Kumada T, Nakashima S, Nakamura Y, Miyata H, Nozawa Y
Department of Otolaryngology, Gifu University School of Medicine, Japan.
Biochim Biophys Acta. 1995 Sep 14;1258(2):107-14. doi: 10.1016/0005-2760(95)00106-m.
The differential implication of protein kinase C (PKC) isozymes in antigen- or PMA-induced phospholipase D (PLD) activation was investigated in rat basophilic leukemia (RBL-2H3) cells. In [3H]oleic acid-labeled cells, both antigen (100 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (100 nM) produced a specific product of PLD activation, [3H]phosphatidylbutanol (PBut) in the presence of butanol. Pretreatment of cells with a selective PKC inhibitor, Ro31-8425 (1-5 microM) inhibited PMA-stimulated PLD activity by 85%. In contrast, the antigen-stimulated PLD activity was much less sensitive to the inhibitor. RBL-2H3 cells express PKC alpha, beta, delta, epsilon and zeta isozymes and down-regulation of PKC by exposure to PMA (20 nM) for 1-2 h caused rapid decrease in PKC alpha and beta isozymes, leaving PKC delta, epsilon and zeta isozymes intact. Apparent decreases in the levels of PKC alpha and beta to about 50% were observed after adding 20 nM PMA for 1 h, when PMA-stimulated PLD activity was inhibited by up to 70%. Decrease in antigen-stimulated PLD activity was evident after 2 h PMA-treatment, when PKC alpha and beta decreased by nearly 70%. These results suggest that in the antigen-mediated PLD pathway PKC may be implicated but not play such a great role as PMA-stimulated pathway which is mediated through PKC alpha or beta. Then, we have examined the involvement of calcium/calmodulin (CaM) in PLD activation by antigen, since the antigen-stimulated PLD activation showed the absolute requirement for extracellular calcium. Preincubation of RBL-2H3 cells with a CaM antagonist W-7 (20 microM) inhibited the antigen-stimulated PLD activity by 90%, but W-5, a chlorine-deficient analogue of W-7 that only weakly interact with CaM, caused little inhibitory effect. Another non-specific CaM antagonist, trifluoperazine (TFP) also inhibited PLD activation. These results suggest that calcium/CaM may be involved in the antigen-stimulated PLD activation.
在大鼠嗜碱性白血病（RBL-2H3）细胞中研究了蛋白激酶C（PKC）同工酶在抗原或佛波酯（PMA）诱导的磷脂酶D（PLD）激活中的差异作用。在[3H]油酸标记的细胞中，抗原（100 ng/ml）和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）（100 nM）在丁醇存在下均产生PLD激活的特异性产物[3H]磷脂酰丁醇（PBut）。用选择性PKC抑制剂Ro31-8425（1-5 microM）预处理细胞可使PMA刺激的PLD活性降低85%。相比之下，抗原刺激的PLD活性对该抑制剂的敏感性要低得多。RBL-2H3细胞表达PKCα、β、δ、ε和ζ同工酶，通过暴露于20 nM PMA 1-2小时使PKC下调导致PKCα和β同工酶迅速减少，而PKCδ、ε和ζ同工酶保持完整。加入20 nM PMA 1小时后，观察到PKCα和β水平明显下降至约50%，此时PMA刺激的PLD活性被抑制高达70%。PMA处理2小时后，抗原刺激的PLD活性明显降低，此时PKCα和β下降近70%。这些结果表明，在抗原介导的PLD途径中PKC可能参与其中，但作用不如通过PKCα或β介导的PMA刺激途径那样大。然后，我们研究了钙/钙调蛋白（CaM）在抗原诱导的PLD激活中的作用，因为抗原刺激的PLD激活显示绝对需要细胞外钙。用CaM拮抗剂W-7（20 microM）预孵育RBL-2H3细胞可使抗原刺激的PLD活性降低90%，但W-5（一种与CaM弱相互作用的W-7缺氯类似物）几乎没有抑制作用。另一种非特异性CaM拮抗剂三氟拉嗪（TFP）也抑制PLD激活。这些结果表明钙/CaM可能参与抗原刺激的PLD激活。
