Banno Y, Okano Y, Nozawa Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
J Biol Chem. 1994 Jun 3;269(22):15846-52.
The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.
利用中国仓鼠卵巢细胞中稳定过表达磷脂酰肌醇特异性磷脂酶C（PLC-δ1）的克隆（PLCδ30细胞），对PLC-δ1的调节机制进行了研究。凝血酶刺激PLCδ30细胞后，总肌醇磷酸（InsP）增加了6.5倍，这显著高于载体转染（V1）细胞（2.0倍）。AIF-4增加了V1和PLCδ30细胞中InsP的积累，百日咳毒素部分阻断了凝血酶刺激的PLCδ30细胞中InsP的积累。与V1细胞相比，鸟苷三磷酸（GTPγS）显著增强了透化的PLCδ30细胞中凝血酶刺激的InsP生成，表明可能有G蛋白参与PLC-δ1的激活。在PLCδ30细胞中，添加EGTA可完全抑制离子霉素诱导的显著InsP生成和凝血酶刺激的InsP生成。此外，PLCδ30细胞中凝血酶加GTPγS的刺激作用比V1细胞对游离钙浓度变化更敏感。12-O-十四酰佛波醇-13-乙酸酯对凝血酶刺激的InsP积累的抑制作用不受Ca2+浓度增加的影响。这些结果表明，凝血酶诱导的PLC-δ1激活是通过G蛋白和钙共同调节的。
