Studies on the regulation of arachidonic acid metabolism using mast cells and neutrophils as model systems

在本项目中，我们研究了各种造血细胞中花生四烯酸（AA）代谢的调节。(1)肥大细胞中的AA代谢。在特异性辅助细胞因子存在下，经IgE和抗原交联的FsepsilonRI刺激小鼠和大鼠肥大细胞，随时间推移显示两种顺序的前列腺素（PG）D_2生物合成反应。胞浆内Ca^2+浓度的瞬时升高可在几分钟内立即产生PGD_2，PGD_2的产生与胞浆磷脂酶A_2（cPLA_2）的瞬时核周易位、磷酸化和活化有关。组成型表达的环氧合酶（考克斯）亚型考克斯-1是参与这种快速反应的主要酶，它将花生四烯酸转化为PGH_2，PGH_2又通过谷胱甘肽依赖的PGD_2合酶代谢为PGD_2。PGD_2的延迟生成，在培养数小时后发生，与考克斯-2的从头诱导和功能有关。德莱德P ...更多信息 GD_2的产生伴随着IIA型分泌型PLA_2（sPLA_2）的诱导，sPLA_2通过增强考克斯-2的表达而与考克斯-2功能性连接。另一种sPLA_2同工酶VsPLA_2也在肥大细胞中表达，并可补偿IIA型sPLA_2。此外，肥大细胞的活化可促进血小板活化因子（PAF）的快速和瞬时产生，其被肥大细胞外切的血浆型PAF-乙酰水解酶灭活，揭示了肥大细胞的抗炎方面。(2)中性粒细胞中的AA代谢。大鼠中性粒细胞在fMLP-奥曲莫赞刺激下AA的释放及随后白三烯B_4和PAF的产生依赖于cPLA_2。cPLA_2的活化部分受磷脂酶D的调节。(3)巨噬细胞中的AA代谢。A23187刺激大鼠腹腔巨噬细胞30-60 min内产生血栓素B2（TX B2），其产生的组成性立即反应是由cPLA 2、考克斯-1和TX合成酶介导的。LPS处理的细胞在培养3-24 h内主要产生PGE_2（延迟反应），其中cPLA_2和IIA型sPLA_2与诱导型考克斯-2协同作用，COX-2又与诱导型PGE_2合酶偶联。LPS致敏12 h后，A23187刺激30 min，细胞产生PGE_2的能力明显强于TXB_2（诱导的立即反应），其中cPLA_2、COX-2和PGE_2合成酶三种诱导酶在功能上相互关联。【结论】这些结果表明，不同的PG生物合成酶在不同的跨膜刺激事件后显示分离的功能偶联，即使在体外催化类似反应的酶在相同的细胞中共存。少

In this project, we have examined the regulation of arachidonic acid (AA) metabolism in various hematopoietic cells.(1) AA metabolism in mast cells. Mouse and rat mast cells stimulated by FsepsilonRI crosslinking via IgE and antigen in the presence of specific accessory cytokines show two sequential prostaglandin (PG) D_2 biosynthetic responses over time. Immediate PGD_2 generation, occurring within a few minutes in response to a transient increase in cytoplasmic Ca^<2+> concentration, is associated with transient perinuclear translocation, phosphorylation and activation of cytosolic phospholipase A_2 (cPLA_2). The constitutively expressed cyclooxygenase (COX) isoform, COX-1, is the dominant enzyme involved in this rapid response, which converts arachidonic acid to PGH_2, which in turn is metabolized to PGD_2 via glutathione-dependent PGD_2 synthase. Delayd PGD_2 generation, occurring overseveral hours of culture, is associated with the de novo induction and function of COX-2. Delayd P … More GD_2 generation is accompanied by the induction of type IIA secretory PLA_2 (sPLA_2), which is functionally linked to COX-2 through enhancing COX-2 expression. Another sPLA_2 isozyme, type VsPLA_2, is also expressedin mast cells and may compensate for type IIA sPLA_2. Furthermore, activation of mast cells elicits rapid and transient production of platelet-activating factor (PAF), which is inactivated by plasma-type PAF-acetylhydrolase exocytosed from mast cells, revealing an anti-inflammatory aspect of mast cells.(2) AA metabolism in neutrophils. Release of AA and subsequent production of leukotriene B_4 and PAF in fMLP-orzymozan-stimulated rat neutrophils depend on cPLA_2. cPLA_2 activation is in part regulated by phospholipase D.(3) AA metabolism in macrophages. Rat peritoneal macrophages stimulated with A23187 produce thromboxane (TX) B_2 in marked preference to PGE_2 within 30-60 min (constitutive immediate response), which is mediated by preexisting cPLA_2, COX-1, and TX synthase. Cells treated with LPS predominantly produce PGE_2 during culture for 3-24 h (delayd response), where cPLA_2 and type IIA sPLA_2 function cooperatively with inducible COX-2, which is in turn coupled with inducible PGE_2 synthase. Cells primed for 12h with LPS and stimulated for 30 min with A23187 produce PGE_2 in marked preference to TXB_2 (induced immediate response), in which three inducible enzymes, cPLA_2, COX2, and PGE_2 synthase are functionally linked.[Conclusion] These results suggest that distinct PG-biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells. Less