|Ki: ||Kd:||Ic 50:||Ec50/Ic50:|
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.. J Zhou; A D Struthers; G A Lyles (1999) Pharmacological research : the official journal of the Italian Pharmacological Society display abstract
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.
Inhibition of the nuclear factor-kappaB activation with pyrrolidine dithiocarbamate attenuating inflammation and oxidative stress after experimental spinal cord trauma in rats.. Giovanni La Rosa; Salvatore Cardali; Tiziana Genovese; Alfredo Conti; Rosanna Di Paola; Domenico La Torre; Fabio Cacciola; Salvatore Cuzzocrea (2004) Journal of neurosurgery. Spine display abstract
OBJECT: The nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in the induction of genes involved in physiological processes and in the response to inflammation. The authors of recent studies have demonstrated that NF-kappaB and oxidative stress contribute to secondary injury after impact-induced spinal cord injury (SCI) in the rat. Dithiocarbamates are antioxidants that are potent inhibitors of NF-kappaB. The authors postulated that pyrrolidine dithiocarbamate (PDTC) would attenuate NF-kappaB-related inflammatory and oxidative events that occur after SCI. METHODS: Spinal cord injury was induced by the application of vascular clips (force of 50 g) to the dura mater after a four-level T5-8 laminectomy. The authors investigated the effects of PDTC (30 mg/kg administered 30 minutes before SCI and 6 hours after SCI) on the development of the inflammatory response associated with SCI in rats. Levels of myeloperoxidase activity were measured as an indicator of polymorphonuclear infiltration; malondialdehyde levels in the spinal cord tissue were determined as an indicator of lipid peroxidation. The following studies were performed: immunohistochemical analysis to assess levels of inducible nitric oxide synthase (iNOS), nitrotyrosine formation, poly([adenosine diphosphate]-ribose) polymerase (PARP) activity; Western blot analysis to determine cytoplasmic levels of inhibitory-kappaB-alpha (IkappaB-alpha); and electrophoretic mobility-shift assay to measure the level of DNA/NF-kappaB binding. The PDTC treatment exerted potent antiinflammatory effects with significant reduction of polymorphonuclear cell infiltration, lipid peroxidation, and iNOS activity. Furthermore, administration of PDTC reduced immunohistochemical evidence of formation of nitrotyrosine and PARP activation in the spinal cord section obtained in the SCI-treated rats. Additionally, PDTC treatment significantly prevented the activation of NF-kappaB (electrophoretic mobility-shift assay and immunoblot analysis). CONCLUSIONS: Overall, the results clearly demonstrate that PDTC-related prevention of the activation of NF-kappaB reduces the development of some secondary injury events after SCI. Therefore, inhibition of NF-kappaB may represent a novel approach in the treatment of SCIs.
In vivo inhibition of nuclear factor-kappa B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock.. S F Liu; X Ye; A B Malik (1997) Journal of immunology (Baltimore, Md. : 1950) display abstract
We determined the in vivo function of LPS-induced nuclear factor-kappa B (NF-kappa B) activation in mediating inducible nitric oxide synthase (iNOS) mRNA and protein expression, and systemic arterial hypotension in a rat model of septic shock. LPS (8 mg/kg i.v.) challenge of rats activated NF-kappa B within 15 min in lung tissue, and the response persisted up to 4 h. NF-kappa B activation preceded the induction of iNOS mRNA. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B effective in cellular studies, prevented NF-kappa B activation in vivo and reduced iNOS mRNA expression and the increase in iNOS activity activated by LPS. At PDTC concentrations of 50, 100, and 200 mg/kg, the reductions of iNOS mRNA were 20, 46, and 48%, and the reductions in iNOS activity were 59, 66, and 75%, respectively. The PDTC concentration-dependent reductions in iNOS activity produced similar decreases in plasma nitrite/nitrate concentrations. PDTC also prevented the decrease in arterial blood pressure induced by LPS. These results demonstrate that activation of NF-kappa B is a critical in vivo regulatory mechanism mediating LPS-induced iNOS expression and the resultant systemic hypotension.