Lee S H, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D
Pennington Biomedical Research Center, Louisiana State University, Baton Rouge 70808.
J Biol Chem. 1992 Dec 25;267(36):25934-8.
Two forms of cyclooxygenase are known to be present in eukaryotic organisms: a cyclooxygenase (COX-1) first purified from ram seminal vesicles encoded by a 2.8-kilobase mRNA, and a newly discovered mitogen-inducible cyclooxygenase (COX-2) encoded by a 4-kilobase mRNA. Expression of these two forms of the enzyme in rat alveolar macrophages stimulated with lipopolysaccharide was investigated by 1) determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin; 2) comparing levels of newly synthesized enzyme proteins in cells treated with or without lipopolysaccharide; and 3) assessing the expression of the mRNAs encoding COX-1 and COX-2. Levels of enzyme proteins were assessed by Western blot analysis and immunoprecipitation of 35S-labeled enzyme using two different antibodies, one specific for COX-2 and the other recognizing both forms of the enzyme but preferentially recognizing COX-1. We report here that the enhanced cyclooxygenase activity induced by the bacterial lipopolysaccharide in rat alveolar macrophages is caused by selective expression of the COX-2. Expression of COX-2 in macrophages stimulated by lipopolysaccharide was completely inhibited by dexamethasone, whereas COX-1 was unaffected. In resting unstimulated macrophages, only COX-1 but not COX-2 was detected. Levels of mRNA for the COX-2 in macrophages were increased, but those of the COX-1 were not affected by lipopolysaccharide as assessed by reverse transcription coupled with polymerase chain reaction. These results indicate that increased synthesis of prostaglandins and thromboxanes in lipopolysaccharide-stimulated macrophages results from selective expression of COX-2.
一种是最初从公羊精囊中纯化得到的环氧化酶（COX-1），由2.8千碱基的mRNA编码；另一种是新发现的由4千碱基的mRNA编码的丝裂原诱导型环氧化酶（COX-2）。通过以下方法研究了这两种形式的酶在脂多糖刺激的大鼠肺泡巨噬细胞中的表达：1）用阿司匹林使内源性酶失活后，测定新合成酶的活性；2）比较用或不用脂多糖处理的细胞中新合成酶蛋白的水平；3）评估编码COX-1和COX-2的mRNA的表达。使用两种不同的抗体通过蛋白质免疫印迹分析和对35S标记酶的免疫沉淀来评估酶蛋白的水平，一种抗体对COX-2具有特异性，另一种抗体能识别两种形式的酶，但优先识别COX-1。我们在此报告，细菌脂多糖在大鼠肺泡巨噬细胞中诱导的环氧化酶活性增强是由COX-2的选择性表达引起的。脂多糖刺激的巨噬细胞中COX-2的表达被地塞米松完全抑制，而COX-1不受影响。在未受刺激的静息巨噬细胞中，仅检测到COX-1而未检测到COX-2。通过逆转录聚合酶链反应评估，脂多糖不影响巨噬细胞中COX-1的mRNA水平，但会使COX-2的mRNA水平升高。这些结果表明，脂多糖刺激的巨噬细胞中前列腺素和血栓素合成增加是由COX-2的选择性表达所致。
