The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO) 2 Fe(L-cysteine) 2 ], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO) 2 Fe(L-cysteine) 2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NO x -species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyliron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO) 2 Fe(Lcysteine) 2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyliron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

We studied the biological aettvity, stability and nteraction of :lnitrosyl-iron(IT)-L-cysteine with vascular tissue. Dinitrosyl-iron(iI)-L-cysteine was a potent activator of purified soluble guanylyl _2~clase (ECso tnM with and 10g} nM without superoJtide dismutase) and relaxed noradrenalin¢~ precontracted segments of endothelium-denuded rabbit fem,.rxl artery (ECso 10 nM superoxide dismutase). Pre-incubation (5 min; 310 K) of cndothelium-denuded rabbit aortic segments witi, dinitrosyl-i c n(II~-L-cysteine (0.036-3.6 raM) resulted in a concentration-dependent formation ofa dinitrosyl-iron(I! complex with protein tbiol g:oups, as l~-tocted by ESR spectroscopy. While the complex with proteins was stable for 2 h at 310 K, dinitrosyi-iron(ll)-L-cysteinc in aqueous ~-olution (3, -360MM) de~0mposed completely within 15 min, as indicated by disappearance of its isotropic ESR signal at g,, = 2.03 (293 K). Aot~i,." segment: :,re-incubated with dinitrosyl-iron(Ill-L-cysteine released a labile vasodilati,g and guanylyl cyclase activating factor. Perfusion of thtse segmer. ,_ with N-aoetyI-L-cysteine resulted in the generation of a low molecular weight dinitrosyl-iron(II)-dithiolate from the dinitrosy'-iT on(II) comp z : with proteins, a~ revealed by the shape change of the ESR signal at 293 K. The low molecular weight dinitrosyl-iromll)-dithiolat¢ accounted ! )-an enhanced guanylyl cyclase activation and vasodilation induced by the aortic effluent. We conctude that nitric oxide (NO) produced by, or z : ins on vasc,Jlar cells can be stabilized and stored as a dinitrosyl-iron(lI) complex with protein thiols, and can be released from cells in the form era low molecular weight dinffrosyl-iron(II)-dithiolate by int-*a-and ¢xtraecllular thiois.Oinitrogyl-irot~(ll'.-dithiolate; Endothelium-derived relaxing f t. tor: Soluble guanyiyl cyclage: N-AcetyI-L-<'ysteine; Nitric oxide: Electron spin re: ~ nanoe spectroscopy

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We investigated whether sodium diethyldithiocarbamate @ETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell mediumpreconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1-l mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.NO synthase; Transcription; NFkappaB; Diethyldithiocarbamate; Bone marrow-derived macrophage 1, INTRODUCTION Intra-and extracellular pathogens, their breakdown products, several cytokines and a plethora of other noxious agents and conditions induce the expression of NO synthase in mammalian cells (for review see [l-3]). NO generated from L-arginine by the inducible NO synthase (iNOS) is regarded as a defense effector molecule with cytotoxic/cytostatic and microbicidal/microbiostatic activity [1,2]. To date little or nothing is known about the transduction cascade that unifies all these pathogenic signals translating them into activation of the iNOS gene. Eukaryotic gene expression is controlled by promotor and enhancer DNA sequences, which are activated by specific protein factors. One of the nuclear transcription factors initially identified in B-cells and monocytes, the multiprotein complex NFkappaB [4], is rapidly activated upon perturbation of cells by conditions similar to those known to induce NOS. NFkappaB is known to mediate the immediate-early gene response by enhancing the transcription of a multitude of genes encoding defense and signalling proteins, such as cytokines and cytokine receptors [4]. Activation of NFkappaB can be specifically prevented by antioxidants, thiols and iron chelators, and especially by dithiocarbamates [4]. To clarify whether NFkappaB participates in the induction of NOS, we assessed the effect of diethyldithiocarbamate (DETC) on iNOS mRNA levels and expression of NOS activity in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages. MATERIALS AND METHODSBone marrow cells from the femurs of Balb/c mice were isolated and cultured in L929-cell conditioned medium as described recently [5]. Cells grown on culture dishes (3.5 cm diameter; 10' cells/dish) were exposed to LPS (0.1 &ml; E. coli serotype 055:B5), DETC (0.01 to 1 mM; both from Sigma, Deisenhofen, Germany) and hemin (Normosang, Leiras, Turku, Finland) according to the protocols described in section 3.Messenger RNA encoding iNOS was assessed by Northern blot technique using a HincIIlSSP I fragment of the mouse iNOS [6] as a cDNA probe. The cDNA probe was a generous gift of Drs. Q. Xie and C.F. Nathan, Cornell University Medi...

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