Introduction Pancreatic acinar cells are polarized epithelial cells that secrete fluid and enzymes in response to stimulation with agonists such as acetylcholine (ACh) and cholecystokinin (CCK) (Palade et al., 1975; Williams et al., 1997; Williams, 2001). Fluid and enzyme secretion in pancreatic acinar cells are Ca 2+ -dependent processes (Petersen, 1992), which rely on agonist-evoked rises in intracellular Ca 2+ that originate within the apical region of the cell (Kasai et al., 1993; Thorn et al., 1993). The apical region contains secretory vesicles termed zymogen granules, which fuse with the apical plasma membrane upon receiving a Ca 2+ signal, releasing their contents into the luminal extracellular environment in a process of exocytosis (Giovannucci et al., 1998; Ito et al., 1997; Palade et al., 1975). In addition, Ca 2+ -dependent Cl – channels present in the apical plasma membrane, thought to be important in regulating fluid secretion, are activated by agonist-evoked rises in intracellular Ca 2+ (Kasai and Augustine, 1990; Park et al., 2001). Increases in intracellular Ca 2+ emanate from regions near the apical plasma membrane, which coincides with the location of inositol (1,4,5)-trisphosphate receptors (IP 3 Rs) (Fogarty et al., 2000a; Lee et al., 1997b; Nathanson et al., 1994; Yule et al., 1997). IP 3 Rs are Ca 2+ -release channels that are embedded in the membrane of the major intracellular Ca 2+ store, namely the endoplasmic reticulum (ER), and are activated by the second messenger IP 3 (Berridge, 1993; Streb et al., 1983). It is likely that the localized distribution of IP 3 Rs to the apical subplasmalemmal region ensures that Ca 2+ -dependent processes that are known to occur within the apical region are able to function effectively. For example, it has been shown that cytosolic Ca 2+ levels within the apical region must reach micromolar concentrations for exocytosis to occur (Ito et al., 1997; Stecher et al., 1992). A previous study indicated that the cytoskeleton has an important role in maintaining Ca 2+ signalling in pancreatic acinar cells. In this study it was shown that local Ca 2+ spiking depends on the microtubular network to position the ER locally, and therefore Ca 2+ release sites, within the apical region of pancreatic acinar cells (Fogarty et al., 2000b). Evidence from studies performed in a variety of cell types, including pancreatic cells, suggests that IP 3 Rs link to the actin cytoskeleton and actin-associated proteins (Bourguignon et al., 1993a; Giovannucci et al., 2000; Joseph and Samanta, 1993; Rossier et al., 1991; Sugiyama et al., 2000; Tuvia et al., 1999). Furthermore, it has been shown that disrupting the actin cytoskeleton with actin depolymerising agents, impairs the ability of cells to raise cytosolic Ca 2+ levels in response to 971 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. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/5/971/DC1 Key words: Acinar, Actin, IP 3 R, Ca 2+ Summary Inositol (1,4,5)-trisphosphate receptor links to filamentous actin are important for generating local Ca 2+ signals in pancreatic acinar cells Matthew R. Turvey 1 , Kevin E. Fogarty 2 and Peter Thorn 1, * 1 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 IPD, UK 2 Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01650, USA *Author for correspondence (e-mail: pt207@cam.ac.uk) Accepted 21 December 2004 Journal of Cell Science 118, 971-980 Published by The Company of Biologists 2005 doi:10.1242/jcs.01693 Research Article Journal฀of฀Cell฀Science JCS ePress online publication date 15 February 2005