NITRIC-OXIDE ACTIVATES CYCLOOXYGENASE ENZYMES
NITRIC-OXIDE ACTIVATES CYCLOOXYGENASE ENZYMES
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DOI：
10.1073/pnas.90.15.7240
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发表时间：
1993-08-01
影响因子：
11.1
通讯作者：
NEEDLEMAN, P
中科院分区：
文献类型：
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作者：
SALVEMINI, D;MISKO, TP;NEEDLEMAN, P
我们评估了一氧化氮(NO)对环氧合酶的构成形式和诱导形式(分别为COX、COX-1和COX-2)活性的作用。大肠杆菌脂多糖(1ug/ml，18h)诱导小鼠巨噬细胞株RAW264.7产生一氧化氮合酶(NOS)和环氧合酶-2(COX-2)，使其产物亚硝酸盐(NO2-)和前列腺素E_2(PGE_2)释放增加。一氧化氮合酶抑制剂N(G)-单甲基-L-精氨酸或氨基胍可阻断亚硝酸根和前列腺素E_2的产生。N(G)-单甲基-L-精氨酸或氨基胍的作用可被NO合成前体L-精氨酸逆转，但不能被D-精氨酸逆转。在无L精氨酸的培养液中，脂多糖刺激RAW264.7细胞18h(通过内源性一氧化氮合酶途径减少NO的产生)，RAW264.7细胞不能释放亚硝酸根，前列腺素E_2的积累量比有L精氨酸的细胞至少少4倍。在L-精氨酸缺乏的培养液中，花生四烯酸处理脂多糖诱导的RAW264.7细胞产生前列腺素E_2的作用时间为15分钟，与含L-精氨酸的培养液中诱导的细胞产生前列腺素E_2的量相比，下降了3倍。为了检测在没有内源性L-精氨酸的情况下诱导型COX的NO活性，首先用白细胞介素1β刺激人胎儿成纤维细胞18h。这些细胞释放PGE2，但不释放NO2-，这与成纤维细胞中COX而不是NOS的诱导一致。外源性NO作为气态溶液或由NO供体硝普钠或三硝酸甘油释放，可使白细胞介素1β刺激的成纤维细胞中的COX活性增加5倍；这些作用可通过与血红蛋白(10微米)共同孵育而取消，血红蛋白可结合并灭活NO，但不能被可溶性鸟苷环化酶抑制剂亚甲蓝所消除。此外，硝普钠(0.25-1 mM)通过小鼠重组COX-1和COX-2增加花生四烯酸刺激的PGE2的产生。这些结果表明，NO通过独立于cGMP的机制增强COX活性，并提示在同时存在NOS和COX系统的情况下，存在NO介导的前列腺炎前列腺素的产生增加，从而可能导致炎症反应的加剧。这些数据表明，NO直接与COX相互作用，导致酶活性增加。
We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 mug/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors N(G)-monomethyl-L-arginine or aminoguanidine. The effects of N(G)-monomethyl-L-arginine or aminoguanidine were reversed by coincubation With L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 muM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.