Articulo 017 FASEB Journal.pdf

The 2 hemiketal (HK) eicosanoids HKD2 and HKE2 are the major products of the biosynthetic crossover of the 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) pathways. HKs result from the rearrangement of a di-endoperoxide intermediate formed in the COX-2-dependent oxygenation of 5S-hydroxyeicosatetraenoic acid (5S-HETE). We analyzed HK biosynthesis in human leukocytes stimulated ex vivo and defined the biosynthetic roles of 5-LOX and COX-2, using inhibitors and incubations with exogenous substrates. Activation of leukocytes with LPS followed by treatment with the calcium ionophore A23187 resulted in the formation of PGE2, 5-HETE, and LTB4 as the principal metabolites of COX-2 and 5-LOX, respectively. The formation of HKD2 and HKE2 was highest after 15 min LPS treatment, and at that time, levels were similar to PGE2, but less than 5-HETE and LTB4 The time course of HK formation paralleled that of 5-HETE and LTB4, implying the availability of the 5S-HETE substrate as a limiting factor in biosynthesis rather than expression levels of COX-2. Specific inhibitors of COX-2 and 5-LOX decreased formation of HKD2 and HKE2 Platelets did not form HKs from exogenous 5S-HETE, implying that COX-1 is not involved. HKs are early products during an inflammatory event and require cells that express 5-LOX and COX-2 for their biosynthesis.-Giménez-Bastida, J. A., Shibata, T., Uchida, K., Schneider, C. Roles of 5-lipoxygenase and cyclooxygenase-2 in the biosynthesis of hemiketals E2 and D2 by activated human leukocytes.

2-半缩酮（HK）二十烷类化合物HKD2和HKE2是5-脂氧合酶（5-LOX）与环氧化酶-2（COX-2）生物合成交叉途径的主要产物。HKs由COX-2依赖性氧化5S-羟基二十碳四烯酸（5S-HETE）过程中形成的双端环氧中间体的重排而产生。我们通过体外刺激人白细胞并使用抑制剂以及与外源底物共同孵育，分析了HK的生物合成过程，并确定了5-LOX和COX-2在生物合成中的生物学作用。使用脂多糖（LPS）激活白细胞，随后用钙离子载体A23187处理，导致COX-2和5-LOX的主要代谢产物PGE2、5-HETE和LTB4的形成。HKD2和HKE2的形成在LPS处理15分钟后达到峰值，此时其水平与PGE2相似，但低于5-HETE和LTB4。HK的形成时间进程与5-HETE和LTB4平行，表明5S-HETE底物的可用性是生物合成的限制因素，而非COX-2的表达水平。COX-2和5-LOX的特异性抑制剂降低了HKD2和HKE2的形成。血小板不能从外源5S-HETE形成HKs，这表明COX-1不参与其中。HKs是炎症事件中的早期产物，其生物合成需要表达5-LOX和COX-2的细胞。-Giménez-Bastida, J. A., Shibata, T., Uchida, K., Schneider, C. 5-脂氧合酶和环氧化酶-2在活化人白细胞生物合成半缩酮E2和D2中的作用。