Effects of phorbol myristate acetate on the synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid by human polymorphonuclear leukocytes.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a newly discovered chemotactic agent for human polymorphonuclear leukocytes (PMNL) which has potent stimulatory effects on cytosolic calcium levels in these cells. Although we have shown that it is synthesized from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by a highly specific microsomal dehydrogenase, little is known about the synthesis of this substance by intact PMNL. In the present study we found that in contrast to PMNL microsomes, intact, unstimulated PMNL produced relatively small amounts of 5-oxo-ETE from 5-HETE, but instead converted 5-HETE primarily to its omega-oxidation product, 5,20-diHETE. However, preincubation of PMNL with phorbol myristate acetate (PMA; EC50, ca. 4 nM) dramatically increased the ratio of 5-oxo-ETE to 5,20-diHETE from 0.07 in its absence to 1.85 in the presence of 100 nM PMA. Both effects were completely reversed by staurosporine, indicated that they were mediated by a protein kinase. PMA also stimulated the formation of 5-oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) from exogenous arachidonic acid. The greatest enhancement was observed for 5-oxo-ETE, which, under all conditions, was produced in greater quantities than LTB4. PMA stimulated the formation of 5-oxo-ETE by PMNL stimulated with either A23187 or zymosan. A23187-stimulated PMNL initially produced more LTB4 than 5-oxo-ETE, but at longer time points, 5-oxo-ETE predominated. These results demonstrate that PMA-activated human PMNL can synthesize substantial amounts of 5-oxo-ETE and raise the possibility that this substance may be an important inflammatory mediator.