In the present study we have employed confocal laser scaning microscopy to monitor the effect of reactive oxygen species (ROS) on mitochondrial activity in mouse pancreatic acinar cells. Previous work has shown that the ROS H₂O₂ induced an increase in [Ca²⁺]_i both in the presence and absence of extracellular Ca²⁺, i.e. H₂O₂ released Ca²⁺ from intracellular stores (Pariente et al. 2001).

Male Swiss mice were used throughout the studies. Animals were killed by rapid cervical dislocation. In our investigations cytosolic ([Ca²⁺]_i) as well as mitochondrial ([Ca²⁺]_m) Ca²⁺ concentrations, mitochondrial inner membrane potential (C{special}_m) and FAD autofluorescence were determined. Following loading of the cells with MitoTracker Green FM, a dye that selectively accummulates in the mitochondria, bright fluorescent spots could be detected, being in principle spread all through the cytosolic area, although the concentration near the zymogen granule area, surrounding the nucleus and beneath the basolateral plasma membrane could be observed too. The same distribution of fluorescence could be observed after loading of cells with rhod-2 and JC-1, for [Ca²⁺]_m and C{special}_m determinations respectively. FAD autofluorescence depicted similar distribution inside the cells compared to that of the fluorescent dyes. ROS used throughout the studies was H₂O₂ (1 mM).

The results show that perfusion of pancreatic acinar cells with H₂O₂ (1 mM) led to an increase in [Ca²⁺]_m that slowly returned to prestimulation level, but remained elevated for at least 10 min. In some experiments step increases in rhod-2 fluorescence were observed, which we have related to the oscillatory pattern of changes in [Ca²⁺]_i induced by H₂O₂. Following H₂O₂ tretament inclusion of CCK (10 nM) in the perfusion medium failed to induce further changes in [Ca²⁺]_m. However, previous stimulation of pancreatic acinar cells with 10 nM CCK, which induced as well an increase in [Ca²⁺]_m, did not block the H₂O₂-induced response.

Increases in [Ca²⁺]_m induced by H₂O₂ correlated with depolarizations of C{special}_m and increases in FAD autofluorescence. Pre-incubation of cells in the presence of 10 µM rotenone, which blocks the mitochondrial electron transport chain, depolarized mitochondria and inhibited the response induced by perfusion of cells with 1 mM H₂O₂ on C{special}_m. Uncoupling of mitochondria led to a decrease in FAD autofluorescence that, however, did not inhibit the effect of H₂O₂. FCCP (100 nM), another mitochondrial uncoupler, which inhibits accummulation of Ca²⁺ by mitochondria, did not block the effect of H₂O₂ on FAD autofluorescence.

Perfusion of cells with thapsigargin (1 µM) led to an increase in [Ca²⁺]_m and FAD autofluorescence, and depolarized C{special}_m.

These changes in [Ca²⁺]_m, C{special}_m and FAD autofluorescence were observed in the absence of extracellular Ca²⁺ as well.

In summary our results show that, in the presence of H₂O₂, changes in mitochondrial activity are observed that might not entirely depend on Ca²⁺ mobilization, in opposition to what has been shown for CCK. This might be due to the oxidant nature of H₂O₂ and, therefore, could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies.

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