Winkler J D, Bolognese B J, Roshak A K, Sung C M, Marshall L A
Department of Immunopharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Biochim Biophys Acta. 1997 Jun 2;1346(2):173-84. doi: 10.1016/s0005-2760(97)00032-5.
Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.
血小板活化因子（PAF）的产生在炎性细胞中受到严格调控。在人类中性粒细胞中，要生成PAF前体1-O-烷基-2-溶血甘油磷脂酰胆碱（lyso-GPC），需要从1-O-烷基-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（GPC）中特异性去除花生四烯酸（AA），这一过程被认为是由非辅酶A依赖性转酰基酶（CoA-IT）介导的。用CoA-IT抑制剂SK&F 98625和SK&F 45905处理A23187刺激的人类单核细胞，可抑制PAF的形成（IC50分别为10和12微摩尔），表明这些细胞产生PAF也需要CoA-IT活性。由于CoA-IT活性将花生四烯酸转移到2-溶血磷脂底物上，其活性依赖于一种sn-2酰基水解酶来形成2-溶血磷脂底物。14 kDa磷脂酶A2（PLA2）的抑制剂SB 203347和85 kDa PLA2的抑制剂AACOCF3，均能抑制A23187刺激的人类单核细胞释放AA。然而，浓度高达3微摩尔的AACOCF3对A23187诱导的PAF形成没有影响。此外，使用反义寡核苷酸（SB 7111，1微摩尔）耗尽85 kDa PLA2对PAF的产生没有影响，表明85 kDa PLA2在PAF生物合成中不起作用。SB 203347和14 kDa PLA2抑制剂scalaradial均能阻断单核细胞中PAF的合成（IC50分别为2和0.5微摩尔），表明14 kDa PLA2在这一过程中起关键作用。此外，A23187刺激的单核细胞产生两种形式的PAF：分别为80%的1-O-烷基-2-乙酰基-GPC和20%的1-酰基-2-乙酰基-GPC，二者均被SB 203347同等程度地抑制。相比之下，使用SK&F 45905（20微摩尔）抑制CoA-IT对1-O-烷基化（-80%）乙酰化物质产生的影响比对1-酰基化（-14%）乙酰化物质产生的影响更大。最后，用外源性人类重组（rh）II型14 kDa PLA2而非rh 85 kDa PLA2处理U937细胞膜，可诱导PAF产生。通过热处理消除膜CoA-IT活性会损害14 kDa PLA2诱导PAF形成的能力。综上所述，这些结果表明，与单核细胞CoA-IT诱导的PAF产生相关的是一种14 kDa PLA2样活性，而非85 kDa PLA2。
