In the present study we have investigated the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity employing confocal laser scanning microscopy. The consequences of changes in [Ca²⁺]_i in response to the hormone on mitochondrial function were assessed.

Male swiss mice were used throughout the studies. After killing by rapid cervical dislocation the pancreas was removed and acinar cells were isolated by collagenase digestion followed by pipetting through tips of decreasing diameters. Cytosolic ([Ca²⁺]_i) as well as mitochondrial Ca²⁺ concentrations ([Ca²⁺]_m), mitochondrial inner membrane potential (C{special}_m) and FAD autofluorescence were determined. Cells were loaded with fluo-3, rhod-2 or JC-1, for [Ca²⁺]_i, [Ca²⁺]_m and C{special}_m determinations, respectively (Gonzalez et al. 2000). Following dye loading of the cells, bright fluorescent spots could be detected, being in principle spread all through the cytosolic area, although concentration near the zymogen granule area, surrounding the nucleus and beneath the basolateral plasma membrane could be observed too (Voronina et al. 2002). FAD autofluorescence depicted similar distribution inside the cells compared to that of the fluorescent dyes. Distribution of fluorescence well correlated with that of MitoTracker Green FM, which selectively accumulates in mitochondria.

Our results show that stimulation of cells with 10 nM CCK led to a transient increase in [Ca²⁺]_i (3 exp/12 cell). The hormone induced as well an increase in [Ca²⁺]_m that remained elevated for a longer time compared to cytosolic responses (4 exp/8 cell/46 mit. area). A depolarization of C{special}_m was observed following stimulation of cells with CCK that partially recovered (6 exp/13 cell/61 mit. area); in addition an increase in FAD autofluorescence was observed following stimulation of cells with the Ca²⁺-mobilizing agonist (7 exp/30 cell). Perfusion of cells with thapsigargin (1 µM) led to an increase in [Ca²⁺]_m (2 exp/5 cell/32 mit. area) and FAD autofluorescence (3 exp/32 cell), and depolarized C{special}_m (2 exp/3 cell/28 mit. area). Pretretament of cells with thapsigargin blocked CCK-evoked changes in these parameters (100 % of cells tested). Preincubation of cells in the presence of 10 µM rotenone, which blocks the mitochondrial electron transport chain, depolarized mitochondria and inhibited the response induced by CCK stimulation on C{special}_m (4 exp/6 cell/17 mit. area). The mitochondrial inhibitor induced as well a decrease in FAD autofluorescence and blocked the CCK-induced increase in FAD autofluoresence (6 exp/19 cell). Monitoring of changes in [Ca²⁺]_m, C{special}_m and FAD autofluorescence were performed in the absence of Ca²⁺ in the perifusion medium to avoid contribution of extracellular Ca²⁺ to cytosolic and mitochondrial signals.

In conclusion, the results presented in this work are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with CCK, which might then match the energy supply neccessary for the cell function during secretion. The changes in [Ca²⁺]_m, C{special}_m and FAD autofluorescence depend on mobilization of Ca²⁺ from intracellular stores and are independent of extracellular Ca²⁺.

This work was supported by DGESIC (BFI2001-0624).