De Vries G W, Amdahl L D, Kramer K D, Wheeler L A
Department of Biological Sciences, Allergan, Inc., Irvine, CA 92713.
Biochem Pharmacol. 1990 Dec 1;40(11):2487-90. doi: 10.1016/0006-2952(90)90090-8.
Incubation of human polymorphonuclear leukocytes (PMNLs) with the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) resulted in a concentration-dependent release of the neutral protease elastase. This response was inhibited by pretreatment of the PMNLs with manoalide (IC50 approximately 0.08 microM). To understand the mechanism of this inhibition, we examined the effect of manoalide on the signal-transduction pathway believed to mediate fMLP stimulation. We observed in fura-2 loaded cells that pretreatment with manoalide blocked fMLP-induced increases in cytosolic free-calcium (IC50 approximately 0.15 microM). However, manoalide had no effect on inositol 1,4,5-trisphosphate (IP3) production at concentrations which completely inhibited the Ca2+ signal. Furthermore, manoalide was approximately 50-fold less potent as an inhibitor of phospholipase C activity in membrane preparations of PMNLs than as an inhibitor of calcium mobilization in whole cells. These data indicate that manoalide can block stimulation of human PMNLs through inhibition of Ca2+ mobilization, but that this occurs at a site beyond phospholipase C activation and inositol phosphate turnover.
人多形核白细胞（PMNLs）与趋化因子N-甲酰-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（fMLP）一起孵育，会导致中性蛋白酶弹性蛋白酶呈浓度依赖性释放。用 manoalide 预处理 PMNLs 可抑制这种反应（IC50 约为 0.08 microM）。为了解这种抑制作用的机制，我们研究了 manoalide 对据信介导 fMLP 刺激的信号转导途径的影响。我们在负载 fura-2 的细胞中观察到，用 manoalide 预处理可阻断 fMLP 诱导的胞质游离钙增加（IC50 约为 0.15 microM）。然而，在完全抑制 Ca2+ 信号的浓度下，manoalide 对肌醇-1,4,5-三磷酸（IP3）的产生没有影响。此外，在 PMNLs 的膜制剂中，manoalide 作为磷脂酶 C 活性抑制剂的效力比作为全细胞钙动员抑制剂的效力低约 50 倍。这些数据表明，manoalide 可通过抑制 Ca2+ 动员来阻断人 PMNLs 的刺激，但这发生在磷脂酶 C 激活和肌醇磷酸周转之后的位点。
