Functional coupling of arachidonate metabolizing enzymes

前列腺素E_2是已知的炎症介质之一，出现在炎症晚期的渗出物中。我们用大鼠腹膜巨噬细胞研究了花生四烯酸级联反应中产生前列腺素E_2的酶的功能偶联，在孵育初期，大鼠巨噬细胞产生TXB_2和PGD_2，并且这些前列腺素的水平在孵育到24小时内没有变化。相反，PGE2水平随着时间的延长而升高，是初始时相的50-60倍，这种增加被选择性COX-2抑制剂如NS-398抑制到初始水平。当外源性花生四烯酸加入刺激的巨噬细胞时，转化为PGE2的活性在12小时达到峰值，24小时活性下降。然而，COX-2的Western印迹显示COX-2水平在孵育至24小时时升高。然后，我们通过评估PGH2向PGE2的转化来检测巨噬细胞裂解物中PGE合成酶的活性。该PGE合成酶活性随孵育时间的延长而增加，在12小时左右达到峰值。因此，我们得出结论，除了COX-2的诱导外，可诱导的PGE合成酶活性的增加可能是内毒素刺激的巨噬细胞产生PGE_2增加的原因。我们还从大鼠和小鼠巨噬细胞中克隆了PGE合成酶的cDNAs，并对分别导入HEK 293细胞的酶进行了鉴定。我们还构建了用于小鼠和大鼠前列腺素E合成酶Northern印迹分析的引物。对大鼠和小鼠巨噬细胞PGE合成酶的Northern印迹分析表明，两种巨噬细胞的PGE合成酶都是COX-2偶联的诱导酶，并产生大量的PGE2。

Prostaglandin E2 is known as one of the inflammatory mediators and appearing in the exudates of late phase of inflammation. We investigated functional coupling of the enzymes working in the arachidonate cascade to produce PGE2 by using rat peritonreal macrophages, when stimulated with lipopolysaccharide of E Coli Rat macrophages produced TXB2 and PGD2 at the initial phase of incubation, and the levels of these PGs did not change throughout the incubation upto 24 hr. On the contrary, PGE2 level increased with time and became 50-60 fold of the initial phase This increase was suppressed by COX-2 selective inhibitors, such as NS-398, to the initial level. When exogenous arachidonic acid was added to the stimulated macrophages, conversion to PGE2 peaked at 12 hr and the activity declined at 24 hr. However, Western blot of COX-2 indicates that COX-2 level increased upto 24 hr of incubation. Then we measured PGE synthase activity in the macrophage lysates by assessing the conversion of PGH2 to PGE2. This PGE synthase activity increased with incubation time and peaked around 12hr. Thus we conclude that an increase in inducible PGE synthase activity may be responsible for the increased PGE2 production in the LPS stimulated macrophages in addition to the induction of COX-2. We also cloned cDNAs of PGE synthases from rat and mouse macrophages and characterized the enzymes transfected into HEK 293 cells, respectively. We also constructed primers for Northern blot analysis of PGE synthases in mouse and rats. Northern blot analysis for PGE synthases of rat and mouse macrophages expressed that the PGE synthases in macrophages from both species were inducible enzyme to couple COX-2 and produce huge amount of PGE2.