Previous studies have shown that microtubules position the endoplasmic reticulum to the apical pole region of pancreatic acinar cells, thus ensuring a polarised Ca²⁺ response that spreads from the apical pole towards the basolateral region of the cell (Fogarty et al. 2000). In our present studies we explore the role of the actin cytoskeleton on Ca²⁺ signalling.

Using the whole-cell patch-clamp configuration with 10 mM Ins(2,4,5)P₃ in the pipette solution, we initiated Ca²⁺-dependent Cl^– current spikes in freshly isolated pancreatic acinar cells from humanely killed mice. The spikes reflect a Ca²⁺ response restricted to the apical pole of the cell. Latrunculin B (Lat B), an agent that inhibits actin polymerisation, at 50Ð90 mM inhibited the current spikes (n = 10). These effects were concentration dependent. At 90 mM, Lat B had completely inhibited spiking within a minute in 3/4 cells. At 50 mM Lat B, spikes were still evident within the time range of 5Ð20 min in four cells, although by the end of each experiment the spikes had either been completely inhibited or reduced in amplitude and altered in profile. In further experiments where we loaded the cells with the Ca²⁺ dye Calcium Green, we demonstrated that this is due to a loss of the Ca²⁺ response and not due to an action on the Cl^– channel (n = 3).

We have also performed immunocytochemistry of actin and IP₃ receptors before and after the addition of Lat B. Both phalloidin and an anti-actin antibody showed actin to be present beneath the plasma membrane throughout the cell but to predominate in the apical pole. Lat B (100 mM) over a time course of up to 1 h, diminished actin levels and disrupted actin distribution. However, in many instances even after prolonged Lat B treatment, some actin remained in the apical pole region. IP₃ receptors, type II and III, were restricted to the apical pole and type II IP₃ receptors were found to colocalise with actin. After treatment with Lat B (100 mM) over a time course of up to an hour, both type II and III IP₃ receptors showed an altered distribution. In experiments where we had labelled type II IP₃ receptors and actin and treated the cells with Lat B (100 mM) the IP₃ receptors colocalised with the remaining actin.

In conclusion, we hypothesise that either a direct or indirect attachment, mediated by an unknown protein, is present between actin and IP₃ receptors in pancreatic acinar cells. This may ensure a polarised Ca²⁺ response, which is probably required for effective secretion in these cells.

M.T. is funded by a BBSRC studentship.

All procedures accord with current UK legislation.