Siddiqui R A, English D, Harvey K, Cui Y, Martin M I, Wentland J, Akard L, Jansen J, Thompson J, Garcia J G
Bone Marrow Transplantation Laboratory, Methodist Hospital of Indiana, Indianapolis 46202, USA.
J Leukoc Biol. 1995 Aug;58(2):189-95. doi: 10.1002/jlb.58.2.189.
This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.
本研究旨在探讨外源性磷脂酸（PA）刺激多形核白细胞释放超氧化物的机制。与膜通透性二辛酰甘油（DiC8-DAG）影响的超氧化物立即释放不同，二辛酰磷脂酸（DiC8-PA）在5-20分钟的延迟期后诱导超氧化物释放。当细胞用亚刺激水平的佛波酯肉豆蔻酸酯（PMA）预处理时，这段时间会显著缩短或消除。颗粒耗尽的中性粒细胞胞质体也对DiC8-PA产生超氧化物爆发反应。在细胞预先暴露于亚刺激水平的PMA后，胞质体超氧化物生成系统对DiC8-PA的激活也明显加快，这表明至少部分预处理机制与PMA诱导的脱颗粒无关。为了进一步研究PMA预处理对PA反应的潜在机制，我们评估了中性粒细胞胞外磷脂酸磷酸水解酶（胞外PA磷酸水解酶）的活性，该酶可从外源性PA生成二酰甘油。PMA预处理对该酶的活性没有明显影响。此外，PA磷酸水解酶抑制剂普萘洛尔并没有选择性地抑制中性粒细胞对DiC8-PA反应的PMA预处理，这表明预处理不是由DiC8-PA水解加速引起的。因此，我们研究了蛋白激酶C激活是预处理反应基础的可能性。几种半选择性蛋白激酶C抑制剂（钙泊三醇C、H-7和酰甲基甘油）对DiC8-DAG和DiC8-PA诱导的超氧化物释放以及PMA预处理反应的抑制程度大致相同。这些结果与以下假设一致，即中性粒细胞对磷脂酸的反应是由胞外PA磷酸水解酶作用产生的甘油二酯介导的。PMA预处理不是由胞外PA磷酸水解酶催化活性增加引起的，而是似乎是由细胞对多种刺激反应中涉及的中间体增强引起的。
