Previous studies have shown that microtubules locally position the endoplasmic reticulum to the secretory pole region of pancreatic acinar cells, thus ensuring a polarised calcium response that spreads from the apical to the basolateral pole (Fogarty et al. 2000). In our present studies we explore the role of the actin cytoskeleton in the calcium response. In particular we were interested to investigate the potential role of myosins in calcium signalling processes. One of the tools we have used to determine this potential interaction was the myosin ATPase inhibitor, 2, 3-butanedione monoxime (BDM).

We used freshly isolated pancreatic acinar cells (from humanely killed mice), loaded with indo-1, to determine single-cell calcium responses. We observed that 10 mM BDM rapidly and reversibly inhibited calcium oscillations induced by acetylcholine (100 nM, n = 3) or sustained calcium responses by higher concentrations of acetylcholine (100 µM, n = 3). The sustained calcium response induced by cholecystokinin (1 nM), another endogenous agonist, was also rapidly abolished (n = 2), indicating that the effects were not agonist specific. In further experiments, BDM did not affect the calcium response induced by thapsigargin (n = 3), suggesting that BDM does not interfere with the capacitative calcium entry process.

Calcium-dependent chloride current spikes were established by infusing 10 µM Ins (2, 4, 5)P₃ through a whole-cell patch pipette in freshly isolated mouse pancreatic acinar cells. BDM (7-10 mM) rapidly abolished the current spikes (n = 6, Fig. 1). These experiments indicate that BDM is not acting at the cell surface receptors or on the mechanisms of calcium entry, but is acting at the level of the calcium store.

We went on to test for an action of BDM on ryanodine receptors, as has been shown in cardiac tissue (Adams et al. 1998). Cells were stimulated with Ins (2, 4, 5)P₃ to induce trains of spikes, recorded by measuring the calcium-dependent chloride currents as before. Ryanodine (25 µM) was then added to block the ryanodine receptors. Preliminary data suggest (n = 2) that this concentration of ryanodine has no acute effect on the spiking. BDM (10 mM) was then added and the spikes were abolished. This indicates that BDM is not acting on ryanodine receptors.

In the final experiments we applied BDM to a resting, unstimulated cell. To our surprise we could observe a rapid calcium response (n = 6/7), with a peak amplitude of 263 ± 8.4 nM (mean ± S.E.M.). In many cases the response was very rapid, suggesting that BDM was not acting to block Ca²⁺-ATPases.

Taken together, these results suggest a potential site of action of BDM on the IP₃ receptor either directly or indirectly (via myosin).

We are grateful to the BBSRC, MRC and the Royal Society for financial support.

Adams, W., Trafford, A. & Eisner, D. (1998). Pflƒgers Arch. 436, 776-781.

Fogarty, K., Kidd, J., Turner, A., Skepper, J., Carmichael, J. & Thorn, P. (2000). J. Biol. Chem. 275, 22487-22494.