Schmidt H H, Warner T D, Nakane M, Förstermann U, Murad F
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Mol Pharmacol. 1992 Apr;41(4):615-24.
In nitrinergic signal transduction, nitrogen oxide (NO) synthases (NOS) (EC 1.14.23) catalyze the conversion of L-arginine to L-citrulline and NO, which in turn activates soluble guanylyl cyclase. Macrophages were reported to contain a single isoform of NOS (type II, soluble, Ca(2+)-independent, 130-kDa) and only upon activation of the cells by interferon-gamma (INF) and lipopolysaccharides (LPS). By a mechanism involving L-type Ca2+ channels, calmodulin, and serine proteases, INF/LPS also induce a cytotoxic activation of macrophages. In RAW264.7 macrophages, NO release was detected upon activation of the cells by INF/LPS but also, although at a 20-fold lower level, in control cells. The latter constitutive NOS activity and NO release were Ca2+ dependent and were decreased in INF/LPS-activated RAW264.7 cells or with increasing passage number. RAW264.7 cells did not express soluble guanylyl cyclase, suggesting other target molecules for NO. In INF/LPS-activated cells, NOS activities and NO release were Ca2+ independent (type II) and coinduced with NADPH-diaphorase activities both in the soluble and in the particulate fractions. The NOS-II activities corresponded to a 130-kDa protein, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was not recognized in a protein immunoblot with anti-NOS-I antibody. The serine protease inhibitor tosyl-lysyl chloromethyl ketone abolished the induction of NOS-II by INF/LPS, by depleting intracellular thiol pools and interfering with protein synthesis. Induction of NOS-II by INF/LPS was transcriptionally based and, for maximal enzyme activity, required increased intracellular tetrahydrobiopterin levels, intracellular Ca2+ mobilization, and activation of non-L-type Ca2+ channels but, unlike the induction of macrophage-mediated cytotoxicity, neither L-type-Ca2+ channels nor calmodulin.
在一氧化氮能信号转导中，一氧化氮合酶（NOS）（EC 1.14.23）催化L-精氨酸转化为L-瓜氨酸和一氧化氮，进而激活可溶性鸟苷酸环化酶。据报道，巨噬细胞含有单一形式的NOS（II型，可溶性，不依赖Ca(2+)，130 kDa），且仅在细胞被干扰素-γ（INF）和脂多糖（LPS）激活时才会出现。通过一种涉及L型Ca2+通道、钙调蛋白和丝氨酸蛋白酶的机制，INF/LPS还可诱导巨噬细胞的细胞毒性激活。在RAW264.7巨噬细胞中，INF/LPS激活细胞后可检测到一氧化氮释放，但在对照细胞中也能检测到，尽管水平低20倍。后者的组成型NOS活性和一氧化氮释放依赖于Ca2+，且在INF/LPS激活的RAW264.7细胞中或随着传代次数增加而降低。RAW264.7细胞不表达可溶性鸟苷酸环化酶，这表明一氧化氮有其他靶分子。在INF/LPS激活的细胞中，NOS活性和一氧化氮释放在可溶性和颗粒部分均不依赖Ca2+（II型），且与NADPH-黄递酶活性共同诱导。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，NOS-II活性对应于一种130 kDa的蛋白质，在用抗NOS-I抗体进行的蛋白质免疫印迹中未被识别。丝氨酸蛋白酶抑制剂甲苯磺酰赖氨酰氯甲酮通过耗尽细胞内硫醇池并干扰蛋白质合成，消除了INF/LPS对NOS-II的诱导。INF/LPS对NOS-II的诱导基于转录，并且为了达到最大酶活性，需要增加细胞内四氢生物蝶呤水平、细胞内Ca2+动员以及非L型Ca2+通道的激活，但与巨噬细胞介导的细胞毒性诱导不同，既不需要L型Ca2+通道也不需要钙调蛋白。
