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

在本项目中，我们研究了花生四烯酸(AA)在不同造血细胞中的代谢调节。(1)肥大细胞中的AA代谢。小鼠和大鼠肥大细胞在特定辅助细胞因子存在下，经IgE和抗原FSepsilonRI交联剂刺激后，随着时间的推移，呈现出两种顺序的前列腺素(PG)D_2生物合成反应。PGD_2的产生与胞浆磷脂酶A_2(CPLA_2)的瞬时核周移位、磷酸化和激活有关，它发生在细胞质内Ca~(2+)浓度短暂升高的几分钟内。结构性表达的环氧合酶(COX)亚型COX-1是参与这一快速反应的主要酶，它将花生四烯酸转化为PGH_2，再通过谷胱甘肽依赖的PGD_2合成酶代谢成PGD_2。延迟的PGD_2产生与COX-2的从头诱导和功能有关，发生在培养数小时以上。Delayd P…更多的GD_2产生伴随着IIA型分泌型磷脂酶A_2(SPLA2)的诱导，它通过促进COX-2的表达而与COX-2功能相关。另一种SPLA2同工酶VsPLA2也在肥大细胞中表达，并可能补偿IIA型SPLA2。此外，肥大细胞的激活引起快速和瞬时的血小板激活因子(PAF)的产生，而PAF被肥大细胞分泌的血浆型PAF-乙酰水解酶失活，揭示了肥大细胞的抗炎作用。(2)中性粒细胞中的AA代谢。在fMLP-orzymozan刺激的大鼠中性粒细胞中，AA的释放以及随后白三烯B_4和PAF的产生依赖于CPLA_2。CPLA_2的激活部分受磷脂酶D的调节。(3)巨噬细胞中AA的代谢。A23187刺激的大鼠腹腔巨噬细胞在30-60分钟内产生血栓烷(TX)B_2，明显优先于PGE_2(结构性即时反应)，这种反应是由预先存在的CPLA_2、COX-1和TX合酶介导的。经内毒素处理的细胞在培养3-24小时内主要产生PGE_2(延迟反应)，其中CPLA_2和IIA SPLA2与诱导型COX-2协同作用，而COX-2又与诱导型PGE_2合成酶偶联。细胞经脂多糖预刺激12h后，经A23187刺激30min后产生前列腺素E_2(PGE_2)，而不是产生TXB_2(诱导即刻反应)，其中CPLA_2、COX2和PGE_2合成酶三种诱导酶在功能上是相连的。较少

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