Diethyldithiocarbamate inhibits induction of macrophage NO synthase

Abstract

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...

Effect of cyclic GMP‐dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP

“…Amongst the 24 consensus sequences that have been identified in the promoter region of the murine iNOS gene are putative binding sites for transcription factors including NF-KB and NFIL-6 whose activity can be stimulated by protein kinase(s) A, protein kinase C and protein tyrosine kinase Ghosh & Baltimore, 1990;Metz & Ziff, 1991;Link et al, 1992). Although the role of NFIL-6 still remains to be clarified, activation of NF-KB seems to be a necessary event for the expression of iNOS in lipopolysaccharide-stimulated macrophages and in IL-lfl-activated vascular smooth muscle cells (Xie et al, 1993;Sherman et al, 1993;MUlsch et al, 1993;Schini-Kerth et al, 1994b). Therefore, the stimulating effect of cyclic AMP on iNOS expression in vascular smooth muscle cells could occur at the level of NF-KB activation.…”

Inhibitors of Inducible Nitric Oxide Synthase Prevent Damage to Human Lens Epithelial Cells Induced by Interferon-Gamma and Lipopolysaccharide

“…15) Aminoguanidine (AG) inhibits the formation of advanced glycosylation end-products (AGE), considered to be a factor in diabetic cataract development, 16) and an inducible nitric oxide synthase (iNOS) inhibitor. 17) We previously reported that the increase in Ca 2ϩ concentration in lenses resulted in cataract development in cataract model rats, and the induction of iNOS protein occurred prior to the elevation of Ca 2ϩ content in the lenses.…”

Effect of Disulfiram Eye Drops on Lipid Peroxide Formation via Excessive Nitric Oxide in Lenses of Hereditary Cataract ICR/f Rats

“…NO 2 Ϫ and NO 3 Ϫ in the supernatant were separated using a reversephase separation column packed with polystyrene polymer (NO-PAK, 4.6ϫ50 mm, Eicom, Kyoto, Japan), and NO 3 Ϫ was reduced to NO 2 Ϫ in a reduction column packed with copperplated cadmium filings (NO-RED, Eicom, Kyoto, Japan). NO 2 Ϫ was mixed with a Griess's reagent to form a purple azo dye in a reaction coil, and placed in a column oven set at 35°C. The absorbance of the color product dye at 540 nm was determined by a flow-through spectrophotometer (NOD-10, Eicom, Kyoto, Japan).…”

“…Diethyldithiocarbamate (DDC), a potent radical scavenger 1) and nitric oxide synthase inhibitor, 2) has been shown to inhibit lipid peroxidation and hydroxyl radicals in rats, 3,4) and has an anti-cataract effect in selenite-treated rats. 5,6) However, DDC is unstable in neutral solution, and is not able penetrate through the cornea into the aqueous humor.…”