Ca2+ signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca2+ channel blockade

Key points Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions. In this study, we have investigated bradykinin‐elicited Ca2+ signal generation in normal mouse pancreatic lobules. We found complete separation of Ca2+ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca2+ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca2+ signals in PACs, never elicited Ca2+ signals in PSCs. The bradykinin‐elicited Ca2+ signals were due to initial Ca2+ release from inositol trisphosphate‐sensitive stores followed by Ca2+ entry through Ca2+ release‐activated channels (CRACs). The Ca2+ entry phase was effectively inhibited by a CRAC blocker. B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. Abstract Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca2+ signals. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The BK‐induced Ca2+ signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca2+ rise in PSCs was due to inositol trisphosphate receptor‐mediated release from internal stores, whereas the sustained phase depended on external Ca2+ entry through Ca2+ release‐activated Ca2+ (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca2+ signal generation in PSCs and this may be helpful in treating acute pancreatitis.


Introduction
It has been known for more than 40 years that pancreatic enzyme secretion is regulated by Ca 2+ signals in the pancreatic acinar cells (PACs) and that the primary event is acetylcholine-(ACh) or cholecystokinin-(CCK) elicited intracellular Ca 2+ release followed by Ca 2+ entry (Case & Clausen, 1973;Matthews et al. 1973;Kondo & Schulz, 1976;Petersen & Ueda, 1976). The ACh-evoked intracellular Ca 2+ release is mediated by inositol trisphosphate (IP 3 ) (Streb et al. 1983;Wakui et al. 1990), whereas the CCK-elicited Ca 2+ release is mediated by nicotinic acid adenine dinucleotide phosphate (Cancela et al. 2000;Gerasimenko et al. 2015). As high K + depolarization of the PAC membrane does not evoke enzyme secretion or Ca 2+ movement (Argent et al. 1971;Matthews et al. 1973), the Ca 2+ entry does not occur through voltage-gated channels (Petersen, 1992), but is due to opening of Ca 2+ release-activated Ca 2+ (CRAC) channels (Parekh & Putney, 2005;Gerasimenko et al. 2013). The acinar Ca 2+ signals also regulate acinar fluid secretion via Ca 2+ -activated Cl − and K + channels (Petersen, 1992;Park et al. 2001). However, the major component of pancreatic fluid secretion is contributed by the ducts and this is regulated by secretin-elicited intra-ductal cyclic AMP formation controlling cystic fibrosis transmembrane conductance regulator (CFTR) channels (Argent, 2006), whose openings are also regulated by the Cl − concentration in the luminal fluid (Broadbent et al. 2015).
The often fatal human disease acute pancreatitis, in which the pancreas digests itself and its surroundings, is initiated by excessive intracellular Ca 2+ release in the PACs followed by excessive Ca 2+ entry, mostly elicited by combinations of alcohol and fatty acids or by bile acids Gerasimenko et al. 2014). Pancreatitis-inducing agents also inhibit ductal fluid and bicarbonate secretion, exacerbating the disease (Pallagi et al. 2011;Maleth et al. 2015).
We have extensive knowledge of the mechanisms generating Ca 2+ signals in the PACs, which has been built up over many decades Gerasimenko et al. 2014). In contrast, the more recently discovered pancreatic stellate cells (PSCs) (Watari et al. 1982), located in the peri-acinar space -with elongated processes around the base of the acinus -have received much less attention and it has essentially been the functional properties of cultured cells that have been investigated (Apte et al. 1998(Apte et al. , 2012Bachem et al. 1998;Wells & Crawford, 1998). The prevailing, and so far unchallenged, view has been that normal PSCs are quiescent and the focus in all published studies has been on the activation of PSCs and their role -in the activated state -in chronic pancreatitis and pancreatic cancer (Bachem et al. 1998;Wells & Crawford, 1998;Sherman et al. 2014). Activation of PSCs -during pancreatic injury or culturing of quiescent PSCs -induces proliferation as well as secretion of extracellular matrix components, thereby playing an important role in the fibrosis that occurs in chronic pancreatitis and pancreatic cancer (Sherman et al. 2014). Work on the so-called quiescent PSCs in culture has shown that they can generate substantial cytosolic Ca 2+ signals in response to stimulation with high concentrations of the blood pressure-lowering nona-peptide bradykinin (BK) and some other substances (Won et al. 2011).
Because BK has long been known as an important player in inflammatory disease, including acute pancreatitis (Ryan et al. 1964;Hirata et al. 2002), we have now investigated BK-induced Ca 2+ signal generation, and its underlying mechanism, in normal PSCs in their normal microenvironment. We find that normal PSCs are much more sensitive to BK than PSCs in culture, generating substantial Ca 2+ signals in response to a BK concentration as low as 100 pM, with maximal effect at 1 nM, orders of magnitude lower than what has been observed in cultured cells (Won et al. 2011). This has important implications, as the threshold for activating normal PSCs (100 pM) is close to the normal plasma level of BK (40-70 pM) (Blais et al. 1999;Hirata et al. 2002) and any increase in the plasma or tissue BK levels, which occurs under several conditions -including acute pancreatitis and use of angiotensin-converting-enzyme (ACE) inhibitors (Liu et al. 1997;Blais et al. 1999;Tsutsumi et al. 1999;Hirata et al. 2002;Su, 2014) -would therefore elicit Ca 2+ signals in PSCs. We have explored the Ca 2+ signalling events and their underlying mechanisms in normal PSCs and show that BK activates bradykinin type 2 (B2) receptors, which causes primary Ca 2+ release from internal stores. This effect can be abolished by phospholipase C inhibition or blockade of IP 3 receptors (IP 3 Rs). Following the initial intracellular Ca 2+ release, there is opening of conventional CRAC channels (Parekh & Putney, 2005;Parekh, 2010) and we show that a recently employed CRAC channel inhibitor, which was protective against the destruction of PACs evoked by agents inducing pancreatitis (Gerasimenko et al. 2013(Gerasimenko et al. , 2014, also reduces BK-induced Ca 2+ signal generation in PSCs. We show that B2 receptor blockade protects against the necrosis evoked by pancreatitis-inducing agents and suggest that the protective effect of CRAC channel blockade against pancreatitis may in part be due to inhibition of Ca 2+ signal generation in PSCs. Overall, our new data indicate that the so far generally accepted notion of normal PSCs being quiescent is potentially misleading as they are in fact exquisitely sensitive to relatively small changes in BK concentrations found in vivo.

Ethical approval
All procedures were carried out in accordance with UK Home Office regulations.

Preparation of pancreatic lobules
Pancreatic lobules and big clusters were isolated from the pancreas of adult C57Bl/6 male mice. Animals were killed according to UK Schedule 1 regulations. The pancreas was rapidly dissected, transferred to collagenase Na-Hepes-based solution (Sigma, Poole, UK) and incubated at 37°C for 5-6 min. After digestion, the tissue was kept in Na-Hepes-based extracellular media, containing (in mM): NaCl, 140; KCl, 4.7; Hepes (KOH), 10; MgCl 2 , 1; glucose 10; CaCl 2 1; pH 7.2. Pancreatic lobules were then incubated with fluorescent dye following the manufacturer's description. All experiments were carried out with freshly prepared pancreatic lobules, attached to the coverslip of the perfusion chamber at room temperature (23°C). Penetration of various substances deep into the pancreatic lobule was highly dependent on the distance from the surface (Fig. 1A, B). It was therefore necessary, in several cases, to use relatively high concentrations (up to 5-fold higher than would have been necessary in experiments on isolated cells or very small cell clusters) and relatively long pre-incubation times.

[Ca 2+ ] measurements in intact cells
Intact cells were loaded with 5 µM Fluo-4 acetoxymethyl ester (AM), for 10 min at room temperature. Cells were transferred to a flow chamber and perfused with the Na-Hepes-based extracellular solution as described above. Cells were visualized using a Leica SP5 MPII two-photon confocal microscope, with a 63× 1.2 NA objective lens. Fluo-4 was excited with a 488 nm Ar laser, at 1-4% power, and emitted light was collected at 510-580 nm. Generally, a series of images was recorded at 512×512 pixel resolution (at a speed of 0.3 frames s −1 ), and analysed using Leica Confocal Software (Leica, Mannheim, Germany). Fluorescence signals were plotted as F/F 0 (F 0 is the initial level of fluorescence). Statistical analysis was performed using ANOVA or Student's t-test.

Measurements of necrosis level in pancreatic lobules
Pancreatic lobules were exposed to 350 mM ethanol, or 500 µM palmitoleic acid ethyl ester (POAEE) or 0.5% sodium choleate for 2 h in the presence or absence of 0.5 µM or 10 µM WIN 64338. Ethanol-or bile-induced pancreatic necrosis was visualized by staining the lobules with propidium iodide (PI) and compared with the control necrosis level (without any treatment). Simultaneously lobules were stained with the nuclear dye Hoechst 33342 to enable cell counting. Both stainings were performed according to the manufacturer's protocols. Fluorescence of Hoechst 33342 and PI was recorded using confocal microscopy, with a 63× 1.2 NA objective lens (excitations 355 and 543 nm; and emissions 390-480 and 570-650 nm, respectively).

Immunocytochemistry
Immunocytochemistry in pancreatic lobules and big clusters was performed as described by Lur et al. (2009) with some modifications. Following blocking with 1% BSA and 10% goat serum, the isolated pancreatic lobules were incubated for 1 h at room temperature with primary antibody (CCK-AR) in 5% goat serum in PBS. The pancreatic lobules were subsequently incubated with CruzFluor (CFL) 594-conjugated secondary antibody for 30 min at room temperature. Cells were attached to the glass coverslips covered with poly-L-lysin. For immunochemical staining with desmin antibody, pancreatic lobules were fixed with 4% paraformaldehyde followed by permeabilization with Triton X100, blocking and incubation with primary (desmin) and then with secondary (CFL) antibodies as described above.

Separate Ca 2+ signalling events in neighbouring PSCs and PACs
It has been shown that desmin is a good marker for PSCs (Apte et al. 1998) and we found that immunochemical staining with desmin antibody identified small elongated cells situated at the periphery of the dominant acinar cells ( Fig. 2A-C). The presence of vitamin A in lipid droplets is another characteristic of PSCs (Bachem et al. 1998) and we could visualize this by intrinsic multiphoton fluorescence ( Fig. 2D, E). Cells situated in the position characteristic of desmin-containing cells ( Fig. 2C) took up the Ca 2+ -sensitive fluorescent probe Fluo-4 much more avidly than PACs ( Fig. 3A-C). In large lobules stained with Fluo-4 ( Fig. 3A, B), it was often possible to observe PSCs as bright 'strings' , whereas in smaller cell clusters, they typically appeared as individual bright cells at the periphery of acinar units (Fig. 3Ca).
Cultured PSCs produce cytosolic Ca 2+ signals in response to a micromolar concentration of BK (Won et al. 2011). In our experiments, normal PSCs in their normal micro-environment typically responded to a short-lasting exposure to a much lower BK concentration (1 nM  , which were not transmitted to the neighbouring PSCs (n = 14 and 17, respectively) (Figs 3Cb and 4A). ATP (100 µM) elicited Ca 2+ signals in a proportion of PSCs (41 of 107 cells), but failed to do so in many others (Fig. 4A). When ATP evoked a [Ca 2+ ] i rise in a PSC, it was never transmitted to the neighbouring PAC (Fig. 4A). Phillips et al. (2010) have suggested that CCK-evoked release of ACh from PSCs could in turn activate PACs and they proposed that this could be the normal mechanism for CCK-elicited secretion from human PACs. However, we did not find any evidence for this hypothesis as CCK never evoked any Ca 2+ signals in PSCs in our preparations (n = 17; at [CCK] = 10 nM) and Ca 2+ signals in PSCs consistently failed to be transmitted to neighbouring PACs (Fig. 4A). Furthermore, although a fluorescent antibody to CCK1 receptors clearly identified the presence of CCK1 receptors on PACs, it failed to do so with regard to PSCs (four experiments) (Fig. 4B).

BK evokes Ca 2+ signals in PSCs at pico-and low nanomolar concentrations
It seemed important to explore the levels of BK needed to evoke Ca 2+ signals (Fig. 5) in relation to what is known about BK concentrations in plasma. From measurements in humans and rats, it is known that normal plasma BK concentrations are in the range 40-70 pM (Blais et al. 1999;Hirata et al. 2002). However, in a bile duct obstruction model of acute pancreatitis induced in rats, it has been shown that the BK plasma concentration rose to ß140 pM (Hirata et al. 2002). As the BK release in acute pancreatitis primarily comes from the pancreas, the local (intra-pancreatic) BK levels could be higher (Ryan et al. 1964;Schachter, 1969). We found that a BK concentration as low as 100 pM (n = 14) could elicit PSC Ca 2+ signals and that maximal responses were obtained at 1 nM (n = 9, in the series of experiments represented by Fig. 5C). Overall, in the course of this investigation, BK responses to 1 nM have been observed in >100 experiments. At the low BK concentrations we used, there was no sign of desensitization (Fig. 5A). Within the time frame of our protocols, the [Ca 2+ ] i elevation was maintained as long as the stimulus was maintained (n = 19) (Fig. 5A) to BK to be able to sense relatively small increases in the surrounding BK level. Although the PSCs in our lobule preparation were much more sensitive to BK than those previously studied in culture (Won et al. 2011), they do have some characteristics in common with quiescent PSCs in culture which, unlike activated PSCs, do not respond to trypsin and thrombin. As seen in Fig. 6 there was no response to thrombin (n = 6) (Fig. 6A) or trypsin (n = 18) (Fig. 6B) in cells that responded to BK with a clear Ca 2+ signal.
The BK-elicited Ca 2+ signals in PSCs are due to activation of B2 receptors The BK receptor sub-type responsible for generating Ca 2+ signals in PSCs has not previously been investigated. We employed the B2 receptor blocker WIN64338 (Hu et al. 2004) and showed that this agent reversibly blocked BK-elicited Ca 2+ signal generation (n = 27) (Fig. 7A). In contrast, the B1 receptor blocker R-715 (Abdouh et al. 2008) failed to inhibit BK-induced Ca 2+ signalling (n = 8) (Fig. 7B). Furthermore, the specific B1 agonist Sar-des-Arg-BK did not evoke any changes in [Ca 2+ ] i of PSCs (n = 8) (Fig. 7C). It would appear that the plateau phase of the BK-induced response to some extent depends on continued B2 receptor activation, because application of WIN64338 immediately after the initial BK-elicited Ca 2+ spike shortened the duration of the plateau phase (Fig. 7D).
As a previous study showed that BK levels increase in acute pancreatitis and that blockade of B2 receptors attenuated the cellular changes underlying the development of acute pancreatitis evoked by obstruction of the pancreatico-biliary duct in rats (Hirata et al. 2002), our results raise the possibility that B2 receptor-mediated  Ca 2+ signals in PSCs contribute to the negative outcome of this disease. We therefore tested whether B2 receptor blockade could protect against cellular changes relevant to the development of acute pancreatitis, which is most frequently related to alcohol abuse or biliary disease . In alcohol-related acute pancreatitis, the pancreas is exposed not only to alcohol, but also to fatty acid ethyl esters (non-oxidative combinations of alcohol and fatty acids), which have been shown to be particularly effective releasers of intracellular Ca 2+ (Criddle et al. 2006;Gerasimenko et al. 2009). In biliary disease, the pancreas will be exposed to high concentrations of bile acids, which have also been shown to be effective liberators of stored Ca 2+ (Voronina et al. 2002;Gerasimenko et al. 2006). We therefore tested the effects of B2 receptor blockade on the level of necrosis elicited by alcohol, POAEE and a bile acid mixture. As seen in Fig. 8, B2 receptor blockade markedly reduced the extent of acinar cell necrosis induced by either a high alcohol concentration (350 mM), POAEE (500 µM) or bile acids (0.5% sodium choleate: a crude ox bile extract which contains the sodium salts of taurocholic, glycocholic, deoxycholic and cholic acids).
BK-elicited Ca 2+ signals in PSCs are primarily due to release of Ca 2+ from internal stores, but is followed by store-operated Ca 2+ entry As shown in Figs 3-5, the BK-elicited Ca 2+ signals consist of a brief transient rise in [Ca 2+ ] i , followed by a prolonged plateau phase of elevated [Ca 2+ ] i . Acute removal of external Ca 2+ did not reduce the initial phase of BK-evoked Ca 2+ signals, but eliminated the following plateau phase (Fig. 9A, B). Re-admission of Ca 2+ resulted in a transient increase in [Ca 2+ ] i and enabled a subsequent BK application to evoke a normal response (Fig. 9A, B). We tested the ability of PSCs to generate store-operated Ca 2+ entry, by using the now 'classical' protocol for assessing this phenomenon (Fig. 9C), employing cyclopiazonic acid (CPA) to block the Ca 2+ pumps in the endoplasmic reticulum (ER). As shown in Fig. 9B and C, re-admission of external Ca 2+ after a period of external Ca 2+ deprivation, and BK stimulation, resulted in a transient rise in [Ca 2+ ] i, but, if the Ca 2+ re-admission occurred after and during a period of continued ER Ca 2+ pump inhibition, the [Ca 2+ ] i elevation was sustained (Fig. 9C). In this situation, Ca 2+ entering store-operated Ca 2+ channels cannot be taken up into the ER, but will only slowly be extruded by the plasma membrane Ca 2+ pumps. We tried to block the store-operated Ca 2+ entry pharmacologically. 2-APB, a well-known, but relatively unspecific, blocker of CRAC channels (Parekh & Putney, 2005), abolished BK-induced Ca 2+ signal generation (Fig. 9D), which may be due to blockade of IP 3 Rs (Ma et al. 2000). We also used a more specific CRAC channel blocker, GSK-7975A, which has recently been shown to block CRAC channel currents in PACs (Gerasimenko et al. 2013). In these experiments, we were able to show that GSK-7975A reversibly blocked the plateau phase of the BK-induced [Ca 2+ ] i elevation, without affecting the initial spike (n = 9) (Fig. 9E). GSK-7975A also blocked the Ca 2+ entry normally occurring when external Ca 2+ was re-admitted after a period of external Ca 2+ deprivation (n = 14) (Fig. 9F).
Finally, we examined the mechanism underlying the initial Ca 2+ spike in response to BK stimulation. Having established that it is due to release of Ca 2+ from internal stores (Fig. 9A), we tested the hypothesis that this Ca 2+ liberation occurs through IP 3 Rs. It is now well stablished that caffeine inhibits opening of IP 3 Rs (Wakui et al. 1990;Ehrlich et al. 1994;Foskett et al. 2007). As seen in Fig. 9G, J Physiol 594.2 caffeine did reversibly block BK-elicited Ca 2+ signalling (n = 29). BK probably activates phospholipase C, as U73122 Smith et al. 1990) blocked the ability of BK to evoke Ca 2+ signals (n = 14) (Fig. 9H).

Discussion
Our results demonstrate for the first time that BK, in concentrations close to those measured in normal plasma (Blais et al. 1999;Hirata et al. 2002), elicits substantial cytosolic Ca 2+ signals in normal PSCs in their normal micro-environment and therefore cast doubt on the hitherto prevailing concept of the quiescent PSC (Apte et al. 1998(Apte et al. , 2012Sherman et al. 2014).
Given that an important role for BK in the development of acute pancreatitis was proposed by Ryan et al. (1964), that there is an increase in the plasma level of BK in acute pancreatitis (Hirata et al. 2002) and that a number of studies have shown that pharmacological blockade of B2 receptors is helpful in suppressing the cellular changes in several pancreatitis disease models (Griesbacher &

. Thrombin and trypsin do not evoke Ca 2+ signals in PSCs
A, thrombin (10 mU ml −1 ) does not evoke a Ca 2+ signal in a PSC, but a subsequent BK application (1 nM) evokes the usual response. B, trypsin (20 µM) fails to evoke a Ca 2+ signal in a PSC, but a subsequent BK application (1 nM) does so. The apparently delayed and somewhat smaller response to BK (although still within the normal range) in B (as compared to A) is probably due to the fact that the cell cluster in this case was relatively large and the PSC deep in the tissue (see Fig. 1). Lembeck 1992; Griesbacher et al. 1993;Hoffmann et al. 1996;Bloechle et al. 1998;Hirata et al. 2002), it would seem important to identify the target for the action of BK in the pancreas and its mechanism of action.

of PAC death elicited by pancreatitis-inducing agents is markedly reduced by B2 receptor blockade
A, treatment of pancreatic lobules with ethanol (350 mM) for 2 h at room temperature increased significantly the percentage of PAC necrosis from the control level of 5.47 ± 0.54% (n = 7) to 17 ± 1% (n = 7) and this was significantly reduced to 7.85 ± 1.3% (n = 3) by 10 µM WIN64338 and to 7.84 ± 0.25% (n = 4) by 0.5 µM WIN64338 ( * P = 0.0025 and * * P = 0.00043, respectively, >1500 cells in each experimental group). B, treatment of pancreatic lobules with POAEE (500 µM) for 2 h at room temperature increased significantly the percentage of PAC necrosis from the control level of 5.46 ± 1.25% (n = 3) to 18.96 ± 1.76% (n = 3) and this was reduced to 7.15 ± 0.67% (n = 3) by 0.5 µM WIN64338 ( * P = 0.003, >990 cells in each experimental group). C, treatment of pancreatic lobules with 0.5% sodium choleate for 2 h at room temperature also increased significantly the percentage of PAC necrosis from the control level of 6.1 ± 0.85% (n = 3) to 14 ± 1% (n = 3) and this was significantly reduced to 7.8 ± 0.8% (n = 3) by 0.5 µM WIN64338 ( * P = 0.0087, >1600 cells in each experimental group). Necrosis was measured by staining of lobules with PI. Cell count was performed using nuclear staining with Hoechst 33342. Bars represent ±SEM. Won et al. (2011) demonstrated that BK -in high (micromolar) concentrations -evoked transient Ca 2+ signals in quiescent and activated PSCs in culture. We focused our attention on the effect of low quasi-physiological concentrations of BK on normal PSCs in their normal micro-environment, the mechanism of action and its consequence. Our results show that whereas BK consistently evokes bi-phasic Ca 2+ signals in PSCs, it consistently fails to do so even in closely neighbouring PACs (Figs 3 and 4). We have therefore now identified a specific cellular site for the action of BK, which may partly or fully explain its importance in pancreatitis. Furthermore, we have established that the BK-elicited Ca 2+ signals in PSCs are due to activation of B2 receptors, providing a plausible explanation for the suppressive effect of B2 blockade on the development of acute pancreatitis (Griesbacher & Lembeck 1992;Griesbacher et al. 1993;Hoffmann et al. 1996;Bloechle et al. 1998;Hirata et al. 2002). We have also shown that PAC necrosis elicited by alcohol, POAEE or bile can be significantly reduced by B2 receptor blockade (Fig. 8).
We failed to observe any effects of CCK on PSCs, although this hormone evoked its usual effect  on neighbouring PACs (Fig. 4A). Therefore, our results do not provide support for the hypothesis that CCK acting on PSCs will increase ACh secretion from these cells, which in turn would activate PACs (Phillips et al. 2010). We worked on normal mouse pancreatic tissue, whereas Phillips et al. (2010) studied ACh secretion from cultured human PSCs. Cultured PSCs clearly have different properties from normal PSCs, because in the normal mouse pancreas we have observed Ca 2+ signal generation in PSCs in response to 0.1 nM BK (Fig. 5), whereas micromolar BK concentrations were required to obtain such responses in cultured mouse PSCs (Won et al. 2011). With regard to the mechanism of CCK action in the human pancreas, the simplest hypothesis remains direct action on PACs, as shown in isolated acinar cell clusters from human pancreas (Murphy et al. 2008).
Although the initiating event elicited by pancreatitis-inducing agents in PACs is Ca 2+ release from intracellular stores (Gerasimenko et al. 2014), we have previously shown that the cellular damage only happens if there is secondary Ca 2+ entry from the extracellular fluid (Raraty et al. 2000). We have recently shown that the CRAC channel blocker GSK-7975A markedly inhibits the store-operated Ca 2+ entry that sustains the [Ca 2+ ] i elevation in PACs evoked by a pancreatitis-inducing agent (Gerasimenko et al. 2013(Gerasimenko et al. , 2014, a finding recently confirmed by Voronina et al. (2015). We have previously shown that the CRAC channel blockade by GSK-7975A provides effective protection of the PACs from alcohol-related intracellular protease activation and necrosis (Gerasimenko et al. 2013) and these results have also very recently been confirmed in J Physiol 594.2 a study employing three different in vivo mouse models of acute pancreatitis (Wen et al. 2015). The BK-elicited Ca 2+ signal generation in PSCs is due to initial release of Ca 2+ from internal stores followed by activation of Ca 2+ entry via store-operated channels. Our new results show that GSK-7975A is also effective in reducing the plateau phase of the BK-elicited [Ca 2+ ] i elevation in PSCs (Fig. 9E). Given that B2 receptor blockade protects against pancreatitis-like cellular changes (Fig. 8), the inhibitory effect of GSK-7975A on BK responses in PSCs  may represent an additional potential benefit of treating cases of severe acute pancreatitis with a CRAC channel blocker. The inhibitory action of GSK-7975A on the BK-induced plateau elevation of [Ca 2+ ] i in PSCs may well have contributed to the impressive protective effects of this agent against acute pancreatitis in vivo (Wen et al. 2015).
Our new data are also relevant when considering the now widespread use of ACE inhibitors in the treatment of hypertension because ACE inhibitors inhibit the breakdown of BK, causing an increase in the tissue and plasma levels of BK (Israili & Hall, 1992;Liu et al. 1997;Tsutsumi et al. 1999;Su, 2014). Previous studies have shown that use of ACE inhibitors is associated with a significantly increased risk of developing acute pancreatitis (Tilkemeier & Thompson, 1988;Eland et al. 2006), but the mechanism was unknown. As we have now shown that any increase in the BK level will elicit Ca 2+ signals in the PSCs via B2 receptors and that blockade of these receptors protects against acute pancreatitis, it is likely that Ca 2+ signal generation in PSCs mediated by BK is at least in part responsible for the increased risk of developing acute pancreatitis during ACE inhibitor treatment.

Translational Perspective
Our work indicates that bradykinin-elicited Ca 2+ signals in pancreatic stellate cells may influence negatively the outcome of acute pancreatitis. We tested the hypothesis that blockage of bradykinin type 2 (B2) receptors would offer protection against the pancreatic acinar cell necrosis caused by pancreatitis-inducing agents, such as alcohol, fatty acid ethyl esters or bile acids. The results showed that specific B2 receptor blockade markedly reduced the extent of necrosis observed after treatment with ethanol, POAEE or a mixture of bile acids. This suggests that B2 receptor blockade in the early stage of acute pancreatitis may be helpful in reducing the severity of the disease. We also show that the bradykinin-elicited sustained elevation of the cytosolic Ca 2+ concentration in pancreatic stellate cells can be inhibited by a specific inhibitor of Ca 2+ release -activated Ca 2+ (CRAC) channels. We have recently shown that CRAC inhibition in PACs offers remarkable protection against necrosis induced by fatty acid ethyl esters. In the intact pancreas, CRAC inhibition would reduce excessive Ca 2+ signal generation both in the acinar cells (brought about by, for example, fatty acid ethyl esters) and in the stellate cells (brought about by bradykinin). Both effects would be beneficial. Our work therefore indicates that combined treatment with a CRAC inhibitor and a B2 receptor -blocking agent should be tested in vivo as a potentially useful therapy against acute pancreatitis.