Grönroos E, Andersson T, Schippert A, Zheng L, Sjölander A
Department of Cell Biology, Faculty of Health Sciences, Linköping University, Sweden.
Biochem J. 1996 May 15;316 ( Pt 1)(Pt 1):239-45. doi: 10.1042/bj3160239.
We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+. In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event. In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells. This reduction was reversed by co-administration of mevalonate (1 mM). The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores. In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein. This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+. A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+. Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.
我们之前已经表明，白三烯D4（LTD4）诱导上皮细胞内Ca2+的动员是由一种G蛋白介导的，该G蛋白与调节随后Ca2+内流的百日咳毒素敏感G蛋白明显不同。在本研究中，我们试图通过研究异戊二烯化途径抑制剂洛伐他汀对这一信号事件的影响，进一步了解LTD4诱导上皮细胞内Ca2+动员所涉及的机制。在预先用10微摩尔洛伐他汀孵育48小时的细胞中，与对照细胞相比，LTD4诱导的细胞内Ca2+动员减少了75%。甲羟戊酸（1毫摩尔）共同给药可逆转这种减少。无论细胞外培养基中是否存在Ca2+，洛伐他汀均有作用，这表明异戊二烯化必须在Ca2+从细胞内储存释放之前发生。据此，我们还发现，在洛伐他汀预处理的细胞中，LTD4诱导的肌醇1,4,5-三磷酸形成以及LTD4诱导的磷脂酶Cγ1（PLCγ1）酪氨酸残基磷酸化均显著降低。这些结果揭示了PLCγ1的激活可能与一种对异戊二烯化途径活性受损敏感的分子有关，例如小的单体G蛋白。肉毒杆菌C3外毒素诱导的Rho蛋白抑制消除了LTD4诱导的细胞内Ca2+动员，这一观察结果支持了这一观点。Rho蛋白在LTD4诱导的PLCγ1激活中的调节作用不太可能通过对细胞骨架的影响间接介导，因为细胞松弛素D对LTD4诱导的Ca2+动员没有主要影响。尽管相互作用的机制仍有待阐明，但目前的研究结果表明，异戊二烯化蛋白如Rho在LTD4诱导的Ca2+信号中起重要作用。
