Zhou J, Struthers A D, Lyles G A
Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK.
Pharmacol Res. 1999 May;39(5):363-73. doi: 10.1006/phrs.1998.0450.
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.
用1或100微克/毫升脂多糖（LPS）处理大鼠主动脉平滑肌细胞（RASMC）20 - 24小时，通过对诱导型一氧化氮合酶（iNOS）蛋白进行蛋白质免疫印迹检测以及测定细胞内亚硝酸盐生成增加，发现会导致iNOS表达。LPS诱导的亚硝酸盐生成几乎完全被以下处理所抑制：将细胞同时用LPS和（a）吡咯烷二硫代氨基甲酸盐（PDTC，25微摩尔）处理，它是一种抑制NF-κB激活的抗氧化剂；（b）N-对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK，20和40微摩尔）处理，它是一种防止NF-κB激活的蛋白酶体抑制剂；（c）去甲二氢愈创木酸（NDGA，10和50微摩尔）处理，它是一种脂氧合酶抑制剂；或（d）鱼藤酮（2、3.5和5毫摩尔）处理，它是一种NADPH氧化酶抑制剂。使用与用于NF-κB的32P标记DNA结合探针孵育的核蛋白提取物进行凝胶迁移试验，检测到两个电泳可分离的包含NF-κB的复合物。当使用LPS处理和未处理的细胞时获得的迁移较快的复合物似乎代表基础或组成型NF-κB活性，而较慢的条带仅在LPS处理后出现。当在存在PDTC（25微摩尔）或TPCK（20微摩尔）的情况下用LPS处理细胞1小时时，后一条带消失，但不受NDGA（50微摩尔）或鱼藤酮（3.5毫摩尔）抑制。基础条带不受任何细胞信号抑制剂的影响。蛋白质免疫印迹的光密度分析表明，后述浓度的PDTC、TPCK、NDGA和鱼藤酮对LPS诱导的iNOS蛋白表达的抑制程度相似（在74%至87%之间）。PDTC和TPCK消除LPS特异性NF-κB激活的能力，同时也对iNOS蛋白表达和亚硝酸盐生成产生相当大的抑制，这表明LPS在RASMC中诱导iNOS涉及NF-κB依赖性转录。然而，NDGA和鱼藤酮未能阻止NF-κB激活，至少在其核积累的早期阶段（长达1小时），这表明这些试剂可能通过另一种待确定的机制影响调节iNOS诱导的细胞信号通路。
