Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs ¹

Abstract

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 (IC50'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 (IC50 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(.).

Regulation of normal cell cycle progression by flavin-containing oxidases

“…To examine the role of p53 and its effector p21 Waf1 (a universal inhibitor of cyclin-dependent kinases (cdk)) in flavin-containing oxidase(s)-mediated events that regulate G 1 to S transition, cellular progression into S phase was examined in sham-manipulated and DPI-treated wild type (wt), p53 À/À and p21 Waf1À/À MEFs. Similar to normal human fibroblasts (Figure 1b The DPI-induced G 1 checkpoint is mediated through NAD(P)H oxidase and not nitric oxide synthase Depending on cellular context and drug concentration, DPI inhibits several flavoprotein oxidoreductases, including NAD(P)H oxidase (Cross and Jones, 1986) and nitric oxide synthase (Stuehr et al, 1991). To examine the role of NAD(P)H oxidase in DPI modulation of G 1 to S transition, progression into S phase was investigated in DPI-treated wt and isogenic MEFs deficient in gp91 phox , the plasma membrane catalytic subunit of NAD(P)H oxidase in fibroblasts (Li et al, 2001).…”

Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function

“…Alternatively, a lowered muscle tissue perfusion could be responsible for a slower PCr recovery by reducing the availability of oxygen and substrate. DPI has been shown to inhibit endothelial nitric oxide synthase (eNOS), an enzyme involved in vascular vasodilation (54). Inhibition of eNOS lowers muscle blood flow during recovery from exercise in humans (8,47) and therefore we cannot exclude that a lower muscle perfusion in DPI-treated animals may have affected k PCr .…”

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

“…DPI, a non-specific NADPH oxidase inhibitor, prevents electron transport within the NADPH oxidase multisubunit complex (O'Donnell et al, 1993;Li and Trush, 1998;Doussiere et al, 1999). Because of its action, however, DPI can also inhibit other electron transporters, like nitric oxide synthase (Stuehr et al, 1991), xanthine oxidase (Doussiere and Vignais, 1992), and mitochondrial complex I (Li and Trush, 1998). APO, which needs activation by peroxidases, inhibits the association of the NADPH oxidase cytosolic subunits (Stolk et al, 1994).…”