Hormone-stimulated calcium release is inhibited by cytoskeleton-disruptingtoxins in AR4-2J cells

We have studied the role of the actin cytoskeleton in bombesin-induced inos itol 1,4,5-trisphosphate (IP3)-production and Ca2+ release in the pancreati c acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca2 concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 mu M), cytochalasin D (10 mu M) or toxin B from Clostridium difficile ( 20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP3-pro duction and Ca2+ release. The toxins had no effect on binding of bombesin t o its receptor, on Ca2+ uptake into intracellular stores and on resting Ca2 + levels. Ca2+ mobilization from intracellular stores, induced by thapsigar gin (100 nM) or IP3 (1 mu M) was not impaired by latrunculin B. In latruncu lin B-pretreated cells inhibition of both, bombesin-stimulated IP3 - produc tion and Ca2+ release was partly suspended in the presence of aluminum fluo ride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP3 and Ca2+ levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin rece ptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP3- production and Ca2+ release. (C) 2000 Har court Publishers Ltd.