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. 2002 Apr 1;540(Pt 1):49–55. doi: 10.1113/jphysiol.2002.017525

Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology

Svetlana Voronina *, Rebecca Longbottom *, Robert Sutton *, Ole H Petersen *, Alexei Tepikin *
PMCID: PMC2290202  PMID: 11927668

Abstract

The effect of the natural bile acid, taurolithocholic acid 3-sulfate (TLC-S), on calcium signalling in pancreatic acinar cells has been investigated. TLC-S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC-S in a calcium-free external solution. TLC-S-induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC-S application, displayed vectorial apical-to-basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC-S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid-induced cellular damage.

Exposure of the pancreas to bile acids is considered to be one of the possible causes of acute pancreatitis. The common duct theory, formulated by Eugene Opie at the beginning of the last century, indicated that when the ampulla of Vater is blocked by a gall stone, bile can penetrate into the pancreas and trigger an attack of acute pancreatitis (Opie, 1901). The ability of bile acids to trigger acute pancreatitis has been confirmed in a number of studies (Niederau et al. 1990; Senninger, 1992), but the cellular mechanism of such bile-mediated injury is not clear.

The cellular mechanisms underlying the toxic bile acid effects have been studied in more detail in liver. In these studies it has been found that the highly toxic monohydroxy bile acids trigger calcium signals in hepatocytes (Anwer et al. 1988; Combettes et al. 1988). These calcium signals were considered to be relevant to the toxic effects of the bile acids (Combettes et al. 1988). It has been documented that the naturally occurring bile acids taurolithocholate (TLC) and its sulfated form (TLC-S) release calcium from intracellular stores in hepatocytes (Combettes et al. 1988, 1990). TLC-S induced calcium release from the same store as IP3, but the release mechanism was different (Combettes et al. 1990). The calcium release induced by TLC-S was independent of extracellular calcium (Combettes et al. 1990). Stimulation with monohydroxy bile acids can trigger calcium oscillations in hepatocytes (Capiod et al. 1991; Marrero et al. 1994) and TLC-S was particularly effective in producing calcium oscillations based on release from internal stores (Marrero et al. 1994).

In the polarized pancreatic acinar cells, the secretory granules are concentrated in the apical part of the cell, whilst the basal region contains the nucleus and the endoplasmic reticulum (Bolender, 1974). Calcium signals trigger and regulate enzyme and fluid secretion (Petersen et al. 1994; Williams, 2001). Low concentrations of cholecystokinin (CCK) and acetylcholine (ACh) evoke oscillatory cytosolic Ca2+ signals, which are mainly confined to the apical secretory pole, by the mitochondrial belt surrounding the granules (Petersen et al. 1994; Tinel et al. 1999). At higher agonist concentrations global Ca2+ waves are initiated in the apical pole, and later spread to the base (Petersen et al. 1994). Abnormal, sustained calcium elevations can cause cellular changes similar to those found in models of in vivo acute pancreatitis (Raraty et al. 2000).

The aim of our study was to investigate the effect of the potentially toxic, naturally occurring bile acid TLC-S on calcium signalling in pancreatic acinar cells.

METHODS

Cell preparation

Pancreata were obtained from adult male mice (CD1) killed by cervical dislocation, in accordance with the Animals (Scientific Procedures) Act, 1986. Fresh mouse pancreatic acinar cells were prepared using collagenase (from Worthington Biochemical Corporation, Lakewood, NJ, USA) digesting for 12–14 min at 37 °C. All experiments were performed at room temperature (23–25 °C) and cells were used within 3–4 h after isolation.

To measure cytosolic calcium ([Ca2+]i) cells were loaded with Fluo 4-AM (2.5 μm) or Fura 2-AM (5 μm) or Fura Red-AM (5 μm) for 20–40 min at room temperature. Fura Red-loaded cells were used in experiments with caffeine, since fluorescence of Fura Red is affected by caffeine to lesser degree than the fluorescence of other indicators.

Solutions

The extracellular solution contained (concentrations in mm): NaCl 140, KCl 4.7, MgCl2 1.13, CaCl2 1, d-glucose 10, Hepes 10 (adjusted to pH 7.3 by NaOH). In some experiments CaCl2 was omitted (calcium free extracellular solution). The concentration of contaminating Ca2+ in such nominally calcium-free solution was estimated using Fura FF. We also measured the calcium concentration in nominally calcium-free solution with 200 μm TLC-S added. We found that the contaminating Ca2+ concentration in these solutions did not exceed 70 μm.

Optical imaging of the cells

Fluorescence imaging of cells loaded with Fura 2 was performed using the QuantiCell system (VisiTech, Sunderland, UK). For excitation, 340 nm and 380 nm filters were used, and for emission a 510 nm filter was used. Confocal imaging of the cells loaded with Fluo 4-AM and Fura Red-AM was performed using a Zeiss LSM510 confocal system. Fura Red was excited by an argon 488 nm laser line and fluorescence was collected through an LP 585 filter. The fluorescence of Fluo 4 was excited using the same 488 nm laser line and emitted light was collected using an LP 505 filter. Images were usually 256 × 256 pixels. The optical section was 5–6 μm. The fluorescence signals of Fluo 4 were expressed as F/Fo ratios where Fo represents the initial level of fluorescence and F is the fluorescence recorded at different time points during experiment. Reverse ratio (Fo/F) was used for Fura Red (for this indicator fluorescence decreases upon calcium binding).

Chemicals

Fluo 4-AM and Fura Red-AM were purchased from Molecular Probes (Eugene, OR, USA). Fura FF was purchased from TefLabs (Austine, TX, USA). Fura 2-AM and all other chemicals (including TLC-S, taurocholate (TC) and taurodeoxycholate (TDC)) were purchased from Sigma (Gillingham, UK) and were of the highest grade available.

RESULTS

TLC-S induced calcium responses in pancreatic acinar cells. Examples of such signals from three different cells are shown in Fig. 1A. Both repetitive calcium transients arising from the baseline and long-lasting calcium elevations were found in cells stimulated with TLC-S. Removal of TLC-S, during a prolonged calcium transient, resulted in fast recovery of [Ca2+]i to the basal level (Fig. 1A). Removal of TLC-S from the external solution during oscillatory signals abolished the oscillations. TLC-S could trigger calcium responses at relatively low concentrations: 25 μm TLC-S induced calcium responses in 11 % of the cells (n = 54), 50 μm induced responses in 37 % of the cells (n = 73), 100 μm triggered responses in 69 % of the cells (n = 71) and almost all cells (94 %, n = 47) responded to 200 μm TLC-S. We also performed a few experiments with higher concentrations of TLC-S. Calcium rises were observed in 16 out of 18 cells in response to application of 300 μm TLC-S and 5 out of 5 cells responded to 500 μm.

Figure 1. TLC-S induces calcium signals in pancreatic acinar cells due to calcium release from internal stores.

Figure 1

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A, patterns of calcium responses induced by 100 μm TLC-S in three individual cells (shown by different colours) from the same acinar triplet. B, calcium responses to TLC-S (200 μm) in calcium-free external solution. C, prolonged TLC-S-induced calcium transients were acutely sensitive to removal of calcium from the extracellular solution. The TLC-S concentration was 500 μm.

The proportion of responding cells that produced a long-lasting calcium plateau was substantial and increased with increasing TLC-S concentration. For example, 50 % of the cells (which responded to 25 μm of TLC-S) displayed an elevated [Ca2+]i plateau (n = 6); this proportion increased to 65 % for 100 μm (n = 48, one cell that responded to TLC-S was excluded from analysis because of an insufficiently stable baseline) and to 100 % for 500 μm (n = 5). If we take into account all the cells tested (not only the responding cells) then the proportion of cells with a long-lasting plateau is 6 % for 25 μm of TLC-S, 44 % for 100 μm and 100 % for 500 μm. No oscillations were found at 500 μm of TLC-S. At this concentration cells responded with just one large calcium transient followed by an elevated calcium plateau (Fig. 1C). The proportion of responding cells with pure oscillatory responses (without detectable long-term calcium elevation, and complete recovery of [Ca2+]i between spikes) decreased with increasing TLC-S concentration: these proportions were 50, 35 and 0 % for, respectively, 25, 100 and 500 μm TLC-S.

Calcium oscillations could be triggered by TLC-S in a calcium-free extracellular solution (Fig. 1B), indicating that the source of the calcium signals is intracellular. However, at most two transients could be generated in this condition. Removal of external calcium during a prolonged calcium elevation induced by sustained TLC-S stimulation, resulted in a rapid return of the calcium concentration to the basal level (Fig. 1C). The ability of TLC-S to trigger calcium signals in a calcium-free extracellular solution indicates that the signals are based on release from internal calcium stores, but the long-term maintenance of calcium signals generated by this bile acid appear to require calcium influx.

The responses triggered by TLC-S were similar to those induced in pancreatic acinar cells by calcium releasing secretagogues. Importantly TLC was shown to activate cholinergic muscarinic receptors in chief cells of guinea pig stomach (Raufman et al. 1998). It was therefore essential to test whether the TLC-S response was mediated by cholinergic receptors. However, in our experiments (n = 6) application of atropine did not prevent TLC-S-induced responses (Fig. 2A). The same concentration of atropine completely blocked responses to 10 μm ACh (n = 6, not shown).

Figure 2. Comparison of ACh- and TLC-S-mediated calcium release.

Figure 2

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A, atropine (100 μm) had no effect on TLC-S (200 μm)-induced release of calcium. B, caffeine (20 mm) blocked the TLC-S-induced response. The TLC-S concentration was 200 μm. Removal of caffeine unmasked the effect of TLC-S. The two traces represent two cells from the same acinar doublet. In this experiment, cells were loaded with Fura Red (unlike other experiments, shown in this and other figures, where Fluo 4 was used as a calcium indicator). The fluorescence of Fura Red is only slightly affected by caffeine. Decrease of Fura Red fluorescence corresponds to an increase in the calcium concentration (please note the changes in the scale for this part of the figure). C, ACh (10 μm) stimulation is able to completely discharge the TLC-S-sensitive calcium store. The TLC-S concentration was 200 μm.

The similarity of the responses elicited by bile acid and secretagogues could indicate involvement of the same calcium store or the same release mechanism. Interestingly, in our experiments, caffeine, which in pancreatic acinar cells completely blocks ACh and IP3-induced calcium signals (Wakui et al. 1990; Toescu et al. 1992), also completely inhibited TLC-S responses. No responses to TLC-S were found in caffeine (20 mm)-containing solutions (n = 6) (Fig. 2B). However, cytosolic calcium signals appeared immediately after removal of caffeine, in the continued presence of TLC-S (Fig. 2B). A similar ‘unmasking’ effect of caffeine removal is well documented for both IP3- and ACh-induced calcium signals (Wakui et al. 1990; Toescu et al. 1992). The similar caffeine effects on both ACh- and TLC-S-elicited responses suggest similar calcium release mechanisms. The ACh response is considered to be primarily IP3 mediated (Cancela et al. 2000) and indeed discharge of the calcium store by supramaximal ACh (10 μm) stimulation abolished the TLC-S response (Fig. 2C, n = 6). It would therefore appear that the same calcium store is involved in generating calcium signals elicited by ACh and TLC-S. Two out of six cells tested in these experiments with ACh-induced store depletion, displayed a small fluorescence decline on addition of TLC-S (Fig. 2C).

At 200 μm, TLC-S did not prevent subsequent calcium release activated by 10 μm of ACh (n = 8, not shown), suggesting that this particular concentration is not able to deplete the ACh-sensitive store completely.

Low concentrations of ACh and CCK activate local calcium signals, confined to the secretory granule (apical) region of the cell (Kasai et al. 1993; Thorn et al. 1993). We found that TLC-S, particularly at a relatively low concentration (100 μm), was indeed capable of generating local apical calcium signals (Fig. 3A, n = 10). Interestingly, some of the TLC-S-induced local calcium transients were very long-lasting - up to 30 s. Local calcium transients of such duration were not reported for stimulation with ACh or CCK.

Figure 3. Local apical calcium responses induced by TLC-S and apical origin of global TLC-S responses.

Figure 3

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The colour of the traces corresponds to the colour of the regions indicated on the transmitted light pictures. Aa, the transmitted light image of the pancreatic acinar triplet and the regions of interest from which the fluorescence was collected. The red trace (Ab) and the red circle (Aa) correspond to the secretory granule region (apical part), the blue colour represents the basal region (Aa,b). Ba, the transmitted light image. The traces shown in Bc were recorded from regions indicated by circles on the transmitted light image. Bb, whole cell response from the top cell in the image. Bc, fluorescence changes in the regions of interest: red trace corresponds to apical region; green trace represents measurements taken from the central part of the cell; blue trace was recorded from the basal region.

The global calcium signals induced by both CCK and ACh originate in the apical region and then spread to the basal region of the cell (Kasai & Augustine, 1990). Global signals induced by TLC-S had the same vectorial pattern. The signals started in the apical region and spread to the basal region with a delay of a few seconds (Fig. 3B).

We also tested other common taurine-conjugated bile acids, namely TC and TDC. Both were much less effective than TLC-S in their ability to trigger calcium signals. However, at millimolar concentrations these bile acids were able to trigger both global and local calcium oscillations (Fig. 4) (n = 9 for TC, n = 7 for TDC). As was the case with TLC-S, some of these local calcium signals were surprisingly long lasting (see Fig. 4).

Figure 4. Local and global responses induced by TC and TDC.

Figure 4

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The colour of the traces corresponds to the colour of the regions indicated on the transmitted light pictures. A, effect of TC. Regions of interests from which measurements were taken are indicated by circles on transmitted light image (Aa). Ab, local (e.g. transients 1, 2, 4, 5…) and global (transients 3 and 6) calcium responses induced by TC. B, effect of TDC. Regions of interests from which measurements were taken are indicated on the transmitted light image (Ba). Bb, global (first transient) and local (transients 2 and 3) calcium responses induced by TDC.

DISCUSSION

Our main finding is that the naturally occurring bile acid TLC-S triggers calcium signals in pancreatic acinar cells. Bile reflux into the pancreas has for a long time been considered a possible cause of acute pancreatitis (Opie, 1901), but the aetiology of acute pancreatitis is still unresolved and there is a wide spectrum of opinions about the activation mechanism (Reber & Mosley, 1980; Niederau et al. 1990; Senninger, 1992; Moody et al. 1993). Abnormally prolonged calcium signals in pancreatic acinar cells, induced by supramaximal CCK stimulation, were recently shown to induce intracellular trypsinogen activation, a critical step in the induction of acute pancreatitis (Raraty et al. 2000; Kruger et al. 2000). The calcium-releasing properties of TLC-S make this component of bile particularly interesting for future studies of bile-mediated acute pancreatitis.

TLC-S is capable of triggering calcium signals at relatively low concentrations, namely a few tens of micromoles per litre. This is approximately ten times lower than the concentration of TLC-S in bile (Cowen et al. 1975; Hofmann, 1976). This means that during bile reflux, even after considerably dilution, the TLC-S concentration in contact with the acini would still be sufficiently high to trigger calcium signals in these cells.

TLC-S was shown to trigger calcium release from intracellular stores in intact hepatocytes. TLC-S could also release calcium from stores in some other cell types (human platelets and neuronal cell line), but only when the plasma membrane was permeabilized with saponin (Coquil et al. 1991). The calcium releasing capabilities of TLC-S therefore depend on its ability to cross the plasma membrane and access the internal calcium stores. Our results show that in intact pancreatic acinar cells TLC-S is as effective at releasing calcium as in hepatocytes, which suggests the presence of an efficient TLC-S transport system in the pancreatic acinar plasma membrane.

Information on calcium signalling induced by bile acids in pancreatic acinar cells has been surprisingly sparse, particularly considering the suspected role of bile in triggering acute pancreatitis. It was reported that TDC and taurochenodeoxycholate (TCDC) induce calcium influx in pancreatic acinar cells, but unlike CCK or ACh had no effect on calcium release (Duan & Erlanson-Albertsson, 1988). The same study reported that TC even at the high concentration of 5 mm had no effect on intracellular calcium in pancreatic acinar cells. This actually contradicts our results. The discrepancy could be explained by the inability in earlier work to record the relatively small local calcium spikes, which were observed in our study. It has been also reported that deoxycholate did not induce calcium release (Takeyama et al. 1986). The calcium releasing effect of monohydroxy bile acids (and in particular TLC and TLC-S) in pancreatic acinar cells has not been investigated before.

In our experiments TLC-S triggered local apical calcium spikes, global transient oscillations and long-lasting calcium elevations (Fig. 1 and Fig. 3). There are three known properties of bile acids that could lead to generation of calcium signals. Monohydroxy bile acids might activate muscarinic cholinergic receptors (Raufman et al. 1998), but in our experiments the TLC-S-induced calcium signals were not blocked by a high concentration of atropine (see Fig. 2A). Taurine-conjugated bile acids have also been shown to act as calcium ionophores (Zimniak et al. 1991) and detergents (Bouscarel et al. 1999). We cannot exclude that an ionophore-like effect of TLC-S contributes to the generation of calcium signals, but the complete inhibition of the TLC-S-mediated response by caffeine (Fig. 2B) argues against this. The inability of TLC-S to trigger calcium signals when the internal calcium store was depleted by ACh, but under conditions when the external solution contained 1 mm calcium (Fig. 2C) also suggests a non-ionophore mechanism of signal generation. The same arguments are applicable for the potentially even less specific detergent-like action of TLC-S.

All bile acids tested were capable of generating local apical calcium signals (Fig. 3A and Fig. 4). In fact bile acids could generate very extended (more than 20 s in duration) local apical signals. The longevity of these signals could indicate relatively slow rates of calcium release from the apical store. Bile-induced formation of global calcium signals also occurred in the same vectorial fashion as for natural hormones and neurotransmitters. The signals originated in the apical part of the cell and then spread as a wave into the basal region (Fig. 3B). The similarity of the signalling patterns suggests that the complex (and not completely understood) arrays of second messengers (IP3, cADP-ribose, NAADP), which are involved in responses to natural secretagogues (Cancela et al. 2000), could also mediate the response to bile acids. In pancreatic acinar cells, as well as in many other cell types, caffeine very effectively inhibits IP3 responses (Wakui et al. 1990; Toescu et al. 1992). Importantly, caffeine does not in itself cause any depletion of calcium stores in pancreatic acinar cells (Wakui et al. 1990). The ability of caffeine to block the TLC-S response (Fig. 2) suggests involvement of IP3 receptors in the generation (or at least in the amplification) of the TLC-S-induced calcium signals. The exact mechanism of TLC-S-mediated calcium release is not known for any cell type and clearly requires further investigation. As for the signals evoked by CCK and ACh, the bile-induced oscillations could be triggered in the absence of external calcium, but depended on external calcium for continuity of signalling (Fig. 1B and C). In this respect it is also important to note that the distribution of bile between the cytosol and the lipid membranes could be calcium dependent (Zimniak et al. 1991). Calcium increases partitioning of bile acids into the hydrophobic core of lipid bilayers (Zimniak et al. 1991). Changes in the external calcium concentration as well as intracellular calcium signals could affect bile acid concentrations in different cellular compartments and therefore provide complex additional feedback for bile-induced calcium signalling.

Long term elevation of the cytosolic calcium concentration (calcium plateau), which depends on continued calcium influx from the external solution, has been shown to be a particularly dangerous signal, promoting activation of trypsinogen inside pancreatic acinar cells (Raraty et al. 2000; Kruger et al. 2000). TLC-S is certainly capable of generating this form of calcium signal (Fig. 1A and C). Oscillatory patterns were more prominent at low doses of agonists, whilst formation of long-lasting calcium plateaus (potentially more damaging signals) were more common at high doses.

The ability of bile acids to produce complex patterns of calcium activity in pancreatic acinar cells adds an important new dimension to studies of the mechanisms of bile-induced acute pancreatitis and suggests calcium toxicity as a possible mechanism for bile-induced injury to pancreatic acinar cells.

Acknowledgments

This study was funded by a programme grant from the Medical Research Council to O.H.P. and A.T. and by a Wellcome Trust grant to R.L. We are also grateful to Michael Ashby for his expert help with confocal microscopy and to Mark Houghton for excellent technical assistance. O.H.P. is a Medical Research Council Professor. R.L. is a Wellcome Trust Prize PhD student.

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