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

Abstract

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 μM), cytochalasin D (10 μM) or toxin B fromClostridium 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 μM) 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 IP3and 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.