Wijkander J, O'Flaherty J T, Nixon A B, Wykle R L
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016, USA.
J Biol Chem. 1995 Nov 3;270(44):26543-9. doi: 10.1074/jbc.270.44.26543.
Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.
当以1-硬脂酰-2-[14C]花生四烯酰磷脂酰胆碱的超声处理囊泡为底物进行测量时，向人中性粒细胞中添加亚微摩尔浓度的花生四烯酸（AA）会使胞质磷脂酶A2（PLA2）的活性增加2倍。用白三烯B4（LTB4）、5-氧代二十碳四烯酸或5-羟基二十碳四烯酸（5-HETE）刺激中性粒细胞也会诱导胞质PLA2活性出现类似增加。LTB4是最有效的激动剂，在1 nM时显示出最大效应。用二十碳四炔酸或齐留通抑制5-脂氧合酶可阻止AA诱导的PLA2活性增加，但对LTB4诱导的反应没有影响。此外，用LTB4受体拮抗剂LY 255283预处理中性粒细胞可阻断AA和LTB4诱导的PLA2激活，但不影响5-HETE的作用。用胰PLA2处理中性粒细胞也会诱导胞质PLA2活性增加；这种反应被二十碳四炔酸或LY 255283抑制。通过蛋白质免疫印迹分析确定，对刺激的反应中PLA2活性的增加与85-kDa PLA2电泳迁移率的变化相关，这表明85-kDa PLA2的磷酸化可能是其催化活性增加的基础。尽管用单个脂氧合酶代谢产物刺激中性粒细胞不会诱导内源性AA的显著动员，但通过质谱分析确定，它们极大地增强了N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸诱导的AA动员。我们的研究结果支持一种正反馈模型，即刺激诱导的AA释放或分泌性PLA2的胞吐作用通过启动LTB4的形成来调节胞质85-kDa PLA2的活性。新生的LTB4随后释放出来作用于LTB4受体，从而促进85-kDa PLA2的进一步激活。由于已知5-HETE和LTB4可引发血小板活化因子的合成，这些研究结果表明85-kDa PLA2在血小板活化因子的合成中起作用。
