitric oxide synthase , sodium nitroprusside , flavoprotein , chemistry , atp synthase , nitric oxide , biochemistry , tetrahydrobiopterin , flavin adenine dinucleotide , nadph oxidase , enzyme , cofactor , organic chemistry
The cofactor requirements of macrophage nitric oxide (NO·) synthase suggest involvement of an NADPH‐dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di‐2‐thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO· synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO· synthesis by mouse macrophages, their lysates, and partially purified macrophage NO· synthase. Inhibition of NO· synthase by these agents was potent (IC 50 ‘s 50–150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP + , or 2'5‘‐ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5‐diphosphoribose, protected NO· synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO· synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine‐induced relaxation of norepinephrine‐preconstricted rabbit aortic rings (IC 50 300 nM). Inhibition of acetylcholine‐induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine‐induced vasoconstriction itself nor on vasorelaxation induced by the NO·‐generating agent sodium nitroprusside. These results suggest that NO∗ synthesis in both macrophages and endothelial cells depends on an NADPH‐utilizing flavoprotein. As a new class of NO· synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO·.—Stuehr, D. J.; Fasehun, O. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.; Nathan, C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 5: 98–103; 1991.