In pancreatic acinar cells, postprandial levels of cholecystokinin (CCK) evoke oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) by releasing calcium from IP₃-operated stores. In addition to IP₃, other factors can modulate Ca²⁺ release through this channel. In several models, including pancreatic acinar cells, the mild oxidant thimerosal can increase the sensitivity of IP₃ receptors (Thorn et al. 1992). It has also been proposed that mitochondria modulate IP₃-induced signals due to the ability to uptake and release Ca²⁺ in the immediate vicinity of the IP₃ receptors (Rizzuto et al. 1993). We have previously shown that mitochondrial inhibitors impair CCK-evoked Ca²⁺ oscillations in pancreatic acinar cells (Camello-Almaraz et al. 2002). Since pretreatment with the antioxidant N-acetyl-cysteine reduced the percentage of cells with an oscillatory response to CCK, we proposed that ROS generated by mitochondria collaborated in Ca²⁺ release by sensitization of IP₃ receptors (Camello et al. 2000). We tested this hypothesis by conventional digital microfluorimetry in fura-2-loaded pancreatic acinar cells freshly isolated from adult mice killed by cervical dislocation. In another sets of experiments we used the patch-clamp technique in the whole-cell configuration to record Ca²⁺-activated Cl^– currents as an indirect method to monitor [Ca²⁺]_i changes.

Electrophysiological experiments showed the mitochondrial inhibitor rotenone does not disrupt oscillations by decreasing cytosolic ATP concentration, given that the internal pipette solution contained 2 mM ATP. To assess the role of mitochondrial reactive oxygen species (ROS), we used 200 µM MnTBAP, a mimetic of superoxide dismutase (SOD) and 1-10 µM MitoQ, an antioxidant ubiquinone targeted to mitochondria by a chemical modification (Kelso et al. 2001). When applied on top of 20 µM CCK-induced [Ca²⁺]_i oscillations, MnTBAP induced partial inhibition of the signal. In the case of MitoQ treatment, most of the cells lost Ca²⁺ oscillations, similar to previous observations for rotenone or FCCP. To assess whether mitochondrial inhibitors disrupted oscillations by cessation of Ca²⁺ uptake into mitochondria, we used the inhibitor of the Ca²⁺ uniporter Ru360. However, this compound had no effects on CCK-evoked oscillations.

Our results indicate that, irrespective of the role of mitochondrial Ca²⁺ uptake, the oxidants generated by mitochondria could play a role in the maintenance of Ca²⁺ oscillations.

This work was supported by SAF-2001-0295. MitoQ was kindly provided by M. Murphy.