Differential effects of some cell signalling inhibitors upon nitric oxide synthase expression and nuclear factor-kappaB activation induced by lipopolysaccharide in rat aortic smooth muscle cells.

Treatment of rat aortic smooth muscle cells (RASMC) with 1 or 100 microg ml-1 lipopolysaccharide (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 microm), an antioxidant inhibitor of NF-kappaB activation; (b) N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 20 and 40 microm), a proteasomal inhibitor which prevents NF-kappaB activation; (c) nordihydroguaiaretic acid (NDGA, 10 and 50 microm), a lipoxygenase inhibitor; or (d) apocynin (2, 3.5 and 5 m m), an inhibitor of NADPH oxidase. Gel-shift assays using nuclear protein extracts incubated with a 32P-labelled DNA binding probe for NF-kappaB detected two electrophoretically separable complexes containing NF-kappaB. A faster migrating complex obtained when using both LPS-treated and untreated cells appeared to represent a basal or constitutive NF-kappaB 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 microm) or TPCK (20 microm), but was not inhibited by NDGA (50 microm) or apocynin (3.5 m m). 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-kappaB activation, while also producing considerable inhibition of iNOS protein expression and nitrite formation, suggests that induction of iNOS by LPS in RASMC involves NF-kappaB-dependent transcription. However, the failure of NDGA and apocynin to prevent NF-kappaB 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.