Calcium signaling in pancreatic ductal epithelial cells: An old friend and a nasty enemy

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Calcium signaling in pancreatic ductal epithelial cells: An old friend and a nasty enemy József Maléth a,b , Péter Hegyi a,∗ a

First Department of Medicine, University of Szeged, Szeged, Hungary Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA b

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 4 February 2014 Accepted 5 February 2014 Available online xxx Keywords: Pancreatic ductal secretion Epithelial Ca2+ signaling Exocrine pancreas

a b s t r a c t Ductal epithelial cells of the exocrine pancreas secrete HCO3 − rich, alkaline pancreatic juice, which maintains the intraluminal pH and washes the digestive enzymes out from the ductal system. Importantly, damage of this secretory process can lead to pancreatic diseases such as acute and chronic pancreatitis. Intracellular Ca2+ signaling plays a central role in the physiological regulation of HCO3 − secretion, however uncontrolled Ca2+ release can lead to intracellular Ca2+ overload and toxicity, including mitochondrial damage and impaired ATP production. Recent findings suggest that the most common pathogenic factors leading to acute pancreatitis, such as bile acids, or ethanol and ethanol metabolites can evoke different types of intracellular Ca2+ signals, which can stimulate or inhibit ductal HCO3 − secretion. Therefore, understanding the intracellular Ca2+ pathways and the mechanisms which can switch a good signal to a bad signal in pancreatic ductal epithelial cells are crucially important. This review summarizes the variety of Ca2+ signals both in physiological and pathophysiological aspects and highlight molecular targets which may strengthen our old friend or release our nasty enemy. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The acinar-ductal functional unit of the exocrine pancreas secretes 1–2 L alkaline, digestive enzymes-rich juice daily [1,2]. The acinar cells produce an acidic, Cl− and protein-rich, high viscosity fluid [3], whereas the ductal epithelial cells (PDEC) secretes high quantity of HCO3 − -rich low viscosity fluid [4]. The final HCO3 − concentration of the pancreatic juice varies among species; importantly human PDEC can produce a maximal intraluminal HCO3 − concentration of 140 mM. The alkaline pancreatic fluid secretion, in response to meal, washes the digestive enzymes out of the pancreatic ductal tree and neutralizes the acidic chyme entering the duodenum. The function of the pancreatic ductal fluid and HCO3 − secretion used to be underestimated, however recent findings suggest that it plays a central role in the physiology and pathophysiology of the pancreas. Importantly, HCO3 − neutralizes protons secreted by the acinar cells and keeps trypsinogen and

∗ Corresponding author at: University of Szeged, Faculty of Medicine, First Department of Medicine, Korányi fasor 8-10, H-6720 Szeged, Hungary. Tel.: +36 62 545 200; fax: +36 62 545 185. E-mail addresses: [email protected], [email protected] (P. Hegyi).

most probably other proteases in an inactive form [5]. Pallagi et al. have recently demonstrated that the autoactivation of trypsinogen is a pH dependent process, with increased activity in acidic environment, which means that HCO3 − secretion prevents the premature trypsinogen activation [5]. We also have to highlight that the most common pathogenic factors for acute pancreatitis (bile acids, ethanol and ethanol metabolites) impair ductal HCO3 − secretion which likely contributes in a major manner to the pancreatic damage [6–8]. The pancreatic ductal HCO3 − secretion is regulated by complex signaling systems, in which both cAMP and intracellular Ca2+ play crucial roles. Agonists (such as secretin or acetylcholine) binding to G protein coupled metabotropic receptors activate adenylyl cyclases and/or release Ca2+ from the intracellular stores in PDEC. On the other hand, some of the molecules do not need membrane receptors to induce an intracellular Ca2+ elevation. Low concentrations of unconjugated bile acids and ethanol also evoke oscillatory intracellular Ca2+ signals and stimulate HCO3 − secretion in PDEC. However, these molecules in high concentrations induce sustained Ca2+ elevations, which inhibit the secretory processes and lead to cell necrosis. These pathogenic steps, which result in the development of toxic, sustained Ca2+ signals, might offer potential therapeutic targets in pancreatic diseases. In this review we summarize the recent advances in pancreatic ductal physiology and

http://dx.doi.org/10.1016/j.ceca.2014.02.004 0143-4160/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: J. Maléth, P. Hegyi, Calcium signaling in pancreatic ductal epithelial cells: An old friend and a nasty enemy, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.02.004

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Fig. 1. Mechanism of pancreatic ductal HCO3 − secretion. Pancreatic ductal cells accumulate HCO3 − across the basolateral membrane via the electrogenic Na+ /HCO3 − cotransporter NBCe1-B. On the luminal membrane PDEC express electrogenic Cl− /HCO3 − exchangers (SLC26A6 and possibly A3) and cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. The operation of these transporters allows the pancreatic ductal cells to create 140 mM maximal HCO3 − concentration during stimulated secretion. The R domain of CFTR interact with the STAS domain of the SLC26 Cl− /HCO3 − exchanger, which increases overall open probability of CFTR. In the proximal ducts, where the intraluminal Cl− concentration ([Cl− ]L )is high, HCO3 − is secreted via the electrogenic Cl− /HCO3 − exchange, driven by the high [Cl− ]L . Under these conditions CFTR functions as a Cl− channel. In the distal ducts, where the [Cl− ]L is low, the low intracellular Cl− concentration ([Cl− ]i ) activates the WNK/SPAK kinases, which phosphorylate CFTR, switching the ion selectivity to HCO3 − . The SLC26 mediated HCO3 − transport is inhibited under these conditions.

pathophysiology, highlighting the dual effects of Ca2+ signaling in PDEC. 2. Bicarbonate secretion in pancreatic ductal cells Pancreatic ductal HCO3 − secretion can be divided to two separate steps, first the accumulation of the HCO3 − ions in the cells via the basolateral membrane and second the secretion into the ductal lumen across the apical membrane (Fig. 1). The basolateral accumulation is carried out by a Na+ /HCO3 − cotransporter (NBCe1-B), which transports 1 Na+ and 2 HCO3 − into the cells, driven by the high intracellular Na+ gradient [9]. Another possible mechanism for the HCO3 − accumulation is the passive diffusion of CO2 trough the cell membrane, followed by the carbonic anydrase mediated conversion of CO2 to HCO3 − [10]. The electroneutral Na+ /H+ exchanger might also contribute to the HCO3 − accumulation, although its role differs among species [11,12], it is essential for intracellular pH (pHi ) homeostasis. On the luminal membrane PDEC express electrogenic Cl− /HCO3 − exchangers (SLC26A6, which operates with a 1 Cl− : 2 HCO3 − stoichiometry and possibly SLC26A3, which transports 2 Cl− : 1 HCO3 − ) [13] and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel [14]. The electrogenic Cl− /HCO3 − exchange allows the pancreatic ductal cells to transport HCO3 − into the ductal lumen and establish the very high (140 mM) maximal intraluminal HCO3 − concentration during stimulated secretion, resulting in an intraluminal HCO3 − level which is ∼5–6 fold higher compared to the cell interior [1,2]. It is important to note that CFTR mutations, which are associated with exocrine pancreatic insufficiency, also establish a major deficiency in the apical CFTR-dependent Cl− /HCO3 − exchange activity [15]. Recent improvements in the field help to

understand the puzzling role of CFTR in HCO3 − secretion. In the proximal pancreatic ducts, where the luminal Cl− concentration ([Cl− ]L ) is high, CFTR functions as a Cl− channel, providing the necessary substrate (luminal Cl− ) for the Cl− /HCO3 − exchange of the SLC26A6 and A3 transporters. In the distal pancreatic ducts however, where the [Cl− ]L and intracellular Cl− concentration ([Cl− ]i ) is low, HCO3 − secretion through CFTR can play an important role. Under these conditions the CFTR Cl− permeability is switched by the With-No-Lysine (WNK)/STE20/SPS1-related proline/alaninerich kinase (SPAK) kinase pathway (which is regulated by [Cl− ]i ), changing CFTR into a HCO3 − permeable channel [16]. Another recently described regulatory protein, named IRBIT, seems to play a crucial role in the regulation of HCO3 − secretion as well. Under resting conditions WNK/SPAK constitutively inhibit the activity of CFTR and NBCe1-B, which is antagonized by IRBIT upon stimulation. Moreover IRBIT promotes the insertion of CFTR into the apical membrane [17]. In addition, IRBIT seems to mediate synergism between Ca2+ and cAMP signaling pathways [18]. The detailed regulation of HCO3 − secretion by IRBIT and by the WNK/SPAK pathway has been reviewed elsewhere and is beyond the scope of this manuscript[1,2,19,20]. PDEC also express Ca2+ activated Cl− channels on the luminal membrane [21], however the molecular identity and contribution to the pancreatic HCO3 − and fluid secretion is unknown at the time. Recently, ANO1 (TMEM16A) was identified on the luminal membrane of the pancreatic acinar cells as the Ca2+ activated Cl− channel, which plays an important role in acinar Cl− secretion [22]. Besides the transporters and channels, which participate in the HCO3 − secretion, PDEC also express aquaporin (AQP) water channels. AQP1 is expressed on both the basolateral and luminal membrane and AQP5 only on the luminal membrane [23]. Moreover, AQP5 was shown to strongly colocalise with CFTR in the

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small pancreatic ducts. This pattern of distribution suggests that the fluid and HCO3 − secretion is coupled in PDEC. 3. Neurohormonal control of pancreatic electrolyte and fluid secretion The secretory functions of PDEC are controlled by multiple stimulatory and inhibitory neural and hormonal mechanisms. In this review we focus on those agonists and antagonists, which exert their effects directly on the ductal HCO3 − secretion. Acetylcholine, the main stimulatory neurotransmitter released from the parasympathic nerve endings, enhances the pancreatic ductal fluid and HCO3 − secretion mostly via M3 metabotropic cholinergic receptor (M3 R) stimulation [24]. The Gq -coupled M3 R stimulation results in the elevation of the [Ca2+ ]i (see below), which in turn stimulates the PDEC function. Another regulator acting via [Ca2+ ]i changes in PDEC is ATP, which can bind to P2Y (metabotropic) and P2X (ionotropic) purinergic receptors expressed on PDEC. The expression of the receptors are polarized; P2Y receptors are expressed on both the apical and basolateral membranes, whereas P2X receptors are on the apical membrane [25,26]. Administration of ATP to either the apical or basolateral membranes of PDEC increases [Ca2+ ]i . The luminal application of ATP stimulates, whereas the basolateral inhibits the pancreatic ductal fluid and HCO3 − secretion [27]. Circulating cholecystokinin (CCK) was described as the major regulator of pancreatic acinar enzyme and fluid secretion, acting via oscillatory [Ca2+ ]i signals [28]. However, in PDEC the role of CCK stimulation differs among species; in humans it has negligible direct effects per se, but remarkably potentiates the stimulatory effect of secretin on the HCO3 − secretion [29]. Moreover, caerulein, an analog of CCK, has no effect on fluid secretion of isolated rat ducts [30]. In contrast, CCK was found to be a potent stimulant of HCO3 − secretion from the in vivo guinea pig pancreas [31]. There are other agonists (angiotensin II, histamine, neurotensin, and bombesin) which have also been shown to stimulate the pancreatic HCO3 − secretion via an increase in the [Ca2+ ]i , however their exact role in pancreatic fluid and HCO3 − secretion (especially in humans) remains elusive [2]. The peptide hormone secretin, which is released by duodenal enteroendocrine cells in response to acidic chyme, is generally considered as the most important regulator of pancreatic fluid and HCO3 − secretion [32]. Stimulation of the adenylyl cyclase coupled secretin receptor results in the elevation of intracellular cAMP levels, which in turn activates protein kinase A (PKA) [33]. Active PKA phosphorylates the CFTR regulatory (R) domain and increases CFTR activity. Vasoactive intestinal peptide and ␤ adrenergic agonists also activate adenylyl cyclase. There are several interactions between the Ca2+ and cAMP signaling [18,19], which will be reviewed elsewhere in this issue. In addition, recent studies suggest that secretion by pancreatic acinar and ductal cells are not only regulated by these neurohormonal stimuli. The cells of the exocrine pancreas seem to behave as an integrated acino-ductal functional unit, where they interact with each other during physiological secretion [3]. Acinar cells secrete ions (Cl− , Ca2+ ) and bioactive molecules (guanylin, uroguanylin, angiotensin II and ATP), which can affect ductal secretion and this ductal secretion also affects acinar cell function [34]. The luminal guanylin and uroguanylin bind to the guanylate cyclase C receptors on PDEC leading to an increase in intracellular cGMP, cGMP-dependent protein kinase II activation and phosphorylation of CFTR [35]. On the other hand the high intraluminal Ca2+ concentration can activate calcium sensing receptors on the apical membrane of PDEC, which stimulate HCO3 − secretion via an elevation in [Ca2+ ]i [36]. Besides the stimulatory effects, inhibitory control of the ductal fluid and HCO3 − secretion is also present; however it is much less

Fig. 2. Intracellular Ca2+ signal generation in PDEC. Agonist binding to G protein coupled receptors (GPCR) activates phospholipase C ␤ (PLC␤), which bind to and deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2 ) and releases inositol trisphosphate (IP3 ). The released IP3 activates IP3 receptors (IP3 R) in the ER membrane and releases the ER Ca2+ . The intracellular Ca2+ elevation stimulates the mitochondrial ATP production, which is necessary for the HCO3 − secretion. In addition, the Ca2+ activates the calmodulin (CaM)/calmodulin dependent kinase (CaMK) pathway, which was shown to stimulate the activity of the acid/base transporters in different cell types. Following agonist stimulation the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) pumps, and the plasma membrane Ca2+ -ATPase (PMCA) pumps move Ca2+ from the cytosol to the ER and the extracellular space, respectively. N: nucleus.

well characterized than the stimulatory pathways. Substance P (SP) is one of the antagonists, which inhibits HCO3 − secretion in guinea pig pancreatic ducts [37] via the activation of G protein coupled neurokinin receptors (NKR). SP binding to NKR activates protein kinase C to mediate the inhibition of HCO3 − secretion [32]. Another negative regulator of ductal secretion is 5-hydroxytryptamine (5HT), which is locally produced by enterochromaffin cells [38]. The inhibition is mediated by the 5-HT3 receptor, which is a ligandgated, non-selective, cation channel. The receptor activation causes an increase in intracellular Na+ , which can inhibit HCO3 − secretion by decreasing the driving force for NBCe1-B and therefore basolateral HCO3 − uptake. 4. Physiological Ca2+ signal generation: an old friend 4.1. Release of intracellular Ca2+ As described above, the secretory functions of PDEC are very rigorously regulated processes, in which intracellular Ca2+ signaling plays a central role (Fig. 2). During stimulation, agonist binding (ACh, ATP and luminal Ca2+ ) to G protein coupled metabotropic receptors activates phospholipase C ␤ (PLC␤), which binds to plasma membrane (PM) phosphoinositides via its pleckstrin homology domain [39]. Activated PLC␤ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2 ) and releases inositol trisphosphate (IP3 ) and diacylglycerol [40]. IP3 binds to and activates IP3 receptors (IP3 R), which are Ca2+ release channels located in the ER membrane [41]. In exocrine glands, IP3 R2 and 3 were described as the major isoforms of IP3 Rs [42,43]. In PDEC, IP3 R2 was shown to localize close to the apical pole of the cells [17]. Besides channel opening and Ca2+ release, binding of IP3 to IP3 R also releases IRBIT [44], which has multiple regulatory roles in PDEC [17,18,45]. The depletion of ER

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Ca2+ stores induces Ca2+ influx through the PM, which could contribute to the intracellular Ca2+ signals during longer stimulations (see below). Since a long sustained intracellular Ca2+ elevation can be toxic, it is necessary to extrude Ca2+ from the cytoplasm. The termination of the Ca2+ signals and the clearance of Ca2+ from the cytoplasm are carried out by two ATP dependent mechanisms. The sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) and the plasma membrane Ca2+ -ATPase (PMCA) move Ca2+ from the cytosol to the ER or to the extracellular space, respectively, and restore basal [Ca2+ ]i . In PDEC, Lee et al. suggested a polarized localization of these pumps with SERCA2b at the apical and SERCA3 at the basal membrane [46]. In pancreatic acinar cells the intracellular Ca2+ signaling has strict spatiotemporal limitations [47,48]; localizing the signal to the apical, secretory pole of the cells. Although not studied in as much detail as acinar cells, in PDEC the overall cell polarity, including the distribution of the acid/base transporters, IP3 receptors and mitochondria [7], suggests that Ca2+ signaling might have similar characteristics to acinar cells. Further studies need to clarify these questions. 4.2. Extracellular Ca2+ entry The role of Ca2+ influx in the plethora of non-excitable cell functions has long been established [49], however its molecular components and exact mechanism remained uncertain for almost four decades. Several years later, Hoth and Penner described that the receptor-mediated depletion of Ca2+ stores induces a sustained, highly Ca2+ selective inward current, which they termed ICRAC (calcium release-activated calcium current) [50]. The breakthrough in the field was brought by the discovery of the ER Ca2+ sensor, stromal interaction molecule 1 (Stim1) [51] and the PM Ca2+ channel Orai1 [52,53]. In resting state, when the ER Ca2+ stores are full, Stim1 distributes evenly in the bulk ER. During stimulation, the ER Ca2+ depletion induces the dissociation of Ca2+ from the Stim1 EF hand domain, which is followed by conformational changes and clustering of Stim1 in specific ER-PM junction, also called puncta formation [54]. The Stim1 Orai1 activation region (SOAR) and the polybasic domains of Stim1, physically interact with the N and C terminal binding sites of Orai1 resulting in Orai1clustering and activation [55]. This process is termed store operated Ca2+ entry (SOCE). Additional possible Ca2+ entry channels, which might play a role in Stim1 mediated SOCE, are the TRPC Ca2+ channels [56]. The mechanism and the role of SOCE in polarized epithelial cells have been extensively studied, mainly focusing on acinar cells from different exocrine glands [57–59], however its role in PDEC, especially in HCO3 − secretion remained elusive. Recently Kim et al. characterized SOCE in pancreatic ductal cells using cultured dog PDEC isolated from the accessory pancreatic duct [60]. They showed that an intracellular Ca2+ elevation, caused by the activation of SOCE might play a role in exocytosis, which is supposed to be the physiological function of this type of PDEC [61]. This, however does not provide evidence for the role of SOCE in other functions of the PDEC, such as HCO3 − secretion. In pancreatic or parotid acinar cells, Orai1 channels are localized to the apical membrane [57,58]. If the same localization applies for the ductal cells one could speculate that the SOCE might play a role in ductal HCO3 − secretion, however until the detailed characterization of the localization and function of store operated Ca2+ channels is performed in PDEC, their role remains elusive. 5. Downstream effects of the intracellular Ca2+ elevation 5.1. Stimulation of the mitochondrial ATP production Mitochondria and the rough ER form close contact sites which are similar to the ER-PM junctions [62]. These close connections

allow the formation of Ca2+ microdomains [63] where the local Ca2+ concentration can be several fold higher compared to the bulk [Ca2+ ]i . During ER Ca2+ release, mitochondria take up Ca2+ via a mitochondrial Ca2+ uniporter [64–66] resulting in an increase in the mitochondrial Ca2+ concentration ([Ca2+ ]m ). Usually, the elevation of [Ca2+ ]m has slower kinetics than that of the [Ca2+ ]i [67,68]. The major route of mitochondrial Ca2+ extrusion are the Na+ /Ca2+ exchangers [69,70]. The activity of the rate limiting enzymes of the tricarboxylic acid cycle are Ca2+ -dependent [71], which helps the mitochondria to adapt to the increased cellular ATP demand [71,72]. Under physiological conditions, the Ca2+ uptake into the mitochondria does not change the mitochondrial membrane potential (()m ) [73]. In pancreatic acinar cells, the intracellular ATP level [ATP]i increased despite the higher rate of ATP consumption during stimulation, which further suggests the adaptation of mitochondrial ATP production to increased cellular ATP demands [74]. The Ca2+ mediated upregulation of the ATP production might have a significant role in PDEC, since pancreatic ductal HCO3 − secretion is a strongly ATP dependent processes. CFTR, also called ABCC7, a member of the ATP-binding cassette transporter superfamily, has two nucleotide binding domain (NBD1 and NBD2). During the activation of CFTR, PKA uses ATP to phosphorylate and activate the R domain of CFTR [75]. This phosphorylation step is followed by the binding of two Mg-ATP molecules on the inter-NBD interface of the NBD domains, leading to the channel gating [76,77]. PKAdependent phosphorylation of the CFTR R domain is also required for the interaction of the R domain with the STAS domain of the SLC26 Cl− /HCO3 − exchangers, which increases the overall open probability and therefore the activity of CFTR [78]. Moreover, evidence suggests that NHE1 acts as an ATP-binding transporter; thus, ATP may directly activate NHE1, however its activity does not require ATP hydrolysis [79]. Recently we demonstrated that [ATP]i depletion, induced by the combined inhibition of the glycolysis and mitochondrial ATP production, by itself completely inhibit pancreatic ductal HCO3 − secretion, which provides further evidence for the importance of the cellular energy balance in HCO3 − secretion [7]. The subcellular distribution of the mitochondria in PDEC seems to serve the ATP demand of the HCO3 − secretion, since mitochondria are localized mostly to the apical region of the cells [7]. 5.2. Activation of other signaling pathways The intracellular Ca2+ elevation was shown to activate multiple downstream signaling events; however its role in the pancreatic ductal physiology or pathophysiology is uncertain at the moment. Here we briefly summarize Ca2+ -dependent events, which might play a role in pancreatic HCO3 − secretion and PDEC physiology. The intracellular Ca2+ elevation activates calmodulin, the ubiquitous calcium-binding protein that controls a wide range of cellular processes. The activated calmodulin interacts with other proteins, such as calmodulin kinases [80], or cyclic AMP response elementbinding protein (CREB) [81]. The role of calmodulin in the physiology and pathophysiology of pancreatic acniar cells was highlighted by Craske et al. [82]. Jung et al. showed that calmodulin physically interacts with the Ca2+ activated Cl− channel ANO1 and induces a change in the anion selectivity, making it permeable to HCO3 − [83]. Although recently another group found that calmodulin does not coimmunoprecipitate with ANO1 and they also showed that it is not required for the channel activation, however the role of calmodulin in modulating the biophysical properties of the channel is still uncertain [84]. As described above, PDEC also express Ca2+ activated Cl− channels, however its contribution to pancreatic HCO3 − secretion is different among species, moreover its molecular identity is also uncertain, yet the fact that Ca2+ /calmodulin complex can regulate ion selectivity of Cl− channels might be relevant

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Fig. 3. The effects of bile acids on PDEC. Low concentration of chenodeoxycholate (CDC) induced repetitive Ca2+ oscillations and stimulated the HCO3 − secretion from the luminal membrane of PDEC, which stimulates the luminal Cl− /HCO3 − exchange. In contrast, high concentration of CDC induced toxic sustained Ca2+ elevation and severe morphological damage of the mitochondria (swelling and disruption of the membrane structure) with a consequent [ATP]i depletion. These changes inhibited acid/base transporters including the basolateral NHE, NBCe1-B and the luminal SLC26 Cl− /HCO3 − exchanger.

for the PDEC physiology. The role of the Ca2+ /calmodulin complex was also highlighted in the stimulatory effect of carbachol on Na+ HCO3 − cotransport activity in murine colonic crypts [85] and in intracellular pH recovery from acidosis via NHE activation [86]. The Ca2+ /calmodulin complex activates the protein phosphatase calcineurin, which plays an important role in numerous cellular processes [87], including the phosphorylation of the transcription factor nuclear factor of activated T cells (NFAT). NFAT is cytoplasmic in resting cells, but enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin [88,89]. However the role of Ca2+ /calmodulin activated signaling events in the regulation of acid/base transporters or gene transcription in PDEC are still uncertain. 6. Pathophysiological Ca2+ signal generation: a nasty enemy It is well established, that physiological Ca2+ signaling has many beneficial effects in PDEC, which help to match stimulatory signals and HCO3 − secretion. However, our group and others have demonstrated, that the most frequent pathogenic factors for acute pancreatitis, such as bile acids, non-oxidative ethanol metabolites and trypsin, induce sustained intracellular [Ca2+ ] elevation both in pancreatic acinar and ductal cells, which inhibits the cellular functions [6,90,91]. These findings suggest, that uncontrolled Ca2+ signal generation plays a central role in the development of cellular damage during acute pancreatitis, the most frequent cause of hospitalization among non-malignant gastrointestinal diseases [92]. Venglovecz et al. demonstrated that the non-conjugated bile acid chenodeoxycholate (CDC), has dose-dependent dual effects on pancreatic HCO3 − secretion (Fig. 3), which might be explained by the type of Ca2+ signals evoked by CDC [6]. Low concentrations (100 ␮M) of CDC induced repetitive, short-lasting Ca2+ oscillations,

which stimulated HCO3 − secretion from the luminal membrane of PDEC. The oscillations were abolished by the IP3 R inhibitor caffeine, or xestospongin C and the PLC inhibitor U73122. Preincubation of the PDEC with the intracellular Ca2+ chelator BAPTA-AM prevented the Ca2+ signals and also abolished the stimulatory effect of 100 ␮M CDC on HCO3 − secretion. In contrast, high concentrations (1 mM) of CDC induced a toxic sustained Ca2+ elevation and severe morphological damage to the mitochondria (swelling and disruption of the membrane structure) with a consequent [ATP]i depletion [7]. These changes inhibited acid/base transporters including the basolateral NHE, NBCe1-B and the luminal CBE [6]. Notably, BAPTAAM preincubation failed to prevent the mitochondrial damage and the inhibitory effect of CDC on the HCO3 − secretion, suggesting the presence of a Ca2+ -independent, direct mechanism underlying the mitochondrial toxicity of bile acids. This toxic effect might be explained by the protonophoric-like effect of the high concentration of CDC that can affect the mitochondrial ATP production. Similarly to PDEC, bile acids induced Ca2+ release from both the ER and acidic intracellular Ca2+ stores through activation of IP3 R and ryanodine receptors in isolated pancreatic acinar cells [93]. Moreover, Voronina et al. showed that taurolithocholicacid 3sulfate (TLC-S) decreased [ATP]i in pancreatic acinar cells [74] and caused the loss of ()m , which was not influenced by BAPTA-AM pretreatment [73]. The other most common causes of acute pancreatitis are the result of exposure to ethanol and its non-oxidative metabolites. Yamamoto et al. showed that a low concentration (1 mM) of ethanol induced a [Ca2+ ]i , elevation and augmented secretinstimulated fluid secretion in guinea pig pancreatic ducts [94]. They also observed a weak inhibition of the stimulated fluid secretion during the administration of 100 mM ethanol. The stimulatory

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Fig. 4. The effects of ethanol and ethanol metabolites in PDEC. Low concentration of ethanol stimulated the HCO3 − secretion in PDEC via Ca2+ release from the ER. In contrast, high concentration of ethanol and fatty acids inhibited acid/base transporters including the basolateral NHE, NBCe1-B and the luminal CFTR, and SLC26 Cl− /HCO3 − exchanger. The inhibitory effect was mediated by toxic sustained Ca2+ overload and impaired [ATP]i production, similarly to the effect of bile acids.

effect of 1 mM ethanol was abolished by BAPTA-AM preincubation, suggesting that it was mediated by the [Ca2+ ]i elevation. In our preliminary experiments, administration of low concentration (10 mM) of ethanol stimulated the HCO3 − secretion via an IP3 mediated [Ca2+ ]i elevation in Capan-1 cells [95] (Fig. 4). In contrast, high concentrations of ethanol (100 mM) and palmitoleic acid (POA) (200 ␮M) inhibited the activities of the apical SLC26 Cl− /HCO3 − exchanger and CFTR Cl− channel and decreased HCO3 − secretion in PDEC. We also showed that ethanol and POA in high concentrations induced a sustained [Ca2+ ]i elevation by releasing Ca2+ from the ER via IP3 and ryanodine receptor activation followed by gadolinium-sensitive extracellular Ca2+ influx. BAPTA-AM preincubation completely abolished the inhibitory effects of ethanol and POA, suggesting that the inhibition was mediated by the sustained [Ca2+ ]i elevation [95]. Moreover, we observed decreased [ATP]i levels and the loss of ()m during the administration of high concentration of ethanol, or POA. In pancreatic acinar cells, similarly to PDEC, non-oxidative ethanol metabolites induced a sustained [Ca2+ ]i elevation leading to necrosis [91,96,97]. Criddle et al. found that a high concentration of POA depolarized the ()m , which was abolished by BAPTA-AM preincubation. Intrapancreatic trypsinogen activation is a hallmark of the pathogenesis of acute pancreatitis. Earlier Pallagi et al. investigated the effects of trypsin on the pancreatic ductal epithelia and showed that trypsin or PAR2 antagonist peptide induced an intracellular Ca2+ elevation and inhibited the luminal acid/base transporters in PDEC when administrated from the luminal membrane. Moreover, the inhibitory effect was abolished by BAPTA-AM preincubation, similarly to the inhibitory effects of ethanol and POA [5].

7. Mitochondrial damage in acute pancreatitis The toxic sustained Ca2+ elevation seems to mediate the inhibitory effect of both ethanol or ethanol metabolites and trypsin and also appears to play an important role in bile acid-induced cellular injury. Sustained [Ca2+ ]i rises can induce mitochondrial injury and ATP depletion, which was shown to be important in the development of acute pancreatitis [98,99]. Despite these observations, the mechanism as to how the sustained elevation of intracellular Ca2+ leads to decreased HCO3 − secretion in PDEC is still not completely resolved. Mitochondria play crucial roles in the spatial and temporal localization of the intracellular Ca2+ signals by acting as Ca2+ buffers (see above) [63]. Prolonged mitochondrial Ca2+ overload can induce the opening of the mitochondrial membrane permeability pore (MPTP) across the inner and outer membranes of mitochondria, resulting in an increased permeability of the mitochondrial membranes to molecules and ions with molecular mass less than 1.5 kDa, including protons and water [100]. Another possible way to disrupt the mitochondrial membrane is following mitochondrial outer membrane permeabilization (MOMP), which is proposed to be a crucial event during apoptosis, causing the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol [101]. Due to the increased membrane permeability the ()m disappears, mitochondria become swollen and mitochondrial membranes rupture, and a consequent drop of ATP production [100,102,103]. This kind of damage is very similar to the alterations observed in PDEC treated with 1 mM CDC [7]. The molecular identity and the exact mechanism of the pore opening remain

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uncertain, however three components were identified which might play role in the set-up of MPTP; the voltage dependent anion channel (VDAC) in the outer mitochondrial membrane, the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane and cyclophilin-D (Cyp-D) in the mitochondrial matrix [104,105]. Prolonged Ca2+ overload might induce conformational changes in VDAC, ANT and Cyp-D producing transmembrane complexes from these components that lead to MPTP formation [104,105]. Moreover, ATP is necessary for the activity of SERCA and PMCA Ca2+ pumps and therefore the drop of [ATP]i can further contribute to the maintenance of the sustained Ca2+ rise [106]. The sustained elevation of [Ca2+ ]i and the resulting mitochondrial damage can lead to a vicious cycle which in turn triggers cell necrosis [90,107]. As detailed above, the activity of several enzymes and transporters, and therefore pancreatic ductal HCO3 − secretion by itself, depends on the accessibility of [ATP]i . The importance of ATP depletion in the inhibitory effect of non-oxidative ethanol metabolites was recently confirmed by Judák et al. This study showed that non-oxidative ethanol metabolites inhibit the CFTR Cl− current in isolated guinea pig PDEC via [ATP]i depletion. Moreover, they demonstrated, that ATP supplementation via a patch pipette significantly restored the inhibited CFTR function [108]. Since toxic cellular Ca2+ overload and mitochondrial damage are intimately related, it is difficult to determine the steps leading to cellular damage during biliary or alcohol-induced pancreatitis. In addition, both bile acids and non-oxidative ethanol metabolites also seem to cause direct mitochondrial damage, highlighting how the mechanisms of damage overlap.

8. Therapeutic targets and closing remarks The role of intracellular Ca2+ signaling in the physiology and pathophysiology of PDEC is still not completely clarified, however the available data suggest that it plays a central role in HCO3 − secretion and also in the pathogenesis of acute pancreatitis. The harmful effects of uncontrolled, toxic Ca2+ rises have been investigated in a plethora of additional other diseases and cell types. Sustained Ca2+ elevations, followed by mitochondrial dysfunction has been reported in neurodegeneration following ischemia [109] and in the pathogenesis of Alzheimer disease [110]. Moreover, cytosolic Ca2+ overload has been highlighted in myocardial ischemia/reperfusion injury [111], and in cardiac hypertrophy and vascular proliferative diseases [112]. Despite the central role of cellular Ca2+ overload and toxicity in a wide range of diseases, the detailed mechanisms, which lead to the transformation of the physiological oscillatory Ca2+ waves to global, sustained Ca2+ rises, are still uncertain. However the prevention of this propagation might be crucial to develop specific therapy, which could help to prevent pancreatic damage during acute pancreatitis. One possible target might be the store operated Ca2+ entry channels. As described above SOCE under physiological conditions helps to restore the intracellular Ca2+ store, depleted during agonist stimulation. Recently the role of SOCE was also suggested in the pancreatitis pathogenesis. In vivo experiments suggest that the pharmacological inhibition [113] or the genetic deletion [114] of the transient receptor potential 3 (TRPC3) reduces SOCE and the severity of acute pancreatitis in mice. Moreover, in vitro inhibition of Orai1 by the pharmacological compound GSK-7975A prevented the sustained Ca2+ elevation, protease activation and cell necrosis caused by 100 ␮M palmitoleic acid ethyl ester (POAEE) in isolated pancreatic acinar cells [115]. These results indicate that the pharmacological inhibition of SOCE might be beneficial during the treatment of acute pancreatitis.

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Conflict of interest The authors have no conflict of interest to declare.

Acknowledgements Our research was supported by the Hungarian National Development Agency grants (TÁMOP-4.2.2.A-11/1/KONV-20120035, TÁMOP-4.2.2-A -11/1/KONV-2012-0052; TÁMOP-4.2.2.A 11/1/KONV-2012 - 0073; TÁMOP-4.2.4.A2-11-1-2012-0001, TÁMOP-4.2.4.A2-SZJÖ-TOK-13-0017), the Hungarian Scientific Research Fund (OTKA NF100677).

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Please cite this article in press as: J. Maléth, P. Hegyi, Calcium signaling in pancreatic ductal epithelial cells: An old friend and a nasty enemy, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.02.004