Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca2+ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca2+]i) signals. Several Ca2+ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca2+ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca2+]i signals by Ca2+ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca2+ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca2+ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca2+]i signals. Together, the data reviewed here indicate that Ca2+-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca2+]i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed.

In the present study we have studied how [Ca2+](i) is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+](i) increase, reaching a stable plateau after 10-15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 microm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 microm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca2+](i). In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+](i) induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.

1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from pancreatic acinar cells with preserved intracellular environments. The calcium dependence of calcium extrusion indicated a strong co-operativity (Hill coefficient, 3 (Tepikin, Voronina, Gallacher & Petersen, 1992a, b;Tepikin, Llopis, Snitsarev, Gallacher & Petersen, 1994).

The droplet technique was used in this study to measure total calcium loss from pancreatic acinar cells due to calcium extrusion. The calcium binding capacity of the cytosol (kc) was measured as the ratio of the decrease in the total calcium concentration of the cytosol of the cell (Δ[Ca]c) and the synchronously occurring decrease in the free calcium ion concentration in the cytosol (Δ[Ca2+]c). The calcium dependency of the calcium binding capacity was determined by plotting values of kc against the corresponding [Ca2+]c. The rise in the cytosolic Ca2+ concentration of pancreatic acinar cells was triggered by stimulation with a supramaximal dose of cholecystokinin (CCK). The recovery of [Ca2+]c during continued exposure to the agonist was due to calcium extrusion from the cell. The calcium binding capacity was about 1500‐2000 for the [Ca2+]c range 150‐500 nM. The mechanism of buffering was not investigated in this study. The calcium binding capacity of the cytosol did not vary significantly with [Ca2+]c in this range. The CCK‐evoked decrease in the total calcium concentration in the lumen of the endoplasmic reticulum (ER) can be estimated from our data, taking into account previously published values for the volume of the ER in pancreatic acinar cells. Comparing the decrease in the total ER calcium concentration with our recently reported values for agonist‐induced reductions in the free Ca2+ concentration inside the ER, we estimate that the calcium binding capacity of the ER is approximately 20. In pancreatic acinar cells we have therefore found a difference of two orders of magnitude in the efficiency of calcium buffering in the cytosol and the ER lumen.