Kadiri C, Cherqui G, Masliah J, Rybkine T, Etienne J, Béréziat G
Départment de Biochimie, URA CNRS 1283, Paris, France.
Mol Pharmacol. 1990 Sep;38(3):418-25.
Various pharmacological effectors were used to investigate the mechanism of arachidonic acid release by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) in guinea pig alveolar macrophages. The fMLP- and PAF-stimulated arachidonic acid release (i) was mimicked by sodium fluoride and inhibited by Bordetella pertussis toxin, suggesting the participation of a guanine nucleotide-binding protein; ii) was mimicked by A23187 but was insensitive to the calmodulin inhibitor R24571, making the involvement of a calmodulin-dependent pathway unlikely; and (iii) was mimicked by 12-O-tetra-decanoyl phorbol 13 acetate (TPA) and was, like the TPA-stimulated release, markedly decreased when protein kinase C (PKC) had been down-regulated by TPA (65% decrease) or inhibited by sphingosine, a diacylglycerol-competitive PKC inhibitor shown to completely abolish the enzyme activity from alveolar macrophages at 40 microM. Moreover, PAF and fMLP, under conditions where they stimulated arachidonic acid release, promoted an appreciable, albeit transient, translocation of PKC, suggesting a possible involvement of the enzyme in the agonist-stimulated process. However, staurosporine, another PKC inhibitor decreasing PKC activity from alveolar macrophages by 60% at 20 nM, failed to alter fMLP- and PAF-stimulated release. These data lead us to suggest that fMLP- and PAF-stimulated arachidonic acid release is mediated by mechanisms involving either a staurosporine-insensitive PKC isoform or a sphingosine-sensitive coupling between a pertussis toxin-sensitive guanine nucleotide-binding protein and phospholipase A2. Finally, the fMLP- and PAF-stimulated arachidonic acid release was inhibited by cholera toxin and was, like A23187-stimulated release, potentiated by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), an exclusive protein kinase A inhibitor in alveolar macrophages, suggesting a negative regulation by protein kinase A.
运用多种药理效应剂研究了N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）和血小板活化因子（PAF）诱导豚鼠肺泡巨噬细胞释放花生四烯酸的机制。fMLP和PAF刺激的花生四烯酸释放：（i）可被氟化钠模拟，并被百日咳博德特氏菌毒素抑制，提示有鸟嘌呤核苷酸结合蛋白参与；（ii）可被A23187模拟，但对钙调蛋白抑制剂R24571不敏感，提示钙调蛋白依赖性途径不太可能参与；（iii）可被12-O-十四烷酰佛波醇-13-乙酸酯（TPA）模拟，并且与TPA刺激的释放一样，当蛋白激酶C（PKC）被TPA下调（降低65%）或被鞘氨醇抑制时，fMLP和PAF刺激的释放明显减少，鞘氨醇是一种二酰基甘油竞争性PKC抑制剂，在40μM时可完全消除肺泡巨噬细胞中的该酶活性。此外，在刺激花生四烯酸释放的条件下，PAF和fMLP促进了PKC的明显但短暂的易位，提示该酶可能参与了激动剂刺激的过程。然而，另一种PKC抑制剂星形孢菌素在20 nM时可使肺泡巨噬细胞中的PKC活性降低60%，但未能改变fMLP和PAF刺激的释放。这些数据使我们推测，fMLP和PAF刺激的花生四烯酸释放是由涉及一种对星形孢菌素不敏感的PKC同工型或百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白与磷脂酶A2之间对鞘氨醇敏感的偶联机制介导的。最后，fMLP和PAF刺激的花生四烯酸释放被霍乱毒素抑制，并且与A23187刺激的释放一样，被N-[2-（甲氨基）乙基]-5-异喹啉磺酰胺二盐酸盐（H8）增强，H8是肺泡巨噬细胞中的一种特异性蛋白激酶A抑制剂，提示蛋白激酶A的负调控作用。
