Cell Calcium 55 (2014) 131–138

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Review

How to win ATP and influence Ca2+ signaling Svetlana Voronina, Emmanuel Okeke, Tony Parker, Alexei Tepikin ∗ Department of Cellular and Molecular Physiology, The Physiological Laboratory, Institute of Translational Medicine, The University of Liverpool, Crown Street, Liverpool L69 3BX, UK

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Article history: Received 3 January 2014 Received in revised form 10 February 2014 Accepted 11 February 2014 Available online 20 February 2014 Keywords: Mitochondria IP3 receptors ROS Store-operated Ca2+ entry Secretory epithelium

a b s t r a c t This brief review discusses recent advances in studies of mitochondrial Ca2+ signaling and considers how the relationships between mitochondria and Ca2+ responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been “How to win ATP and influence Ca2+ signaling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells”. But “brevity is a virtue” and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Among these mechanisms are mitochondrial regulation of Ca2+ entry and the role of mitochondria in the formation of localized Ca2+ responses. The roles of Ca2+ signaling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed. © 2014 Published by Elsevier Ltd.

1. Introduction (or why mitochondria are effective Ca2+ scavengers) 1.1. MCU/MICU and other Ca2+ transport pathways Mitochondrial transmembrane voltage ( , approximately −160 mV with respect to the cytosol) provides considerable driving force for Ca2+ entry into the mitochondrial matrix. Mitochondrial calcium uniporter (MCU) is a protein complex localized in the inner mitochondrial membrane and responsible for this Ca2+ entry. By the end of the 20th century it was well established that Ca2+ influx into the mitochondria is not linked to translocation of other ions (thus ‘uniporter’) and that it can be effectively inhibited by submicromolar or low-micromolar concentrations of Ruthenium Red (RuRed) and its analog Ruthenium 360 (Ru360) (see [1] for review of studies that characterized the processes involved in the mitochondrial Ca2+ uptake)). Recently papers from V. Mootha’s and R. Rizzuto’s groups identified the channel-forming component of mitochondrial uniporter (MCU) [2,3]. The notion that a channel represents the primary mechanism of Ca2+ entry into mitochondria is important since the influx rate via a channel can be 3–5 orders of magnitude higher than that mediated by a transporter. The biophysical properties of MCU have been extensively characterized by Y. Kirichok and colleagues in D. Clapham’s laboratory [4]. In their elegant electrophysiological study the authors observed Ruthenium 360 (Ru360)

∗ Corresponding author. Tel.: +44 01517945351. E-mail addresses: [email protected], [email protected] (A. Tepikin). http://dx.doi.org/10.1016/j.ceca.2014.02.010 0143-4160/© 2014 Published by Elsevier Ltd.

sensitive channel in the inner mitochondrial membrane with considerable conductance and high Ca2+ selectivity. The current density of 55 ± 19 pA pF-1 recorded in mitoplasts (mitochondria with removed outer membrane and intact inner membrane) suggests that biophysically mitochondria are designed as formidable Ca2+ transporting machines [4]. Notably, the high affinity for Ca2+ and inverse rectification of the current [4] indicate its evolutionary adaptation, optimal for serving as the mitochondrial Ca2+ import mechanism in physiological conditions. Purified MCU protein incorporated in a planar lipid bilayer produced Ca2+ conducting channel activity inhibitable by Ruthenium Red [3]. Very recently MCU−/− mouse model (i.e. animals that do not have MCU) was produced and characterized [5]. The phenotype of this model is perhaps somewhat less severe than expected (e.g. no obvious developmental abnormalities) but mitochondria of these animals certainly have drastically diminished Ca2+ influx and the animals have significantly reduced peak performance of skeletal muscle [5]. The drastic decrease in Ca2+ uptake displayed by mitochondria from MCU−/− animals [5] is another proof of the dominant role played by this mechanism in normal physiologically relevant Ca2+ transport. The MCU protein is an essential channel component of the Ca2+ entry complex but Ca2+ sensing of the complex also involves another protein – mitochondrial calcium uptake 1 (MICU1) – an EF hand protein which was discovered and characterized by F. Perocchi and colleagues in V. Mootha’s laboratory [6]. The discovery of MICU1 was also crucially important for the later identification of MCU as a Ca2+ channel protein [2,3]. It seems that the role of MICU1 is to serve both as gatekeeper and facilitator [7]. Specifically, MICU1 is a gatekeeper at low near-resting Ca2+ concentrations [7,8] but it confers positive

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cooperativity on the mitochondrial Ca2+ entry and therefore serves as a facilitator for larger (e.g. agonist-induced) Ca2+ signals [7]. This research area is exceptionally dynamic and the number of other MCU interacting partners involved in the formation of Ca2+ import complex have been recently identified, including MCUb [9], MICU2 [10], Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) [11] and Essential MCU Regulator (ERME) [12]. It should be noted that, although the MCU complex seems to be the main Ca2+ transporting mechanism in mitochondria, other pathways could also play a role: in mitochondria from MCU−/− animals some Ca2+ influx was detected (particularly at higher Ca2+ concentrations); a recent electrophysiological study from W. Graier’s laboratory identified two calcium currents in mitoplasts (extra large conductance Ca2+ current and bursting Ca2+ current) which are not attributable to MCU [13]. Uncoupling protein 2 is among the proteins potentially involved in non-MCU mediated mitochondrial Ca2+ import [14]. 1.2. The end of temporary storage (NCLX and MPTP) As an organelle without an obvious access to the extracellular environment, mitochondria had to develop an export mechanism to remove Ca from the matrix.1 The Na+ /Ca2+ exchange process in mitochondria was discovered in the 70s [15]. The molecular identity of the protein responsible for the Na+ /Ca2+ exchange (NCLX protein) was discovered later by I. Sekler’s group in 2009 [16]. The remarkable capability of mitochondrial Ca2+ export was demonstrated in the example of cardiomiocytes where mitochondrial calcium was significantly elevated and returned to baseline by Na+ /Ca2+ exchanger in rapid ‘beat-to-beat’ oscillations [17,18]. In addition to NCLX some cell types also express Ca2+ /H+ antiporter [19,20]. The role of this mechanism in mitochondrial Ca2+ extrusion is however not clear. The recently discovered Ca2+ /H+ antiporter Letm1, identified by genome-wide RNAi screen [19], mediates Ca2+ entry into mitochondria in physiologically relevant conditions; however it should reverse in conditions of mitochondrial Ca2+ overload [19,21]. Mitochondria, overloaded by Ca can also release Ca2+ into the cytosol by opening a large pore (permeable to molecules with molecular weight up to 1500 Da) termed Mitochondrial Permeability Transition Pore (MPTP). The opening of such a large channel is accompanied by the loss of mitochondrial membrane potential and consequently ability to retain Ca2+ in the mitochondrial matrix. Ca2+ -sensitivity is one of the fundamental properties of MPTP induction (reviewed in [22–24]). The opening of the pore can be transient (in this case mitochondria restore  and consequently the ability to import Ca2+ from the cytosol). MPTP can also initiate programmed cell death; as prolonged opening of the pore and the loss of mitochondrial membrane potential is usually detrimental and can result in necrotic cell death [23,24]. A number of different components of the pore have been suggested including adenine nucleotide translocase, cyclophilin D, and voltage-dependent anion-selective channel (reviewed in [22,24]). A recent study indicated that the MPTP can be formed by dimers of F0 /F1 ATP synthase – the very protein that is responsible for ATP synthesis [22,25]. Another recent candidate is the mitochondrial phosphate carrier [26,27]. 1.3. Strategic location and privileged access Mitochondria are capable of forming contacts with the ER. The tethers utilized by the mitochondria to form the contacts have been

1 One cannot exclude that damaged, calcium-overloaded mitochondria can be digested by an autophagolysosomes and their calcium content excreted out of the cells by secretory lysosomes but this hypothetical process is not usually considered in discussions of the mitochondrial Ca2+ transport.

visualized [28] and some of the proteins involved in the formation of the junctions (notably Mitofusin 2) have also been identified [29]. In some cell types a fraction of mitochondria undergo rapid movement [30]. These mitochondria are unlikely to form ‘static’ contacts with the ER, but even motile mitochondria are able to sense Ca2+ gradient and accumulate in the vicinity of Ca2+ -releasing channels by slowing their movement in high [Ca2+ ] environment [30]. By arranging ‘static’ or ‘dynamic’ contacts mitochondria can have privileged access to Ca2+ released from the ER via inositol trisphosphate receptors (IP3 Rs), Ryanodine receptors (RyRs) or to Ca2+ entering the cell via the plasma membrane channels. The access is privileged because mitochondrial Ca2+ transporters are exposed to much higher Ca2+ concentration than would be their ‘fair share’ (i.e. averaged cytosolic [Ca2+ ]). The discrepancy between ‘privileged’ and ‘fair’ resulted in decades of confusion caused by experiments recreating an ‘averaged’ cytosolic [Ca2+ ] environment in egalitarian conditions of a cuvette containing isolated mitochondria. The conclusion from these experiments was that mitochondria are unlikely to significantly import Ca2+ and participate in [Ca2+ ] regulation in physiological conditions. This conclusion was revised at the end of the 90s partially because of the development of specific protein-based mitochondria-targeted Ca2+ probes [31] and the visualization of close proximity between mitochondria and the ER [32]. The history of mitochondrial Ca2+ research is reviewed in detail by E. Carafoli [1] who was the champion for mitochondria in the Ca2+ signaling field in both the ‘dark ages’ and the beginning of the mitochondrial Ca2+ ‘renaissance’. The importance of mitochondrial positioning, specifically of contact sites for the efficiency of Ca2+ transfer into the mitochondria was recently demonstrated in an elegant study which employed inducible linkers to modulate ER-mitochondria contacts. This study revealed large localized Ca2+ transients in the contact regions [33]. Large [Ca2+ ] hotspots in ER–mitochondria junctions were also visualized in another study that utilized Ca2+ probes targeted to the mitochondrial surface [34]. Privileged access to cytosolic [Ca2+ ] hotspots, large driving force for Ca2+ uptake, efficient channel-based Ca2+ influx mechanism and multiple Ca2+ efflux mechanisms all contribute to the ability of mitochondria to influence cytosolic Ca2+ responses. In the following sections we will consider how the general Ca2+ -transporting properties of mitochondria, described in Section 1, allow these organelles to shape Ca2+ signals in polarized epithelial cells.

2. Mitochondrial firewall and localized Ca2+ transients in secretory epithelia The ability to form tight signaling microdomains is the trademark of cytosolic Ca2+ responses and is essential for the function of this ion as a prominent second messenger. In pancreatic acinar cells such microdomains are preferentially formed in the apical secretory region of the cell [35–37] reviewed in [38]. The apical region has a high density of Ca2+ releasing channels – IP3 receptors [39–42] which decorate the subplasmalemmal stands of ER in this region. This is an efficient and ‘economical’ way to organize signaling – to produce localized Ca2+ transients exactly in the area where they are needed for triggering exocytosis of secretory granules and opening channels involved in the fluid secretion [39,43,44]. A surprising observation was that some of these localized Ca2+ transients are very long-lived. During such events a steep [Ca2+ ] gradient of a few hundreds of nM per ␮m (measured along a line connecting apical and basal region of an acinar cell) can form and persist for more than 5 s [35]. Correlating localized Ca2+ responses with the map of cellular organelles (by simultaneously using fluorescent Ca2+ indicators and fluorescent probes for cellular organelles) revealed that localized Ca2+ responses terminate in the perigranular mitochondrial belt – the region of the acinar cells which is highly enriched

S. Voronina et al. / Cell Calcium 55 (2014) 131–138

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Fig. 1. Ca2+ exchange between mitochondria and the ER: privileged access and the formation of a microdomain.

with mitochondria [45]. The perigranular belt was observed in the acinar cells by a number of research groups using both fluorescent probes [46–49] and electron microscopy [50]. Importantly, depolarizing mitochondria by protonophores prevented formation of local Ca2+ transients as signals can no longer be retained in the apical region and once initiated they spread to the basal region in cells stimulated by Ca2+ -releasing agonists [49] and in cells where Ca2+ responses were triggered by uncaging of IP3 [48]. It is likely that the strategic positioning of mitochondria on the boundary between apical and basal regions, and the high Ca2+ influx rate due to efficient uptake via MCUs are essential for retaining Ca2+ responses in the apical region of acinar cells i.e. for the formation of the typical ‘physiological’ Ca2+ responses in this cell type. However, we cannot exclude that mitochondria in this region have other functions that are important for shaping Ca2+ responses. These could include the reduction in ER strand density because of the necessity to accommodate high density of mitochondria (and therefore some ‘thinning’ of excitable medium) as well as the conditioning of IP3 Rs and/or Ca2+ pumps in the ER by ROS, ATP or other substances released from mitochondria. It should be noted however that robust mitochondrial Ca2+ transients were recorded specifically in the perigranular mitochondria during localized apical Ca2+ transients, suggesting that active transport by mitochondria is indeed involved in shaping local apical Ca2+ events [47]. Further evidence for the importance of specific localization of mitochondria for the formation of localized Ca2+ responses and propagating (apical-to-basal) Ca2+ waves came from the study conducted on Mist1 null mice. Mist1 plays an important role in determining the polarity of pancreatic acinar cells and the specific positioning of mitochondria. The loss of ‘mitochondrial belt’ in Mist1−/− animals resulted in drastic changes of the Ca2+ signaling pattern in pancreatic acinar cells. Importantly, the cells not only lost the ability to form local apical Ca2+ responses and propagating apical-to-basal Ca2+ waves; but the ability to generate [Ca2+ ] oscillations was also diminished [51]. These observations clearly supported the notion that mitochondria are important for shaping physiologically relevant local Ca2+ signals. Similar conclusion about the ability of mitochondria to shape localized IP3 R-mediated Ca2+ responses was reached following experiments on hepatocytes [52]. The role of mitochondria in influencing IP3 R-mediated Ca2+ release is summarized in Fig. 1. In addition to IP3 Rs, the propagation of Ca2+ signaling in pancreatic acinar cells and formation of global Ca2+ transients require functioning RyR [48,53–55]. RyR also play an important role in the pathophysiology of acinar cells [55]. Unlike IP3 Rs that are mainly

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concentrated in the apical regions, RyRs can be found in all parts of the acinar cells, but a particularly high density is observed close to the mitochondrial belt [48]. Mitochondrial Ca2+ uptake modulates Ca2+ concentration in the vicinity of RyRs and, by this mechanism influences the propagation of Ca2+ signals in the acinar cells [48]. Perigranular mitochondria (i.e. ‘mitochondrial belt’) is not the only prominent grouping of mitochondria observed in the acinar cells; subplasmalemmal and perinuclear groups have also been identified [47,50]. Interestingly, global Ca2+ uncaging (using photolysis of NP-EGTA) resulted in similar Ca2+ uptake in all groups of mitochondria in acinar cells (perigranular, perinuclear and subplasmalemmal), suggesting similar Ca2+ transporting abilities of mitochondria in these groups [47]. Mitochondria and mitochondrial Ca2+ uptake were also shown to be essential for shaping Ca2+ signaling and forming segregated Ca2+ signaling domains in human airway epithelia [56]. In addition to a complete functional separation of Ca2+ signals in specific regions (i.e. formation of microdomains) mitochondria can impose less drastic ‘temporary’ limitations on Ca2+ signals. An example of such effect is a delay in nuclear Ca2+ responses due to the activity of perinuclear mitochondria in parotid acinar cells observed in a study by J. Bruce and colleagues [57]. Mitochondria shape Ca2+ transients not only by Ca2+ uptake but also by modulating Ca2+ release mechanisms with mitochondriaproduced metabolites such as reactive oxygen species (ROS) and ATP. For example, depletion of ATP in pancreatic acinar cells by inhibitors of oxidative phosphorylation results in a very effective suppression of Ca2+ oscillations induced by acetylcholine [58] or its analog carbamylcholine [59]. This form of agonist-induced Ca2+ signaling is particularly dependent on the generation of IP3 and activation of IP3 Rs. All 3 types of IP3 Rs are expressed in pancreatic acinar cells [39–42], in which type 2 and 3 are functionally dominant forms [60]. Both these receptor types are ATP-sensitive [61–63]. H. Park and colleagues from D. Yule’s laboratory demonstrated that the observed suppression of Ca2+ signals in the pancreatic acinar cells by mitochondrial inhibitors [58,59] occurs primarily because of ATP-sensitivity of IP3 R type 2 [59]. Mitochondria are important sites of ROS generation (reviewed in [64]). A number of studies reported mitochondrial ROS production in response to Ca2+ releasing agonists in exocrine secretory cells (e.g. [65,66]). An elegant study by C. Camello-Almaras and colleagues revealed a critical role of ROS produced by mitochondria for normal physiological oscillatory Ca2+ signaling in pancreatic acinar cells [66]. In particular mitochondrial ROS scavenger MitoQ effectively inhibited Ca2+ oscillations induced by a physiological concentration of CCK [66]. Combining the information described in this section one can conclude that mitochondria utilize a plethora of mechanisms to support Ca2+ signals and to shape their special properties.

3. Mitochondria and Ca2+ influx via the plasma membrane of polarized epithelial cells A particularly intriguing area of research deals with the relationship between mitochondria and Ca2+ influx channels in the plasma membrane. Secretory epithelial cells are usually nonexcitable, and Ca2+ signals in these cells are initiated by Ca2+ release from the intracellular stores via IP3 Rs [39,52,59,60,67–69] or RyRs [48,53–55]. The depletion of the intracellular store induces Ca2+ influx; such store-operated Ca2+ (SOC) influx plays a significant role in both physiological and pathophysiological Ca2+ responses of these cell types [58,70,71]. The concept of SOC entry (SOCE) (also termed capacitive Ca2+ entry) was originally developed following experiments conducted on secretory epithelial cells (reviewed

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Fig. 2. Mitochondria and store-operated Ca2+ entry.

in [72,73]). The current via SOC channels termed ICRAC (calcium release activated calcium current) is the electrophysiological manifestation of this Ca2+ entry process [74,75], reviewed in [72]. The recently identified proteins mediating SOCE include ER-localized Ca2+ -sensing protein stromal interaction molecule 1 (STIM1) and pore-forming plasma membrane protein Orai1 ([76–80] reviewed in [81]). The role of other STIM (STIM2) and Orai (Orai2 and Orai3) isoforms and their involvement in SOCE was characterized later (e.g. [82,83] reviewed in [73,81]). STIM1 was also shown to activate another type of Ca2+ – conducting channels – TRPC channels (reviewed in [84]). The physiological necessity of a substantial Ca2+ entry in pancreatic acinar cells was illustrated in droplet technique experiments that revealed significant Ca loss (few hundreds of ␮M of cellular Ca per minute) even at modest ‘physiological’ stimulation that induce oscillatory cytosolic [Ca2+ ] response [85,86]. Clearly to ensure long-term uninterrupted secretion of the gland the cells need to import equivalently significant amounts of Ca. A number of studies revealed that mitochondria (specifically those that are functionally energized and capable of Ca2+ uptake) are essential for efficient SOC entry [87–92]. In these studies SOCE was inhibited by maneuvers that dissipate mitochondrial membrane potential and notably by the inhibition of mitochondrial Ca2+ uniporter. The hypothesis, which received considerable support, suggests that Ca2+ uptake into mitochondria suppresses Ca2+ -dependent fast inactivation of SOCE channels and therefore effectively potentiates SOCE. To fulfill this SOCE-modulating function for a significant period of time and to facilitate the reloading of the Ca2+ store mitochondria have to transfer its Ca2+ to the ER (or SR). To achieve this trans-mitochondrial Ca2+ flux (involving the co-ordination of SOC channels, MCUs, mitochondrial NCLXs and sarcoplasmic/endoplasmic reticulum Ca2+ pumps) is required (see Fig. 2). Mitochondria participating in SOCE regulation are strategically localized near the plasma membrane in the proximity to the SOCE channels [93]. Artificial redirection of mitochondria away from the plasma membrane resulted in substantial reduction in SOCE [94], suggesting a strategic role of the close contacts between the organelles and the membrane for this form of Ca2+ entry. A study by A. Quintana and colleagues from the M. Hoth’s laboratory revealed that mitochondria can be actively recruited to the plasma membrane by a Ca2+ -dependent process involving kinesinmediated traffic [92]. Notably, subplasmalemmal mitochondria can also facilitate Ca2+ reloading of the ER by locally competing for Ca2+ with plasma membrane Ca2+ ATPases (PMCAs) and therefore reducing Ca2+ export [95]. In migrating cells ER–PM junctions (which serve as platforms for interaction between STIM and Orai, and eventually for SOCE) undergo the processes of continuous movement,

formation and dissolution [97]. One could hypothesize that to attain full Ca2+ transporting capacity the STIM–Orai competent junctions need to attract mitochondria. Such putative co-ordination of migration (of cells, ER–PM junctions and mitochondria) is an exciting area for further investigations. The close proximity of mitochondria to the plasma membrane and specifically to plasma membrane regions containing STIM1-decorated ER–PM junctions was recently demonstrated in secretory epithelial cells using both fluorescent and electron microscopy [93,96]. Inhibitors of the respiratory chain and of ATP synthase reduced ATP concentration and suppressed SOCE induced by Ca2+ -releasing secretagogues in pancreatic acinar cells [58]. During ATP depletion translocation of STIM1 to ER–PM junctions and its co-localization with Orai is not compromised (in fact ATP depletion can trigger STIM1 translocation to ER–PM junctions because of loss of Ca2+ from the ER) but SOCE is inhibited [98]. The specific mechanism(s) involved in this phenomenon are debated and can potentially include the inhibition of mitochondria Ca2+ uptake; the loss of local ATP-dependent Ca2+ buffering; changes in the phosphorylation status of proteins involved in SOCE; and the loss of particular species of phospholipids. Independently of the specific mechanism, the phenomenon, which was also observed in a number of other cell types [87–90,92,94,99–101], signified an important feedback process designed to avoid (or at least reduce) Ca2+ overload in cells with compromised bioenergetics. Interestingly, whilst SOCE in pancreatic acinar cells was effectively inhibited by ATP depletion, Ca2+ influx induced by bile acid tauro-lithocholic acid-3-sulfate was not suppressed by mitochondrial inhibitors [58]. A possible reason for this is that in addition to SOCE, extensively characterized in this cell type (e.g. [40,70,71,93]), TLC-S could directly activate a different type of Ca2+ entry pathway. Indeed, TLC-S-induced Ca2+ current, with electrophysiological properties different from that of ICRAC, has been reported in this cell type [102]. TLC-S is one of the inducers of acute pancreatitis [103,104]. TLC-S was also shown to effectively depolarize mitochondria [65,105] and reduce cellular ATP concentration, suggesting that it can be a particularly potent inducer of Ca2+ toxicity [106].

4. Positive and negative feedbacks of Ca2+ signals and mitochondrial functions in secretory epithelia One can reverse the question ‘What can mitochondria do for Ca2+ signaling?’ and ask ‘What Ca2+ signaling can do for mitochondria?’ The answer to the second question is – “It can adjust ATP production to meet increased energy demand”. Pioneering work from R. Denton’s group (Bristol) revealed that Ca2+ upregulates the activity of dehydrogenases of Krebs cycle (specifically of pyruvate dehydrogenase, isocitrate dehydrogenase and oxoglutarate dehydrogenase; reviewed in [107]). NAD(P)H is an important reducing equivalent produced as a result of Krebs cycle. NAD(P)H is fluorescent and can be dynamically measured in living cells [108]. At single cell level spectacular correlation between Ca2+ responses and NAD(P)H transients was shown in hepatocytes [109]. Notably reduction in FAD (which also reflects acceleration of the Krebs cycle) also mirrored NAD(P)H increases [109]. Measurements on individual isolated pancreatic acinar cells revealed oscillatory NAD(P)H responses and showed a clear correlation between cytosolic [Ca2+ ], mitochondrial [Ca2+ ] and NAD(P)H response (reflecting the rate of the Krebs cycle) [110]. Prominent increase of NADH autofluorescence was also recorded in parotid acinar cells stimulated with calcium releasing agonist carbachol [57]. Ca2+ signals can not only increase the rate of Krebs cycle but also cytosolic and mitochondrial ATP levels, signifying a considerable increase in ATP production [111]. This seems to be the case also for pancreatic acinar cells, in which Ca2+ – releasing secretagogues (e.g. CCK and

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ACh) increased total cytosolic and mitochondrial ATP content in spite of significantly enhanced ATP consumption [106,112]. The energized mitochondria (with accelerated NAD(P)H and ATP production) should be able to effectively contribute to shaping Ca2+ responses. This status of the organelles is favorable for prolonged Ca2+ signaling observed in many secretory epithelia cell types. The relationships between Ca2+ signaling and mitochondria can therefore be classed as an efficient physiological adaptation. In addition to the adjustment of bioenergetics to immediate physiological functions mitochondrial Ca2+ signals were recently shown to be essential for regulating a long term tissue adaptation–hepatocytes proliferation and liver regeneration [113]. The essential role of basal mitochondrial Ca2+ uptake for cell survival was recently reported by J.K. Foskett’s laboratory [114]. This study demonstrated that without basal mitochondrial uptake of Ca2+ delivered by IP3 Rs cells undergo starvation (and activate autophagy) even in the presence of nutrients [114]. This ‘mitochondria-lubricating’ basal Ca2+ uptake is an interesting concept that highlights the importance of the Ca2+ release at basal IP3 concentration and perhaps the specific role of low physiological concentrations of Ca2+ releasing secretagogues in maintaining viable cellular bioenergetics. Significant Ca2+ load of the mitochondria initiates opening of MPTP that can lead to cell death. This phenomenon was documented in many cell types including secretory epithelial cells [115–120]. In pancreatic acinar cells and hepatocytes ROS also seems to be critically important for MPTP opening [65,117,121]. Interestingly, this MPTP-inducing ROS can be also generated by the mitochondria [65,117]. Mitochondrial injury is considered as the mechanism responsible for cellular damage and death in a number of models of diseases affecting secretory epithelia [65,116,117,120,122–124]. The damaged mitochondria can release cytochrome c (via MPTP) and initiate activation of caspases – events leading to apoptosis. In this pathology-relevant condition mitochondria can impose a different form of control over the Ca2+ signals involving caspase-mediated cleavage of components of Ca2+ signaling cascade (e.g. cleavage by caspase-3 of PMCA [125] and of IP3 Rs [126]), and modulation of IP3 Rs by cytochrome c [127].

5. Concluding remarks Specifically localized mitochondria utilize Ca2+ influx channels and significant driving force for Ca2+ entry to shape Ca2+ signaling responses by direct Ca2+ uptake. In polarized secretory epithelial cells such mitochondria can restrict Ca2+ signals to specialized parts of the cell (e.g. in basal or apical region, secretory granule region or the region immediately adjacent to the plasma membrane). In addition to direct Ca2+ uptake mitochondria also use the ‘soft power’ of ROS production and the changing of ATP/ADP ratio to modify Ca2+ channels and pumps to ultimately adjust Ca2+ signaling and metabolic status. Complex system of feedback regulations operates between Ca2+ signaling and mitochondrial functions. Ca2+ entering mitochondria upregulates the rate of Kebs cycle and ATP production which in turn can potentiate both Ca2+ release and Ca2+ removal from the cytosol. Finally in pathological conditions relationships between mitochondria and calcium signaling determine if the cell is going to survive or die (and if to die then by what specific cell death mechanism). In life and death relationships between the organelle and the signaling cascade are conceptually important. The recent discoveries of the principle players in mitochondrial Ca2+ uptake (MCU, MICU1 and NCLX) will give a strong impetus to further detailed characterization of these relationships. Ca2+ released from the ER by IP3 R is transferred into closely juxtaposed mitochondrion via mitochondrial calcium uniporter (MCU), which is regulated by Mitochondrial Calcium Uniporter

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Regulator 1(MICU1). MCU/MICU1 complex is exposed to increased Ca2+ microdomain formed in the vicinity of IP3 R. This arrangement is essential for the efficient mitochondrial Ca2+ influx. Ca2+ is then extruded from mitochondria by Na+ /Ca2+ exchangers (NCLXs) and partially reloaded into the ER by sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). Mitochondrial Ca2+ uptake prevents local Ca2+ -dependent activation of ryanodine receptors (RyRs) and IP3 Rs and therefore restricts Ca2+ microdomain. Decrease of [Ca2+ ] in the ER triggers store-operated Ca2+ entry (SOCE) via channels formed by interacting proteins STIM1 and Orai1. Part of the Ca2+ flux entering the cell is rapidly taken into mitochondria. This process reduces inhibition of SOCE by cytosolic Ca2+ and facilitates Ca2+ entry. Ca2+ enters mitochondria via mitochondrial calcium uniporter (MCU), which is regulated by Mitochondrial Calcium Uniporter Regulator 1. Ca2+ is then exported from mitochondria by Na+ /Ca2+ exchanger (NCLX) and is actively imported into ER via sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). Some of the cytosolic Ca2+ is extruded from the cell by plasma membrane Ca2+ ATPase (PMCA). Acknowledgements The work of laboratory is supported by Medical Research Council (UK) grant MR/K012967/1 and by the National Institute for Health Research (UK) grant to the NIHR Liverpool Pancreas Biomedical Research Unit. E.O. and T.P. are Wellcome Trust – funded PhD students (grant numbers 092790/Z/10/Z and 092792/Z/10/Z). References [1] E. Carafoli, The interplay of mitochondria with calcium: an historical appraisal, Cell Calcium 52 (2012) 1–8. [2] J.M. Baughman, F. Perocchi, H.S. Girgis, M. Plovanich, C.A. Belcher-Timme, Y. Sancak, X.R. Bao, L. Strittmatter, O. Goldberger, R.L. Bogorad, V. Koteliansky, V.K. Mootha, Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter, Nature 476 (2011) 341–345. [3] S.D. De, A. Raffaello, E. Teardo, I. Szabo, R. Rizzuto, A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter, Nature 476 (2011) 336–340. [4] Y. Kirichok, G. Krapivinsky, D.E. Clapham, The mitochondrial calcium uniporter is a highly selective ion channel, Nature 427 (2004) 360–364. [5] X. Pan, J. Liu, T. Nguyen, C. Liu, J. Sun, Y. Teng, M.M. Fergusson, I.I. Rovira, M. Allen, D.A. Springer, A.M. Aponte, M. Gucek, R.S. Balaban, E. Murphy, T. Finkel, The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter, Nat. Cell Biol. 15 (2013) 1464–1472. [6] F. Perocchi, V.M. Gohil, H.S. Girgis, X.R. Bao, J.E. McCombs, A.E. Palmer, V.K. Mootha, MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake, Nature 467 (2010) 291–296. [7] G. Csordas, T. Golenar, E.L. Seifert, K.J. Kamer, Y. Sancak, F. Perocchi, C. Moffat, D. Weaver, P.S. de la Fuente, R. Bogorad, V. Koteliansky, J. Adijanto, V.K. Mootha, G. Hajnoczky, MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca(2)(+) uniporter, Cell Metab. 17 (2013) 976–987. [8] K. Mallilankaraman, P. Doonan, C. Cardenas, H.C. Chandramoorthy, M. Muller, R. Miller, N.E. Hoffman, R.K. Gandhirajan, J. Molgo, M.J. Birnbaum, B.S. Rothberg, D.O. Mak, J.K. Foskett, M. Madesh, MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival, Cell 151 (2012) 630–644. [9] A. Raffaello, S.D. De, D. Sabbadin, E. Teardo, G. Merli, A. Picard, V. Checchetto, S. Moro, I. Szabo, R. Rizzuto, The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit, EMBO J. 32 (2013) 2362–2376. [10] M. Plovanich, R.L. Bogorad, Y. Sancak, K.J. Kamer, L. Strittmatter, A.A. Li, H.S. Girgis, S. Kuchimanchi, G.J. De, L. Speciner, N. Taneja, J. Oshea, V. Koteliansky, V.K. Mootha, MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling, PLoS ONE 8 (2013) e55785. [11] K. Mallilankaraman, C. Cardenas, P.J. Doonan, H.C. Chandramoorthy, K.M. Irrinki, T. Golenar, G. Csordas, P. Madireddi, J. Yang, M. Muller, R. Miller, J.E. Kolesar, J. Molgo, B. Kaufman, G. Hajnoczky, J.K. Foskett, M. Madesh, MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism, Nat. Cell Biol. 14 (2012) 1336–1343. [12] Y. Sancak, A.L. Markhard, T. Kitami, E. Kovacs-Bogdan, K.J. Kamer, N.D. Udeshi, S.A. Carr, D. Chaudhuri, D.E. Clapham, A.A. Li, S.E. Calvo, O. Goldberger, V.K. Mootha, EMRE is an essential component of the mitochondrial calcium uniporter complex, Science 342 (2013) 1379–1382.

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