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Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.

Agonist-evoked cytosolic Ca 2⍣ 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 Ca 2⍣ concentration using simultaneous imaging of Ca 2⍣ -sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca 2⍣ -releasing messenger inositol 1,4,5-trisphosphate (IP 3 ), the local repetitive Ca 2⍣ responses in the granule area were transformed into a global rise in the cellular Ca 2⍣ concentration. In the absence of IP 3 , CCCP had no effect on the cytosolic Ca 2⍣ levels. Antimycin and antimycin ⍣ oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca 2⍣ diffusion from the primary Ca 2⍣ 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 Ca 2⍣ signals spread all over the cytosol.

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.

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 Ca(2+) oscillations initiated in the granular area triggered mitochondrial Ca(2+) uptake mainly in the perigranular area. The most intensive uptake occurred in the mitochondria close to the apical plasma membrane. Store-operated Ca(2+) influx through the basolateral membrane caused preferential Ca(2+) uptake into sub-plasmalemmal mitochondria. The perinuclear mitochondria were activated specifically by local uncaging of Ca(2+) in the nucleus. These mitochondria could isolate nuclear and cytosolic Ca(2+) 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 Ca(2+) signals and can therefore participate in the local regulation of Ca(2+) homeostasis and energy supply.

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.

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.