S.E.M. The dose-response curve for current inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) was fitted with the equation:
I = (Imin - Imax)/(1 + ([DIDS]/IC50)nH) + Imax,
where Imax is maximal current inhibition, Imin is minimal current inhibition, IC50 is half-maximal current inhibition and nHis the Hill coefficient.
Fura-2 measurements
Simultaneous measurement of whole-cell currents and the cytosolic Ca2+ concentration was performed by combining patch-clamp techniques and photomultiplier-based fluorimetry (TILL Photonics, Planegg, Germany). Cells were preincubated with 5 µM of the acetoxymethyl ester of the Ca2+-sensitive dye fura-2 in the culture medium for 30 min at 37°C. Patch pipettes were filled with the pipette potassium glutamate solution (see Solutions and material) supplemented with 70 µM fura-2 pentapotassium salt to prevent washout of the dye in the fast whole-cell configuration. Voltage ramps from -100 to +80 mV were applied every 4 s. Between the voltage ramps fura-2 fluorescence was determined at 340 and 380 nm excitation wavelength and the ratio was calculated.
Reverse transcription (RT)
Total RNA was prepared from pancreas and kidney of one 5-day-old and one adult rat by the method of Chomczynski & Sacci (1987). The adult rat was killed by inhalation of CO2 followed by decapitation. RNA samples were pre-treated with DNase I (amplification grade, Gibco) prior to the RT reaction to ensure that the polymerase chain reaction (PCR) products were not amplified from genomic DNA contaminations. Five micrograms of total RNA and 20 pmol of oligo-dT-primer were heated at 70°C for 10 min in 10 µl water, placed on ice and added to 8 µl of 'first strand cocktail', containing 50 mM Tris-HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 1 mM desoxynucleotide triphosphates and 10 mM DTT. After incubation at 37°C for 4 min 200 U M-MuLV-reverse transcriptase (Gibco) was added. The mixture was incubated for 2 h at 37°C and the RT reaction was terminated at 70°C for 15 min.
Amplification, cloning and sequencing of ClC-3 cDNA
The amplification of the obtained cDNA was processed in a total volume of 50 µl, using 200 µM desoxynucleotide triphosphates, 10 pmol primers, 20 mM Tris-HCl (pH 8·4), 50 mM KCl, 2 mM MgCl2, 1 U Taq DNA polymerase (Gibco) and 2 µl of RT-cDNA. The PCR cycles were performed with a Landgraf thermocycler (Langenhagen, Germany).
The following amplification primers were deduced from the rat ClC-3 cDNA sequence (accession number: D17521): for-1: 5'-(466)-TATGCCTCTGAGCTGCAAGG-(485)-3'; rev-1: 5'-(2802)-CTCTCTCCTCATCTACAGG-(2784)-3'; for-2: 5'-(2266)-TTCACTCATACAACCCTGG-(2284)-3'; rev-2: 5'-(2783)-ACTCAGTTGAACATTATTGAAGC-(2761)-3'. The first amplification step was carried out for thirty-five cycles with the primer pair for-1/rev-1. For the second, nested amplification step, forty cycles were chosen using the primer pair for-2/rev-2. As target in negative PCR controls we used water and RT-RNA samples without reverse transcriptase. To control the integrity of the used cDNA target, we amplified a cDNA fragment of the ubiquitously expressed p24A protein (Blum et al. 1996; accession number: X92098) with the primers for-p24A: 5'-(328)-ATGACTCCAAAAATAGTGATGTT-(350)-3' and rev-p24A: 5'-(593)-TAGATCTGTCCCAATGTCAT-(574)-3'. PCR-DNA aliquots were separated by agarose gel electrophoresis. The 517 bp cDNA band of ClC-3 obtained from the nested amplification was cloned in the pCR2.1 vector (Invitrogen) and characterized by DNA sequencing using the T7 sequencing kit (Pharmacia). cDNA sequences were analysed using the Wisconsin Sequence analysis package from the Genetics Computer Group (Madison, WI, USA).
Solutions and material
Patch pipettes were filled with either a KCl (mmol l-1: 145 KCl, 1 MgCl2, 0·1 EGTA, 10 glucose, 10 Hepes; pH 7·1 adjusted with KOH) or a potassium glutamate solution (mmol l-1: 126 potassium glutamate, 15·5 NaCl, 1 MgCl2, 0·1 EGTA, 10 glucose, 10 Hepes; pH 7·1 adjusted with KOH). In some experiments 2 mM MgATP was added to the pipette solution. The standard NaCl bath solution consisted of (mmol l-1): 140 NaCl, 4·7 KCl, 1 MgCl2, 1·3 CaCl2, 10 glucose, 10 Hepes; pH 7·4 adjusted with NaOH. Hyposmolar solutions were produced by omitting 40 mmol l-1 NaCl from the standard NaCl bath solution. Hyperosmolar solutions were obtained by addition of 100 mmol l-1 sucrose to the standard NaCl bath solution. For chloride substitution equimolar amounts of methanesulphonate, iodide, bromide or fluoride instead of chloride were added to the bath solution. Cation substitution experiments were performed by replacing NaCl with an equimolar concentration of N-methyl-D-glucamine chloride (NMDG-Cl).
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RESULTS |
Cultured pancreatic acinar cells lose the typical morphological polarization known from freshly prepared acinar cells. The cells in primary culture become flattened and the zymogen granules, which in freshly prepared cells are restricted to the luminal cell pole, become arranged near the nucleus in the form of clusters or parallel strings (Anderson & McNiven, 1995; Schmid et al. 1997). Furthermore, pancreatic acinar cells in primary culture lose their normal responsiveness to the secretagogues ACh, cholecystokinin and bombesin. Whereas hormonal stimulation of freshly prepared acinar cells from adult animals leads to an increase in [Ca2+]i and thereby to an activation of Ca2+-dependent chloride and cation currents (Petersen, 1992), stimulation of pancreatic acinar cells from early postnatal rats in primary culture neither elicits intracellular Ca2+ signals nor does it produce activation of Ca2+-dependent membrane currents (data not shown).
Activation of cell volume-sensitive currents
When cultured acinar cells were stimulated by intracellular application of GTP
S (40-100 µM), which is assumed to bypass receptor-dependent activation of G-proteins, a transient increase in the membrane conductance could be observed in more than 90 % of the experiments (n > 50) (Fig. 1). Analysis of GTPS-induced whole-cell currents revealed activation of outward currents at 0 mV (Erev for monovalent cations, Ecat) and inward current at -54 mV (Erev for Cl-, ECl) clamp potential. Within 2-5 min of establishing the whole-cell configuration a maximum membrane conductance was reached and the whole-cell conductance then slowly declined to the level which was observed at the beginning of the experiment. The GTPS-induced currents could be rapidly abolished by application of a hyperosmolar (+ 100 mM sucrose) bath solution (n = 7) (Fig. 1B). On the other hand, when the cells were exposed to a hyposmolar bath solution (100 mM NaCl instead of 140 mM NaCl) a similar current activation could be observed (n = 22) as after application of GTPS (Fig. 1A). The current response due to hyposmolar challenge was not dependent on the presence of intracellular GTPS (n = 12). The cell volume-sensitive currents could also be activated by application of positive pressure (5 cmH2O) to the pipette interior (n = 26) (Fig. 1B). This method of current activation also did not require the presence of GTPS in the pipette solution (n = 16). GTPS- as well as pressure-induced current activation could be observed in the absence (n = 22) and presence of 2 mM MgATP (n = 16) in the patch pipette. The maximal currents evoked by the different activation methods were of the same order of magnitude. With potassium glutamate in the pipette solution stimulation of the cells with GTPS produced a current maximum at 15·4 ± 1·9 pA pF-1 (n = 27) at 0 mV clamp potential. The pressure-induced currents peaked at 16·7 ± 2·7 pA pF-1 (n = 22) and hyposmolar treatment of the cells elicited maximal currents of 14·4 ± 2·5 pA pF-1 (n = 10). When the hydrolysable nucleotide GTP (1 mM) was used instead of GTPS only in 1 of 7 experiments a weak current activation could be observed.
Ion selectivity of cell volume-sensitive currents
Stimulation of freshly prepared pancreatic acinar cells from adult mice and rats with intracellular GTPS leads to activation of a chloride and monovalent cation current with the occurrence of the chloride current preceding the occurrence of the cation current (Kasai & Augustine, 1990; Schmid & Schulz, 1996). Therefore, the observation that in cultured pancreatic acinar cells GTPS induces outward currents at Ecat and inward currents at ECl raises the question whether in cultured cells there is also successive activation of both, a chloride- and cation-selective conductance. When I-V curves were continuously recorded during current activation (Fig. 2A) and inactivation all I-V curves intersected almost at the same point indicating that there is either only one prominent current component or there is no temporal difference in the activation and inactivation of chloride and monovalent cation currents, respectively.
To distinguish between these two possibilities, experiments were performed with KCl in the pipette and NMDG-Cl in the bath (Fig. 2B). Since the Ca2+-dependent non-selective cation channel in epithelia is only poorly permeable to NMDG+ (Siemer & Gögelein, 1992), activation of a non-selective cation conductance under these ionic conditions should cause cell hyperpolarization and, at 0 mV clamp potential, K+ outward currents. However, the reversal potential of the GTPS-evoked whole-cell currents under these ionic conditions was near 0 mV (n = 8) indicating that the activated conductance is highly selective for anions over cations. Furthermore, these experiments exclude a significant GTPS-induced activation of a potassium conductance. In consequence, the outward currents at a clamp potential of 0 mV observed with a potassium glutamate solution in the pipette and a NaCl solution in the bath must be due to chloride influx (Figs 1 and 2A).
Anion substitution experiments (Fig. 2C) showed that the cell volume-sensitive membrane conductance was about equally permeable for Cl- and Br- (n = 4), whereas substitution of Cl- with F- (n = 5) or methanesulphonate- (n = 10) in the bath solution led to a decrease in the membrane conductance and to a cell depolarization due to the lower permeability of these anions. This leads to a permeability sequence of Cl- 
Br- > F- > methanesulphonate- > glutamate-. Interestingly, when I- was used instead of Cl-, outward currents carried by anion influx decreased but at the same time a small hyperpolarization could be observed (n = 5) indicating a higher permeability but lower conductance of the membrane for I- compared with Cl-. With a potassium glutamate solution in the pipette (17·5 mM Cl-) and NaCl in the bath the mean reversal potential of the cell volume-sensitive currents was -37·7 ± 0·7 mV (n = 10). To avoid artefacts by changes in the intracellular chloride concentration due to massive chloride influx during current activation, the reversal potentials were determined only in experiments in which voltage ramps were applied from a holding potential of -60 mV. Assuming that the permeability of the cell volume-sensitive conductance for cations can be neglected a permeability ratio Pglutamate/PCl = 0·12 ± 0·01 (n = 10) could be calculated. The permeability ratios (PX/PCl) for the other anions were: Br- = 1·01 ± 0·02 (n = 4), I- = 1·22 ± 0·05 (n = 5), F- = 0·67 ± 0·06 (n = 5) and methanesulphonate- = 0·46 ± 0·03 (n = 10). The respective conductance ratios (gX/gCl) determined in the voltage range from -30 to 0 mV were: Br- = 0·99 ± 0·03 (n = 4), I- = 0·83 ± 0·06 (n = 5), F- = 0·29 ± 0·04 (n = 5) and methanesulphonate- = 0·25 ± 0·02 (n = 10).
Cell volume-sensitive chloride currents in freshly prepared pancreatic acinar cells
Application of GTPS on freshly prepared pancreatic acinar cells from adult mice causes oscillatory activation of a Ca2+-dependent chloride current, whereas in acinar cells kept in primary culture GTPS induces long-lasting activation of a cell volume-sensitive chloride current. To find out whether this difference in the effect of intracellular GTPS is due to the culturing of the cells or whether it reflects different stages of development in newborn and adult animals, we applied GTPS also to freshly prepared pancreatic acinar cells from 5- to 7-day-old animals. With potassium glutamate (+ 17·5 mM Cl-) in the pipette solution, GTPS (100 µM, n = 8) induced transient activation of outward currents at 0 mV and inward currents at -54 mV clamp potential (Fig. 3A), similar to the current activation observed on cultured cells. The somewhat smaller magnitude of the current maximum in freshly prepared cells might be due to the smaller membrane surface area of these cells (cell capacitance Cm = 9·1 ± 0·7 pF, n = 14) compared with cells kept in primary culture (day 1-5: Cm = 17·7 ± 1·1 pF, n = 59). In freshly prepared cells (day 0) volume-sensitive chloride currents showed a maximal current of 100·0 ± 19·3 pA (n = 14) at 0 mV clamp potential. This corresponds to a normalized current of 10·6 ± 1·9 pA pF-1. In cells cultured for 1-5 days a maximal current of 263·05 ± 25·18 pA (n = 59) at 0 mV could be measured and the normalized cell volume-sensitive current was 15·7 ± 1·4 pA pF-1, which is not significantly different from the value found in freshly prepared cells.
The GTPS-induced current in freshly prepared acinar cells showed, similar to the current in cultured cells, outward rectification and a reversal potential near -37 mV (Fig. 3B). The outwardly rectifying currents could also be activated by positive pressure applied to the pipette interior (n = 6) and by hyposmolar bath solutions (n = 4). These data indicate that, concerning the volume-sensitive chloride currents, on cells from 5- to 7-day-old rats the same results could be obtained regardless of whether they were freshly prepared or kept in primary culture.
Effect of cytosolic Ca2+ concentration and protein kinase C (PKC)
Since in adult animals activation of chloride currents is strictly correlated to a rise in [Ca2+]i, we tested with fura-2 measurements and synchronous current recordings whether cytosolic Ca2+ signals are also involved in activation of the cell volume-sensitive chloride currents in cultured cells. These experiments showed that during development of GTPS-evoked chloride currents no rise in [Ca2+]i could be detected (n = 5) (Fig. 4A). On the other hand, when the cytosolic Ca2+ concentration was artificially elevated by depletion of intracellular Ca2+ stores with 100 nM thapsigargin (n = 11) there was no increase in the whole-cell conductance (Fig. 4A and B). Moreover, cell swelling induced by a hyposmolar bath solution led, also in the presence of elevated [Ca2+]i, to activation of the cell volume-sensitive chloride current (Fig. 4B and C).
The cytosolic Ca2+ concentration can also be increased by addition of Ca2+ to the pipette solution. In a previous study on pancreatic acinar cells from adult mice we have shown that addition of 100 µM free Ca2+ to the pipette solution causes activation of whole-cell chloride currents (Pfeiffer et al. 1995). Application of the same experimental protocol to cultured cells from early postnatal rats failed to elicit chloride currents (n = 5, data not shown). Furthermore, Ca2+-independent activation of GTPS-induced chloride currents could be shown in the presence of 1 mM of the Ca2+ chelator EGTA (n = 6) in the pipette solution. These results clearly demonstrate that, in contrast to the observations on cells from adult animals, activation of chloride currents in cultured pancreatic acinar cells from early postnatal rats does not correlate to changes in [Ca2+]i.
In previous studies it has been shown that cell volume-dependent chloride currents can be downregulated by activation of PKC (for review see: Kawasaki et al. 1994; Nilius et al. 1997a; Duan et al. 1997). Consistent with these observations, we found in experiments on pancreatic acinar cells in primary culture that cell volume-sensitive chloride currents elicited by positive pressure (5 cmH2O) were diminished by treatment of the cells with the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 µM). Application of OAG for 2 min caused a reduction in the pressure-induced conductance (determined in the range of 0 to -30 mV) to 30·2 ± 3·7 % (n = 6).
Effect of chloride channel blockers
Several blockers were applied to characterize the pharmacological properties of the GTPS-evoked chloride current. First we tested the stilbene derivatives SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) and DIDS (Fig. 5A-F). Both substances, applied at a concentration of 100 µM, reduced the GTPS-induced whole-cell conductance significantly (Fig. 5D). SITS and DIDS inhibited the chloride current in a voltage-dependent manner, being more effective at positive than at negative clamp potentials (Fig. 5D). SITS was less potent than DIDS. Voltage pulse protocols (Fig. 5B) revealed that outward rectification of GTPS-induced chloride currents (Fig. 5A) was due to a transient depolarization-dependent activation. In particular, at clamp potentials more positive than +60 mV there was a time-dependent current inactivation (Fig. 5B). DIDS inhibited the fast current peak as well as the plateau phase (Fig. 5C). When the current fraction which was inhibited by DIDS at a clamp potential of +40 mV was plotted against the DIDS concentration a dose-response curve could be fitted yielding an IC50 value of 81 µM and a Hill coefficient of 1·5 (Fig. 5E). The voltage dependence of the current inhibition by DIDS is shown in Fig. 5F. At +40 mV DIDS inhibited 51·2 ± 7·3 % (mean ± S.D., S.E.M. = 1·6) of the GTPS-evoked current, whereas at -100 mV clamp potential only 20·7 ± 8·7 % (mean ± S.D., S.E.M. = 2·1) of the current was inhibited. The effects of SITS and DIDS occurred immediately after application of the substance and were fully reversible.
Quinine (500 µM, n = 5), quinidine (500 µM, n = 5), NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid; 100 µM, n = 4) and the antioestrogen tamoxifen (10 µM, n = 6) also inhibited the cell volume-sensitive chloride currents, but had a much slower time course of current inhibition than DIDS and SITS. A maximal inhibition by these substances was achieved about 30 s after drug application. Furthermore, current inhibition by quinine, quinidine, NPPB and tamoxifen was voltage independent (Fig. 6A-D).
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Figure 5. DIDS and SITS inhibit the GTPS-induced cell volume-sensitive current in a voltage-dependent manner
Voltage ramps from -100 to +40 mV were applied in the absence (control) or presence of 100 µM DIDS. Under control conditions the current-voltage curve of the rat pancreatic acinar cell in primary culture (day 6) shows outward rectification. In the presence of DIDS in particular outward currents were reduced (A). Pulse protocols (-100 to +100 mV, incremental step +20 mV) from a clamp potential of 0 mV revealed time-dependent current inactivation at voltages >= +60 mV (B). Outward peak currents as well as outward plateau currents were effectively inhibited by 100 µM DIDS, whereas inward currents were only slightly affected (C). Application of SITS (100 µM) also led to a voltage-dependent reduction in the GTPS-induced whole-cell current. DIDS was shown to be the more effective blocker (D). The inhibitory effect of DIDS is given in a dose-response relationship (E). The current fraction inhibited by DIDS is plotted against the DIDS concentration. The data points are fitted with a logistic function yielding a IC50 concentration for DIDS of 81 µM and a Hill coefficient of 1·5. Current inhibition by 100 µM DIDS as a function of clamp potential is given in F.
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Figure 6. Cell volume-sensitive chloride currents in rat pancreatic acinar cells in primary culture are inhibited by quinine, quinidine, NPPB and tamoxifen
The pipette was filled with potassium glutamate (17·5 mM Cl-) and the bath contained the standard NaCl solution. Volume-sensitive currents were evoked either by intracellular application of 100 µM GTPS (A) or by positive pressure (5 cmH2O) applied to the pipette interior (B-D). The cell volume-sensitive whole-cell currents were reversibly inhibited by 500 µM quinine (A), 500 µM quinidine (B), 100 µM NPPB (C) and 10 µM tamoxifen (D) in a voltage-independent manner.
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Detection of ClC-3 transcripts in pancreas from early postnatal rats
There is growing evidence that the molecular mechanism underlying cell volume-sensitive chloride currents is encoded by ClC-3, a member of the ClC chloride channel family (Duan et al. 1997; Yamazaki et al. 1998). To verify the expression of rat ClC-3 in pancreas of 5-day-old rats, we performed a comparative PCR study. Primers were chosen according to the known ClC-3 cDNA sequence which had been isolated from rat kidney (accession number D17521; Kawasaki et al. 1994). Using a two-step amplification protocol, we were able to amplify a 517 bp cDNA fragment from pancreas and kidney of 5-day-old rats and as positive control from kidney of adult rats (Fig. 7). However, in pancreas from adult rats ClC-3 could not be amplified. To verify the integrity of the cDNA used in the ClC-3-specific PCR, we amplified in all preparations a 266 bp fragment of the ubiquitously expressed p24A cDNA (Blum et al. 1996) from the corresponding cDNA preparations. The 517 bp amplification product from the pancreas of 5-day-old rats was cloned and analysed by DNA sequence analysis. The cloned pancreatic cDNA was identical to the previously published ClC-3 sequence from rat kidney (Kawasaki et al. 1994). The RNA samples were proven not to be contaminated with genomic DNA. The comparative PCR study shows that ClC-3 is expressed in pancreas of 5-day-old rats, but it could not be detected in pancreas cDNA of adult rats.
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Figure 7. PCR-based detection of ClC-3 in rat pancreatic tissue from early postnatal rats
cDNA was produced from RNA preparations from pancreas and kidney of 5-day-old and adult rats. cDNA amplification was performed with primers specific for rat kidney ClC-3 (upper panel) and rat p24A (lower panel). RT-PCR amplification products were separated by standard agarose gel electrophoresis and stained with ethidium bromide. In controls, primers without cDNA (water control) as well as DNase I-treated RNA samples from 5-day-old and adult rats were used in the PCR reaction mixture. Using a two-step amplification protocol ('nested PCR') a 517 bp fragment of ClC-3 cDNA could be amplified from kidney and pancreas of 5-day-old rats, as well as from the kidney of adult rats (upper panel). The integrity of the pancreas cDNA preparation from adult animals could be shown by amplification of a 266 bp cDNA fragment of the ubiquitously expressed protein p24A (lower panel). Molecular weight (MW) markers were added to the first and last lane.
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DISCUSSION |
Outwardly rectifying cell volume-sensitive chloride currents (for review see Nilius et al. 1997a) have been found in numerous species in a large variety of cell types such as mouse T lymphocytes (Lewis et al. 1993), rat parotid acinar cells (Arreola et al. 1995), rat pancreatic duct cells (Verdon et al. 1995), M-1 mouse cortical collecting duct cells (Meyer & Korbmacher, 1996), bovine non-pigmented ciliary epithelial cells (Wu et al. 1996), cultured bovine pulmonary artery endothelial (CPAE) cells (Voets et al. 1996), BC3H1 myoblasts (Voets et al. 1997), antral gastric myocytes (Xu et al. 1997) and atrial myocytes of the guinea-pig (Sakaguchi et al. 1997). Most of these studies reported that cell volume-sensitive chloride currents are quiescent at cell resting conditions and become activated when the cell is challenged with hyposmolar bath solutions. On the other hand, hyperosmolar bath solutions in most cases led to inactivation of the cell volume-sensitive chloride current. With respect to these features the cell volume-sensitive chloride conductance now found in pancreatic acinar cells from early postnatal rats can be classified into this family of chloride conductances.
The properties of cell volume-sensitive chloride conductances in different species and tissues are not entirely uniform. There are some variabilities concerning the anion permeability and conductance sequence, the dependence on the presence of intracellular ATP and the voltage dependence. In our experiments we found a permeability ratio for PCl/Pglutamate of 8·3 : 1 and a permeability sequence of I- > Br- Cl- > F- > methanesulphonate- > glutamate-, which is similar to the anion permeability sequence I- > Br- 
Cl- >> glutamate- described for most other members of the conductance family (Lewis et al. 1993; Nilius et al. 1994; Arreola et al. 1995; Meyer & Korbmacher, 1996). A new aspect in the characterization of cell volume-sensitive chloride currents was the observation that in rat pancreatic acinar cells the conductance for I- was smaller than the conductance for Cl-. A smaller anion conductance ratio (gI/gCl = 1·12) compared with the permeability ratio (PI/PCl = 1·35) was also found e.g. in T lymphocytes (Lewis et al. 1993).
The family of cell volume-sensitive chloride conductances can be divided into two classes, one showing fast current inactivation upon large membrane depolarization and the other showing little or no voltage-dependent inactivation (Lewis et al. 1993; Arreola et al. 1995; Jackson & Strange, 1995; Meyer & Korbmacher, 1996; Voets et al. 1997). Our experiments on rat pancreatic acinar cells showed current inactivation at membrane potentials +60 mV indicating that the volume-sensitive current in pancreatic cells belongs to the depolarization-inactivated chloride currents.
A variety of observations exist concerning the ATP dependence of cell swelling-induced chloride currents. In studies on atrial myocytes or M-1 cortical collecting duct cells it has been described that intracellular ATP is absolutely necessary for current activation (Meyer & Korbmacher, 1996; Sakaguchi et al. 1997). Sakaguchi et al. (1997) found that the effect of ATP could be mimicked by addition of the non-hydrolysable ATP analogue adenylyl imidophosphate (AMP-PNP) to the pipette solution. In contrast, Meyer & Korbmacher (1996) described that ATP could be replaced with ATPS but not with AMP-PNP. They concluded that a phosphorylation step is necessary for current activation. On the other hand, in mouse T lymphocytes (Lewis et al. 1993) and bovine non-pigmented ciliary epithelial cells (Wu et al. 1996), swelling-induced chloride currents could be activated without ATP in the pipette solution within about 10 min after establishing the whole-cell configuration. After this time a run-down in current activation could be observed which could be prevented by addition of ATP to the pipette solution. In several other cell types e.g. bovine chromaffin cells (Doroshenko & Neher, 1992) and rat parotid acinar cells (Arreola et al. 1995) there was no evidence for the requirement of ATP for activation of cell volume-sensitive chloride currents. This is consistent with our findings that ATP is not necessary for current activation in rat pancreatic acinar cells in primary culture.
A further common feature of the cell volume-sensitive chloride current in different cell types is the reversible and voltage-dependent inhibition by the stilbenes DIDS and SITS (Lewis et al. 1993; Arreola et al. 1995; Meyer & Korbmacher, 1996; Wu et al. 1996; Xu et al. 1997). In all studies, including ours, it has been observed that DIDS and SITS were more effective at depolarizing than at hyperpolarizing clamp potentials. A dose-response relationship for DIDS determined on T lymphocytes (Lewis et al. 1993) revealed a half-maximal current inhibition by DIDS at a concentration of 17 µM, which is only slightly lower than the IC50 value determined in our experiments (81 µM). Furthermore, we have shown that the chloride current in rat pancreatic acinar cells can also be inhibited by NPPB, quinidine, quinine and tamoxifen, which are also widely used as blockers of cell volume-sensitive chloride currents (Lewis et al. 1993; Nilius et al. 1994; Meyer & Korbmacher, 1996; Voets et al. 1996; Nilius et al. 1997a).
Activation of cell volume-sensitive chloride currents by GTPS has been shown for some cell types (Doroshenko, 1991; Doroshenko & Neher, 1992; Nilius et al. 1997b) whereas in other cell systems GTPS inhibited activation of volume-sensitive chloride currents (Robson & Hunter, 1997). The mechanism by which GTPS can modulate channel activity is still unclear (Nilius et al. 1997a). It is assumed that heterotrimeric as well as small GTP-binding proteins could play a role. In our experiments we found that GTPS but not GTP leads to current activation. This argues against a direct nucleotide-dependent channel activation and also favours a model which involves participation of heterotrimeric G-proteins in current activation.
The combined measurements of whole-cell currents and of changes in the cytosolic Ca2+ concentrations showed that the chloride current described in the present study is not dependent on cytosolic Ca2+ signals. These data are consistent with the findings on several other cell types which also showed no correlation between cytosolic Ca2+ signals and development of cell volume-sensitive chloride currents (Doroshenko, 1991; Arreola et al. 1995; Meyer & Korbmacher, 1996; Wu et al. 1996). In conclusion, we can say that with respect to the biophysical, pharmacological and physiological characterization the chloride current now found in rat pancreatic acinar cells clearly belongs to the large family of volume-sensitive chloride currents found in a variety of cell types from different species.
In the present study we have shown that pancreatic acinar cells from early postnatal rats respond to stimulation with intracellularly applied GTPS with activation of a cell volume-sensitive chloride current. This chloride conductance is, in contrast to the chloride conductance known from experiments on pancreatic acinar cells from adult mice and rats (Kasai & Augustine, 1990; Osipchuk et al. 1990; Petersen, 1992; Thorn et al. 1993), not dependent on the cytosolic Ca2+ concentration. Moreover, our experiments showed that in pancreatic acinar cells from early postnatal rats an artificial elevation of the cytosolic Ca2+ concentration with the Ca2+-ATPase inhibitor thapsigargin or by addition of Ca2+ to the pipette solution does not lead to activation of Ca2+-dependent chloride currents. Similar to this observation it has been reported that submandibular acinar cells from newborn and early postnatal rats are also lacking Ca2+-dependent chloride currents during the first 2-3 weeks of life (Fatherazi et al. 1996). It has been suggested that the lack of Ca2+-dependent chloride currents in submandibular acinar cells from very young rats can explain the reduced ability of the immature gland to secrete saliva. Unfortunately, only little is known about electrolyte and enzyme secretion from the immature rat exocrine pancreas. Studies on enzyme secretion showed that stimulation of pancreatic acinar cells from 1-day-old rats with cholecystokinin induced amylase release, whereas fetal acinar cells showed no hormone response (Chang & Jamieson, 1986). This indicates that after birth the immature rat pancreas is able to secrete enzymes. A correlation between weaning and pancreatic fluid and enzyme secretion was found in a study on young pigs (Pierzynowski et al. 1993). In this study it has been shown that during the suckling period pancreatic fluid and enzyme secretion remained low, whereas, after weaning juice secretion, output of different hydrolases and the cofactor colipase markedly increased postprandially. The disappearence of the cell volume-sensitive chloride conductance and the occurrence of a Ca2+-dependent chloride conductance during the postnatal development of rats might therefore reflect an adaptation of the electrophysiological properties of the exocrine acinar cell to differences in the function of the exocrine pancreas during suckling and after weaning.
Using RT-PCR-based methods we have demonstrated the presence of mRNA encoding ClC-3 chloride channels in pancreas of 5-day-old rats whereas the same protocol failed to detect ClC-3 transcripts in pancreas from adult rats. The latter observation is consistent with the findings of Kawasaki et al. (1994) who did not find ClC-3 expression in pancreatic tissue from adult rats with Northern blot analysis. The molecular data therefore suggest that the ClC-3 gene product might be the molecular mechanism underlying volume-sensitive chloride currents in pancreatic acinar cells from early postnatal rats. This hypothesis is supported by our electrophysiological findings which showed current inhibition by DIDS and tamoxifen as well as by activation of PKC. Also the biophysical properties of the current, i.e. outward rectification and current inactivation upon large membrane depolarization, argue in favour of this hypothesis. Only our observation that I- had a higher permeability but lower conductance than Cl- is partly in contrast to the data described for ClC-3 transfected NIH/3T3 cells (Duan et al. 1997). However, at the moment the possibility cannot be excluded that ClC-3 gene products form heteromeric complexes, e.g. with other ClC subunits. In such heteromeric complexes the conductance behaviour could be influenced by the supplementary subunit.
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REFERENCES |
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Acknowledgements
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB-246/A9). We thank Bärbel Kohler and Eva Pinter-Schmid for excellent technical assistance and Professor Dr Irene Schulz, Dr Peter Feick and Dr Fatima Pfeiffer for helpful suggestions and critical discussions.
Corresponding author
A. Schmid: 2. Physiologisches Institut, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
Email: schmid{at}med-ph.uni-sb.de
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Top
Introduction
100 nM) oscillatory changes in [Ca2+]i can be observed. The oscillatory cytosolic Ca2+ signals are often restricted to the luminal cell pole (Thorn et al. 1993) where they trigger the activation of a Ca2+-dependent chloride conductance in the luminal plasma membrane. Similar Ca2+ signals and current responses as evoked by low hormone concentrations can be elicited by intracellular application of GTP
. All potentials refer to the cytosolic side of the cell membrane with the bath solution connected to ground. Measurements were carried out at room temperature (20-25°C). If not indicated otherwise mean values are given as the mean ± 
i/-54 mV) was calculated. The pipette was filled with a potassium glutamate solution containing 17·5 mM Cl- and supplemented with 40 µM GTP
) and during current activation (
). The hyposmolar-induced chloride current (
