Store-mediated calcium entry (SMCE) is one of the main pathways for calcium influx in non-excitable cells, such as pancreatic acinar cells; however the nature of the mechanism involved in this process remains unclear. Recently, a secretion-like coupling model has been proposed (Patterson et al. 1999; Rosado et al. 2000a), which is based on a physical and reversible interaction between the endoplasmic reticulum (ER) and the PM modulated by the actin cytoskeleton (Rosado et al. 2000a). In pancreatic acinar cells tyrosine kinases, such as p60^src, are involved in many cellular events. p60^src activity has been suggested to be important for the activation of SMCE in human platelets (Rosado et al. 2000b). In the present study, the involvement of p60^src in the activation of SMCE in pancreatic acini has been investigated.

Donor mice were humanely killed by rapid cervical dislocation. Pancreatic acinar cells were isolated and fluorescence measurement and Western blotting performed as previously described (Rosado et al. 2000b; Pariente et al. 2001). Thapsigargin (TG)-induced SMCE was estimated as the integral of the rise in [Ca²⁺]_i for 2.5 min after addition of Ca²⁺. In a Ca²⁺-free medium, treatment of pancreatic acinar cells with 1 µM TG induced release of Ca²⁺ from internal stores. The subsequent addition of Ca²⁺ (2 mM) to the medium resulted in a rapid increase in [Ca²⁺]_i indicative of SMCE. Treatment of pancreatic acinar cells with PP1, a specific inhibitor of p60^src, reduced the activation of SMCE by 16 ± 4, 48 ± 6 and 71 ± 7 % at 3, 10 and 50 µM, respectively (means ± S.E.M.; n = 4-7, P < 0.05, ANOVA), without having any effect on its maintenance. In addition, we have found that depletion of the intracellular calcium stores, either using TG or the physiological agonist CCK-8 (1 nM), induced activation and cytoskeletal association of p60^src, an event that occurs independently of Ca²⁺ entry. Store depletion-induced activation of p60^src was prevented by treatment with cytochalasin D, suggesting that this process requires the integrity of the actin cytoskeleton.

These findings provide evidence for the activation and cytoskeletal association of p60^src upon Ca²⁺ store depletion in pancreatic acinar cells, a process that might be important for the activation of SMCE in these cells.

This work was supported by DGESIC (BFI2001-0624) and Programa Propio of UEX. P.C.R. is supported by a DGESIC fellowship.