DIFFERENTIAL EFFECTS OF SOME CELL SIGNALLING INHIBITORS UPON NITRIC OXIDE SYNTHASE EXPRESSION AND NUCLEAR FACTOR-κB ACTIVATION INDUCED BY LIPOPOLYSACCHARIDE IN RAT AORTIC SMOOTH MUSCLE CELLS

Abstract

Treatment of rat aortic smooth muscle cells (RASMC) with 1 or 100 μg ml−1lipopolysaccharide (LPS) for 20–24 h led to expression of the inducible form of nitric oxide synthase (iNOS) as detected by Western blotting for iNOS protein, and by determination of increased cellular nitrite formation. LPS-induced nitrite production was inhibited almost completely by concomitant treatment of cells with LPS and either (a) pyrrolidine dithiocarbamate (PDTC, 25 μm), an antioxidant inhibitor of NF-κB activation; (b)N-tosyl-l-phenylalanine chloromethyl ketone (TPCK, 20 and 40 μm), a proteasomal inhibitor which prevents NF-κB activation; (c) nordihydroguaiaretic acid (NDGA, 10 and 50 μm), a lipoxygenase inhibitor; or (d) apocynin (2, 3.5 and 5 mm), an inhibitor of NADPH oxidase. Gel-shift assays using nuclear protein extracts incubated with a32P-labelled DNA binding probe for NF-κB detected two electrophoretically separable complexes containing NF-κB. A faster migrating complex obtained when using both LPS-treated and untreated cells appeared to represent a basal or constitutive NF-κB activity, whereas a slower band was found only after LPS-treatment. The latter band was abolished when using cells treated for 1 h with LPS in the presence of PDTC (25 μm) or TPCK (20 μm), but was not inhibited by NDGA (50 μm) or apocynin (3.5 mm). The basal band was unaffected by any of the cell signalling inhibitors. Densitometry of Western blots indicated that LPS-induced iNOS protein expression was inhibited to a similar extent (between 74 and 87%) by the latter concentrations of PDTC, TPCK, NDGA and apocynin. The ability of PDTC and TPCK to abolish LPS-specific NF-κB activation, while also producing considerable inhibition of iNOS protein expression and nitrite formation, suggests that induction of iNOS by LPS in RASMC involves NF-κB-dependent transcription. However, the failure of NDGA and apocynin to prevent NF-κB activation, at least during early stages (up to 1 h) of its nuclear accumulation, suggests that these agents may affect cell signalling pathways which regulate iNOS induction by another mechanism to be determined.