Aberrant Ca(2+) signalling through acidic calcium stores in pancreatic acinar cells.

Pancreatic acinar cells possess a very large Ca(2+) store in the endoplasmic reticulum, but also have extensive acidic Ca(2+) stores. Whereas the endoplasmic reticulum is principally located in the baso-lateral part of the cells, although with extensions into the granular area, the acidic stores are exclusively present in the apical part. The two types of stores can be differentiated pharmacologically because the endoplasmic reticulum accumulates Ca(2+) via SERCA pumps, whereas the acidic pools require functional vacuolar H(+) pumps in order to maintain a high intra-organellar Ca(2+) concentration. The human disease acute pancreatitis is initiated by trypsinogen activation in the apical pole and this is mostly due to either complications arising from gall bladder stones or excessive alcohol consumption. Attention has therefore been focussed on assessing the acute effects of bile acids as well as alcohol metabolites. The evidence accumulated so far indicates that bile acids and fatty acid ethyl esters - the non-oxidative products of alcohol and fatty acids - exert their pathological effects primarily by excessive Ca(2+) release from the acidic stores. This occurs by opening of the very same release channels that are also responsible for normal stimulus-secretion coupling, namely inositol trisphosphate and ryanodine receptors. The inositol trisphosphate receptors are of particular importance and the results of gene deletion experiments indicate that the fatty acid ethyl esters mainly utilize sub-types 2 and 3.

Decoding glutamate receptor activation by the Ca2+ sensor protein hippocalcin in rat hippocampal neurons.

Hippocalcin is a Ca(2+)-binding protein that belongs to a family of neuronal Ca(2+)sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor-dependent depolarization and following Ca(2+)influx via voltage-operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site due to direct Ca(2+)influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin-YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back-propagating action potentials. The translocation to spines required Ca(2+)influx via synaptic NMDA receptors in which Mg(2+) block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1-2 microm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.

Fatty acids, alcohol and fatty acid ethyl esters: toxic Ca2+ signal generation and pancreatitis.

Pancreatitis, a potentially fatal disease in which the pancreas digests itself as well as its surroundings, is a well recognized complication of hyperlipidemia. Fatty acids have toxic effects on pancreatic acinar cells and these are mediated by large sustained elevations of the cytosolic Ca(2+) concentration. An important component of the effect of fatty acids is due to inhibition of mitochondrial function and subsequent ATP depletion, which reduces the operation of Ca(2+)-activated ATPases in both the endoplasmic reticulum and the plasma membrane. One of the main causes of pancreatitis is alcohol abuse. Whereas the effects of even high alcohol concentrations on isolated pancreatic acinar cells are variable and often small, fatty acid ethyl esters--synthesized by combination of alcohol and fatty acids--consistently evoke major Ca(2+) release from intracellular stores, subsequently opening Ca(2+) entry channels in the plasma membrane. The crucial trigger for pancreatic autodigestion is intracellular trypsin activation. Although there is still uncertainty about the exact molecular mechanism by which this Ca(2+)-dependent process occurs, progress has been made in identifying a subcellular compartment--namely acid post-exocytotic endocytic vacuoles--in which this activation takes place.

Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspects.

Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca(2+) store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P(3) receptors [Ins(1,4,5)P(3)Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P(3)Rs by Ins(1,4,5)P(3). We have also investigated the possible consequences of nuclear calcium signals: the role of Ca(2+) in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca(2+)-mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca(2+): the IEG expression level was lowest in Ca(2+)-free solution, increased at the physiological level of 1 mm [Ca(2+)](o) and was maximal at 10 mm [Ca(2+)](o), i.e.: 102 +/- 22% and 163 +/- 15% for c-fos; c-myc -73 +/- 13% and 106 +/- 24%; c-jun -49 +/- 8% and 59 +/- 9% at 1 and 10 mm of extracellular Ca(2+) respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca(2+) together with nuclear Ca(2+) signals induced by CCK play important roles in the induction of IEG expression.

Movement of calcium signals and calcium-binding proteins: firewalls, traps and tunnels.

In the board game 'Snakes and Ladders', placed on the image of a pancreatic acinar cell, calcium ions have to move from release sites in the secretory region to the nucleus. There is another important contraflow - from calcium entry channels in the basal part of the cell to ER (endoplasmic reticulum) terminals in the secretory granule region. Both transport routes are perilous as the messenger can disappear in any place on the game board. It can be grabbed by calcium ATPases of the ER (masquerading as a snake but functioning like a ladder) and tunnelled through its low buffering environment, it can be lured into the whirlpools of mitochondria uniporters and forced to regulate the tricarboxylic acid cycle, and it can be permanently placed inside the matrix of secretory granules and released only outside the cell. The organelles could trade calcium (e.g. from the ER to mitochondria and vice versa) almost depriving this ion the light of the cytosol and noble company of cytosolic calcium buffers. Altogether it is a rich and colourful story.

Generation and modulation of cytosolic Ca2+ signals in pancreatic acinar cells: techniques and mechanisms.

Calcium is a ubiquitous signalling molecule, known to control a vast array of cellular processes. In order to retain stimulus fidelity, the cell encodes the increases in intracellular calcium in the form of oscillations that are regulated both temporally and spatially. Here, we review recent work, using the pancreatic acinar cell as a model system, on the mechanisms employed to generate and modulate cytosolic Ca(2+) signals, and the technical advances that have made these studies possible.

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells.

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.

The distribution of the endoplasmic reticulum in living pancreatic acinar cells.

Studies on pancreatic acinar cells provided the original evidence for the Ca(2+) releasing action of inositol 1,4,5-trisphosphate (IP(3)). Ironically, this system has presented problems for the general theory that IP(3) acts primarily on the endoplasmic reticulum (ER), because the IP(3)-elicited Ca(2+) release occurs in the apical pole, which is dominated by zymogen granules (ZGs) and apparently contains very little ER. Using confocal and two-photon microscopy and a number of different ER-specific fluorescent probes, we have now investigated in detail the distribution of the ER in living pancreatic acinar cells. It turns out that although the bulk of the ER, as expected, is clearly located in the baso-lateral part of the cell, there is significant invasion of ER into the granular pole and each ZG is in fact surrounded by strands of ER. This structural evidence from living cells, in conjunction with recent functional studies demonstrating the high Ca(2+) mobility in the ER lumen, provides the framework for a coherent and internally consistent theory for cytosolic Ca(2+) signal generation in the apical secretory pole, in which the primary Ca(2+) release occurs from ER extensions in the granular pole supplied with Ca(2+) from the main store at the base of the cell by the tunnel function of the ER.

Calcium leak from intracellular stores--the enigma of calcium signalling.

Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca(2+)] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca(2+) for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.

Local uncaging of caged Ca(2+) reveals distribution of Ca(2+)-activated Cl(-) channels in pancreatic acinar cells.

In exocrine acinar cells, Ca(2+)-activated Cl(-) channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl(-) uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca(2+), was used to reveal the Cl(-) channel distribution in mouse pancreatic acinar cells, where approximately 90% of the current activated by Ca(2+) in response to acetylcholine was carried by Cl(-). When caged Ca(2+) in the cytosol was uncaged locally in the apical pole, the Cl(-) current was activated, whereas local Ca(2+) uncaging in the basal or lateral areas of the cell had no effect. Even when Ca(2+) was uncaged along the whole inner surface of the basolateral membrane, no Cl(-) current was elicited. There was little current deactivation at a high cytosolic Ca(2+) concentration ([Ca(2+)](c)), but at a low [Ca(2+)](c) there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca(2+)-activated Cl(-) channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca(2+)](c) and membrane potential. Ca(2+)-activated Cl(-) channels do not mediate Cl(-) uptake at the base, but acetylcholine-elicited local [Ca(2+)](c) spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.

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 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.

ER calcium and the functions of intracellular organelles.

Cytosolic calcium has long been known as a second messenger of major significance. Recently it has become apparent that calcium stored in cellular organelles can also be an important regulator of cellular functions. The endoplasmic reticulum (ER) is usually the largest store of releasable calcium in the cell. The diverse signalling functions of calcium populating the endoplasmic reticulum and its interactions with other organelles are illustrated in Figure ?? and described in this paper.

The endoplasmic reticulum as one continuous Ca(2+) pool: visualization of rapid Ca(2+) movements and equilibration.

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca(2+) store or is composed of separate subunits by monitoring movements of Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) concentration throughout the cell, but repetitive cytosolic Ca(2+) spikes, induced by a low ACh concentration, hardly reduced the ER Ca(2+) level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca(2+) liberation. Ca(2+) released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate.

Hormones and neurotransmitters mobilize Ca(2+) from the endoplasmic reticulum via inositol trisphosphate (IP(3)) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca(2+) responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca(2+) spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca(2+) signals. We show that IP(3), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca(2+) spiking by activating common oscillator units composed of IP(3) and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP(3) 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 Ca(2+) release, indicating that the three types of Ca(2+) release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.

The relationship between acetylcholine-evoked Ca(2+)-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells.

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.

Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca(2+) signals.

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.

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.

Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse pancreatic acinar cells.

1. 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 (Delta[Ca]c) and the synchronously occurring decrease in the free calcium ion concentration in the cytosol (Delta[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. 2. 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.

Hormone-induced secretory and nuclear translocation of calmodulin: oscillations of calmodulin concentration with the nucleus as an integrator.

Many important enzyme activities are regulated by Ca2+-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca2+-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca2+ signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. We loaded fluorescent-labeled CaM into pancreatic acinar cells and monitored the fluorescence in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoked a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that bound Ca2+ translocated. At a lower hormone concentration, evoking Ca2+ oscillations, regular spikes of increased CaM concentration were seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus was able to integrate the Ca2+ spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein.