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 (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.
Bozem
M
M
Department of Physiology, University of the Saarland, Homburg-Saar, Germany.
Kuhlmann
S
S
Blum
R
R
Feick
P
P
Schulz
I
I
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D001839:Bombesin / Q000378:metabolism / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D006728:Hormones / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010190:Pancreatic Neoplasms; D051381:Rats; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism
D017607:Aluminum Compounds; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D006728:Hormones; D013844:Thiazoles; D053778:Thiazolidines; C031815:toxB protein, Clostridium difficile; D015638:Cytochalasin D; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; C037068:latrunculin B; D001839:Bombesin; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride