Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

Agonist‐evoked cytosolic Ca2+ spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria‐specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca2+ concentration using simultaneous imaging of Ca2+‐sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m ‐chlorophenylhydrazone (CCCP) was applied in the presence of the Ca2+‐releasing messenger inositol 1,4,5‐trisphosphate (IP3), the local repetitive Ca2+ responses in the granule area were transformed into a global rise in the cellular Ca2+ concentration. In the absence of IP3, CCCP had no effect on the cytosolic Ca2+ levels. Antimycin and antimycin + oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca2+ diffusion from the primary Ca2+ release sites in the granule‐rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca2+ signals spread all over the cytosol.

We have identified three distinct groups of mitochondria in normal living pancreatic acinar cells, located (i) in the peripheral basolateral region close to the plasma membrane, (ii) around the nucleus and (iii) in the periphery of the granular region separating the granules from the basolateral area. Three‐dimensional reconstruction of confocal slices showed that the perigranular mitochondria form a barrier surrounding the whole of the granular region. Cytosolic Ca2+ oscillations initiated in the granular area triggered mitochondrial Ca2+ uptake mainly in the perigranular area. The most intensive uptake occurred in the mitochondria close to the apical plasma membrane. Store‐operated Ca2+ influx through the basolateral membrane caused preferential Ca2+ uptake into sub‐plasmalemmal mitochondria. The perinuclear mitochondria were activated specifically by local uncaging of Ca2+ in the nucleus. These mitochondria could isolate nuclear and cytosolic Ca2+ signalling. Photobleaching experiments indicated that different groups of mitochondria were not luminally connected. The three mitochondrial groups are activated independently by specific spatiotemporal patterns of cytosolic Ca2+ signals and can therefore participate in the local regulation of Ca2+ homeostasis and energy supply.

Cytosolic Ca²⁺ signals are crucial for the control of fluid and enzyme secretion from exocrine glands. The highly polarized exocrine acinar cells have evolved sophisticated and complex Ca²⁺ signaling mechanisms that exercise precise control of the secretory events occurring across the apical plasma membrane bordering the gland lumen. Ca²⁺ stores in the endoplasmic reticulum, the secretory granules, the lysosomes, and the endosomes all play important roles in the generation of the local apical Ca²⁺ spikes that switch on Cl⁻ channels in the apical plasma membrane as well as exocytotic export of enzymes. The mitochondria are crucial not only for ATP generation but also for the physiologically important subcellular compartmentalization of the cytosolic Ca²⁺ signals.

Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca²⁺]_C) implicated in pancreatitis. We sought to define the mechanisms of this elevation. Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca²⁺]_C (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca²⁺]_ER, Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10–100 μmol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. Intracellular delivery of ester induced oscillatory increases of [Ca²⁺]_C and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca²⁺]_ER, not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca²⁺]_C, although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. Fatty acid ethyl ester increases [Ca²⁺]_C through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.