Functional coupling of arachidonate metabolizing enzymes

前列腺素E2是炎症介质之一，出现在炎症晚期的渗出物中。我们利用大肠杆菌脂多糖刺激大鼠腹膜巨噬细胞，研究了花生四烯酸级联产生PGE2的酶的功能偶联，大鼠巨噬细胞在孵育初期产生TXB2和PGD2，并且这些PGs的水平在整个孵育过程中长达24小时没有变化。相反，PGE2水平随着时间的增加而增加，达到初始阶段的50-60倍，这种增加被COX-2选择性抑制剂如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，PGE2的转化在12小时达到顶峰，24小时活性下降。然而，COX-2的Western blot显示，COX-2水平在孵育24小时后升高。然后，我们通过评估PGH2向PGE2的转化来测量巨噬细胞裂解物中PGE合成酶的活性。PGE合成酶活性随孵育时间增加而增加，在12h左右达到峰值。因此，我们得出结论，除了诱导COX-2外，LPS刺激的巨噬细胞中诱导的PGE2合成酶活性的增加可能是导致PGE2生成增加的原因。我们还从大鼠和小鼠巨噬细胞中克隆了PGE合成酶的cdna，并分别鉴定了转染到HEK 293细胞中的酶。我们还构建了引物用于小鼠和大鼠PGE合成酶的Northern blot分析。对大鼠和小鼠巨噬细胞的PGE合成酶进行Northern blot分析表明，两种巨噬细胞的PGE合成酶都是诱导酶偶联COX-2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.