Functional polarity of Na+/H+ and Clminus /HCOminus 3 exchangers in a rat cholangiocyte cell line

Carlo Spirlì1,2, Anna Granato1, Àkos Zsembery1, Franca Anglani1, Lajos Okolicsànyi2, Nicholas F. LaRusso3, Gaetano Crepaldi1, and Mario Strazzabosco1

1 Institute of Internal Medicine, University of Padova, 35100 Padova; 2 Chair of Gastroenterology, University of Parma, 43100 Parma, Italy; and 3 Mayo Clinic, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intrahepatic bile duct cells (cholangiocytes) play an important role in the secretion and alkalinization of bile. Both Na+/H+ exchange (NHE) and Cl-/HCO-3 exchange (AE) contribute to these functions, but their functional distribution between the apical and basolateral membrane domains remains speculative. We have addressed this issue in a normal rat cholangiocyte cell line (NRC-1), which maintains a polarized distribution of membrane markers. Gene expression of AE and NHE isoforms was studied by RT-PCR. For functional studies, cells were placed in a chamber that allowed separate perfusion of the apical and basolateral aspect of the epithelial sheet; intracellular pH (pHi) was measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorometry. In HCO-3-CO2free medium and in the presence of apical amiloride, pHi recovery from an acid load was Na+ dependent and was inhibited by basolateral amiloride and by HOE-642 (10 µM), consistent with basolateral localization of the NHE1 isoform, which had clearly expressed mRNA. Apical Na+ readmission induced a slow pHi recovery that was inhibited by apical administration of 1 mM HOE-642 or amiloride. Among the apical NHE isoforms, NHE2 but not NHE3 gene expression was detected. The AE1 gene was not expressed, but two different variants of AE2 mRNAs (AE2a and AE2b) were detected; pHi experiments disclosed AE activities at both sides of the membrane, but only apical AE was activated by cAMP. In conclusion, these studies provide the first functional description of acid-base transporters in a polarized cholangiocyte cell line. NHE1, NHE2, AE2a, and AE2b isoforms are expressed and show different membrane polarity, functional properties, and sensitivity to inhibitors. These observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes.

intracellular pH; reverse transcription-polymerase chain reaction

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AFTER ITS SECRETION BY hepatocytes, bile flows through a progressively merging network of conduits lined by epithelial cells (cholangiocytes). Cholangiocytes modulate bile flow and alkalinity by secreting and reabsorbing fluid and electrolytes, mainly Cl- and HCO-3 (32), following stimulation with secretin and other hormones modulating intracellular concentrations of cAMP.

A number of ion channels and acid-base carriers have been functionally identified in the biliary epithelium (32). Among the acid-base carriers, Na+/H+ exchange (NHE) and Na+-HCO-3 symport mediate acid extrusion from the cells, whereas Na+-independent Cl-/HCO-3 (anion) exchange (AE) functions as an acid loader. Current models for cholangiocyte acid-base transport propose that NHE (together with carbonic anhydrase) and Na+-HCO-3 symport increase intracellular HCO-3 concentration, whereas AE mediates HCO-3 efflux. In concert with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, AE is responsible for cholangiocyte HCO-3 secretion induced by cAMP and secretin (3, 32).

Although epithelial cell function is strictly dependent on the polarized distribution of ion carriers (13), the above model is derived from studies in unpolarized cells in which vectorial transport properties of normal cholangiocytes are lost or from polarized duct preparations in which the apical membrane is not accessible. Thus the allocation of acid-base transporters to the apical or basolateral plasma membrane domains and their putative physiological role remains speculative. In addition, three isoforms of the Cl-/HCO-3 exchanger (AE1, AE2, AE3) (27) and four isoforms of the Na+/H+ exchanger (NHE1, NHE2, NHE3, NHE4) (37) have been cloned in different tissues. These isoforms differ in terms of functional properties, sensitivity to inhibitors, regulatory mechanisms, tissue distribution, and membrane polarity (1, 23, 27, 37, 45). NHE1 and AE1 are involved in homeostatic functions such as intracellular pH (pHi) regulation and cell volume control. AE2 and NHE2 and NHE3 are involved in HCO-3 secretion and Na+ reabsorption, respectively.

Using a normal rat cholangiocyte cell line (NRC-1) that, when grown on semipermeable membrane inserts, maintains differentiated phenotypes and a polarized distribution of membrane markers (39), we studied the functional membrane distribution of the different Na+/H+ exchanger and Cl-/HCO-3 exchanger isoforms expressed in the intrahepatic biliary epithelium.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. HEPES, amiloride, DMSO, nigericin, DIDS, D-gluconolactone, sodium gluconate, potassium gluconate, calcium gluconate, gluconic acid, choline chloride, choline bicarbonate, ammonium bicarbonate, sodium propionate, epidermal growth factor, dexamethasone, triiodothyronine, EDTA, high-activity collagenase, forskolin, N6,2'-O-dibutyryl-cAMP (DBcAMP), and IBMX were purchased from Sigma Chemical (Milano, Italy). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM was purchased from Molecular Probes (Eugene, OR). Culture medium DMEM, Ham's F-12, fetal bovine serum, MEM nonessential amino acids solution, glyceryl monostearate, chemically defined lipid concentrate, MEM vitamin solutions, trypsin inhibitor soybean, penicillin-streptomycin, gentamicin, trypsin-EDTA, and glutamine were purchased from GIBCO (Grand Island, NY). NuSerum and bovine pituitary extract were from Beckton Dickinson (Milano, Italy). Membrane inserts were purchased from Nunc (Mascia Brunelli, Milano, Italy); RNAzol B solution from Biotex (Milano, Italy); Maloney murine leukemia virus (MMLV) RT was from Perkin-Elmer (Milano, Italy). Qiagen QIAquick PCR purification kit and Qiagen QIAquick gel extraction kit were purchased from Qiagen (Ilden, Germany). 4-Isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (HOE-642) was a kind gift from Drs. A. Weichert and H. J. Lang (Hoechst Marion Russel, Frankfurt/Main, Germany).

Cell culture. NRC-1 cells were routinely grown on the top of rat tail collagen in culture flasks (Corning, NY) in DMEM-Ham's F-12 medium as described by Vroman and LaRusso (39). Experiments were performed in cells cultured for at least 1 wk after confluence was reached over collagen-coated semipermeable membrane inserts (Nunc Anophore, 0.2 µm pore-size). As described previously (39), these culture conditions allow the establishment of confluent monolayers with apical microvilli and polarized distribution of phenotypic and functional markers. Establishment of a confluent monolayer with competent tight junction was routinely checked by measuring transepithelial resistance and membrane potential difference (Millicell ERS system). In cells used for functional studies, transepithelial resistance was above 800 Omega  · cm2 and membrane potential difference was -6.77 ± 1.5 mV (n = 87). (Values obtained with collagen-coated inserts alone were subtracted from values recorded with monolayers.) In preliminary studies, we showed that the percentage of experiments in which apical recovery could be recorded increased from 33% at day 3 to 100% at day 8. Thus, to reduce variability due to culture conditions, cells were studied 8 days postconfluency.

pHi measurement. pHi was measured with the fluorescent pHi indicator BCECF (33). Cells were incubated with the cell permeant tetraacetoxymethyl ester BCECF-AM (12 µmol/l) for 30 min at 37°C followed by a 10-min wash in BCECF-free medium. NRC-1 cell membrane inserts containing monolayers of proper transepithelial resistance (see above) were transferred into a thermostated (37°C) perfusion chamber placed on the stage of a Nikon (Galileo, Siscam, Florence, Italy) inverted microscope. The chamber was modified to allow separate perfusion of the apical vs. basolateral aspects of the inserts. HEPES solutions were in the nominal absence of HCO-3; in HEPES experiments, the cells were equilibrated for 50 min in nominally HCO-3-free HEPES before starting the experiments. In experiments with HCO-3-CO2-buffered Ringer medium, solutions were continuously gassed with 5% CO2-95% O2; perfusion tubes were made with CO2-impermeant materials. The equipment and procedures were essentially as described previously (33). Briefly, the microscope was connected to an SPEX-AR-CM microsystem (Spex Industries, Edison, NJ, or ISA Instruments, Milan, Italy) equipped with a 150 W xenon lamp, and the sample was excited at 495 and 440 nm. Emitted light was then captured by a Nikon ×40 Achromat LWD with a 1.3 numerical aperture objective and read by a photon counting photometer. Cell autofluorescence was not higher than background values of collagen-coated inserts, which, at the end of each experiment, was subtracted from fluorescence readings. Signal-to-background ratio at 440 nm was ~40:1. The 495 nm-to-440 nm fluorescence ratio data were converted to pHi values using a calibration curve generated at the end of each experiment by exposing cells to the K+-H+ ionophore nigericin (12 µmol/l) in a Na+-free medium containing a high K+ concentration (135 mM) and buffered at three different pHi values (6.8, 7.2, and 7.6) (34).

Cellular intrinsic buffering power (beta i) was determined at different pHi values by exposing cells to various stepwise decreasing concentrations of a permeant weak base (NH4Cl) as described (7, 34, 44). To exclude all transport systems able to counterregulate pHi, experiments were performed in the absence of Na+ and HCO-3. The beta i values measured from seven experiments were pooled and correlated to the respective pHi values using a best-fit program (Graph-Pad, Biosoft, Cambridge, UK). Buffering power data are shown in Fig. 1. Similar relationships between beta i and pHi have also been reported in previous studies in isolated rat cholangiocytes (3, 34). The total intracellular buffering power (beta tot) in the presence of the open buffering system (H2CO3-CO2) was then calculated from beta i as beta tot = beta i + 2.302 × [HCO-3]i, where intracellular HCO-3 concentration ([HCO-3]i) is derived from the measured pHi with the Henderson-Hasselbach equation. Rates of pHi changes in the alkaline or acidic directions (Delta pHi/Delta t) were measured by hand drawing a tangent from the experimental plot. Transmembrane H+ fluxes (JH+) were calculated from beta i and Delta pHi/Delta t as JH+ = beta i × Delta pHi/Delta t (6, 33).


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Fig. 1.   pH dependence of intrinsic buffering power (beta i) in NRC-1 cells (). An inverse relationship between beta i and intracellular pH (pHi) is clearly shown [best fit gave the following polynomial curve: 4,178 + (-1,108)x + 73.69x2]. Curve with filled triangles describes the calculated total buffering power [polynomial curve: 6,787 + (-1,108)x + 134x2]. See text for methodological details.

The composition of perfusion buffers (all in mmol/l) used for pHi studies was essentially as described (6, 33). HEPES-buffered Ringer solution contained 135 NaCl, 4.7 KCl, 1.2 KH2PO4, 1 MgSO4, 1.5 CaCl2, 10 HEPES, 5 glucose, and 1 sodium pyruvate and was titrated to pH 7.4 with NaOH. In NaHCO3-CO2-buffered Ringer solution (KRB), NaCl was 115 mmol/l and 25 mmol/l NaHCO3 substituted for HEPES. In Cl--free KRB, equimolar gluconate substituted for Cl-. In Na+-free KRB and HEPES, equimolar choline substituted for Na+. In KRB and HEPES used to acid load cells, 30 mmol/l NH4Cl substituted for equal amounts of NaCl. In propionate KRB, 50 mmol/l sodium propionate substituted for 50 mmol/l NaCl. BCECF was prepared as 1 mmol/l stock solution dissolved in DMSO. Amiloride was dissolved in DMSO and then added to the different solutions at the desired concentration, whereas nigericin was solubilized in ethanol. HOE-642 was solubilized directly into the solutions at the desired concentrations.

NHE and AE gene expression. NHE and AE gene expression was assessed by RT-PCR. Total RNA from NRC-1 cells and rat kidney (used as positive control) (17, 27, 37) was isolated using the RNAzol B solution (Biotex) according to manufacturer's instructions on the basis of the guanidinium thiocyanate-phenol chloroform method (8). The quantity of total RNA was measured by spectrophotometry, and 1 µg was electrophoresed on 2% NuSieve 3:1 agarose gel with ethidium bromide to check the RNA integrity.

Total RNA (1 µg) was reverse transcribed with MMLV RT (2.5 U/µl) in a final volume of 20 µl containing buffer (500 mM KCl and 100 mM Tris · HCl, pH 8.3), MgCl2 (5 mM), dNTPs (1 mM), random examers (2.5 µM), and RNase inhibitor (1 U/µl). cDNA synthesis was performed in a thermalcycler (M. J. Research, M-Medical, Firenze, Italy); tubes were incubated for 10 min at room temperature and then for 30 min at 42°C. At the end of the incubation period, RT was inactivated by heating at 99°C for 5 min.

To examine the expression of AE and NHE isoforms, PCR was performed with the specific primers reported in Table 1. To increase the specificity and the efficiency of the PCR reaction, the hot start procedure was applied by using a Taq-specific antibody (Clontech). Two units of Taq DNA polymerase from a freshly prepared 28:1 mixture of Taq antibody and Taq polymerase were added to a final volume of 50 µl. For AE isoforms, the thermalcycler profile for AE1 consisted of an initial denaturation at 95°C per 5 min, followed by 35 cycles, with denaturation at 94°C for 1 min, primer annealing at 55°C, and primer extension at 72°C for 1 min. For the AE2 isoform and its variants AE2a and AE2b, PCR conditions were as follows: denaturation at 94°C for 1 min for AE2 and 45 s for AE2a and AE2b, annealing at 60°C for 1 min for AE2 and 2 min for AE2a and AE2b, primer extension at 72°C for 1 min for AE2 and 2 min for AE2a and AE2b. A final extension of 7 min was terminated by rapidly cooling to 4°C after 32 cycles for AE2 and AE2a and 35 cycles for AE2b. For NHE isoforms, after the initial denaturation (95°C for 5 min), the thermalcycler profile consisted of a denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and primer extension 72°C for 45 s. PCR conditions for each isoform are summarized in Table 2. In all amplifications, contamination by genomic DNA was ruled out by running samples without a previous reverse transcription phase.

                              
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Table 1.   Primers used for AE and NHE isoform expression

                              
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Table 2.   Amplification conditions

Amplification products were electrophoresed on 7% acrylamide gel and visualized by ethidium bromide and then silver stained to enhance the sensitivity of the detection system. To confirm their identity, PCR products were then sequenced. To this aim, PCR samples were purified using either the Qiagen QIAquick PCR purification kit or gel purified using the Qiagen QIAquick gel extraction kit. Purified PCR samples were sequenced on an ABI 373A Stretch automated sequencer using the PRISM dye terminator cycle sequencing kit according to the manufacturer's instructions. Approximately 10 ng per 100 bp of cDNA were used in each sequencing reaction. Sequences were analyzed using the Perkin-Elmer sequencing analysis program 2.1.2.

Statistical analysis. Results of continuous variables are shown as means ± SD. Paired and unpaired t-tests were carried out using STATGRAPH software (STSC).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Gene expression of AE and NHE isoforms. Gene expression of Na+/H+ exchanger isoforms (NHE1, NHE2, NHE3) and of Cl-/HCO-3 exchanger isoforms (AE1 and AE2) was tested by RT-PCR using total RNA extracted from NRC-1 cells and rat kidneys, which were used as positive controls (17, 27, 37). As expected, RT-PCR of rat kidney RNA produced amplicons of the proper size for all isoforms tested on agarose gel. On the contrary, as shown in Fig. 2, only NHE1, NHE2 (Wang sequence) (40), and AE2 were expressed in NRC-1 cells. Identity of the amplification products obtained from kidney and NRC-1 RNA was confirmed by sequence analysis. These findings are in agreement with those of Marti et al. (21) who detected NHE1 and NHE2 in freshly isolated, partially purified cholangiocytes and with those of Martinez-Ansò et al. (22) who reported expression of AE2, but not AE1, in RNA extracted from human liver biopsies. Given the presence of functional Cl-/HCO-3 exchange both at the apical and basolateral membranes (see Cl-/HCO-3 exchange activity), we also looked for expression of two variants of AE2 (AE2a and AE2b), which are generated by different promoters. These two variants differ at their NH2-terminal sequence and may be differently targeted and regulated (1). As shown in Fig. 3, kidney and NRC-1 RNA amplification products of the proper size were detected for AE2a and AE2b using RT-PCR.


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Fig. 2.   Anion exchanger (AE) and Na+/H+ exchanger (NHE) isoform gene expression in NRC-1 cells and rat kidneys. Total RNAs (1 µg) from NRC-1 cells and rat kidneys were reverse transcribed with random primers and then PCR amplified with NHE1, NHE2, and NHE3 primers and with AE1 and AE2 primers (see Table 1 for primer sequences and Table 2 for amplification conditions); 5 µl of the amplification products were directly stained with ethidium bromide and then silver stained on 7% acrylamide gel. Lane 1: DNA marker phiX 174/Hae III. Lane 2: rat kidney NHE1. Lane 3: NRC-1 NHE1. Lane 4: rat kidney NHE2 (Wang et al.). Lane 5: NRC-1 NHE2 (Wang et al.). Lane 6: rat kidney NHE2 (Collins et al., Ref. 10). Lane 7: NRC-1 NHE2 (Collins et al.). Lane 8: rat kidney NHE3. Lane 9: NRC-1 NHE3. Lane 10: rat kidney AE1. Lane 11: NRC-1 AE1. Lane 12: DNA marker phiX 174/Hae III. Lane 13: rat kidney AE2. Lane 14: NRC-1 AE2. Contrary to kidney, NRC-1 cells express only AE2, NHE1, and the 12-transmembrane domain NHE2 transcripts cloned by Wang et al. (40).


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Fig. 3.   Expression of AE2 gene variants in NRC-1 cells. Total RNAs (1 µg) from NRC-1 cells and rat kidneys were reverse transcribed with random primers and then PCR amplified with AE2a and AE2b primers (see Table 1 for primer sequences and Table 2 for amplification conditions); 5 µl of the amplification products were directly stained with ethidium bromide and then silver stained on 7% acrylamide gel. Lanes from left to right are as follows. Lane 1: NRC-1 genomic AE2a. Lane 2: rat kidney genomic AE2a. Lane 3: NRC-1 AE2a. Lane 4: rat kidney AE2a. Lane 5: NRC-1 AE2b. Lane 6: rat kidney AE2b. Lane 7: DNA marker phiX 174/Hae III. Lane 8: NRC-1 genomic AE2b. Lane 9: rat kidney genomic AE2b. RT-PCR detected amplification products of the proper size for both AE2a and AE2b.

Na+/H+ exchange activity. Mechanisms responsible for H+ extrusion were studied by measuring pHi recovery after intracellular acidification in nominally HCO-3-CO2-free medium (HEPES) (Fig. 4). Removal of basolateral Na+ (substitution with the impermeant monovalent cation choline), in the presence of the Na+/H+ exchange inhibitor amiloride (1 mM) on the apical side, induced a rapid acidification of pHi (from pHi 7.13 ± 0.12 to pHi 6.58 ± 0.19). After readmission of basolateral Na+, still in the presence of apical amiloride, cells recovered to basal pHi, extruding protons [JH+ = 27.5 ± 9.6 mmol · l-1 · min-1 and Delta pH/Delta t = 0.392 ± 0.123/min at pHi 6.7 (n = 8) and JH+ = 9.79 ± 5.6 mmol · l-1 · min-1 and Delta pH/Delta t = 0.297 ± 0.172/min (n = 11) at pHi 7.0]. In another set of experiments, basolateral amiloride was added at different concentrations simultaneously with Na+ readmission; pHi recovery (at pHi 6.7) was inhibited in a dose-dependent way (with 0.1 mM amiloride: 85% inhibition; 0.5 mM amiloride: 94% inhibition; 1 mM amiloride: 95% inhibition). To further characterize the NHE isoform expressed at the basolateral membrane, we tested the effects of HOE-642, a new benzoyl-guanidinium inhibitor similar to HOE-694 (30). Because of its specific and potent inhibitory activity toward NHE1, HOE-642 can be used to discriminate between different NHE isoforms (30). As shown in Fig. 5A, cells were acidified by pulsing with 30 mM NH4Cl and thereafter perfused from both sides with Na+-free HEPES medium. The basolateral side was then exposed to various concentrations of HOE-642 in the presence of Na+-containing HEPES medium. As shown in Table 3 and Fig. 5C, JH+ was exquisitely sensitive to HOE-642, being almost completely inhibited by a concentration of 10 µM [apparent inhibition constant (Ki) = 1 µM]. This high sensitivity to HOE-642 clearly identifies NHE1 (23, 30) as the isoform functionally expressed at the basolateral membrane of cholangiocytes.


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Fig. 4.   Representative experiments showing functional evidence for basolateral NHE1. Apical medium changes are shown above the tracing; basolateral changes are shown below. In HCO-3-free medium (HEPES), removal of basolateral Na+ (substitution with the impermeant monovalent cation choline) in the presence of apical amiloride (1 mM) acidified pHi (1), which rapidly recovered following basolateral Na+ readmission; pHi recovery induced by Na+ readmission was inhibited by basolateral amiloride (1 mM) (2).


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Fig. 5.   Effect of HOE-642 on basolateral and apical NHE activities. A: representative experiments showing basolateral Na+-dependent H+ extrusion after intracellular acid load. In HCO-3-free medium (HEPES), administration and withdrawal of 30 mM NH4Cl induced an acute intracellular acidification (1). Cells were perfused at both sides with Na+-free HEPES buffer (2); when basolateral Na+ was then readmitted, a complete recovery at baseline pHi was achieved (3), indicating basolateral NHE exchanger. HOE-642 inhibition curve is shown in C. B: representative experiments showing apical Na+-dependent H+ extrusion after intracellular acid load. In HCO-3-free medium (HEPES), administration and withdrawal of 30 mM of NH4Cl induced an acute intracellular acidification (1). Cells were perfused at both sides with Na+-free HEPES buffer (2), while the basolateral side was also exposed to 0.5 mM HOE-642. Na+ was then readmitted at the apical side. At this point, a partial recovery was evident (3). C: dose-dependent effect of HOE-642 on apical (open bars) and basolateral (solid bars) NHE activities. HOE-642 was administered 1 min before Na+ readmission at the apical or basolateral aspect of the cells following the protocol described in A and B. Data are expressed in % with respect to NHE activity in control experiments. Clearly, basolateral NHE is much more sensitive to HOE-642 than apical NHE. See also Table 3.

                              
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Table 3.   Effect of HOE-642 on basolateral and apical NHE activity in NRC-1 cells

In Na+-reabsorbing gastrointestinal epithelia, including the gallbladder (11, 43), an Na+/H+ exchanger belonging to the NHE2 or NHE3 isoform is located at the apical pole of the cell. Given the presence of the NHE2 message in NRC-1 cells, we looked for functional Na+/H+ exchanger activity also on the apical membrane. In a preliminary report by Singh et al. (31), an apical Na+-dependent, amiloride-sensitive H+ mechanism, active from pH 6.2 and 6.5, was described in microperfused bile ducts. To achieve a comparable acidification, cells were pulsed with 30 mM NH4Cl and then perfused with Na+-free HEPES buffer (Fig. 5B), with average nadir pHi of 6.38 ± 0.13 (n = 12), while the basolateral side was perfused with an Na+-free HEPES containing 0.5 mM HOE-642. When Na+ was readmitted at the apical side, a slow but measurable pHi recovery (JH+ = 3.57 ± 1.34 mmol · l-1 · min-1, Delta pHi/Delta t = 0.027 ± 0.016/min at pHi 6.5) was present. Using a similar experimental protocol, but in the presence of 1 mM basolateral amiloride, we also evaluated the effects of apical amiloride (1 mM). A concentration of 1 mM amiloride fully inhibited pHi recovery [JH+ = 3.71 ± 1.36 mmol · l-1 · min-1, Delta pHi/Delta t = 0.045 ± 0.015/min at pHi 6.5 (n = 5)] in controls (not shown), indicating that H+ efflux was mediated by an Na+/H+ exchange isoform. In another set of experiments (see Fig. 5B), 1 min before apical Na+ readmission, the apical side of the monolayer was also exposed to HOE-642 at different concentrations. As shown in Table 3 and Fig. 5C, apical pHi recovery was inhibited by HOE-642 concentrations 100 times higher than basolateral NHE (apparent Ki = 100 µM). Although NHE3 is ten times less sensitive to HOE-642, with a reported apparent Ki of 1 mM (30), these data are consistent with the presence of NHE2 at the apical membrane of cholangiocytes.

Cl-/HCO-3 exchange activity. In NRC-1 cells, AE2 but not AE1 RNA amplicons were detected. In the human liver, monoclonal antibodies directed toward a synthetic peptide specific for the AE2 isoform of Cl-/HCO-3 exchange decorates the apical membrane of intrahepatic cholangiocytes (22); thus we looked for the functional presence of AE2 at the apical pole of NRC-1 cells. Removal of apical Cl- in NRC-1 cells pretreated with basolateral DIDS (1 mM per 40 min) did not cause a significant pHi alkalinization, even in the presence of agents raising intracellular cAMP levels (not shown). Although Cl- removal may be incomplete following this maneuver, or the activity of the acid loader AE2 may be too low at the baseline pHi, mechanisms of HCO-3 extrusion were also investigated by measuring recovery after an intracellular alkalinization induced by administration and withdrawal of the permeant weak acid propionate (6) (Fig. 6) in cells pretreated with basolateral DIDS (1 mM). As expected, cell pH alkalinized following propionate withdrawal (from pHi 7.11 ± 0.175 to pHi 7.37 ± 0.181); pHi recovered to baseline pHi [JOH- = 7.5 ± 2.43 mmol · l-1 · min-1, Delta pH/Delta t = 0.116 ± 0.05/min (n = 17) at pHi 7.3] by a mechanism that was inhibited by apical Cl- removal [JOH- = 1.08 ± 1.63 mmol · l-1 · min-1, Delta pH/Delta t = 0.018 ± 0.02/min (n = 9) at pHi 7.3] and by pretreatment with apical DIDS (1 mM) [JOH- = 3.12 ± 1.43 mmol · l-1 · min-1, Delta pH/Delta t = 0.05 ± 0.02/min (n = 5)] (Table 4), consistent with the operation of Cl-/HCO-3 exchange. Administration of agents increasing intracellular cAMP levels (100 µM DBcAMP, 3 µM forskolin, and 100 µM IBMX) (3, 5, 14) significantly increased the rate of pHi recovery [JOH- = 12.65 ± 4.27 mmol · l-1 · min-1, Delta pH/Delta t = 0.216 ± 0.07/ min at pHi 7.3 (n = 12); P < 0.001 vs. controls]. Taken together, these data indicate that a cAMP-activated Cl-/HCO-3 exchanger is located at the apical pole of cholangiocytes expressing AE2 transcripts.


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Fig. 6.   Representative tracings of pHi recovery after acute intracellular alkalinization in cells pretreated with basolateral (BL) DIDS. In the presence of HCO-3-CO2 (KRB), administration and withdrawal of propionate (prop; 50 mM), in cells pretreated basolaterally with DIDS, induced an intracellular alkalinization. pHi recovery (1) was inhibited by apical (AP) Cl- removal (2) and partly inhibited by apical DIDS pretreatment (3). After agents that raise intracellular cAMP concentration [100 µM dibutyryl-cAMP (DBcAMP), 3 µM forskolin, 100 µM IBMX 100] were administered, HCO-3 efflux increased significantly (4).

                              
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Table 4.   Cl-/HCO-3 exchange activity

To investigate the functional presence of a basolateral Cl-/HCO-3 exchange in NRC-1 cells, pHi transients were monitored during acute removal of extracellular Cl-. This maneuver, in the presence of an active Cl-/HCO-3 exchange, causes intracellular alkalinization because HCO-3 enters the cells in exchange with intracellular Cl-, which is forced to exit (34). In the presence of HCO-3-CO2 (KRB buffer), in cells pretreated with 1 mM DIDS on the apical side, removal of basolateral Cl- induced a rapid pHi alkalinization (Fig. 7) (from pHi 7.21 ± 0.12 to pHi 7.54 ± 0.12) [JOH- = 21.6 ± 13.46 mmol · l-1 · min-1, Delta pH/Delta t = 0.368 ± 0.22/min (n = 19) at 7.3], followed by a quick recovery when Cl- was readmitted [JOH- = 38.01 ± 14.52 mmol · l-1 · min-1, Delta pH/Delta t = 0.707 ± 0.42/min (n = 19) at pHi 7.3]. This alkalinization was inhibited by basolateral DIDS (1 mM for 40 min) pretreatment [JOH- = 4.61 ± 3.1 mmol · l-1 · min-1, Delta pH/Delta t = 0.062 ± 0.03/min (n = 6)], indicating that Cl-/HCO-3 exchange is also located at the basolateral membrane. Contrary to the apical anion exchanger, an increase in intracellular cAMP concentration did not stimulate base fluxes during the alkalinization phase [JOH- = 22.6 ± 10.31 mmol · l-1 · min-1, Delta pH/Delta t = 0.397 ± 0.17/min (n = 19) at pHi 7.3] or the pHi recovery phase [JOH- = 38.36 ± 16.6 mmol · l-1 · min-1, Delta pH/Delta t = 0.767 ± 0.42/min (n = 19) at pHi 7.3] (Table 4).


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Fig. 7.   Representative experiments demonstrating the functional Cl-/HCO-3 exchanger on the basolateral aspect of the monolayer. In the presence of HCO-3-CO2, in cells pretreated apically with DIDS, removal of basolateral Cl- induced a rapid pHi alkalinization (1), which was not increased by cAMP (that is, 100 µM DBcAMP, 3 µM forskolin, 100 µM IBMX) (2); pHi recovery was inhibited by basolateral DIDS (3).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Epithelial cell function depends on the structural and functional diversity of its apical and basolateral plasma membrane domains. Establishment of a polarized distribution of membrane carriers and other proteins involved in specialized epithelial function is a fundamental characteristic of epithelia that enables the vectorial transport of ions, fluid, and other constituents from one compartment to another (13). In addition, loss of membrane polarity is an important pathogenetic mechanism in a number of diseases, including cholestasis (32).

A number of studies have addressed the mechanisms of cholangiocyte ion transport. However, acid-base transport studies in cholangiocytes have, thus far, been restricted largely to nonpolarized cell preparations or to cell preparations in which the apical side is not easily accessible. Recently, a differentiated normal rat cholangiocyte cell line (NRC-1) that maintains a polarized distribution of a number of membrane markers has become available (39). The NRC-1 cell line is positive for cholangiocyte phenotypic markers such as gamma -glutamyltranspeptidase, CK-7, and CK-19 and is negative for the mesenchymal markers vimentin and desmin. Ultrastructural examination of cells grown over semipermeable membrane inserts reveals competent tight junction, apical microvilli and cilia, basolaterally restricted nuclei, and membrane interdigitations; somatostatin receptors are expressed basolaterally, staining for gamma -glutamyltranspeptidase decorates the apical membrane (39). NRC-1 cells absorb bile acids and glucose at their apical domain via the ileal Na+-dependent bile acid transporters (19) and the Na+-glucose cotransporter SGLT-1 (20), respectively. Activation of purinergic P2Y2 receptors located at the apical membrane stimulated short-circuit currents and produced a basolateral-to-apical Cl- efflux that is inhibited by apical administration of Cl- channel blockers (28). Expression of CFTR protein, in NRC-1 cells, has been demonstrated in apical plasma membrane vesicles by immunoblotting (36).

We have thus exploited this cell model to investigate the functional topographic distribution and gene expression of two cholangiocyte acid-base carriers, the Na+/H+ exchanger and the Cl-/HCO-3 exchanger. Our results show that, in NRC-1 cells, functional NHE1 activity is restricted at the basolateral membrane, whereas an NHE2 isoform is likely to be active on the side facing the lumen. Furthermore, two transcripts (AE2a and AE2b) (1) of the AE2 gene are expressed in NRC-1 cells, consistent with the functional presence of Cl-/HCO-3 exchanger activities with different sensitivity to stimulation by cAMP and inhibition by DIDS at the apical and basolateral side of the monolayer.

The presence of the basolateral Na+/H+ exchanger is suggested by the decrease in pHi after basolateral Na+ removal in the presence of apical amiloride (1 mM) and by the basolateral amiloride-inhibitable pHi recovery recorded when basolateral Na+ was readmitted. JH+ was also inhibited by HOE-642 (30), a recently developed compound that is not derived from amiloride but possesses a highly specific and potent inhibitory activity toward the NHE1 isoform (Fig. 5C). The high sensitivity of basolateral pHi recovery to amiloride and HOE-642 is consistent with basolateral expression of the mitogen-activated isoform of the Na+/H+ exchanger NHE1, whose mRNA was clearly expressed (14, 23, 33). In fact, NHE2 is known to be much less sensitive to HOE-642 (see below). NHE1 is an electroneutral carrier that extrudes H+ from the cell, energized by the transmembrane Na+ gradient. In epithelial cells, NHE1 is usually located at the basolateral membrane where it is involved in homeostatic functions such as pHi, control of cell volume, and regulation of cellular ionic milieu following stimulation with growth factors (15). In addition, NHE1 participates in transepithelial HCO-3 fluxes by loading HCO-3 into the cell. HCO-3 uptake in rat cholangiocytes is, in fact, performed by two mechanisms, which has a still not clear relative importance: intracellular CO2 diffusion followed by carbonic anhydrase-catalized CO2 hydration and backward transport of H+ via NHE1 or direct uptake of HCO-3 by the electrogenic Na+-HCO3(n) cotransport (4, 32).

Recently, Marti et al. (21) have reported the presence of NHE2 transcripts in partially purified rat cholangiocytes. Among the two different published cDNAs coding for rat NHE2, the one reported by Collins et al. (10) encodes for a protein possessing 10 hydrophobic transmembrane domains; primers specific for this truncated NHE2 isoform that misses the 356 NH2-terminal amino acids (16, 26) gave negative results in NRC-1 cells. On the other hand, the set of primers used by Marti et al. (21), recognizing also a 12-transmembrane domain NHE2 isoform (40), produced amplimers of the expected size and sequence. In addition, in a preliminary report, Singh et al. (31) showed that, in microperfused bile ducts, an apical Na+/H+ exchanger functions as a pHi regulator at very acidic pHi values. In this study, we have shown that apical Na+ readmission induces a slow but significant pHi recovery in cells acidified by exposition to NH4Cl and perfused in Na+-free medium. We were also able to consistently show inhibition by high concentrations of apical amiloride and HOE-642 (see Table 3 and Fig. 5C). As expected for the NHE2 isoform (30), apical pHi recovery was ~100 times less sensitive to HOE-642 with respect to basolateral recovery. NHE3 is known to be inhibited only by HOE-642 concentrations 10 times higher than NHE2 (30); thus, given the clear expression of NHE2 mRNA, it seems reasonable to conclude that NHE2 is the isoform expressed at the apical membrane of NRC-1 cells. The discrepancy between our data and the preliminary report of Singh et al. (31) may be explained by the known heterogeneity of cholangiocyte transport systems along the biliary tree (2). In addition, the slow and incomplete pHi recovery performed by NHE2 is consistent with the idea that the primary function of the NHE2 and NHE3 isoforms, expressed on the apical membrane of gastrointestinal epithelia including gallbladder (11), is Na+ reabsorption rather than pHi regulation. The incomplete recovery mediated by NHE may indicate that cellular acid production matches NHE activity at pH 6.7 so that, if basolateral NHE1 is inhibited, further recovery can be mediated by NHE2. NHE2 is likely involved in Na+ reabsorption and biliary acidification also in cholangiocytes, and it is interesting to note that luminal pH recorded in isolated ductules microinjected with BCECF-dextran (pH 7.8) (28) is higher than the pH of bile recorded from bile fistula rats (pH 7.3) (35). In agreement with current views on morphofunctional heterogeneity of cholangiocytes (2), we speculate that a segment of the biliary tree may indeed be devoted to biliary acidification and fluid reabsorption.

The presence of the Cl-/HCO-3 exchanger at the apical side is demonstrated by the pHi recovery from an acute alkali load, which is inhibited by apical Cl- removal and by application of DIDS on the apical side (Fig. 6). In addition, the DIDS-inhibitable alkalinization during basolateral Cl- removal in cells treated with apical DIDS suggests that Cl-/HCO-3 exchange activity is also expressed on the basolateral side (Fig. 7). Expression of the anion exchange in both plasma membrane domains has been described in other epithelial cells, such as interlobular pancreatic ducts (45) and kidney beta -intercalated cells (12, 42), where the AE1 is present either apically or basolaterally, depending on the plating density of the cells (38). In our experiments, basolateral Cl-/HCO-3 exchange activity was present in cells plated at both low and high density (not shown). The reported negative immunohistochemistry for AE1 or AE2 on the basolateral membrane in human liver biopsies is not surprising; for example, whereas all cells in the kidney outer cortical collecting duct possess basolateral Cl-/HCO-3 exchange activity, antibodies to band-3 proteins label only a subset of cells, indicating that there can be immunological heterogeneity for Cl-/HCO-3 exchange within a single region of the collecting duct (12). In spite of the presence of AE activity at both sides of the monolayer, only transcripts for AE2 were detected in NRC-1 cells. It is known that AE genes can generate a variety of transcripts able to perform different physiological functions as a result of their tissue-specific expression and subcellular location. Recently, the AE2 gene has been shown to contain three different promoters that lead to the production of mRNAs encoding for three variants of the exchanger encoded with different sorting properties (41). By RT-PCR, we have shown that both AE2a and AE2b are expressed in NRC-1 cells; these two variants are known to differ in their NH2-terminal sequences, where the AE2a but not AE2b sequence contains a potential phosphorylation site for protein kinase A (41). Interestingly, on the basis of their tissue distributions, it has been proposed that AE2a may be apically targeted (41). Clarke and Harline (9) have reported data suggesting that cAMP-stimulated HCO-3 transport in the duodenum involves two mechanisms: electrogenic secretion via CFTR-mediated HCO-3 conductance and electroneutral secretion involving carbonic anhydrase and CFTR-dependent Cl-/HCO-3 exchange. The functional location of AE2 on the apical membrane and its sensitivity to stimulation by cAMP suggest it may play a role in biliary HCO-3 secretion (5, 22, 32) similar to that proposed by Novak and Greger (24) for pancreatic duct cells in which, following cAMP stimulation, CFTR recycles Cl- to a luminal Cl-/HCO-3 exchange resulting in cAMP-stimulated HCO-3 secretion. On the other hand, the physiological function of basolateral Cl-/HCO-3 exchanger in cholangiocytes is unclear and it may be limited to pHi and volume regulation. Availability of the NRC-1 cell line will facilitate further studies of this important aspect of epithelial cell physiology.

In conclusion, we have provided functional evidence in a polarized rat cholangiocyte cell line that different NHE and AE isoforms are expressed at different plasma membrane domains. NHE1 is expressed on the basolateral membrane where it is likely involved in HCO-3 cell loading as well as in pHi and volume housekeeping functions. On the other hand, NHE2 appears to be located at the apical membrane where it may serve to reabsorb Na+ and acidify the biliary fluid. Two different AE2 transcripts were detected in NRC-1 cells, consistent with the observation that Cl-/HCO-3 exchange activity was present at both the apical and basolateral sides. Apical AE was activated by cAMP, consistent with its proposed role in HCO-3 extrusion, whereas basolateral AE is likely involved in pHi homeostatic functions. To the extent that data on cell lines can be relevant to the in vivo situation, these observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes. This study also indicates that the NRC-1 cell line can be a useful tool to investigate the mechanisms of transport protein sorting in the biliary epithelium.

    ACKNOWLEDGEMENTS

We are indebted to James L. Boyer, M.D., Elena Ossi, M.D., and J.F. Medina, M.D., for helpful discussion. À. Zsembery thanks Prof. Làszlò Rosivall for helpful discussion and his constant support.

    FOOTNOTES

This work was sponsored by Grant 96.0344.04 from Consiglio Nazionale delle Ricerche. The financial support of Telethon (Grant E-430) is gratefully acknowledged. This work was also supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-24031 to N. F. LaRusso.

À. Zsembery was the recipient of an International Fellowship from the University of Padova.

Part of this work was presented at the 47th Annual Meeting of the American Association for the Study of Liver Diseases in Chicago, IL, November 8-12, 1996, and at the 32nd Annual Meeting of the European Association for the Study of the Liver in London, UK, April 9-12, 1997, and published in abstract form (Hepatology 24: 146, 1996; J. Hepatol. 26: 63, 1997).

Present address of À. Zsembery: Institute of Pathophysiology, University of Medicine, Budapest, Hungary.

Address for reprint requests: M. Strazzabosco, Institute of Internal Medicine, Univ. of Padova, Via Giustiniani, 2, I-35100 Padova, Italy.

Received 6 November 1997; accepted in final form 30 July 1998.

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