In non-excitable cells depletion of the intracellular Ca²⁺ stores induces Ca²⁺ influx across the plasma membrane (PM), but the mechanism underlying store-mediated Ca²⁺ entry (SMCE) remains unclear. Hypotheses have considered both direct and indirect coupling mechanisms. Recently, a secretion-like coupling model, which shares properties with the direct coupling, has been proposed (Patterson et al. 1999; Rosado et al. 2000). This mechanism involves a physical and reversible interaction between the endoplasmic reticulum (ER) and the PM where the actin cytoskeleton plays an important role (Rosado et al. 2000; Rosado & Sage, 2001). In the present study, the nature of the mechanism underlying SMCE in pancreatic acinar cells has been investigated through a combination of cytoskeletal modifications.

Donor mice were humanely killed by rapid cervical dislocation. Pancreatic acinar cells were isolated and fluorescence measured as previously described (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, TG induced a transient increase in [Ca²⁺]_i due to Ca²⁺ release 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 the cytoskeletal disrupters cytochalasin D (CD; 10 µM) and latrunculin A (Lat A; 3 µM) significantly reduced the activation of SMCE by 78.8 ± 8.8 and 46.6 ± 9.8 %, respectively (S.E.M.; n = 5-10, P < 0.01, Student’s paired t test). In addition, when CD or Lat A was added once SMCE had been activated, Ca²⁺ entry was reduced by 50.5 ± 4.4 and 48.2 ± 1.0 %, respectively (n = 5, P < 0.01). To investigate the role of the apical cytoskeleton in SMCE we induced stabilisation of a cortical actin barrier using jasplakinolide (JP). Treatment of pancreatic acinar cells with 10 µM JP reduced the activation of SMCE by 52.5 ± 3.8 % (n = 5-10, P < 0.01) without having any effect on the maintenance. JP also reduced Ca²⁺ entry stimulated by 1 nM CCK-8, a physiological agonist, without having any effect on the release of Ca²⁺ from the stores, suggesting that it is unlikely that a diffusible molecule mediates SMCE in these cells. Such a factor could reach the PM after cytoskeletal modifications since InsP₃, generated by CCK-8, was able to stimulate Ca²⁺ release. These findings suggest that, as for secretion, the actin cytoskeleton plays a double role in SMCE, acting as a negative clamp, preventing the interaction between the ER and PM, but also is required for this mechanism since the cytoskeleton disrupters impaired Ca²⁺ entry.

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