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REVIEW
Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage S. Voronina, M. Sherwood, S. Barrow, N. Dolman, A. Conant and A. Tepikin Physiological Laboratory, University of Liverpool, Liverpool, UK
Received 2 June 2008, accepted 8 July 2008 Correspondence: A. Tepikin, Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK. E-mail:
[email protected]
Abstract Ca2+ is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca2+ signalling and homeostasis. Downstream effects of Ca2+ signalling have been and continue to be an important research avenue. The primary functions regulated by Ca2+ in pancreatic acinar cells – exocytotic secretion and fluid secretion – have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca2+ in cellular pathology and the related question of the interplay between Ca2+ signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review. Keywords ATP, calcium signalling, endosomes, mitochondria, pancreatitis, vacuoles.
In the initial part of the review we will give a brief introduction to Ca2+ signalling in pancreatic acinar cells. The second part will discus how calcium signals are shaped by mitochondria and vice versa how Ca2+ changes modify mitochondrial responses. Finally in the last part of this review we will deal with the Ca2+dependent damage to pancreatic acinar cells with specific emphases on vacuole formation.
Physiological and pathological calcium signals The secretagogues acetylcholine and cholecystokinin are involved in the calcium signalling cascade triggering and regulating secretion in pancreatic acinar cells. Two main types of physiological calcium signals are induced by physiological doses of these secretagogues –local and global transients of cytosolic [Ca2+] (Kasai et al. 1993, Thorn et al. 1993, reviewed in Petersen & Tepikin 2008). Pathological calcium signals are prolonged plateaus of elevated cytosolic [Ca2+]. These signals can be induced by supramaximal doses of secretagogues, by bile acids and by non-oxidative derivatives of ethanol
(Raraty et al. 2000, Kim et al. 2002, Voronina et al. 2002a, Criddle et al. 2004). Local calcium transients originate in the apical part of the acinar cell and spend their entire lifetime (usually 1–10 s) in this cellular region (Kasai et al. 1993, Thorn et al. 1993, Gerasimenko et al. 1996, reviewed in Petersen & Tepikin 2008). Very steep gradients of cytosolic Ca2+ concentration ([Ca2+]c) of a few hundreds of nanomoles per micrometre form at the peak of the calcium response between the apical and basal parts of the cell (Gerasimenko et al. 1996). Global calcium responses also usually originate in the apical region but rapidly propagate to the basal part of the cell and invade the nucleoplasm (Kasai et al. 1993, Thorn et al. 1993, Gerasimenko et al. 1996). Local and global calcium responses were recorded in isolated cells, cellular clusters and in individual cells of undissociated pancreatic tissues (Ashby et al. 2003). Structurally the localization of inositol 1,4,5-triphosphate (InsP3) receptors in the apical part of the cell (Nathanson et al. 1994, Lee et al. 1997, Turvey et al. 2005) is probably the most important determinant of local apical [Ca2+]
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responses. All three types of ryanodine receptors (RyRs) are present in acinar cells (Fitzsimmons et al. 2000), the RyRs and corresponding Ca2+ release were found in both the basal and apical region (Fitzsimmons et al. 2000, Straub et al. 2000) with a predominant concentration of RyRs in the boundary between the apical and basal region (Straub et al. 2000). There are different opinions on the mechanism of the formation of local calcium responses in acinar cells (Ashby et al. 2003, Li et al. 2004). Intra patch pipette uncaging, a technique developed by our group, supports the notion of longdistance communication between the receptors located in the basal region and calcium-releasing channels preferentially localized in the apical compartment (Ashby et al. 2003). In the framework of this model the second messengers are produced in the basal region and diffuse for a distance of approx. 15 lm to stimulate the release channels on the other side of the cell. It is interesting to note that the formation of calcium responses in acinar cells could involve both InsP3 receptors and RyRs (Fitzsimmons et al. 2000, Straub et al. 2000, Gerasimenko et al. 2003). Calcium-induced calcium release is an important mechanism for the spread of the calcium wave from the apical to the basal parts of the cell. Paradoxically it was not possible to trigger a calcium wave in the basal region by the local elevation of cytosolic calcium, induced by local calcium uncaging, whilst propagating calcium signals were observed when calcium was uncaged in the apical part of the cell (Ashby et al. 2002). Local apical calcium signals should be the most economical way of organizing this signalling process. Indeed secretory granules that rely on calcium-dependent exocytosis are localized in this region, as are calcium-activated Cl) channels, necessary to produce fluid secretion and therefore clear intrercalated pancreatic ducts from secreted enzymes and zymogens. Both exocytosis and activation of Cl) channels can be induced by local apical [Ca2+] responses (Maruyama et al. 1993, Park et al. 2001b). To form this useful and economical local Ca2+ responses the cell needs to solve a number of complicated puzzles. The first is presented by the small size of pancreatic acinar cells. It seems relatively straightforward to make local calcium signals in the vast space of oocytes or cardiomyocytes but pancreatic acinar cells are only 15–20 lm in diameter and creating prolonged local responses in such cells is a very difficult task. The first property that a small cell needs to develop to produce local calcium responses is to generate substantial calcium buffering. Pancreatic acinar cells have solved this task successfully – they have developed one of the highest calcium buffering capacities in the cellular world – approx. 1500 (this means that out of 1500 calcium ions added to the cytosol of the cell one will remain free and the rest will be
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scavenged by buffers) (Mogami et al. 1999). Another challenge is presented by the necessity to load the calcium store, from which Ca2+ is released, during each calcium response, and to re-load the store again after each calcium transient. The spatial arrangement of organelles in pancreatic acinar cells is such that there is very little endoplasmic reticulum (ER) in the apical part of the acinar cell. This is clearly problematic for a cell that needs to retain frequent calcium oscillations in the apical region. The pancreatic acinar cell however, successfully and ingeniously solves this problem by connecting relatively small projections of apical ER, from which calcium is released during local calcium signals to the very substantial reservoir of basal and lateral ER, which in this case serves as a Ca2+ storage reservoir and Ca2+-transporting conduit for replenishing the losses of apical ER (Mogami et al. 1997, Park et al. 2000). All these arrangements would not be sufficient to retain such a substantial calcium gradient between the apical and basal parts of the pancreatic acinar cell for such a considerable period of time [local calcium transients produced by secretagogues can last up to 14 s, even more prolonged local calcium transients were produced by stimulating these cells with bile acids (Voronina et al. 2002a)]. We need a cellular organelle equipped with powerful calcium uptake mechanisms that would provide a barrier to inhibit the spread of calcium responses from the apical to basal parts of the cell. In the opinion of our laboratory this function is played by mitochondria.
The role of mitochondria in the formation of calcium signals and the role of calcium signals in adjusting the mitochondrial metabolism Mitochondria form three distinct groups in pancreatic acinar cells – perigranular, perinuclear and sub-plasmalemmal (Tinel et al. 1999, Straub et al. 2000, Park et al. 2001a, Johnson et al. 2003). Mitochondria could be found outside these regions (Johnson et al. 2003) but the density is very low (these ‘orphan’ mitochondria are most probably organelles in transition from one group to another). Local calcium uncaging and local photobleaching experiments revealed some connectivity between mitochondria belonging to individual groups but no connections were found between mitochondria from different groups (Park et al. 2001a). The largest clustering of mitochondria (see Fig. 1a) was found on the border between the apical and basal regions and was termed the perigranular mitochondrial belt (Park et al. 2001a). An unexpected recent finding in our group was that mitochondria surround the Golgi apparatus in acinar cells from the basal and lateral regions and form remarkably strong connections with this organelle (Dolman et al. 2005). This places the Golgi apparatus
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(a)
(b)
(c)
Figure 1 Mitochondria and vacuoles in pancreatic acinar cells. (a) Localization of mitochondria in a triplet of pancreatic acinar cells. Left panel shows transmitted image, right panel shows staining with mitochondrial probe Mito Tracker Deep Red. Note the high density of mitochondria in the region adjacent to secretory granules (‘perigranular belt’ of mitochondria). (b) Endocytic vacuoles. Left panel shows transmitted image of a triplet of pancreatic acinar cells. Central image is the confocal section of these cells placed in solution containing fluorescent dextran (Texas red dextran). Endocytic vacuoles formed in these cells as a result of stimulation with a high dose of CCK are shown on the right panel (adapted with modifications from Sherwood et al. 2007). (c) Simultaneous imaging of endocytic and non-endocytic vacuoles. Transmitted image of two cells is shown on the left panel. Central panel shows fluorescence image of Dextran Alexa Fluor 488 (green colour) which was added to the extracellular solution. Large endocytic vacuole, formed as a result of CCK stimulation, is clearly visible on this image. Right panel shows overlay of Dextran Alexa Fluor 488 fluorescence (green) and Dextran Texas Red fluorescence (red). Dextran Texas Red was infused into the upper cell using a patch pipette in the whole-cell configuration. Dark imprints on the background of Dextran Texas Red fluorescence reveal non-endocytic vacuoles (this part of the figure was adapted with modifications from Voronina et al. 2007).
on the apical side of the mitochondrial belt and within the range of [Ca2+] elevations occurring during the local apical calcium responses. The profile of calcium changes during a local calcium response reveals that the Golgi is indeed immersed in elevated [Ca2+] whilst the calcium transient dissipates into the mitochondrial belt (Gerasimenko et al. 1996, Dolman et al. 2005). During a local calcium transient elevated [Ca2+] does not reach
the nucleus and the regions of the cytosol located on the basal side of the mitochondrial belt (Gerasimenko et al. 1996, Dolman et al. 2005). This suggests that mitochondria play an important ‘firewall’ role preventing the propagation of Ca2+ signals from the basal to the apical part of the cell and effectively divide the cell into two distinct signalling compartments. Indeed inhibition of mitochondrial calcium uptake or depolarizing
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mitochondria using protonophores or with inhibitors of the electron transport chain (this also prevents mitochondrial calcium accumulation) results in the formation of calcium signals spreading into the basal part of the cell (Tinel et al. 1999, Straub et al. 2000). The importance of the proper positioning of mitochondria in the formation of local calcium responses and calcium oscillations was highlighted by a study by Luo et al. (2005) who observed drastic alterations to calcium signalling in Mist1 null mice. The reason for the disappearance of normal ‘physiological’ forms of calcium signalling was mislocalization of mitochondria with a consequent reduction in Ca2+ uptake by these organelles (Luo et al. 2005). Even at physiological levels cholecystokinin (CCK) produces not only local but also global calcium signals (reviewed in Petersen & Tepikin 2008). The need for such global signalling responses is probably explained by the necessity to co-ordinate the secretion of enzymes and fluid with a more prolonged regulation of gene expression and protein processing. The influence of mitochondria on global calcium responses was revealed by comparing the shape of global calcium transients under conditions in which mitochondria were intact or inhibited by appropriate mitochondrial blockers. The effect of mitochondria on global calcium responses in the acinar cells was measurable but much less impressive than the influence of these organelles on the local transients (Johnson et al. 2002). These experiments however allowed us to estimate total calcium accumulation by mitochondria, which was evaluated using the known ratio of mitochondrial and cytosolic volumes (Bolender 1974) and the known calcium binding capacity of the cytosol (Mogami et al. 1999). Our estimation indicated that during global calcium responses mitochondria can increase their total calcium concentration by hundreds of micromoles. Such a significant increase in total calcium content is paralleled by only moderate increases in mitochondrial [Ca2+] demonstrating the high calcium binding capacity of these organelles (Johnson et al. 2002). Measurements of mitochondrial calcium concentration were conducted in our laboratory using rhod-2, which in its acetoxymethylester (AM) form contains a delocalized positive charge and preferentially accumulates in mitochondria (this allows preferential mitochondrial cleavage and retention of the indicator). We observed that low levels of stimulation with calcium-releasing secretagogues induced the preferential response of mitochondria in the perigranular belt (Park et al. 2001a). It is interesting to note that particularly substantial Ca2+ responses were usually observed in mitochondria located in extreme lateral regions of the belt (Park et al. 2001a). These mitochondria are not ‘shielded’ by the Golgi and are most probably located very close to the regions of
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calcium release or influx (Park et al. 2001a, Dolman et al. 2005). With an increase in the intensity of stimulation all groups of pancreatic mitochondria display robust calcium accumulation (Park et al. 2001a). Sub-plasmalemmal mitochondria were found to be the most responsive to calcium influx (Park et al. 2001a). This group of mitochondria should be particularly susceptible to sustained pathological [Ca2+] responses as the influx of Ca2+ is the essential contributing mechanism of such responses. The perinuclear mitochondria in pancreatic acinar cells (Park et al. 2001a) and other exocrine cells (Bruce et al. 2004) delay and reduce calcium transfer from the cytosol to the nucleoplasm. A convenient way to visualize a perinuclear group of mitochondria, loaded with a calcium probe, is by local calcium uncaging in the nuclear region of the cell (Park et al. 2001a). Calcium influx into mitochondria influences other important functions of these organelles. The mitochondrial membrane potential provides an essential driving force for ATP synthesis. The influx of positively-charged Ca2+ ions via the mitochondrial uniporter is expected to depolarize these organelles. In our early experiments we failed to detect mitochondrial depolarization during calcium responses induced by a physiological or supramaximal concentration of calcium-releasing secretagogues (Raraty et al. 2000). In these experiments we utilized the low concentration mode of the application of the fluorescent mitochondrial probes tetramethylrhodamine ethyl ester (TMRE) or tetramethyl-rhodamine methyl ester (TMRM) (Raraty et al. 2000). In this experimental configuration the distribution of the indicators, which are positively charged yet membrane permeable, is expected to follow the Nernst equation (i.e. depolarization of mitochondria should result in loss of the fluorescent probe from the mitochondrial matrix) (Duchen et al. 2003). The lack of success in our initial measurements of the changes in mitochondrial membrane potential was surprising, considering that both cytosolic calcium measurements and more direct mitochondrial calcium measurements clearly indicated substantial accumulation of this divalent cation in mitochondria of pancreatic acinar cells. Another technique for the measurement of mitochondrial membrane potential is referred to as dequench mode (or quench/ dequench mode) (Duchen et al. 2003, Voronina et al. 2004). In this technique the cells are loaded with relatively high concentrations of the indicators (e.g. TMRE or TMRM). These positively-charged membrane permeant indicators accumulate in mitochondria to such an extent that they undergo self-quenching. In this case depolarization of mitochondria results in escape of the indicator from the ‘quenching’ interior of the mitochondria into the cytosol. In this way the indicator becomes dequenched and the overall fluorescence of the
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cell increases (Duchen et al. 2003, Voronina et al. 2004). The direct comparison of the two modes of measurement of mitochondrial membrane potential, using different concentrations of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), indicated that the dequench mode is substantially more sensitive than the low concentration mode (Voronina et al. 2004). Indeed using the dequench mode we were able to characterize depolarization of mitochondria induced by physiological and supramaximal CCK concentrations (Voronina et al. 2004) as well as by bile acids (taurolithocholic acid 3-sulphate, taurodeoxycholic acid and taurocholic acid) (Voronina et al. 2004). CCK-induced depolarization of mitochondria in pancreatic acinar cells was also reported by Gukovskaya et al. (2002). Calcium signals that are regulated by mitochondria are sampled by these organelles and utilized to adjust cellular energetics. One of the parameters which is easily measurable in pancreatic acinar cells is NAD(P)H content. NAD(P)H is an important output of the Krebs cycle (citric acid cycle) and the main reducing equivalent for the mitochondrial electron transport chain cascade. NAD(P)H is fluorescent when excited in nearUV wavelength ranges. When excited with a 364 nm laser line NAD(P)H is the main contributor to the autofluorescence of pancreatic acinar cells in the ‘bluegreen’ part of the emission spectrum. This autofluorescence has a clear mitochondrial distribution, as was revealed by co-localization with other mitochondrial probes. NAD(P)H autofluorescence provides useful information about the status of the Krebs cycle in the mitochondria of the cells (Voronina et al. 2002b). We found that cytosolic calcium signals which are more than 2.5 s in duration produce measurable changes in NAD(P)H levels. Triple combined recordings of cytosolic calcium (measured using the calcium-dependent Cl) current), mitochondrial calcium (using rhod-2 indicator) and NAD(P)H revealed the sequence of events leading to the NAD(P)H response, which was slower to develop and usually substantially more prolonged than its initiating calcium transient (Voronina et al. 2002b). This is a very useful arrangement because accelerated energy production, which outlasts the calcium transient, will allow the restoration of all ionic gradients disturbed by the calcium responses. This is particularly important considering the well-developed Cl) calcium-dependent current in acinar cells (reviewed in Petersen 1992) and significant changes in intracellular [Na+] recorded during stimulation with secretagogues and bile acids (Voronina et al. 2005). CCK-induced oscillations of cytosolic [Ca2+] resulted in spectacular oscillations of mitochondrial NAD(P)H (Voronina et al. 2002b). Oscillatory responses triggered by acetylcholine (ACh) were also accompanied by oscillations in
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NAD(P)H levels, whilst the supramaximal concentration of both agonists (CCK and ACh) resulted in just one transient elevation of NAD(P)H concentration followed by a decline to pre-stimulation levels (or slightly below) (Voronina et al. 2002b). This emphasizes the usefulness of the oscillatory mode of calcium signalling, triggered by physiological doses of secretagogues, which allows multiple activation of the Krebs cycle and therefore efficient adaptation to increased ATP consumption. Supramaximal stimulation with calciumreleasing secretagogues clearly lacks such provision. A decrease in the efficiency of the microcirculation, a decline in oxygen content in the pancreas and a decrease in ATP levels were reported for some experimental models of acute pancreatitis (Luthen et al. 1995, Plusczyk et al. 1997, Kinnala et al. 2002). So how pancreatic acinar cells adapted to such potentially damaging conditions? In our recent experiments we compared the calcium signals and cytosolic ATP levels. The ATP concentration in these experiments was monitored using a luciferase construct delivered to the cytosol of these cells using infection with replication-deficient adenoviruses (Barrow et al. 2008). We observed that ATP levels imposed a very tight control over the cytosolic calcium responses. For example, a relatively modest decrease in cytosolic ATP concentration (approx. twofold with respect to the resting level) results in complete inhibition of ACh-induced calcium oscillations, which occur due to the inhibition of Ca2+ release from the intracellular calcium store (Barrow et al. 2008). Even more unexpected was a very efficient inhibition of calcium influx by ATP depletion. The decline in Ca2+ influx via storeoperated calcium channels was already observed at a moderate decrease in ATP concentration (corresponding to an approx. 20% decrease in bioluminescence) and develops to nearly complete inhibition in remarkable synchrony with the decline in Ca2+ extrusion (Barrow et al. 2008).
Endocytic and non-endocytic vacuoles induced by Ca2+-releasing secretagogues Prominent features of pancreatic acinar cells, which develop during stimulation with the supramaximal levels of calcium-releasing secretagogues, are cytosolic vacuoles. These structures have been observed and characterized using both visible and electron microscopy (Watanabe et al. 1984, Saito et al. 1987, Lerch et al. 1995, Otani et al. 1998, Otani & Gorelick 2000, Raraty et al. 2000, Sherwood et al. 2007). A significant proportion of such vacuoles is of endocytic origin (Sherwood et al. 2007). The process of exocytosis in live pancreatic acinar cells was recently visualized in works from laboratories of P. Thorn and H. Kasai and it involves the formation of large W-shaped
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post-exocytic structures (Nemoto et al. 2004, Thorn et al. 2004). These W-shaped post-exocytic structures develop as a result of compound exocytosis. The formation of large post-exocytic structures was observed at both physiological and supramaximal levels of stimulation (Nemoto et al. 2004, Thorn et al. 2004, Sherwood et al. 2007) but under conditions of supramaximal stimulation these structures were shown to disconnect from the apical plasma membrane and convert to giant (2–6 lm in diameter) endocytic vacuoles (Sherwood et al. 2007). The formation of endocytic vacuoles was revealed using two-photon microscopy and fluorescent-labelled dextrans (Sherwood et al. 2007). The process involves the movement of the post-exocytic structure towards the centre of the cell and the formation of an extended ‘neck’ between the main part of the structure and the exterior of the cell, which eventually ruptures releasing the giant endosome. The process of vacuole formation proceeds via the internalization of a complete post-exocytic structure. It is important to note that the process occurs without involvement of clathrin and it therefore represents a novel mechanism of endocytosis. The formation of giant endocytic vacuoles was also observed following stimulation with bile acids, and the inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) thapsigargin (Sherwood et al. 2007). A common feature of all these inducers is that they trigger elevation of cytosolic [Ca2+]. Therefore the rise in [Ca2+]c is most probably the essential factor in the formation of giant endocytic vacuoles. The endocytic vacuoles always originated in the apical part of the cell and usually translocated to the basal region (Fig. 1b) where they were frequently found adjacent to the basal plasma membrane. We have not observed the fusion of vacuoles with the basal membrane. We have however observed fusion between different endocytic vacuoles and endocytic vacuoles with other small unidentified organelles (possibly lysosomes). The process of formation and translocation of vacuoles was visualized using fluorescentlabelled dextrans which were maintained in the extracellular solution during the period of vacuole formation. The endocytic vacuoles were mildly acidic (pH approx. 5.9) and had a [Ca2+] concentration of approx. 40 lm (Sherwood et al. 2007). The low calcium concentration is explained by a fast leak of Ca2+ from the vacuole. These conditions are not optimal but permissive for activation of trypsinogen (Szmola & Sahin-Toth 2007) and indeed trypsinogen activation was observed in these large endocytic structures (Sherwood et al. 2007). The intracellular activation of trypsinogen is considered as an important mechanism of pancreatic acinar cell damage in pancreatitis. Both visible and electron microscopy identi-
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fied the intracellular activation of trypsinogen in different cellular compartments and in the cytosol (Halangk et al. 1997, Hofbauer et al. 1998, Kruger et al. 1998, 2000, Otani et al. 1998, Raraty et al. 2000); endocytic vacuoles could be now considered as a site of trypsinogen activation and possibly the initial place of cell damage (Sherwood et al. 2007). Endocytic vacuoles are not the only type of vacuoles formed in this cell type. Using contrasting indicators delivered into the cytosol of cells via patch pipette, we observed the formation of another type of vacuoles – non-endocytic vacuoles (Fig. 1c). The formation of non-endocytic vacuoles can be also triggered by supramaximal doses of CCK. These structures reveal themselves as dark imprints on the fluorescence background (sometimes we refer to these organelles as ‘dark vacuoles’). A combination of the two fluorescent probes – intracellular and extracellular – allowed us to monitor simultaneously the formation of correspondingly non-endocytic (dark) and endocytic vacuoles (Voronina et al. 2007)). The non-endocytic vacuoles usually originate in the vicinity of the Golgi apparatus and are frequently seen to disrupt this structure. The non-endocytic vacuole can grow very rapidly to a few micrometres in diameter (Voronina et al. 2007). We currently do not know the nature of these organelles. Autophagosomes and inappropriately developed condensing vacuoles (Watanabe et al. 1984, Willemer et al. 1990, Hammel et al. 1999) are possible candidates.
The travel plan of vacuoles and its consequences Both endocytic and non-endocytic vacuoles form in the intracellular environment packed with different types of cellular organelles. Both types of vacuoles disrupt the highly organized ‘layered’ arrangement of cellular organelles. On their way from the apical to the basal plasma membrane massive endocytic vacuoles must split the Golgi compartments, pierce the perigranular mitochondrial belt and somehow get through the very tightly packed layers of the rough ER. Non-endocytic vacuoles were shown to form in the vicinity of the Golgi and change the positioning of parts of the Golgi. The chaos in organelle positioning, induced by Ca2+dependent formation and translocation of vacuoles is unlikely to be beneficial to the cells. For example, disturbance of the perigranular mitochondrial belt could lead to the inability of the cell to form local calcium signals and global prolonged calcium responses, generated in such conditions, could in turn produce positive feedback and intensify the formation of vacuoles. The interplay of calcium signalling, vacuoles and mitochondria remains an important focus of investigation in our laboratory.
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Conflict of interest We do not have any conflict of interest for this study.
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