O. H. Petersen

Perinuclear, perigranular and sub‐plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport

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.

Spatial dynamics of second messengers : IP3 and cAMP as long-range and associative messengers

Recent imaging experiments have revealed the distinct spatial dynamics of second-messenger actions. In general, actions of Ca2+ tend to be local, whereas those of other messengers such as inositol 1,4,5-trisphosphate (IP3) and cAMP are long range. In pancreatic acinar cells, IP3 generated at the base can diffuse across the cell and evoke a spatially confined Ca2+ signal in the apical pole, triggering enzyme and fluid secretion. Similar mechanisms might also operate in other cell types. We propose that the distinct dynamics of messengers might be relevant to neuronal function: IP3 and cAMP could convey signals over long distances along neurites, and serve as mediators for association and co-operation, for example, during learning.

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP‐gamma‐S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura‐2) and measurement of the Ca2(+)‐dependent Cl‐ current (patch‐clamp whole‐cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1‐0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)‐dependent Cl‐ current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl‐ current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP‐gamma‐S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl‐ current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2+-induced Ca2+ release

Receptor-mediated inositol 1,4,5-trisphosphate (Ins-(1,4,5)P3) generation evokes fluctuations in the cytoplasmic Ca2+ concentration ([Ca2+]i). Intracellular Ca2+ infusion into single mouse pancreatic acinar cells mimicks the effect of external acetylcholine (ACh) or internal Ins(1,4,5)P3 application by evoking repetitive Ca2+ release monitored by Ca2(+)-activated Cl- current. Intracellular infusion of the Ins(1,4,5)P3 receptor antagonist heparin fails to inhibit Ca2+ spiking caused by Ca2+ infusion, but blocks ACh- and Ins(1,4,5)P3-evoked Ca2+ oscillations. Caffeine (1 mM), a potentiator of Ca2(+)-induced Ca2+ release, evokes Ca2+ spiking during subthreshold intracellular Ca2+ infusion. These results indicate that ACh-evoked Ca2+ oscillations are due to pulses of Ca2+ release through a caffeine-sensitive channel triggered by a small steady Ins(1,4,5)P3-evoked Ca2+ flow.

The endoplasmic reticulum as one continuous Ca2+ pool: visualization of rapid Ca2+ movements and equilibration

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca2+ store or is composed of separate subunits by monitoring movements of Ca2+ and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca2+ and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca2+ store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca2+ concentration throughout the cell, but repetitive cytosolic Ca2+ spikes, induced by a low ACh concentration, hardly reduced the ER Ca2+ level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca2+ liberation. Ca2+ released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.

Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells

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.

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine‐sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP‐ribose (cADP‐ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP‐ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.

Intracellular ADP activates K+ channels that are inhibited by ATP in an insulin-secreting cell line

the effect of ADP on ATP‐sensitive K+ channels in the insulin‐secreting RINm5F cell line has been investigated with the help of single‐channel current recording from saponin‐permeabilized cells. ADP (100–500 μM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP‐β‐S cannot mimick this effect. During sustained ATP (500 μM)‐evoked inhibition of K+ channel opening, 500 μM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP‐activated channels. It is suggested that the physiological control of K+ channel opening in the insulin‐secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.

Calcium uptake via endocytosis with rapid release from acidifying endosomes

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.

Two different but converging messenger pathways to intracellular Ca2+ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

Hormones and neurotransmitters mobilize Ca2+ from the endoplasmic reticulum via inositol trisphosphate (IP3) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca2+ responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca2+ spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca2+ signals. We show that IP3, cyclic ADP‐ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca2+ spiking by activating common oscillator units composed of IP3 and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP3 receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP‐ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated specifically by cyclic ADP‐ribose receptors. In the apical pole there is a common early activation site for Ca2+ release, indicating that the three types of Ca2+ release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.