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

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP3)-production and Ca2+ release in the pancreatic 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-production and Ca2+ release. The toxins had no effect on binding of bombesin to its receptor, on Ca2+ uptake into intracellular stores and on resting Ca2+ levels. Ca2+ mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP3 (1 mu M) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP3 - production and Ca2+ release was partly suspended in the presence of aluminum fluoride, 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 receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP3- production and Ca2+ release. (C) 2000 Harcourt Publishers Ltd.