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J. Biol. Chem., Vol. 280, Issue 3, 1764-1770, January 21, 2005

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Bile Acids Induce a Cationic Current, Depolarizing Pancreatic Acinar Cells and Increasing the Intracellular Na⁺ Concentration^*

Svetlana G. Voronina, Olexyi V. Gryshchenko, Oleg V. Gerasimenko, Anne K. Green¶, Ole H. Petersen, and Alexei V. Tepikin||

From the Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom

Received for publication, September 7, 2004 , and in revised form, November 8, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na⁺ concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca²⁺-dependent and Ca²⁺-independent components of the Na⁺ concentration increase. The Ca²⁺-independent component was resolved in conditions when the cytosolic Ca²⁺ level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca²⁺-dependent component of intracellular Na⁺ increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca²⁺ oscillations the recovery of cytosolic Na⁺ was much slower than the recovery of Ca²⁺, creating a possibility for the summation of Na⁺ transients. The bile-induced Ca²⁺-independent current was found to be carried primarily by Na⁺ and K⁺, with only small Ca²⁺ and Cl^– contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 µM). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bile acids can get access to pancreatic acinar cells in certain pathological conditions either as a result of bile reflux into the pancreatic duct (due to blocked ampulla of Vater) or via interstitial leakage. Bile is considered a putative trigger of acute pancreatitis (1, 2), although issue is still debated (3–6). Bile acids have been shown to increase significantly the permeability of the ductal mucosal barrier in the pancreas (7–9), and recently two investigations demonstrated that bile acids are able to induce prolonged toxic cytosolic calcium signals in pancreatic acinar cells (1, 10).

In our previous study we found that low concentrations of taurolithocholic acid 3-sulfate (TLC-S)¹ produce preferentially local apical calcium signals, whereas higher concentrations of this bile acid produce sustained global calcium elevations. Other bile acids, taurochenodeoxycholic acid (TC) and taurocholic acid, also initiated cytosolic calcium responses (10). Similar results were reported by Kim et al. (1). Furthermore, bile acids have been shown to increase membrane permeability to ions in a number of model cell lines (11–13).

The membrane potential of pancreatic acinar cells is dependent on the activity of the Na⁺/K⁺ pump and is influenced by electrogenic transporters (e.g. Na⁺-amino acid co-transporters) and ionic channels (14–18). The ionic channels expressed in pancreatic acinar cells include Ca²⁺-activated non-selective cationic channels (permeable to Na⁺ and K⁺) (18–20) and prominent Ca²⁺-activated Cl^– channels (18, 20–23).

Bile acids were reported to enter into pancreatic acinar cells via an anionic HCO^–₃-dependent exchanger and a Na⁺-dependent co-transporter (1). The Na⁺/bile co-transporters have been shown to be electrogenic in hepatocytes (24) and ileal epithelial cells (25). Therefore, bile acids could potentially affect membrane voltage by activation/formation of ionic channels and by activation of electrogenic bile transporters. Activation of ionic conductances and the depolarizing effect on the transmembrane potential could be contributing factors to the pathological action of bile on pancreatic acinar cells. The electrophysiological effects of bile have not been characterized before in pancreatic acinar cells. This became a focus of our study. The largely unexpected results of this investigation are described below.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Preparation—Pancreata were obtained from adult mice (CD1) killed by cervical dislocation in accordance with the Animal (Scientific Procedure) Act of 1986. Pancreatic acinar cells were prepared by injecting 1 ml of 200 units ml^–1 collagenase (Worthington Lakewood, NJ) and digesting for 16–17 min at 37 °C with constant agitation. After digestion the pancreas was agitated manually to release single cells or small clusters in solution. Cells were washed three times by centrifugation in standard Na-HEPES buffer. All experiments were performed at room temperature (22–25 °C), and cells were used within 3–4 h after isolation.

Solutions—The standard extracellular solution (Na-HEPES buffer) used for cell preparation and for perfusion of cells during experiments contained 140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl₂, 1 mM CaCl₂, 10 mM D-glucose, 10 mM HEPES, pH 7.4. Sodium and potassium-free solutions contained 150 mM N-methyl-D-glucamine (NMDG⁺). In some experiments chloride was replaced by aspartate (Asp). During standard whole-cell recordings, the pipette solution contained 140 mM KCl, 1.5 mM MgCl₂, 2 mM MgATP, 10 mM HEPES, 0.1 mM EGTA, pH 7.2. In some experiments calcium was heavily buffered by the addition of 10 or 20 mM BAPTA and 2 mM CaCl₂. In K⁺-free pipette solution, K⁺ was replaced by NMDG⁺; in Cl^– low pipette solution KCl was replace by K-Asp. For perforated whole-cell recordings, the pipette solution contained 15 mM KCl, 100 mM K₂SO₄, 10 mM NaCl, 7 mM MgCl₂, 10 mM HEPES, pH 7.2.

The cells were placed on a cover glass coated with poly-L-lysine (0.01%), and the cover glass was attached to an open perfusion chamber. Solutions were perfused using a gravity-fed system.

Patch Clamp Recording—The whole cell configuration of the patch clamp technique was used to record currents from single pancreatic acinar cells. Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, Edenbridge, Kent, UK) and fire-polished. The pipettes had a resistance of 3 to 5 megaohms when filled with an intracellular solution (containing 140 mM KCl). Whole cell currents were sampled at 10 KHz using an EPS-8 amplifier and Pulse software (HEKA, Lambrecht, Pfalz, Germany). In the standard protocol the membrane voltage was clamped at –30mV, and voltage steps to +10mV (test potential) were applied for 50-ms durations twice per second. The averaged currents at the holding potential and the test potential were calculated. For investigations of current-voltage relationships voltage ramps were applied from –50 to +40 mV (the slope was 300 or 400 mV/s). In current clamp experiments we utilized the perforated patch mode; in these experiments amphotericin B was added to the patch pipette solution at a concentration of 300 µg ml^–1.

Measurements of Intracellular Sodium and Calcium Concentrations—To monitor changes of sodium concentration in the cells we initially attempted to use the cell-permeable forms of the indicators Sodium Green (Sodium Green tetraacetate) and SBFI (SBFI, AM). The intracellular distribution of the indicators in these experiments was not uniform with patches of the indicators found in the perigranular area. With this loading we did not manage to obtain the expected responses to applications of monensin, gramicidin, and ouabain. Loading of the cell-impermeant Sodium Green, tetramethylammonium salt (40–50 µM) through the patch pipette was much better; the distribution was uniform, and reasonable changes in fluorescence were observed. The fluorescence was monitored using the Zeiss LSM-510 confocal microscope. Sodium Green was exited by an argon laser line at 488 nm, and fluorescence was collected using a BP 505–550 emission filter. Sodium Green was calibrated in separate experiments conducted using a fluorimeter LS 50B (PerkinElmer Life Sciences). The wavelengths of excitation/emission light were similar to that used in confocal experiments. To increase the concentration of Na⁺ in calibration solutions, NaCl was introduced into the solution in exchange for KCl. In our experiments the estimated dissociation constant of Sodium Green to Na⁺ (K_d = 23 mM) was similar to that reported by Molecular Probes (K_d = 21 mM) for the indicator in K⁺-containing solution (www.probes.com/media/pis/mp06900.pdf).

We also attempted intracellular calibration of Sodium Green in the whole cell patch clamp configuration using gramicidin. In these experiments we recorded the expected changes of Sodium Green fluorescence but had difficulties in attaining a plateau of fluorescence for both sodium-free and high sodium solutions (possibly due to diffusion of Na⁺ into the patch pipette). We, therefore, used the results of the cell-free calibration for the estimations of rates of sodium change. The intracellular calcium concentration was measured with Fluo-4 dye. 3 µM Fluo-4 AM was loaded into the cell for 20–25 min at room temperature. The fluorescence of Fluo-4 was excited using the 488-nm laser line, and the emitted light was collected using a LP505 filter.

Chemicals—Sodium Green tetramethylammonium salt and Fluo-4 AM were purchased from Molecular Probes, Inc. (Eugene, OR). 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 2 Na (DIDS) was purchased from Calbiochem. TLC-S, taurochenodeoxycholic acid (TCDC), TC, sodium salt of bile acid mixture from ox bile (BA), N-methyl-D-glutamine (NMDG) and other chemicals were purchased from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Voltage Changes Induced by TLC-S
Fig. 1 shows an example of perforated patch clamp experiments (current clamp mode) in which we observed voltage changes induced by the bile acid TLC-S and by the calcium-releasing secretagogue acetylcholine (ACh). Amphotericin B was used as a perforating agent; this preserves the normal composition of the cytosol of the intact cell, since only very small molecules and monovalent ions can be exchanged through amphotericin pores. The resting membrane potential recorded from isolated acinar cells in our experiments ranged from –16 to –30 mV (n = 26), values expected for single isolated cells, that are usually somewhat depolarized (26). The application of a low concentration of the bile acid (25 µM) induced a slow depolarization, whereas ACh applications produced oscillatory hyperpolarizations (Fig. 1A). ACh-induced hyperpolarizations are consistent with activation of the calcium-dependent chloride current (the reversal potential for Cl^– under the condition of these experiments was approximately –35mV). The TLC-S-induced depolarization implies activation of Na⁺ or Ca²⁺-conducting channels. In experiments with a low concentration of TLC-S (25 µM) a slow depolarization was observed in 22 of 24 cells tested. The amplitude of TLC-S-induced depolarization ranged from 4 to 23 mV and tended to be higher in cells with a more negative resting membrane potential. In one out of these 22 cells the slow depolarization was preceded by an initial fast hyperpolarization (not shown), probably reflecting a cytosolic calcium concentration rise that could be observed sometimes even at this low concentration of the bile acid (10).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Effect of the bile acid TLC-S and the neurotransmitter ACh on the membrane potential of single isolated acinar cells. The figure shows the results of perforated patch clamp experiments conducted in the current clamp mode. Agonists were applied by perfusion in the extracellular solution. A, application of 25 µM TLC-S depolarizes the membrane of a single acinar cell, suggesting activation of bile-induced inward currents. ACh (50 nM) induces the opposite effect; that is, a hyperpolarization due to activation of Ca2+-dependent Cl– current. B, simultaneous measurements of membrane potential (upper trace) and normalized Fluo-4 fluorescence (F/Fo, lower trace) in a single acinar cell. The depolarization induced by TLC-S (25 µM) was accompanied by only a very small increase of Fluo-4 fluorescence. Transient hyperpolarizations triggered by ACh (50 nM) occur synchronously with substantial Ca2+ signals. C, a higher concentration of TLC-S (100 µM) induces a response composed of transient hyperpolarizations (most probably due to calcium transients) superimposed on an elevated membrane voltage. ACh (50 nM) induced transient hyperpolarizations.

We previously reported that higher doses of TLC-S induce calcium signals in the majority of pancreatic acinar cells (10). In a perforated patch clamp experiment, application of 100 µM TLC-S triggered a complex response in which fast hyperpolarizing transients were superimposed on a slow depolarization (Fig. 1C, n = 4).

We hypothesized that the bile-induced depolarization reflects calcium-independent activation of cationic, most probably Na⁺-permeable channels. One way to verify this hypothesis was to measure sodium changes induced by TLC-S.

Changes of [Na⁺]_c Induced by TLC-S— [Na⁺]_c was measured using the indicator Sodium Green. We loaded the indicator into the cell through a patch pipette and conducted experiments in the whole cell mode, since loading with a cell-permeant form of this probe was ineffective (see "Experimental Procedures"). In experiments where cytosolic calcium was not buffered with BAPTA, we were able to record both current and sodium changes induced by TLC-S and ACh (Fig. 2, A and B). In these experiments we used equal Cl^– concentrations in the patch pipette and in the extracellular solution; the chloride equilibrium potential should, therefore, be close to 0 mV. The calcium oscillations under this condition are reflected by transient activations of an inward chloride current (currents were recorded at –30mV).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Simultaneous measurements of changes in [Na+]c and membrane currents stimulated by the bile acid TLC-S and by ACh. Ionic currents were recorded using the whole cell patch clamp configuration at a holding potential of –30mV. Sodium Green was loaded into the cells via the patch pipette (40 µM in the experiments shown in A and B and 50 µM in the experiment shown in C). A, TLC-S (50 µM) activated an inward current (upper trace) accompanied by an elevation of the Sodium Green fluorescence (lower trace, the arrow points to the corresponding fluorescence intensity axis). Subsequent application of 100 µM TLC-S induced transients of Ca2+-activated Cl– current, superimposed on the plateau of inward current; the lower trace shows stepwise increases of sodium concentrations during the inward current transients. B, recording of Ca2+-activated Cl– current (upper trace) and changes of Sodium Green fluorescence (lower trace) induced by application of 50 nM ACh. C, in this experiment the patch pipette contained 20 mM BAPTA. The upper trace shows the development of the calcium-independent inward current upon application of 100 µM TLC-S. The lower trace shows simultaneous changes of the Sodium Green fluorescence.

It is interesting to note that during the ACh-induced oscillations and after removal of the agonist, the recovery of [Na⁺]_c was much slower than the recovery of the calcium-dependent current. This provides an opportunity for the summation of sodium responses (Fig. 2B) and could result in sodium overload (if the [Ca²⁺]_c spike frequency is too high).

The kinetics of the current transients and [Na⁺]_c changes strongly suggest that the sodium responses induced by ACh are calcium-dependent. The two components of the TLC-S induced [Na⁺]_c changes could be interpreted as calcium-independent (the slow rise induced by a low dose of bile) and calcium-dependent (stepwise increases of [Na⁺]_c during calcium transients). To reveal the calcium-independent component of the bile-induced sodium changes, we conducted an experiment in which [Ca²⁺]_c was effectively buffered by a calcium chelator, BAPTA, added to the patch pipette solution (and used at a very high concentration, 10 or 20 mM). In this condition application of TLC-S induce an increase in Sodium Green fluorescence accompanied by an inward current in cells held at –30 mV (Fig. 2C). These experiments (n = 5) strongly suggest the existence of calcium-independent sodium influx and a calcium-independent cationic current. The properties of this TLC-S-induced calcium-independent current were further investigated.

TLC-S-induced Currents in BAPTA-buffered Cells
Na⁺/K⁺ Current—In the experiments described in this section we used high concentrations of the calcium chelator BAPTA (10 or 20 mM) in the patch pipette (similarly to the experiments described above and shown in Fig. 2C) to prevent activation of calcium-dependent currents. Even a very low concentration of TLC-S (10 µM) induced a measurable current (22 ± 5 pA, n = 14 recorded at –30 mV, see Fig. 3 and the left inset of A). Subsequent increases of the bile concentration to 25 µM and then to 100 µM produced further slowly developing increases of currents (inward at –30 mV and outward at +10 mV). The averaged amplitude of currents induced by different concentrations of TLC-S (recorded at –30 mV) are shown in the right inset of Fig. 3A. Removal of the bile acid resulted in recovery of the currents to the prestimulated values (Fig. 3A). The recordings at –30 and +10 mV were interrupted by ramp protocols at the indicated time points. The reversal potential for bile-induced currents was close to 0 mV (Fig. 3B). The current-voltage relationship was largely linear for the range of voltages used in our experiments. The results shown in Fig. 3 suggest activation or formation of non-selective cationic channels. To verify this hypothesis we carried out substitutions of Na⁺ with NMDG⁺ in the extracellular solution (Fig. 4A) and K⁺ with NMDG⁺ in the intracellular (patch pipette) solution (Fig. 4B). In the NMDG⁺-containing extracellular solution, application of TLC-S produced only a small inward current (most probably carried by NMDG⁺), and the amplitude of the inward current increased drastically when NMDG⁺ was replaced by Na⁺ (Fig. 4A, n = 15), suggesting that this current is preferentially carried by Na⁺. Substitution of K⁺ by NMDG⁺ in the intracellular (pipette) solution largely abolished the outward current recorded at +10 mV (Fig. 4B, n = 25), indicating that the outward current is mainly carried by K⁺.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Bile acid (TLC-S) induces calcium-independent cationic currents in isolated acinar cells. To eliminate the activation of Ca2+-dependent channels patch clamp pipettes were filled with a solution containing 20 mM BAPTA. A, this part shows currents induced by increasing concentrations of TLC-S. The experiment was performed using the whole-cell patch clamp configuration in voltage clamp condition. Inward current (lower trace) was recorded at –30 mV, and outward current (upper trace) was recorded at +10 mV. The left inset shows the initial part of the inward current on an expanded current scale (the time scale is the same as in the main figure). The right inset shows averaged amplitudes (±S.E.) of currents (recorded at –30mV, n = 14) induced by different concentrations of TLC-S. B, representative I/V curves were obtained at different concentrations of TLC-S (shown on the graph) using a voltage ramp protocol (0.4 V/s) from –50 mV to +40 mV. The time points of the voltage ramp experiments are indicated by asterisks in panel A of this figure.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effect of substitution of small monovalent cations with NMDG+ on bile-induced transmembrane currents. The Patch pipette solution contained 10 mM BAPTA to eliminate calcium-dependant currents. A, in sodium-free (NMDG+ containing) solution, the bile acid TLC-S induced only a small inward current, probably mediated by NMDG+ (lower trace). Substitution of bath NMDGCl with NaCl resulted in a strong rise of the inward current (lower trace) recorded at –30mV but had only a small effect on the outward current measured at +10mV (upper trace). B, comparison of TLC-S-induced outward currents (measured at +10 mV) in potassium-containing and potassium-free intracellular solution. The current traces were recorded in different cells. The upper trace shows the current recorded using standard patch pipette solution containing 140 mM K+. The lower trace depicts the outward current when K+ in the patch pipette solution was replaced by NMDG+; only a very small current can be recorded in these conditions.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Contribution of Ca2+ and Cl– to the TLC-S-induced current. A, Ca2+ component of TLC-S-induced current. The intrapipette solution was supplemented with 20 mM BAPTA to prevent activation of Ca2+-dependent currents. The bars in the upper part of the figure show periods of TLC-S (100 µM) applications, Na+/NMDG+ substitution, and changes of Ca2+ concentration in extracellular solution. In this experiment K+ was replaced by NMDG+ in the patch pipette solution. The bar labeled NMDG indicates the period of time when all Na+ in the extracellular solution was substituted by NMDG+. The figure shows the current recorded at –30 mV. The amplitude of the TLC-S-induced inward current was compared in sodium-containing extracellular solution, sodium-free extracellular solution (Na+ substituted with NMDG+, [Ca2+]o = 1 mM), and sodium-free extracellular solution with a high concentration of Ca2+ (40 mM). B, Cl– component of total current induced by application of the bile acid TLC-S. The intrapipette solution was supplemented with 10 mM BAPTA to prevent activation of Ca2+-dependent currents. A change of [Cl–]o from 5 to 145 mM produced small changes of ionic current, recorded in Na+-free (NMDG+-containing) extracellular solution. The dashed line shows the extension of the current plateau level, attained before the change in chloride concentration. At the end of the experiment the normal sodium concentration (140 mM) in the extracellular solution was restored in the continued presence of TLC-S. This resulted in a large increase of inward current.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Inhibition of TLC-S induced current by DIDS. A, DIDS effect in Cl–-containing solutions. A single cell was patched with a pipette containing KCl and 10 mM BAPTA. 1 mM DIDS inhibited both inward and outward currents stimulated by 100 µM TLC-S. B, DIDS effect in low Cl– solution. A single cell was patched with a pipette containing a low Cl– concentration (KCl in the pipette was replaced by K-Asp and supplemented with 10 mM BAPTA). The bile acid TLC-S (100 µM) was applied in a low Cl– extracellular solution (NaCl was substituted by Na-Asp). 200 µM DIDS inhibits inward and outward currents.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Currents induced by TCDC, TC, and bile. The current were recorded at +10 mV and –30 mV. The intrapipette solution contained 10 mM BAPTA. A, inward and outward currents stimulated by application of 0.1 or 0.25 mM TCDC. B, currents stimulated by applications of 0.5 and 1 mM TC. C, currents induced by sequential application of 0.1, 0.25, and 0.5 mg/ml. BA, bile acid mixture from ox bile.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We report that bile acids induce Ca²⁺-independent currents mainly carried by Na⁺ and K⁺. The contributions of Cl^– and Ca²⁺ to these currents are small. The activation of cationic current explains the depolarization of the cells recorded in current clamp experiments. The inward sodium current creates Na⁺ influx and increase [Na⁺]_c, resolved in our experiments with the fluorescent indicator Sodium Green. This should also be accompanied by a loss of K⁺ from the cytosol of the cell.

In a study on the T84 colonic cell line, Devor et al. (11) characterized the electrophysiological effects of taurodeoxycholate and taurocholic acid. The authors reported activation of K⁺ and Cl^– channels. These channels were Ca²⁺-dependent; incubation of the cells with BAPTA-AM eliminated the currents through these channels (11). The pathway leading to activation of Cl^– and K⁺ currents involved taurodeoxycholic acid-induced accumulation of inositol 1,4,5-trisphosphate and calcium release from internal stores. Interestingly, this study also described a taurodeoxycholic acid-induced nonselective cation current that could be blocked by replacing Na⁺ in the extracellular solution with NMDG⁺. There are, however, some differences between the nonselective cationic current characterized by Devor et al. (11) and the one found in our study. The nonselective current described by Devor et al. (11) in T84 cells was mediated by infrequent openings of very high conductance channels (channel openings were visible even in whole-cell experiments). This is clearly different from the current found in pancreatic acinar cells. There were other differences in kinetics and relative amplitude of bile-mediated cationic currents in T84 and pancreatic acinar cells (e.g. development of the cationic current was much slower in acinar cells, whereas the amplitude was larger). An important finding in the study of Devor et al. (11) that is in complete agreement with our work (Ref. 10 and this study) is that bile acids do not induce a leak of fluorescent indicators from the cytosol of the cells. Good retention of the small molecular weight fluorescent indicator in the cytosol indicates that the detergent effect of bile acids is minimal.

It is possible that activation of Ca²⁺-independent cationic currents also occur in other cell types exposed to elevated concentrations of bile in pathological conditions. We conducted a preliminary investigation of the effect of TLC-S on voltage-clamped and BAPTA-loaded rat hepatocytes. We resolved a calcium-independent cationic current induced by application of 100 µM of TLC-S but had difficulties reversing this current by the removal of TLC-S (n = 4, not shown). The effect of TLC-S on hepatocytes and other cell types that could be exposed to this toxic bile acid deserves a separate detailed investigation.

Two types of bile acid transporters were recently described in pancreatic acinar cells; they are a Na⁺-dependent co-transporter and a HCO^–₃-dependent exchanger. It is interesting to note that the outward potassium current in BAPTA-buffered cells develops effectively in a bicarbonate-free solution and in the complete absence of extracellular Na⁺. This suggests that Na⁺-dependent and HCO^–₃-dependent transports are not essential for activation of nonselective currents by TLC-S.

In hepatocytes the amplitude of the current generated by the Na⁺/bile exchanger is only a few pA at a physiological membrane potential (24). This is much smaller than the amplitude of currents induced by TLC-S and other bile acids in pancreatic acinar cells. It is possible, therefore, that the small current of the exchanger is masked in our experiments by the much larger bile-induced cationic current. Our experiments also represent an important warning regarding measurements of the electrophysiological effects of the Na⁺/bile exchanger, since even small activation of nonselective cationic channels by bile acids could drastically affect such measurements.

The development of substantial non-selective cationic currents should result in changes of ionic concentration and would inevitably put additional stress on the energy homeostasis of the cells. The bile-induced Na⁺ flux is substantial. Our estimations from experiments with Sodium Green show that the calcium-independent sodium influx results in an increase of [Na⁺]_c by a few mM/min (e.g. 2 mM/min for the experiment shown in Fig. 2A and 8 mM/min for the one shown in Fig. 2C). Even more drastic sodium changes are observed during calcium oscillations. We found that during a single calcium transient [Na⁺]_c can increase by more than 5 mM. Both the calcium-dependent and the calcium-independent sodium influx induced by bile acids should put a very considerable pressure on ATP turnover in the acinar cell. A decrease of the transmembrane sodium gradient could also have an adverse effect on sodium dependent transporters responsible for delivery of amino acids into the cell (27) or for regulation of ionic concentrations (28–30). It is also interesting to note that for calcium-dependent sodium responses, the recovery of sodium transients is much slower than the recovery of the underlying calcium transients. This creates the possibility for summation of sodium transients and formation of a sustained elevated sodium level during calcium oscillations. This phenomenon is, however, beyond the scope of this paper and deserves a separate investigation. Another important effect of bile acids will be on fluid secretion. Bile-induced depolarization of the plasma membrane of pancreatic acinar cells, due to increased Na⁺ permeability, will inevitably decrease the driving force for Cl^– efflux across the apical membrane into the luminal space. The decreased Cl^– exit (and consequently water transport) into the luminal space will suppress the initial stage of transport of secreted enzymes through the pancreatic ducts. Retention of zymogens in the vicinity of the acinar cells could increase the probability of inappropriate activation of digestive enzymes within the pancreas.

It is important to emphasize that TLC-S induces activation of Ca⁺-independent currents even when applied at a very low concentration; 10 µM concentrations of this bile acid are sufficient to produce measurable changes in current in the majority of cells. This concentration is almost 2 orders of magnitude smaller than that found in bile (31, 32). Therefore, even minor reflux of this toxic bile component into the pancreas will most probably have deleterious effects on pancreatic functions. The low level of TLC-S, which has been found in this study to activate the Ca²⁺-independent current, is close to the concentrations of sulfated lithocholic acid conjugates detected in serum in different pathological conditions (33, 34). The concentration of TLC-S necessary to induce cationic current is even smaller than that required to trigger cytosolic calcium responses (in our previous work we found that a higher concentration of TLC-S (25 µM) produced calcium responses in only 11% of the cells tested) and similar to the threshold concentration that has been reported to induce transient mitochondrial depolarization (35). Therefore, activation of non-selective calcium-independent conductances could be one of the primary effects of bile on pancreatic acinar cells and could conceivably occur not only due to pancreatic reflux of the concentrated bile acid but also due to the action of bile accumulated at a much lower concentration in serum. The potency of bile acids in terms of their ability to induce non-selective current in BAPTA-loaded cells, decreased with increasing number of hydroxyl groups and a decrease of hydrophobicity. TLC-S was more effective than TCDC, which in turn was more potent than TC. This suggests that the mechanism of activation of the non-selective currents requires lipid partitioning of bile acids. However, trihydroxy (such as TC) and dihydroxy (such as TCDC) bile acids are present in bile at higher concentrations than monohydroxy bile acids (e.g. TLC-S) and could, therefore, make a substantial contribution to the total non-selective currents and membrane depolarizations in the case of interstitial leakage or bile reflux.

	FOOTNOTES

 
^* This work was supported by a Medical Research Council (MRC) program grant (to O. H. P., O. V. Gerasimenko, and A. V. T.) and an MRC Professorship (to O. H. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors have made equal contributions to this study.

Current address: Dept. of Physiology of the Nervous System, Bogomoletz Institute of Physiology, Bogomoletz Street 4, Kiev-24, GSP 01024,Ukraine.

^¶ Current address: BioMedical Research Institute, Dept. of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.

^|| To whom correspondence should be addressed. Tel.: 44-151-794-53-51; Fax: 44-0-151-794-53-27; E-mail: a.tepikin{at}liv.ac.uk.

¹ TLC-S, taurolithocholic acid 3-sulfate; TCDC, taurochenodeoxycholic acid; TC, taurocholic acid; NMDG, N-methyl-D-glutamine; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; Ach, acetylcholine; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 2 Na; [Ca²⁺]_c, cytosolic Ca²⁺ concentration; [Na⁺],_c cytosolic Na⁺ concentration.

	ACKNOWLEDGMENTS

 
The technical help of Mark Houghton is gratefully acknowledged. We also thank Nicholas Dolman, David Criddle, and Robert Sutton for useful suggestions and fruitful discussions.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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