Hu S Y, Song S L, Ho M Y, Chueh S H
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
J Neurochem. 1997 Feb;68(2):846-54. doi: 10.1046/j.1471-4159.1997.68020846.x.
Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.
用包括冈田酸和花萼海绵诱癌素A在内的磷酸酶抑制剂预先处理NG108-15细胞，可抑制缓激肽诱导的胞质Ca2+浓度（[Ca2+]i）升高约63%。这种抑制作用依赖于冈田酸的浓度，IC50为0.15 nM。无论细胞外Ca2+是否存在，冈田酸处理仅降低了[Ca2+]i升高的最大反应，而对缓激肽的EC50值没有影响。冈田酸处理既不降低细胞内非线粒体Ca2+储存中45Ca2+的积累能力，也不降低毒胡萝卜素诱导的[Ca2+]i升高幅度。相反，相同的磷酸酶抑制剂处理抑制了缓激肽诱发的肌醇1,4,5-三磷酸（IP3）生成、Mn2+内流以及线粒体Ca2+积累能力。此外，冈田酸预处理抑制了IP3在Ca2+释放中的敏感性。我们的结果表明，通过促进蛋白质磷酸化降低缓激肽诱导的[Ca2+]i升高，归因于磷脂酶C活性降低、对IP3敏感性降低以及Ca2+内流速率减慢。因此，磷酸化在NG108-15细胞中缓激肽敏感的Ca2+信号级联反应中发挥作用。
