Clminus -dependent secretory mechanisms in isolated rat bile duct epithelial units

Satish K. Singh1,2, Albert Mennone1, Alessandro Gigliozzi1, Flavia Fraioli1, and James L. Boyer1

1 Liver Center, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8019; and 2 Evans Biomedical Research Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118-2518


    ABSTRACT
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INTRODUCTION
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Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several Cl--secreting epithelia, Na+-K+-2Cl- cotransport is a basolateral Cl- uptake pathway facilitating apical Cl- secretion. To determine if cholangiocytes possess similar mechanisms independent of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, we assessed Cl--dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, forskolin (FSK) stimulated secretion entirely dependent on Na+ and Cl- and inhibited by Na+-K+-2Cl- inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. In contrast, FSK-stimulated secretion was inhibited ~85% by K+ channel inhibitor Ba2+ and blocked completely by bumetanide plus Ba2+. IBDU Na+-K+-2Cl- cotransport activity was assessed by recording intracellular pH during NH4Cl exposure. Bumetanide inhibited initial acidification rates due to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry in the presence and absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In contrast, when stimulated by FSK, a 35% increase in Na+-K+-2Cl- cotransport activity occurred without CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. These data suggest a cellular model of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent secretion in which Na+-K+-2Cl- cotransport maintains high intracellular Cl- concentration. Intracellular cAMP concentration increases activate basolateral K+ conductance, raises apical Cl- permeability, and causes transcellular Cl- movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial Cl--dependent fluid secretion independent of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Bumetanide-sensitive Na+-K+-2Cl- cotransport, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, and Ba2+-sensitive K+ channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium.

ammonia; cholangiocyte; bumetanide; barium; pH


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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ALTHOUGH HEPATOCYTES ARE THE primary source of bile, ~10% of basal bile flow in rats and up to 40% in humans originates from the bile duct epithelium. Cholangiocytes comprising the small intralobular bile duct segments are capable of absorptive and secretory functions that modify the composition of canalicular bile (9). These segments are the site of injury in several primary cholestatic disorders (40). After common bile duct ligation in the rat, bile duct epithelial cells proliferate with potentiation of spontaneous bile flow. In this model, hormonal stimulation by secretin, which increases biliary cAMP, increases both bile flow rate and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> content by ~50% (2).

In cholangiocytes, secretion stimulated by cAMP is believed to occur largely via coupling of apical Cl- conductance(s) and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (3, 33). Evidence for this model of secretion comes from normal rat biliary epithelial cells in which secretin stimulates Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, facilitated by a cAMP-regulated increase in Cl- conductance, consistent with recycling of Cl- at the apical membrane via Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (3, 13). Thus, in an epithelial model in which active Cl- secretion drives a component of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, mechanisms that maintain intracellular Cl- concentration ([Cl-]i) above electrochemical equilibrium would facilitate secretory functions (18). Indeed, in several Cl--secreting epithelia, a basolateral, electroneutral, loop-diuretic-sensitive Na+-K+-2Cl- cotransporter (20, 26), energized by Cl- and Na+ gradients (23), functions to maintain high [Cl-]i (48). Bumetanide-sensitive Na+-K+-2Cl- cotransport processes have been described in a number of epithelial and nonepithelial cells. In addition to their function in epithelial salt transport, Na+-K+-2Cl- cotransporters participate in cell volume homeostasis (17) and regulation of cell proliferation (37). In certain epithelia, e.g., rat parotid acinar cells, basolateral Na+-K+-2Cl- cotransport, in combination with linked Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers, contributes to cellular Cl- uptake (18, 41). Typically, in salt-secreting epithelia, activation of cAMP- and/or Ca2+-sensitive apical Cl- channels increases apical membrane Cl- permeability, resulting in active movement of Cl- from the cell into the luminal space; Na+ and water follow passively along electrochemical and osmotic gradients, respectively (48, 49). Secretion can be potentiated in certain tracheal and gut epithelia by basolateral cAMP- and/or Ca2+-activated K+ channels; activation of these K+ channels hyperpolarizes the cell, driving potential-dependent Cl- secretion (30, 47). Certain biliary epithelial cell lines have been described to possess bumetanide-inhibitable Rb uptake (7).

Isolated bile duct units (IBDUs) are intact polarized epithelial structures derived from intralobular ducts maintained in short-term culture. IBDUs have been exploited as a model biliary epithelium in which relative secretion can be quantitated using videomicroscopy (35). In previous studies (35), stimuli that increased intracellular cAMP concentration resulted in substantial fluid secretion in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. However, modest forskolin-stimulated fluid secretion could be observed in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> as well (35). Although secretion observed in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was attributed to transport of ambient and/or endogenously generated CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, this observation also raised the possibility that cholangiocytes possess CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent, cAMP-stimulated vectorial Cl- secretion.

In the present communication, using videomicroscopy to assess the volume of fluid secreted by IBDUs, we have identified a role for Cl- that is separate from the well-appreciated role of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in cAMP-stimulated fluid secretion by cholangiocytes. We found that forskolin-stimulated secretion, in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, depended entirely on extracellular Cl-. Cl--dependent secretion was, in turn, sensitive to serosal Na+ and to bumetanide (50 µM), consistent with participation of Na+-K+-2Cl- cotransport. Furthermore, Cl--dependent secretion in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was inhibited by Ba2+, consistent with a role for basolateral cAMP-activated K+ channels in potential-driven Cl- secretion. Intracellular pH (pHi) records of cholangiocytes comprising IBDUs revealed bumetanide-sensitive NH<UP><SUB>4</SUB><SUP>+</SUP></UP> uptake, supporting the constitutive presence of Na+-K+-2Cl- cotransport. In addition, Na+-K+-2Cl- cotransport activity was stimulated by forskolin and inhibited by bumetanide. In the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, forskolin-induced secretion was entirely Cl--dependent as well, but only partly stilbene sensitive and not inhibited at all by bumetanide. When combined, the anion exchange inhibitor DIDS and bumetanide together blocked forskolin-stimulated secretion completely, consistent with Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange functioning as a parallel Cl- uptake pathway. These results, derived largely from functional assays, identify the importance of extracellular Cl- to secretion and suggest a key role for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent Cl- secretion by biliary epithelium.


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Isolation of bile duct units. With the full approval of the Yale University School of Medicine Animal Care and Use Committee, we obtained male Sprague-Dawley rats weighing 200-250 g from Camm Laboratory Animals (Wayne, NJ). Bile duct fragments (30-100 µM) were enzymatically isolated from rat livers and plated on Matrigel-coated (Collaborative Biomedical, Bedford, MA) glass coverslips and incubated at 37°C in a 5% CO2-air atmosphere with supplemented alpha -MEM (GIBCO BRL, Grand Island, NY), as described previously (35). The culture medium was exchanged at 24 h, and adherent IBDUs were utilized in experiments by 48-60 h after initial plating.

Quantitation of secretion by video imaging. Coverslips with adherent IBDUs were placed in a temperature-controlled superfusable chamber and transilluminated on the stage of an inverted microscope equipped with Nomarski optics. Coverslips were screened for single spherical IBDUs possessing a sharply defined luminal border at a focal plane that delineated the greatest luminal area. A television camera and computerized imaging system were used to obtain and store magnified digital images of superfused IBDUs on an optical disk. IBDUs were equilibrated with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing or CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free Ringer saline solutions for a minimum of 10 min before experimental perturbations to achieve steady state. Images of IBDUs were acquired at 5-min intervals after the equilibration period. The volume of secreted fluid was assessed, as described previously (4, 10, 35, 36), with the IBDU lumen modeled as a sphere. In previous work (4, 10, 35, 36), we validated the use of cross-sectional area (CSA) to assess the volume of fluid secreted by comparing increases in CSA to increases in secreted volume assessed by three-dimensional reconstruction of Z-sections through the luminal space. Percent increases in volume were found to be virtually identical to percent increases in CSA in the same IBDUs. For this reason, and because two-dimensional images are readily and rapidly obtainable, we obtained measurements of CSA alone. As such, the total volume of fluid contained in the IBDU lumen (VL) is related to the greatest measured CSA of the lumen by VL = 0.7523(CSA)1.5. At the conclusion of experiments, software-based image analysis was used to measure CSA at successive 5-min intervals from stored images. Measured CSA values were then normalized to, and expressed as, a percentage of the CSA immediately before the addition of forskolin. At the conclusion of the experiments, cell viability was assessed by exclusion of a 4% solution of trypan blue. Experiments were discarded if <95% of cells excluded trypan blue.

Clearly, the IBDU preparation is subject to many factors that could lead to heterogeneous responses, and CSA tends to vary between short-term culture preparations. Variations in luminal CSA are not unexpected, as the mechanics of luminal expansion are, in part, dependent on the initial volume of the IBDU lumen, the compliance and H2O permeability of its luminal border, and the net of absorptive and secretory processes present. Thus measurements of IBDU luminal CSA do not provide absolute quantitative rates of fluid secretion but rather rates of secretion relative to IBDUs from the same preparation maintained under identical culture conditions. As such, we compare experimental perturbations to day-matched controls from the same "batch" of IBDUs in short-term culture.

Measurements using fluorescent dyes. Cholangiocytes comprising IBDUs were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) by incubation with 10 µM of the membrane-permeant AM (BCECF-AM, Molecular Probes, Eugene, OR) for 10 min at 37°C. Coverslips containing IBDUs were transferred to a temperature-controlled chamber (37°C) on the stage of an inverted microscope and superfused with either 1) CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free Ringer saline, pH 7.4, buffered with 32 mM HEPES and gassed with 100% N2 or 2) Krebs-Ringer-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (KRB) solution, pH 7.4, containing 22 mM NaHCO3, equilibrated with 5% CO2-95% O2. Light from a mercury arc lamp was used to alternately excite intracellular BCECF at 440 and 495 nm. Fluorescence images were obtained using a Cohu charge-coupled device camera (San Diego, CA) through a 510-nm bandpass filter at a rate of two to six images per minute. Fluorescence emission intensity consistently exceeded background autofluorescence by >45-fold. The 495 nm-to-440 nm fluorescence intensity ratio data were converted to pHi values by using a nigericin calibration curve, as described previously (45). Over the pHi range of 6.8-7.6, fluorescence ratios varied linearly with extracellular pH.

In experiments designed to assess the integrity of intercellular junctions, we identified and counted all IBDUs on a coverslip. We then exposed the IBDUs to 1 mM Texas red-dextran (40,000 MW, Molecular Probes) for 10 min. Subsequently, Texas red-dextran was washed away and IBDUs were illuminated with 590 nm light with fluorescence images obtained through a 615-nm bandpass filter. IBDUs containing detectable dye in their lumens were then counted. At the conclusion of the experiments, IBDUs were again incubated with Texas red-dextran and recounted in an identical manner.

Assessment of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport from pHi records. Exposing IBDUs to 20 mM NH4Cl, produced an initial rapid alkalization due to NH3 diffusion into the cell, followed by a slower acidification from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry via a variety of pathways, including Na+-K+-ATPase (16, 28), K+ channels (8), and Na+-K+-2Cl- cotransport (15, 27, 38) where NH<UP><SUB>4</SUB><SUP>+</SUP></UP> substitutes for K+. The initial rate of this acidification has been used previously (5, 6, 38, 46) to assess the rate of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> uptake. For our experiments, we exposed IBDUs to two successive 20 mM NH4Cl pulses. The NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced pHi change over the initial 120 s of acidification (instantaneous rate of pHi change) was expressed in pH units per minute. H+ fluxes (JH+) were determined from a previously determined intrinsic buffering power vs. pHi titration curve (44). Data are expressed as means ± SD. Paired and unpaired t-tests were used to compare rates between the first and second NH4Cl prepulses, with P < 0.05 indicating statistical significance.

General experimental procedures. All experiments were performed at 37°C under isotonic (290 mosM) conditions. The compositions of the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing Ringer and HEPES-Ringer were identical to those described previously (35). To ensure that HEPES-buffered solutions were free of ambient CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> but not O2 free, solutions were gassed with N2 then passed via gas-permeable tygon tubing to a superfusion chamber open to the atmosphere. Analysis of a typical N2-gassed air-equilibrated HEPES-buffered solution sampled from the superfusion chamber revealed pH of ~7.4, O2 partial pressure of ~170 mmHg, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration <1 mM, which is below the detection range of the gas analyzer. In experiments requiring isosmolar ion replacement, gluconate was substituted for Cl- and choline or NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was substituted for Na+ as required. All experimental perturbations were compared with controls from the same IBDU preparation. All data from measurements of maximum CSA and pHi are presented as arithmetical means ± SE. Paired Student's t-tests were used to determine statistical significance.


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As shown in Fig. 1, we first assessed the role of extracellular Cl- in forskolin-induced secretion in IBDUs. We added 10 µM forskolin to IBDUs superfused with Cl--free HEPES-Ringer. We observed no effect on secretion until Cl- (132 mM) was returned to the HEPES-Ringer solution. Thereafter, luminal CSA doubled in 10 min and increased further to 150% above baseline within 20 min. Thus cAMP-stimulated secretion requires basolateral Cl- and occurs in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.


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Fig. 1.   Forskolin (FSK)-stimulated secretion in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is dependent on extracellular Cl- and is not due to secretion of intracellular CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Effect on isolated polarized bile duct unit (IBDU) lumen area of FSK (10 µM) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free bathing solution before (points a to b) and on introduction of 132 mM Cl- (points b to d) (control). Effect of the cell-permeant carbonic anhydrase inhibitor ethoxyzolamide (10 µM) using the identical experimental protocol. Superfusion solutions were HEPES buffered and rendered CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free by equilibration with 100% N2 gas. Values are means ± SE; error bars are omitted where smaller than symbols. CSA, cross-sectional area.

Cellular respiration generates a nominal amount of CO2 that can be hydrated to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the presence of carbonic anhydrase (24). To rule out that cAMP-stimulated secretion represented secretion of endogenously generated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the same experimental protocol was repeated under anoxic conditions and in the continuous presence (after 20 min preincubation) of ethoxyzolamide (10 µM), a cell-permeant inhibitor of carbonic anhydrase. As shown in Fig. 1, ethoxyzolamide, in the complete absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, did not affect cAMP-stimulated Cl--dependent secretion. Acetazolamide (500 µM), another carbonic anhydrase inhibitor, also did not inhibit secretion in similar experiments (not shown). These results indicate that neither ambient CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> nor endogenously generated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> could account for the forskolin-stimulated Cl--dependent secretion observed in this experiment.

To determine if Cl--dependent secretion in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was dependent on basolateral Na+, as would be expected for a Na+-K+-2Cl- cotransporter, IBDUs were superfused with HEPES-Ringer free of Cl-, Na+, and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. As depicted in Fig. 2, addition of 10 µM forskolin to IBDUs superfused with Cl-, Na+, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES-Ringer did not result in secretion when Cl- was added to the solution in the absence of Na+. However, after introduction of Na+ (125 mM) to the Cl--containing solution, we observed a rapid rate of secretion to 40% above baseline. Thus HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent, cAMP-stimulated secretion requires a coupled basolateral Cl- and Na+ transport process.


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Fig. 2.   FSK-stimulated secretion in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> requires the presence of both extracellular Cl- and Na+. Effect on IBDU lumen area of FSK (10 µM) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free bathing solution in the absence of both Cl- and Na+ (points a to b), in the presence of 132 mM Cl- without Na+ (points b to c), and in the presence of 125 mM Na+ and 132 mM Cl- (points c to d). Superfusion solutions were HEPES buffered and rendered CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free by equilibration with 100% N2 gas. Choline replaced Na+. Values are means ± SE (n = 10 experiments); error bars are omitted where smaller than symbols.

As such, we next examined the effects of bumetanide (50 µM), a specific inhibitor of Na+-K+-2Cl- cotransport on secretion. As shown in Fig. 3, under Cl-- and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions, 10 µM forskolin caused no net secretion. When Cl- was added to the solution, luminal CSA increased 100% within 20 min. In contrast, when 50 µM bumetanide was present (Fig. 3), forskolin-stimulated secretion was inhibited by 55% (P < 0.01).


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Fig. 3.   Bumetanide, in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, inhibits FSK-stimulated Cl--dependent secretion. Effect on IBDU lumen area of FSK (10 µM) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free bathing solution before (points a to b) and on introduction of 132 mM Cl- (points b to c) to the bathing solution (control). Effect of the inhibitor of Na+-K+-2Cl- cotransport, bumetanide (50 µM), on the identical experimental protocol. Superfusion solutions were HEPES buffered and rendered CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free by equilibration with 100% N2 gas. Values are means ± SE; error bars are omitted where smaller than symbols.

NH<UP><SUB>4</SUB><SUP>+</SUP></UP> can substitute for K+ on the Na+-K+-2Cl- cotransporter (15, 27, 39), and pHi measurements were used to characterize Na+-K+-2Cl- cotransport activity in the presence and absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. For each experimental protocol, a 20 mM NH4Cl prepulse was performed. After recovery to resting pHi, a second prepulse was performed in the presence of 50 µM bumetanide, either in the absence or presence of 10 µM forskolin. During each prepulse, an initial acute alkalinization due to NH3 entry was observed, followed by a slower phase of acidification as NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entered the cell. In CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES solution, bumetanide (50 µM) inhibited the initial rate of acidification rate by 24% (JH+ = 1.97 ± 0.71 vs. 1.54 ± 0.39 mM/min, P < 0.001; Fig. 4A and Table 1). In CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing KRB solution, bumetanide inhibited the initial rate of acidification by 25% (JH+ = 5.52 ± 1.61 vs. 4.15 ± 0.61 mM/min, P < 0.001; Fig. 4B and Table 1). In contrast to the effects of bumetanide, 10 µM forskolin stimulated the initial acidification rate by 35% (JH+ = 1.77 ± 0.29 vs. 2.34 ± 0.45 mM/min, P < 0.001) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions (Table 1). Forskolin induced significant changes in initial rates of acidification in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing KRB solution as well (JH+ = 5.06 ± 1.73 vs. 5.53 ± 2.85 mM/min, P < 0.50). In both the absence and presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, bumetanide (50 µM) completely inhibited forskolin-induced stimulation of initial acidification rates; in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, there was in fact a further 13% inhibition (JH+ = 1.49 ± 0.90 vs. 1.31 ± 0.77 mM/min, P < 0.001). These suggest that forskolin stimulates Na+-K+-2Cl- cotransport both in the absence and presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.


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Fig. 4.   Bumetanide, both in the absence and presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, inhibits NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry. Overlaid representative intracellular pH (pHi) tracings of the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-induced acidification phases during sequential NH4Cl (20 mM) prepulses, the first performed in the absence and the second in the presence of bumetanide (50 µM) in HEPES-Ringer solution (A) and Krebs-Ringer-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (KRB) solution (B). Whether in the absence or presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, bumetanide significantly decreased the initial rate of acidification during the phase of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry (see Table 1).


                              
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Table 1.   Initial recovery rate from NH4Cl-induced alkaline load in HEPES-Ringer and KRB solutions

We next examined the role of a possible basolateral cAMP-activated K+ conductance on forskolin-stimulated fluid secretion in IBDUs (49) by examining the effects of Ba2+. As shown in Fig. 5, addition of 10 µM forskolin to IBDUs superfused with Cl--free HEPES-Ringer did not lead to net secretion after 5 min. When Cl- was returned to the HEPES-Ringer solution, luminal CSA increased to 150% above baseline within 30 min. In other experiments, the identical protocol was repeated, but after 5 min of preincubation with, and in the continuous presence of, 1 mM BaCl (Fig. 5). As in the control experiments without Ba2+ (Fig. 5), exposure to forskolin in the virtual absence of Cl- did not induce secretion until 132 mM Cl- was introduced to the bathing solution. However, Ba2+ (1 mM) alone decreased forskolin-stimulated, Cl--dependent secretion by one-half (Fig. 5) compared with controls (P < 0.05). Furthermore, when the identical protocol was repeated in the presence of both 1 mM Ba2+ and 50 µM bumetanide (Fig. 5), net forskolin-stimulated, Cl--dependent secretion was not observed after 30 min.


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Fig. 5.   Barium, in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, inhibits FSK-stimulated Cl--dependent secretion. Effect on IBDU lumen area of FSK (10 µM) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free bathing solution before (points a to b) and on introduction of 132 mM Cl- (points b to c) to the bathing solution (control). Effect of the inhibitor of K+ channels, BaCl (1 mM), and the combination of bumetanide (50 µM) and BaCl (1 mM) on the identical experimental protocol. Superfusion solutions were HEPES buffered and rendered CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free by equilibration with 100% N2 gas. Values are means ± SE; error bars are omitted where smaller than symbols.

Given the apparent importance of Cl- to secretion in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, we examined the role extracellular Cl- plays in cAMP-stimulated fluid secretion under physiological CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conditions. As shown in Fig. 6, forskolin-stimulated secretion required both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-. Surprisingly, bumetanide had no effect on secretion in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>; this is in contrast to the bumetanide-sensitive secretion observed in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (see Fig. 3) and the bumetanide-sensitive NH<UP><SUB>4</SUB><SUP>+</SUP></UP> uptake observed in the absence (see Fig. 4A) as well as in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (see Fig. 4B). Thus a pathway other than Na+-K+-2Cl- cotransport appears to function in the basolateral uptake of Cl-. Previous studies (3, 4) in IBDUs revealed a stilbene-sensitive Cl-/HCO3 exchange stimulated by secretin. Thus to determine if a Cl-/HCO3 exchange process participates in Cl- uptake, the effect of the anion exchange inhibitor DIDS was examined. In the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, DIDS (1 mM) alone blocked 25% of forskolin-stimulated Cl--dependent secretion (n = 13, P < 0.03 vs. control, data not shown). However, as shown in Fig. 7, when combined with bumetanide, DIDS completely blocked forskolin-stimulated secretion in the presence of Cl- and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.


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Fig. 6.   FSK-stimulated secretion in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> requires Cl- and is not inhibited by bumetanide. Effect on IBDU lumen area of FSK (10 µM) in the absence (points a to b) and on sequential introduction of 5% CO2/22 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (points b to c) then 121 mM Cl- (points c to d) to the bathing solution (control). Effect of the inhibitor of Na+-K+-2Cl- cotransport bumetanide (50 µM) on the identical experimental protocol. CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free superfusion solutions were HEPES buffered and equilibrated with 100% N2 gas. Values are means ± SE; error bars are omitted where smaller than symbols.



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Fig. 7.   FSK-stimulated Cl--dependent secretion in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is ablated by the combination of bumetanide and DIDS. Effect on IBDU lumen area of FSK (10 µM) in the continuous presence of 50 µM bumetanide. Secretion observed in the absence (points a to b) and on sequential introduction of 5% CO2/22 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (points b to c) and 121 mM Cl- (points c to d) in the absence and presence of the anion transport inhibitor DIDS (1 mM). CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free superfusion solutions were HEPES buffered and equilibrated with 100% N2 gas. Values are means ± SE; error bars are omitted where smaller than symbols.

Our assessments of secretion may have been confounded if paracellular fluid movement was markedly affected during experiments. To determine if paracellular permeability was increased by the removal and/or readmission of Cl-, forskolin, and/or bumetanide, we performed a series of experiments using Texas red-dextran (40,000 MW) in the bathing solution. Compared with a 20-min control period in HEPES-Ringer (after which 88% of initially intact IBDUs continued to exclude Texas red-dextran from their lumens, n = 17) there was no significant difference in Texas red-dextran entry after 1) 15 min in Cl--free HEPES-Ringer followed by 25 min in Cl--containing HEPES-Ringer (94% exclusion, n = 16), 2) 15 min in Cl--free HEPES-Ringer followed by 25 min in Cl--containing HEPES-Ringer in the presence of 10 µM forskolin during both periods (95% exclusion, n = 20), or 3) 15 min in Cl--free HEPES-Ringer followed by 25 min in Cl--containing HEPES-Ringer with 10 µM forskolin and 50 µM bumetanide present during both periods (89% exclusion, n = 24).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cholangiocytes play an important role in modifying the composition of canalicular bile through secretory and reabsorptive processes (1, 42). Bile is particularly enriched with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> compared with Cl-, and cAMP-induced HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is predominantly driven by a luminal Cl- recycling mechanism that couples an apical Cl- conductance with a Cl-/HCO3 exchange (3, 33). However, in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, forskolin-induced fluid secretion was observed in IBDUs (35). In the present study, we again exploited IBDUs as a model intrahepatic biliary epithelium from rat liver that secretes fluid in response to elevations in [cAMP]i (4, 10, 35). In the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, we observed that forskolin-stimulated secretion was Cl- and Na+ dependent, consistent with transport of Na+ and Cl-. Bumetanide, a loop diuretic inhibitor of Na+-K+-2Cl- cotransport, decreased cAMP-stimulated secretion in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by 55%. Experiments with Texas red-dextran suggest that paracellular permeability is not affected by forskolin, bumetanide, or absence of Cl-. Nonetheless, ~ 45% of stimulated secretion was not blocked by bumetanide, consistent with either the presence of 1) other Na+-dependent, but HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-independent pathways for basolateral Cl- uptake and/or 2) potential-driven, serosal-to-mucosal Cl- (and Na+, when present) movement due to activation of a basolateral K+ efflux pathway. Basolateral Ba2+, an inhibitor of K+ channels, decreased stimulated secretion by ~50%, consistent with activation of basolateral K+ conductance(s). A basolateral K+ channel activated by cAMP and/or Ca2+ would permit exit and recycling of K+ taken up by basolateral Na+-K+-2Cl- cotransport and Na+-K+-ATPase (34). Stimulated fluid secretion was entirely inhibited by the combination of bumetanide and Ba2+, indicating that in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> the dual action of these transport processes supports virtually all cAMP-stimulated secretion. Of note, despite the presence of 1 mM extracellular Cl- in experiments in which BaCl was used, this concentration of Cl-, in the face of inhibited K+ channels, did not support forskolin-stimulated secretion (Fig. 5). Furthermore, in pHi studies of nonstimulated IBDUs, bumetanide inhibited ~25% of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport into cells, consistent with a bumetanide-sensitive K+ transport pathway. Although bumetanide-inhibitable Na+-Cl- cotransport has been described in other epithelia, bumetanide is a relatively specific inhibitor of Na+-K+-2Cl- cotransporters (26), and the finding of bumetanide-sensitive NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport (substituting for K+ on the cotransporter) is consistent with the presence of Na+-K+-2Cl- cotransport.

We used forskolin to evaluate cotransporter activity during cAMP-induced secretion. Both in the absence and presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, forskolin significantly increased NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx reflected by a more rapid rate of cellular acidification (see Table 1). This increase in acidification rate was completely abolished by 50 µM bumetanide, suggesting that the observed increase in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx was related to stimulation of Na+-K+-2Cl- cotransport. Thus Na+-K+-2Cl- cotransport activity is increased during forskolin-induced secretion, more so in the absence than in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Na+-K+-2Cl- cotransport presumably functions as a Cl- uptake mechanism that helps maintain [Cl-]i above electrochemical equilibrium.

Previous studies in several other secretory epithelia have demonstrated that secretagogues activate Na+-K+-2Cl- cotransport either directly (12, 32, 39) or as secondary responses to apical Cl- secretion (21, 22, 29). Normally, in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, cholangiocyte HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is driven by the coupling of an apical Cl- conductance to an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (3, 33). Thus cAMP-induced Cl- secretion results largely from recycling of secreted Cl- by apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. This recycling of Cl- may explain why we do not observe an increase in the Na+-K+-2Cl- cotransporter activity after forskolin stimulation in IBDUs perfused with KRB, because under CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing conditions Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange may be sufficient to recycle Cl-, keeping [Cl-]i relatively high. Alternatively, cAMP may also stimulate basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport (3), counteracting NH<UP><SUB>4</SUB><SUP>+</SUP></UP> influx-mediated acidification and/or other Cl- uptake systems that might be present.

Because in other secretory epithelia Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange also contributes to Cl- uptake (18), we examined the effect of bumetanide and DIDS, an inhibitor of anion transport (19), on secretion under physiological CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conditions. Because bumetanide did not inhibit forskolin-stimulated secretion in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-, alternative CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent pathways for Cl- uptake must be present. DIDS alone inhibited Cl--dependent secretion by only 25%, whereas bumetanide together with DIDS ablated stimulated secretion, strongly suggesting that a stilbene-sensitive Cl- uptake pathway, i.e., a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and/or Cl- channel may be pathways for Cl- uptake in cholangiocytes. Although previously described cAMP- or Ca2+-activated Cl- channels in biliary epithelial cells (13) might function as alternate pathways for Cl- uptake, we did not observe Cl--dependent secretion in the absence of Na+ and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the perfusate (Fig. 2). It is also possible that bumetanide incompletely inhibits Na+-K+-2Cl- cotransport under CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conditions, but our observation that bumetanide blocks the forskolin-induced increase in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry rates in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> argues against bumetanide being inactive under CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conditions (see Table 1).

The present findings are most consistent with basolateral Na+-K+-2Cl- cotransport that exists in parallel with basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. This Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange may be a dominant, parallel pathway for Cl- entry across the basolateral membrane. However, although IBDUs are a model epithelium in which cell polarity and intercellular junctions are preserved, thus excluding rapid entry of solutes such as DIDS into the luminal solution, DIDS inhibition of apical Cl- exit and entry cannot be entirely ruled out. Nevertheless, because Na+/H+ exchange is present on the basolateral membrane in cholangiocytes (11, 31, 43), we speculate that, as in parotid acinar cells, a basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange may be coupled to Na+/H+ exchange. Indeed in Na+-K+-2Cl--deficient suckling mice, there is no deficit in stimulated small intestinal fluid secretion, and it has been proposed that Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+/H+ exchange function as alternative mechanisms of basolateral Cl- and Na+ uptake (14).

On the basis of these findings, we propose an integrative model for cAMP-stimulated Cl- secretion in cholangiocytes (Fig. 8). In this model, basolateral Na+-K+-2Cl- cotransport and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (the latter possibly linked to Na+/H+ exchange) function as active Cl- uptake mechanisms that maintain [Cl]i above electrochemical equilibrium. Increases in [cAMP]i lead to both an increase in apical membrane permeability to Cl- and activation of a basolateral membrane K+ conductance. As a result, electromotive forces move Cl- into the lumen with attendant movement of Na+ and water. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entry in the cell occurs via Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport and is transported across the apical membrane into the lumen of the bile duct via Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange.


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Fig. 8.   Speculative integrated model of cAMP-stimulated secretion by intralobular bile duct epithelium. Intracellular Cl- concentration is maintained above electrochemical equilibrium by secondary active transport via basolateral Na+-K+-2Cl- cotransport and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (possibly linked to Na+/H+ exchange). Increases in intracellular cAMP concentration, perhaps via intracellular Ca2+ concentration, activate apical pathways for Cl- permeation as well as basolateral pathways for K+ exit that result in net secretion of Cl- into the lumen. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> can then be secreted via apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. Basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport as well as Na+/H+ exchange may function as cellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loaders that support the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange processes.

In summary, the present study provides evidence for the presence of a secretory Na+-K+-2Cl- cotransport process in IBDUs from rat liver. Because it is feasible to isolate enriched populations of intrahepatic biliary epithelial cells (25), molecular identification of the specific isoform(s) of a bumetanide-sensitive cotransporter should be possible (26).


    ACKNOWLEDGEMENTS

We thank Michelle Pate for assistance with cell isolation.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants KO8-DK-02410 (S. K. Singh) and R01-DK-25636 (J. L. Boyer), a Junior Faculty Research Award from Yale School of Medicine (S. K. Singh), and a Career Development Award from the Crohn's and Colitis Foundation of America (S. K. Singh). Cholangiocytes were isolated in the Cell Isolation and Culture Core of the Yale Liver Center supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant P30-DK-34989.

Address for reprint requests and other correspondence: S. K. Singh, Dept. of Medicine, Boston Univ. School of Medicine, Ste. 504, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118-2518 (E-mail: satish.singh{at}bmc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 February 2000; accepted in final form 21 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpini, G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, Lasater J, and LeSage GD. Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion. Am J Physiol Gastrointest Liver Physiol 272: G1064-G1074, 1997[Abstract/Free Full Text].

2.   Alpini, G, Lenzi R, Sarkozi L, and Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 81: 569-578, 1988[ISI][Medline].

3.   Alvaro, D, Cho WK, Mennone A, and Boyer JL. Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 92: 1314-1325, 1993[ISI][Medline].

4.   Alvaro, D, Mennone A, and Boyer JL. Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in cholangiocytes. Am J Physiol Gastrointest Liver Physiol 273: G303-G313, 1997[Abstract/Free Full Text].

5.   Amlal, H, Paillard M, and Bichara M. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport pathways in cells of medullary thick ascending limb of rat kidney. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> conductance and K+/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (H+) antiport. J Biol Chem 269: 21962-21971, 1994[Abstract/Free Full Text].

6.   Amlal, H, and Soleimani M. K+/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> antiporter: a unique ammonium carrying transporter in the kidney inner medulla. Biochim Biophys Acta 1323: 319-333, 1997[ISI][Medline].

7.   Basavappa, S, Middleton J, Mangel AW, McGill JM, Cohn JA, and Fitz JG. Cl- and K+ transport in human biliary cell lines. Gastroenterology 104: 1796-1805, 1993[ISI][Medline].

8.   Bleich, M, Schlatter E, and Greger R. The luminal K+ channel of the thick ascending limb of Henle's loop. Pflügers Arch 415: 449-460, 1990[ISI][Medline].

9.   Boyer, JL. Bile duct epithelium: frontiers in transport physiology. Am J Physiol Gastrointest Liver Physiol 270: G1-G5, 1996[Abstract/Free Full Text].

10.   Cho, WK, Mennone A, Rydberg SA, and Boyer JL. Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion. Gastroenterology 113: 311-321, 1997[ISI][Medline].

11.   Elsing, C, Kassner A, and Stremmel W. Sodium, hydrogen antiporter activation by extracellular adenosine triphosphate in biliary epithelial cells. Gastroenterology 111: 1321-1332, 1996[ISI][Medline].

12.   Evans, RL, and Turner RJ. Upregulation of Na+-K+-2Cl- cotransporter activity in rat parotid acinar cells by muscarinic stimulation. J Physiol (Lond) 499: 351-359, 1997[Abstract].

13.   Fitz, JG, Basavappa S, McGill J, Melhus O, and Cohn JA. Regulation of membrane chloride currents in rat bile duct epithelial cells. J Clin Invest 91: 319-328, 1993[ISI][Medline].

14.   Flagella, M, Clarke LL, Miller ML, Erway LC, Giannella RA, Andringa A, Gawenis LR, Kramer J, Duffy JJ, Doetschman T, Lorenz JN, Yamoah EN, Cardell EL, and Shull GE. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J Biol Chem 274: 26946-26955, 1999[Abstract/Free Full Text].

15.   Garvin, JL, Burg MB, and Knepper MA. Active NH<UP><SUB>4</SUB><SUP>+</SUP></UP> absorption by the thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 255: F57-F65, 1988[Abstract/Free Full Text].

16.   Garvin, JL, Burg MB, and Knepper MA. Ammonium replaces potassium in supporting sodium transport by the Na-K-ATPase of renal proximal straight tubules. Am J Physiol Renal Fluid Electrolyte Physiol 249: F785-F788, 1985[Abstract/Free Full Text].

17.   Geck, P, and Pfeiffer B. Na-K-2Cl cotransport in animal cells: its role in volume regulation. Ann NY Acad Sci 456: 166-182, 1985[Abstract].

18.   Greger, R. The membrane transporters regulating epithelial NaCl secretion. Pflügers Arch 432: 579-588, 1996[ISI][Medline].

19.   Grinstein, S, Ship S, and Rothstein A. Anion transport in relation to proteolytic dissection of band 3 protein. Biochim Biophys Acta 507: 294-304, 1978[ISI][Medline].

20.   Haas, M. The Na-K-Cl cotransporters. Am J Physiol Cell Physiol 267: C869-C885, 1994[Abstract/Free Full Text].

21.   Haas, M, and McBrayer DG. Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i. Am J Physiol Cell Physiol 266: C1440-C1452, 1994[Abstract/Free Full Text].

22.   Haas, M, McBrayer DG, and Yankaskas JL. Dual mechanism for Na-K-Cl cotransport regulation in airway epithelial cells. Am J Physiol Cell Physiol 264: C189-C200, 1993[Abstract/Free Full Text].

23.   Haas, M, Schmidt WF, and McManus TJ. Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral Na-K-2Cl co-transport. J Gen Physiol 80: 125-147, 1982[Abstract].

24.   Henry, RP. Multiple roles of carbonic anhydrase in cellular transport and metabolism. Annu Rev Physiol 58: 523-538, 1996[ISI][Medline].

25.   Ishii, M, Vroman B, and LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97: 1236-1247, 1989[ISI][Medline].

26.   Kaplan, MR, Mount DB, and Delpire E. Molecular mechanisms of NaCl cotransport. Annu Rev Physiol 58: 649-668, 1996[ISI][Medline].

27.   Kinne, R, Kinne-Saffran E, Schutz H, and Scholermann B. Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na-K-Cl cotransporter. J Membr Biol 94: 279-284, 1986[ISI][Medline].

28.   Kurtz, I, and Balaban RS. Ammonium as a substrate for Na-K-ATPase in rabbit proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 250: F497-F502, 1986[Abstract/Free Full Text].

29.   Lytle, C, and Forbush BF. Is intracellular chloride the switch controlling Na-K-2Cl cotransport in shark rectal gland? (Abstract). Biophys J 61: A34, 1992.

30.   Mandel, KG, McRoberts JA, Beuerlein G, Foster ES, and Dharmsathaphorn K. Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers. Am J Physiol Cell Physiol 250: C486-C494, 1986[Abstract/Free Full Text].

31.   Marti, U, Elsing C, Renner EL, Liechti-Gallati S, and Reichen J. Differential expression of Na+,H+-antiporter mRNA in biliary epithelial cells and in hepatocytes. J Hepatol 24: 498-502, 1996[ISI][Medline].

32.   Matthews, JB, Smith JA, Tally KJ, Awtrey CS, Nguyen H, Rich J, and Madara JL. Na-K-2Cl cotransport in intestinal epithelial cells. Influence of chloride efflux and F-actin on regulation of cotransporter activity and bumetanide binding. J Biol Chem 269: 15703-15709, 1994[Abstract/Free Full Text].

33.   McGill, JM, Basavappa S, Gettys TW, and Fitz JG. Secretin activates Cl channels in bile duct epithelial cells through a cAMP-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 266: G731-G736, 1994[Abstract/Free Full Text].

34.   McRoberts, JA, Beuerlein G, and Dharmsathaphorn K. Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line. J Biol Chem 260: 14163-14172, 1985[Abstract/Free Full Text].

35.   Mennone, A, Alvaro D, Cho W, and Boyer JL. Isolation of small polarized bile duct units. Proc Natl Acad Sci USA 92: 6527-6531, 1995[Abstract].

36.   Nathanson, MH, Burgstahler AD, Mennone A, Dranoff JA, and Rios-Velez L. Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver. J Clin Invest 101: 2665-2676, 1998[Abstract/Free Full Text].

37.   Panet, R, Markus M, and Atlan H. Bumetanide and furosemide inhibited vascular endothelial cell proliferation. J Cell Physiol 158: 121-127, 1994[ISI][Medline].

38.   Paulais, M, and Turner RJ. Activation of the Na-K-2Cl cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors. J Biol Chem 267: 21558-21563, 1992[Abstract/Free Full Text].

39.   Paulais, M, and Turner RJ. Beta-adrenergic upregulation of the Na-K-2Cl cotransporter in rat parotid acinar cells. J Clin Invest 89: 1142-1147, 1992[ISI][Medline].

40.   Roberts, SK, Ludwig J, and Larusso NF. The pathobiology of biliary epithelia. Gastroenterology 112: 269-279, 1997[ISI][Medline].

41.   Robertson, MA, and Foskett JK. Membrane crosstalk in secretory epithelial cells mediated by intracellular chloride concentration. Jpn J Physiol 44 Suppl: S309-S315, 1994[ISI][Medline].

42.   Sellinger, M, and Boyer JL. Physiology of bile secretion and cholestasis. Prog Liver Dis 9: 237-259, 1990[Medline].

43.   Singh, SK, Boron WF, Cavestro GM, and Boyer JL. Distinct apical and basolateral Na-H exchangers in isolated perfused intrahepatic bile duct segments (Abstract). Gastroenterology 112: A1384, 1997[ISI].

44.   Strazzabosco, M, and Boyer JL. Ion transporters that regulate intracellular pH and secretion in bile duct epithelial cells. In: Biliary and Pancreatic Ductal Epithelia: Pathobiology and Pathophysiology, edited by Sirica AE, and Longnecker DS.. New York: Marcel Dekker, 1997, p. 85-106.

45.   Strazzabosco, M, Mennone A, and Boyer JL. Intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 87: 1503-1512, 1991[ISI][Medline].

46.   Tseng, H, and Berk BC. The Na/K/2Cl cotransporter is increased in hypertrophied vascular smooth muscle cells. J Biol Chem 267: 8161-8167, 1992[Abstract/Free Full Text].

47.   Welsh, MJ. Evidence for basolateral membrane potassium conductance in canine tracheal epithelium. Am J Physiol Cell Physiol 244: C377-C384, 1983[Abstract].

48.   Welsh, MJ. Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloride-secreting epithelium. J Clin Invest 71: 1392-1401, 1983[ISI][Medline].

49.   Weymer, A, Huott P, Liu W, McRoberts JA, and Dharmsathaphorn K. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J Clin Invest 76: 1828-1836, 1985[ISI][Medline].


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