Grönroos E, Schippert A, Engström M, Sjölander A
Department of Cell Biology, Linköping University, Sweden.
Cell Calcium. 1995 Mar;17(3):177-86. doi: 10.1016/0143-4160(95)90032-2.
Leukotriene D4 (LTD4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD4 receptor. The tyrosine kinase inhibitors genistein and herbimycin. A both reduced the LTD4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD4 was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.
白三烯D4（LTD4）已被发现可在肠上皮细胞系Int 407中诱导钙信号传导，并且该作用涉及不同的GTP结合蛋白（G蛋白）和γ亚型磷脂酶C（PLC-γ）的激活。基于这一认识，我们研究了蛋白酪氨酸激酶活性在LTD4受体钙信号系统中可能的调节作用。酪氨酸激酶抑制剂染料木黄酮和除莠霉素A在细胞外钙存在的情况下刺激Int 407细胞时，均使LTD4诱导的钙信号降低了70%，但在无钙培养基中刺激细胞时对该信号没有影响。与这些发现一致，用酪氨酸激酶抑制剂预处理也能阻断毒胡萝卜素诱导的细胞钙内流。这些抑制剂对细胞内钙的动员没有影响，这一发现得到了支持，即即使存在一种酪氨酸激酶抑制剂，LTD4仍能够诱导PLC-γ酪氨酸磷酸化增加。关于染料木黄酮对LTD4诱导的钙内流的影响，可能令人感兴趣的是，在用LTD4刺激的细胞中用PLC-γ抗体获得的免疫沉淀物中检测到两条主要的酪氨酸磷酸化蛋白带。这些与PLC-γ相关的蛋白估计分子量分别为84 kD和97 kD。用染料木黄酮预孵育完全消除了LTD4诱导的主要97 kD条带酪氨酸磷酸化增加，而84 kD蛋白带，与PLC-γ条带一样，在LTD4刺激下酪氨酸残基的磷酸化仍增加。当细胞用染料木黄酮的无活性类似物大豆苷元预孵育时，既不会诱导这种效应，也不会诱导染料木黄酮的任何其他效应。目前的结果表明，LTD4在上皮细胞中诱导的钙信号传导不仅涉及PLC-γ的酪氨酸磷酸化，还涉及酪氨酸激酶依赖性步骤，该步骤发生在PLC-γ激活的下游，直接参与激动剂介导的钙内流调节。
