Bozem M, Kuhlmann S, Blum R, Feick P, Schulz I
Department of Physiology, University of the Saarland, Homburg-Saar, Germany.
Cell Calcium. 2000 Aug;28(2):73-82. doi: 10.1054/ceca.2000.0133.
We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release. The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels. Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.
我们研究了肌动蛋白细胞骨架在蛙皮素诱导胰腺腺泡肿瘤细胞系AR4-2J产生肌醇1,4,5-三磷酸（IP(3)）及释放Ca(2+)过程中的作用。使用Fura-2测定细胞悬液中的细胞内和细胞外游离Ca(2+)浓度。用拉特肌醇B（10 microM）、细胞松弛素D（10 microM）或艰难梭菌毒素B（20 ng/ml）预处理细胞5 - 29小时，破坏肌动蛋白细胞骨架，导致蛙皮素刺激的IP(3)产生和Ca(2+)释放均受到抑制。这些毒素对蛙皮素与其受体的结合、Ca(2+)摄入细胞内储存库以及静息Ca(2+)水平均无影响。毒胡萝卜素（100 nM）或IP(3)（1 microM）诱导的细胞内储存库Ca(2+)动员不受拉特肌醇B影响。在经拉特肌醇B预处理的细胞中，在G蛋白激活剂氟化铝存在的情况下，蛙皮素刺激的IP(3)产生和Ca(2+)释放的抑制作用部分被解除。氟化铝对对照细胞和经毒素预处理细胞的基础IP(3)和Ca(2+)水平无影响。我们得出结论，肌动蛋白细胞骨架的破坏损害了蛙皮素受体与其G蛋白的偶联，导致磷脂酶C活性受到抑制，随后IP(3)产生和Ca(2+)释放减少。
