Production of nitric oxide and other iron-containing metabolites during the reductive metabolism of nitroprusside by microsomes and by thiols

Abstract

Sodium nitroprusside is used as a hypotensive agent because of its ability to produce nitric oxide (NO), although direct demonstration of this has not been reported in a biological system. Nitroprusside (NP) nitroxide radical anion, the first reduction product of NP generated in the presence of microsomes and NADPH, was found to undergo further metabolism. One of the products produced during this reductive metabolism was shown to be NO. By using N-methylglucamine dithiocarbamate-FeCl2 complex [(NMGD)2Fe(II)] as the NO trap, we have detected and characterized the mononitroso bis(N-methylglucamine dithiocarbamato) iron (II) complex (MNBI) (g = 2.040, and A(14N) = 13.3 G) as the product of NO trapping. The production of NO during the reductive metabolism of NP by submitochondrial particles and a human HepG2 hepatoblastoma cell line was also demonstrated using (NMGD)2Fe(II). In addition to MNBI, two other mononitrosyl iron complexes, the NP nitroxide radical anion and a second species designated as Fe(NO)(X)(Y) (g = 2.032, and A(14N) = 14.3 G), and additional unidentified paramagnetic products containing iron were also detected. Thiol compounds such as glutathione, cysteine, and cysteamine reduce NP to generate NP nitroxide radical anion and a paramagnetic species characterized as a dithiolated dinitroso iron complex (DDIC), Fe(NO)2(RS)2 g = 2.030, A(14N) = 2.2 G(2N), and A(1H) = 1.1 G(4H). At 77 K, DDIC generated from cysteine and NP has an axial symmetry, with g perpendicular = 2.040, and g parallel = 2.014. Two additional paramagnetic products, designated as species C (g = 2.020, linewidth = 4.8 G) and species D (g = 2.008, linewidth = 6.1 G), were also formed during NP reduction by thiol compounds. The characterization of these complexes has been hampered by the lack of hyperfine features in the ESR spectra. The production of NO during cysteamine reduction of NP was demonstrated by using (NMGD)2-Fe(II) as the spin-trap. These results directly demonstrate the production of NO during the reduction of NP by microsomes plus NADPH or by thiols. (NMGD)2-Fe(II) is a particularly useful spin-trap for the detection of NO in a strong reducing environment.