latrunculin-b and inositol-2-4-5-trisphosphate

We explored a potential structural and functional link between filamentous actin (F-actin) and inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) in mouse pancreatic acinar cells. Using immunocytochemistry, F-actin and type 2 and 3 IP(3)Rs (IP(3)R2 and IP(3)R3) were identified in a cellular compartment immediately beneath the apical plasma membrane. In an effort to demonstrate that IP(3)R distribution is dependent on an intact F-actin network in the apical subplasmalemmal region, cells were treated with the actin-depolymerising agent latrunculin B. Immunocytochemistry indicated that latrunculin B treatment reduced F-actin in the basolateral subplasmalemmal compartment, and reduced and fractured F-actin in the apical subplasmalemmal compartment. This latrunculin-B-induced loss of F-actin in the apical region coincided with a reduction in IP(3)R2 and IP(3)R3, with the remaining IP(3)Rs localized with the remaining F-actin. Experiments using western blot analysis showed that IP(3)R3s are resistant to extraction by detergents, which indicates a potential interaction with the cytoskeleton. Latrunculin B treatment in whole-cell patch-clamped cells inhibited Ca(2+)-dependent Cl(-) current spikes evoked by inositol (2,4,5)-trisphosphate; this is due to an inhibition of the underlying local Ca(2+) signal. Based on these findings, we suggest that IP(3)Rs form links with F-actin in the apical domain and that these links are essential for the generation of local Ca(2+) spikes.

Topics: Actin Cytoskeleton; Actins; Animals; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Channels; Calcium Signaling; Cell Membrane; Cytoskeleton; Detergents; Electrophysiology; Gelsolin; Green Fluorescent Proteins; Immunohistochemistry; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Pancreas; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidines