Na+-dependent and -independent Cl?/HCO3? exchange mediate cellular HCO3? transport in cultured human...

Na/-Dependent and -Independent Cl0/HCO03 Exchange Mediate

Cellular HCO03 Transport in Cultured Human Intrahepatic

Bile Duct Cells

MARIO STRAZZABOSCO,1 RUTH JOPLIN,2 AKOS ZSEMBERY,1 LORRAINE WALLACE,2 CARLO SPIRLI,1 LUCA FABRIS,1

ANNA GRANATO,1 ANDREA ROSSANESE,1 CARLO POCI,1 JAMES M. NEUBERGER,2 LAJOS OKOLICSANYI,3

AND GAETANO CREPALDI1

able. Administration of agents raising intracellularBiliary epithelial cells (cholangiocytes) modulate bilecAMP concentrations increased both Na/-dependentfluidity and alkalinity absorbing and/or secreting fluidand Na/-independent Cl0/HCO0

3 exchange activity. Stim-and electrolytes, particularly HCO03 and Cl0. Mecha-

ulation of Cl0/HCO03 exchange activity was not pre-nisms responsible for transepithelial H//HCO0

3 secretionvented by the Cl0 channel inhibitor 5*-nitro-2(2)-phe-in human cholangiocytes are largely unknown. Humannylpropyl-amino-benzoate(NPPB). In conclusion, hu-cholangiocytes isolated by enzymatic digestion and im-man cholangiocytes possess two acid extruders (Na//H/munomagnetic purification from normal liver tissue ob-exchanger and Na/-dependent Cl0/HCO0

3 exchange) andtained from reduced grafts used for pediatric liver trans-an acid loader (Cl0/HCO0

3 exchange), whereas no evi-plantation were cultured in the presence of humandence was found for cAMP activated H/-ATPase. Bicar-hepatocyte growth factor. Maintenance of cholangiocytebonate influx is thus mainly mediated by Na-dependentphenotypic features was assessed using markers suchCl0/HCO0

3 exchange, whereas Na/:HCO03 cotransport isas cytokeratin 19, g-glutamyltranspeptidase, vimentin,

not active in the physiological range of pHi. Stimulationfactor VIII-related antigen, desmin, epithelial mem-of Na/-independent Cl0/HCO0

3 exchanger by cAMP doesbrane antigen (EMA), and human epithelial antigennot require activation of Cl0 conductances. These mech-(HEA) 125. Intracellular pH (pHi) transients were mea-anisms may underlay hormone-regulated biliary HCO0

3sured microfluorimetrically 2*7*-Bis(2-carboxyethyl)-5,6,secretion in the human biliary tree. (HEPATOLOGYcarboxyfluorescein-acetossimethylester (BCECF). In1997;25:976-985.)the absence of HCO0

3 , pHi recovery from an intracellularacid load (ammonia pre-pulse technique) was Na/-de-pendent and amiloride-inhibitable. No Na/-independent

Interest in cholangiocyte physiology and pathophysiologyrecovery was recorded even after stimulation withhas been stimulated by the observation that the biliary epi-agents raising intracellular cyclic adenosine monophos-thelium is the primary target in several chronic cholestaticphate (cAMP) concentrations. In the presence ofliver disorders, including primary biliary cirrhosis, primaryHCO0

3 , recovery from an intracellular acid load requiredsclerosing cholangitis, and liver allograft rejection.1-5 Inter-Na/, but was only partly inhibited by amiloride. In theseestingly, in cystic fibrosis, a disease of ion transporting epi-conditions H/ extrusion was inhibited by 4,4,-diisothio-thelia, hepatobiliary complications occur in approximatelycyan atostilben-2,2,-disulfonic acid (DIDS) and by intra-30% of cases6 and ultrastructural bile duct cell damage cancellular Cl0 depletion. Acute removal of extracellular Cl0be shown well before hepatocellular damage.7 In this condi-induced a pHi alkalinization that was inhibited by DIDS.tion, low rates of ductular HCO0

3 and Cl0 secretion may leadpHi recovery from an intracellular alkaline load (isohy-to increased viscosity of bile and ‘‘ductular’’ cholestasis.7,8

dric CO2 changes) was Cl0-dependent and DIDS-inhibit-Hepatocellular bile is, in fact, extensively modified while

flowing through the biliary tree. The epithelial cells lining theintrahepatic bile ducts (cholangiocytes) modulate bile fluidity

Abbreviations: cAMP, cyclic adenosine monophosphate; NPPB, 5*-nitro-2(2)-phenylpro-and alkalinity absorbing or secreting fluid and electrolytes,pyl-amino-benzoate; Hepes, N-2-hydroxyethylpiperazine-N*-2-ethanesulfonic acid; DIDS,

4,4,-diisothiocyanatostilben-2,2,-disulfonic acid; DBcAMP, N6,2*-O-dibutyryladenosine particularly HCO03 and Cl0, under the influence of digestive

3*:5*-cyclic monophosphate; IBMX, 3-isobutyl-1-methyl-xanthine; BCECF-AM, 2*:7*-Bis(2- hormones such as secretin and somatostatin.1,3-5,9,10 Studiescarboxyethyl)-5,6, carboxyfluorescein-acetossimethylester; HEA, human epithelial antigen; in short-term cultured intrahepatic rat cholangiocytes haveg-GT, g-glutamyltranspeptidase; pHi, intracellular measurements; Bi, intrinsic buffering

characterized a number of membrane carriers and ion chan-power; KRB, bicarbonate buffered ringer.From the 1Institute of Internal Medicine, University of Padova and Azienda Ospedaliera nels possibly involved in transepithelial fluid and electrolyte

di Padova, Padua, Italy; 2Liver Unit, University Hospital, Birmingham, UK; and 3Chair of transport11,12 and suggested that, in the rat biliary epithe-Gastroenterology, University of Parma, Parma, Italy. lium HCO0

3 secretion was performed by an apical Cl0/Received June 25, 1996; accepted December 16, 1996.

HCO03 exchange, whereas HCO0

3 entry through the basolat-Sponsored by grant 94.02908.CT04 and ‘‘Progetto Nazionale Invecchiamento’’ from Con-siglio Nazionale delle Ricerche (Italy) and by ‘‘Centro per lo studio dell’invecchiamento’’, eral membrane was mediated by the electrogenic Na/:University of Padova. The financial support of Telethon-Italy (Grant MD.E-430) is gratefully HCO0

3 cotransport.11 On the other hand, studies in pig chol-acknowledged. Dr. Akos Zsembery was a recipient of an International Fellowship for the angiocytes suggested a role for cyclic adenosine mono-University of Padova.

phosphate (cAMP)-activated H/-ATPase in the generation ofPart of this work has been presented at the 95th Annual Meeting of the AmericanGastroenterological Association (New Orleans, LA, 1994) and published in Abstract form intracellular HCO0

3 .in Gastroenterology 106:A990, 1994. Because of the presence of a number of species-specific vari-

Address reprint requests to: Mario Strazzabosco, M.D., Institute of Internal Medicine, ations in bile physiology13,14 the relevance of the above trans-University of Padova, Via Giustiniani, 2, 35100 Padova, Italy. Fax: 39-49-8212151.

porters in human physiology is still uncertain. For example,Copyright q 1997 by the American Association for the Study of Liver Diseases.0270-9139/97/2504-0031$3.00/0 while, in the rat, bile secretion is relatively unresponsive to

976

AID Hepa 0023 / 5p1e$$$441 03-12-97 09:42:41 hpta WBS: Hepatology

HEPATOLOGY Vol. 25, No. 4, 1997 STRAZZABOSCO ET AL. 977

5 mg/mL, and penicillin/streptomycin). Human cholangiocytes prolif-hormonal stimulation, humans have developed the abilityerate and form a confluent monolayer within 2 weeks; these cellsto acutely and dramatically modify bile composition duringcan be resuspended and reinseminated or frozen in liquid nitrogenphases of increased digestive activity.15 To provide a modelto be studied later. Cells were studied between the second and thefor ductular secretory function relevant to human physiologythird passage. The protocol was approved by the local ethical commit-and diseases, we have studied acid/base regulatory mecha- tees.

nisms in primary cultures of human bile duct cells, and in Characterization of Human Cholangiocyte Cultures. The mainte-bile duct cell lines of human origin. nance of phenotypic features of human cholangiocytes was assessed

Our results show that human cholangiocytes possess three by staining for HEA 125, epithelial membrane antigen, vimentin,desmin, factor VIII-related antigen, and the specific bile duct cellmain acid/base transporters: Na//H/ exchange, Na/-depen-markers g-glutamyltranspeptidase (g-GT) and cytokeratin 19, as de-dent Cl0/HCO0

3 exchange, and Cl0/HCO03 exchanger,

scribed.14whereas, no evidence was found for a cAMP-stimulated H/-In the initial isolate the purity determined by the above mentionedATPase. Both anion exchangers are activated by cAMP (se-

markers, was higher than 95% and viability tested by Trypan bluecretin’s second messenger). Cl0/HCO03 exchanger activation

exclusion was 95%. The cholangiocyte phenotypic markers, cytokera-by cAMP is not inhibited by the Cl0channel inhibitor 5*-nitro- tin 19 and g-GT were maintained throughout the culture, whereas2(2)-phenylpropyl-amino-benzoate (NPPB). Thus, in human the cells remained negative for desmin and factor VIII-related anti-cholangiocytes hormone-stimulated transepithelial HCO0

3 in- gen. As already described,14 after the fourth week, cells began to loseflux is mainly mediated by Na/-dependent and -independent HEA 125 positivity, and the percentage of vimentin-positive cells

progressively increased from 5% to 100% in the latest cultures (5thanion exchangers.passage).

MATERIALS AND METHODS Culture of Biliary Cell Lines. Two biliary cell lines (SK-Cha1 andMZ-Cha1) (kindly provided by Dr. A. Knuth, Mainz University,

Materials Mainz, Germany), both established from human biliary adenocarci-nomas, were cultured in aMEM medium supplemented with 10%N-2-hydroxyethylpiperazine-N*-2-ethanesulfonic acid (Hepes),fetal calf serum and penicillin/streptomycin (100 U-0.1 mg/mL) in aamiloride, dimethylsulfoxide, nigericin, 4,4,-diisothiocyan atostilben-humidified 95% air, 5% CO2 atmosphere.20 Two to three days before2,2,-disulfonic acid (DIDS), D-gluconolactone, Na/-gluconate, K-glu-the experiment, cells were suspended by washing with Ca2/-Mg2/-conate, Ca-gluconate, gluconic acid, N-methyl-D-glucamine, cholinefree phosphate-buffered saline containing 0.68 mmol/l ethylenedi-chloride, choline bicarbonate, ammonium bicarbonate, Na/-propio-aminetetraacetic acid plated over glass coverslip fragments con-nate, epidermal growth factor, fibronectin, insulin, hydrocortisone,tained into tissue culture plastic wells (Falcon) and cultured withcholera toxin, triiodothyronin, ethylenediaminetetraacetic acid, for-the same aMEM medium, until confluent. These cells have beenskolin, N6,2*-O-dibutyryladenosine 3*:5*-cyclic monophosphateextensively used as a model biliary cell line21-24 and are positive for(DBcAMP), and 3-isobutyl-1-methyl-xanthine (IBMX) were pur-the bile duct cell markers g-GT and cytokeratin 19.22

chased from SIGMA Chemical Company (20127 Milano, Italy). 2*:7*-Bis ( 2 - carboxyethyl ) - 5 , 6 , carboxyfluorescein -acetossimethylester

pHi Measurement(BCECF-AM) was purchased from Molecular Probes Inc. (97402 Eu-gene, Oregon). NPPB was a generous gift from Dr. Greger (Freiburg,

For intracellular pH (pHi) measurements cells were inseminatedGermany). Culture media (a-minimum essential medium, Dulbecco’sat high density onto fibronectin-coated semipermeable membranemodified Eagle medium, HAM F12, fetal bovine serum, penicillin/inserts (Falcon 0.45 mm pore size) or over glass coverslip fragmentsstreptomycin, trypsin/ethylenediaminetetraacetic acid, glutamine)and cultured until subconfluent. Twenty-four hours before the exper-were from Gibco (14072 Grand Island, New York). Percoll was pur-iment medium was changed to quiescent medium (HAM F12 supple-chased from Pharmacia (20093 Cologno Monzese, Italy). Human he-mented with 3% fetal calf serum and penicillin/streptomycin, butpatocyte growth factor was obtained from the Mitsubishi Corporationwithout, insulin, epidermal growth factor, cholera toxin, triiodothy-(Yokohama 227, Japan).16 The monoclonal Ab human epithelial anti-ronine, and hydrocortisone).gen (HEA) 125 was purchased from Progen Biotechnik GmbH (69120

pHi was measured with the fluorescent dye BCECF24,25 that wasHeidelberg, Germany), and Dynabeads from Unipas (20127 Milano,loaded into the cells by incubation for 10 to 20 minutes at 377C withItaly). Antibodies against epithelial membrane antigen, vimentin,a concentration 12 mmol/L of its tetracetoxymethylester derivativedesmin, factor VIII-related antigen, and cytokeratin 19 were pur-(BCECF-AM). At the end of the incubation period, to remove thechased from Dako Corporation (DK2600 Glostrup, Denmark).extracellular dye, cells were washed for 10 minutes at 377C in aBCECF-free medium. Cultured human cholangiocytes easily took upCell Isolation and Culturethe dye that showed an even distribution in the cytoplasm. Leakageand photobleaching were negligible; in fact the experiment could beIsolation and Culture of Cholangiocytes. Human cholangiocytes

were isolated from fragments obtained from six livers used for re- performed for 40 minutes without significant loss of 440-nm signal.At the end of the loading procedure, cholangiocytes were transferredduced size orthotopic liver transplantation as recently de-

scribed.14,17,18 Briefly, 30 g of liver tissue were finely minced and into a thermostated (377C) perfusion chamber, placed on the stageof an inverse microscope Nikon TDM (Florence, Italy). Gassing ofdigested with collagenase (1%) for 3 hours at 377C. The tissue was

then filtered to eliminate the undigested debris. Nonparenchymal perfusion media (100% O2, for Hepes-buffered solutions, and 95%O2/5% CO2 mixture for solutions containing 25 mmol/L HCO0

3 ) wasliver cells were then separated from hepatocytes, red blood cells, anddebris by centrifugating on a discontinuous gradient of Percoll at performed by an artificial lung. The set-up has been previously de-

scribed.23 Briefly, the microscope is connected with a microfluori-800g for 30 minutes. The cells banding at the density interface 1.04-1.09 were harvested and further purified by immunomagnetic sepa- meter Spex (Spex Industries, Edison, NJ; Barletta Strumenti Scien-

tifici, Milano, Italy) equipped for continuous dual excitation ratio.ration with a monoclonal Ab (HEA 125) against a 34 kD membraneglycoprotein (egp 34) that, in the liver, is expressed only by the biliary The sample is excited by the light emitted by 150W xenon lamp

passing through two interferential filters, with an emission peak atepithelium.14,17-19 Nonparenchymal liver cells were incubated for 30minutes at 377C with 5 mg/mL HEA 125 mAb, followed by 30 minutes 495 nm or at 440 nm and the light intensity is then lowered by a

0.1% neutral density filter, to reduce both photodynamic damage toat 47C incubation with sheep anti-mouse IgG1 conjugated Dyna-beads. The cells recognized by HEA 125 mAb were then separated the cell and photobleaching of the tracer. Emitted light is then cap-

tured by a Nikon 40x Fluor N.A. 1.3 objective and read by a photonfrom contaminating cells by applying a Magnetic Particle Concentra-tor (MPCTM-1, Dynal, Oslo, Norway) for 2 minutes followed by two counting photometer. The analysis field is limited by a pin hole sur-

rounding a circular area enclosing about 5 to 10 cells. For each excita-washes with culture medium.The human cholangiocytes were resuspended and inseminated in tion wavelength data were acquired for 500 msec every 1.5 seconds,

averaged, and displayed as a single experimental point. Since cholan-Falcon plastic flasks and incubated with Dulbecco’s modified eaglemedium HAMF12 medium14 containing 10 ng/mL of hHGF, a growth giocyte autofluorescence was not higher than background, at the

end of each experiment, background fluorescence was determined byfactor that has been previously shown to stimulate cholangiocytegrowth in culture (other constituents were FBS 3%, epidermal measuring a cell-free area of the same size and subtracting from

fluorescent intensity readings.11,26 Signal to background ratio at 440growth factor 10 ng/mL, hydrocortisone 0.4 mg/mL, cholera toxin 10ng/mL, transferrin 5 mg/mL, tri-iodothyronine 21 1009 mol/L, insulin nm was approximately 110:1.


978 STRAZZABOSCO ET AL. HEPATOLOGY April 1997

Fluorescence 495/440 ratio data were converted to pHi values by sible for steady state pHi maintenance, in the absence ofgenerating a calibration curve at the end of each experiment. Cells HCO0

3 , is a Na//H/ exchange. In presence of HCO03 in the

were exposed to the K//H/ ionophore nigericin (12 mmol/L) in a Na/- perfusion medium (KRB), basal pHi also appeared to befree medium containing high K/ concentrations and buffered at three maintained by a Na/-dependent mechanism, because re-different pH values, as previously described.11,24,27 Cellular intrinsic moval of extracellular Na/ decreased pHi by 0.32 { 0.09 pHbuffering power (bi) was determined at different pHi, by expos-

units; n Å 4. As in rat cholangiocytes, administration of 1ing cells to stepwise decreasing NH4Cl concentrations, as de-mmol/L amiloride, in the presence of HCO0

3 (KRB), did notscribed.11,24,26,28 The total intracellular buffering power in the pres-cause a significant acidification (0.04 { 0.02); n Å 4. Lack ofence of the open buffering system ([H2CO3/CO2]) was then calculated

from bi as: btot Å bi / 2.302 1 [HCO03 ]i, where intracellular acidification following amiloride suggests, that in the pres-

HCO03 concentration is derived from the measured pHi using the ence of HCO0

3 the metabolic acid load is lower, or, most likely,Henderson-Hasselbach equation. Rates of pHi changes in the alka- that a second Na/-dependent, amiloride-insensitive acid ex-line or in the acid direction were measured as dpHi/dt by hand- truder, is activated. In cells preincubated for 40 minutes withdrawing a tangent from the experimental plot.29 Transmembrane H/

0.5 mmol/L DIDS, pHi was lower than controls (6.91 { 0.18fluxes (JH/) were calculated from bi and dpHi/dt as JH/ Å intrinsic n Å 18, P õ .02) thus suggesting that this additional DIDS-buffering power 1 dpHi/dt.11,24,30

inhibitable HCO03 -dependent carrier is active at basal pHi.

Demonstration of Na//H/ Exchange. To better clarify theSolutionsmechanisms responsible for H/ extrusion in human cholangio-

Buffers for pHi experiments were essentially as described.11,24,30-32

cytes, mechanisms of pHi recovery after acute intracellularBriefly, bicarbonate-free ringers (Hepes) were buffered with Hepes acid load, induced by rapid administration and removal ofat pH 7.4 and contained the following salts (mol/L): NaCl 135, KCl

NH4Cl,11,24,30 were studied. As shown in Fig. 1A and Table 1,4.7, MgSO4 1, KH2PO4 1.2, CaCl2 1.5, Hepes 10, Glucose 5, and Pyru-cholangiocyte pHi rapidly increased after administration ofvate 1, titrated to pH 7.4 with NaOH. Bicarbonate buffered ringer20 mmol/L NH4Cl and decreased after NH4Cl withdrawal.(KRB) contained: NaCl 115, KCl 4.7, KH2PO4 1.2, MgSO4 1, CaCl2

1.5, NaHCO3 25, Glucose 5, and Pyruvate 1 and was equilibrated When this maneuver was performed in cells perfused withwith 5% CO2. In KRB and Hepes used to acid load cells, 20 mmol/L Hepes medium, cholangiocyte pHi decreased to 6.47 { 0.08.NH4Cl substituted for equal amounts NaCl. In Na/-free Hepes or Cells recovered from this acid load extruding protons at aKRB, equimolar choline substituted for Na/. In Cl0-free KRB 115 rate (JH/) of 12.9 { 5.6 mmol/L/min, (measured at pHi 6.6,mmol/L isethionic acid and 6.2 mmol/L. Gluconic acid substituted n Å 8). pHi did not recover if, after NH4Cl removal, cells werefor Cl0. In Cl0-free solutions used to acid load the cells 25 mmol/L perfused with a Na/-free medium. As shown in Fig. 1B, inNH4HCO3 substituted for NaHCO3. In Na/- and Cl0-free KRB 115

these conditions pHi acidified to 6.22{ 0.04 and H/ extrusionmmol/L N-methyl-D-glucamine and 25 mmol/L choline substitutedwas blocked (JH/ Å 1.33 { 0.89 mmol/L/min, at pHi 6.3 n Åfor Na/. In isohydric alkaline pulses, KRB contained 50 mmol/L12 P õ .01), until Na/ was readmitted. In another set ofNaHCO3 (NaCl was 90 mmol/L) and was equilibrated with 10% CO2.

BCECF-AM was dissolved in DMSO as a 1 mmol/L stock solution. experiments (n Å 7), at the moment of NH4Cl withdrawal,Amiloride was dissolved in dimethylsulfoxide as a 1 mmol/L stock cells were perfused with the Na//H/ exchange inhibitor, amil-solution, while Nigericin was solubilized in ethanol. oride (1 mmol/L). As shown in Fig. 1C, in the presence of

amiloride, cells reached a more acid pHi as respect to controlsStatistical Analysis (6.24 { 0.04) and H/ extrusion was significantly inhibited

(JH Å 1.83 { 0.03, at pHi 6.6 n Å 7, P õ .01). These dataResults are shown as arithmetical averages { SD. Paired andunpaired t test analyses were performed using Statgraph software are consistent with baseline pHi data and indicate that in(STSC, Inc. Rockville, MD). the nominal absence of HCO0

3 human cholangiocytes recoverfrom an intracellular acid load by a Na/-dependent, amiloride

RESULTS sensitive mechanism (Na//H/ exchanger).Demonstration of Na/-Dependent Cl0/HCO0

3 Exchange. ToCellular bi. Rather than being a constant value, in manycell types, intrinsic cellular bi is inversely related to pHi. bi characterize mechanisms that mediate H/ extrusion in the

presence of HCO03 cells perfused with KRB were acutely acidi-was therefore determined at different pHi by exposing the

cells to step-wise decreasing concentrations of a permeant fied by pulsing with NH4Cl, as described above. After NH4Clremoval, cholangiocytes acidified to 6.48 { 0.03 and thenweak base (NH4Cl) as described.11,26,28 To exclude all trans-

port systems able to counter-regulate pHi, experiments were rapidly recovered basal pHi (Fig. 2A and Table 1). JH/ mea-sured at pHi 6.6 in these conditions was 15.8 { 3.5 mmol/L/performed in the absence of Na and HCO0

3 . bi values mea-sured in six different experiments were grouped and related min, n Å 7. In the absence of extracellular Na/ (substitution

with choline) cells reached a more acidic pHi (6.20 { 0.10)with the respective pHi using a best-fit program (Enz-Fitter,Elsevier Biosoft, Cambridge, UK). bi was 35.10 mmol/L/min and acid extrusion was blocked (JH/ Å 0.4 { 0.3 mM/min,

at pHi 6.3; n Å 4, 97% inhibition, P õ .001) until Na/ wasat pHi 6.7, a value very close to that reported for many othercell types, including rat hepatocytes and cholangiocytes. The readmitted (JH Å 17.6 { 9.9 mmol/L/min at pHi 6.7) (Fig.

3A). In contrast, proton extrusion was only partly inhibitedbi value varied from 62.57 mmol/L/min at pHi 6.5 to 14.47mmol/L/min at pHi 7.0. A similar inverse relationship be- by administration of 1 mmol/L amiloride. In these conditions

amiloride inhibited transmembrane H/ flow only by 56%tween bi and pHi has also been reported for rat cholangio-cytes.11,12 Buffering power of SK-ChA-1 and MZ-ChA-1 cell (JH/ Å 8.65 { 3.86, at pHi 6.6; n Å 10, P õ .01) suggesting

that in the presence of HCO03 part of acid extrusion is medi-lines had already been determined.24

Basal pHi. Basal pHi of cultured human cholangiocytes ated by an Na/-dependent mechanism, other than Na//H/

exchange (Fig. 2B).measured in the nominal absence of HCO03 from the perfusion

medium (Hepes buffer) was 6.85 { 0.15; n Å 35. This value This HCO03 -dependent transport system is inhibited by

DIDS. In six experiments, pHi recovery from an intracellularwas significantly lower than that measured in presence ofHCO0

3 (KRB): 7.03 { 0.14; n Å 69 (P õ .01). When cells acid load was evaluated in cells pretreated for 40 minuteswith 0.5 mmol/L DIDS and then washed for 15 minutes inincubated in Hepes were exposed to Na/-free medium (re-

placed with the impermeant monovalent cation choline) basal DIDS-free KRB; after the acid load, cells were exposed toamiloride, to avoid the contribution of Na//H/ exchange. AcidpHi rapidly decreased by 0.35{ 0.06 pH units; nÅ 4. Similar

effects were recorded after administration of 1 mmol/L amilo- extrusion was significantly reduced in comparison with con-trols in the presence of amiloride, but without pretreatmentride, an inhibitor of Na//H/ exchange, in HCO0

3 -free media(Hepes). In these conditions, pHi diminished by 0.135 { 0.05 with DIDS (JH/ Å 2.45 { 0.86, at pHi 6.6; n Å 6 vs. 8.65 {

3.86, at pHi 6.6; n Å 10, P õ .01) (Fig. 2C).pH units; n Å 4, thus suggesting that the mechanism respon-



TABLE 1. H/ Extrusion Rates (JH/) UnderDifferent Experimental Conditions

TracingAt in

Nadir pHi JH/ pHi n Figure

Hepes 6.47 { 0.08 12.9 { 5.60 6.6 8 1aHepes Na-free 6.22 { 0.04 1.33 { 0.89 6.3 12 1bHepes amiloride 6.24 { 0.04 1.83 { 0.03 6.6 7 1cKRB 6.48 { 0.03 15.8 { 3.50 6.6 7 2aKRB Na-free 6.20 { 0.10 0.40 { 0.30 6.3 4 3aKRB amiloride 6.26 { 0.89 8.65 { 3.86 6.6 10 2bKRB DIDS / amiloride 6.31 { 0.16 2.45 { 0.86 6.6 6 2cKRB recovery from Na-free 6.20 { 0.10 17.6 { 9.90 6.7 4 3a/amiloride 6.18 { 0.06 9.40 { 3.73 6.5 13 3b0 Cl / amiloride 6.30 { 0.10 3.36 { 1.98 6.5 30 3c

Carriers capable of mediating DIDS-sensitive, amiloride-insensitive Na/-, and HCO0

3 -dependent acid extrusion, in-clude Na/-dependent Cl0/HCO0

3 exchange and Na/:HCO03 co-

transport.30 Because these carriers can be distinguished onthe basis of their Cl0-dependance, we measured acid extru-sion in cells that had been depleted of intracellular Cl0 by40 minutes of exposure in Cl0-free KRB medium. After ad-ministration/withdrawal of 25 mmol/L NH3/NH4 and follow-ing removal of extracellular Na/, pHi decreased to 6.30 {0.10. The cells were then exposed to amiloride, in a mediumcontaining Na/, but not Cl0. In these conditions, H/ extrusionwas slower than that measured in the presence of Cl0 andamiloride (JH/ at pHi 6.5 was 3.36 { 1.98 mmol/L/min; n Å30 vs 9.40{ 3.73 mmol/L/min at pHi 6.5; nÅ 13, respectively,P õ .02) (Fig. 3C). The Cl0-dependance of acid extrusion ratesuggests that the mechanism mediating Na/ and HCO0

3 de-pendent acid extrusion is an Na/-dependent Cl0/HCO0

3 ex-change. Although pHi recovery was strongly inhibited in theabsence of Cl0, as shown in Figs. 3C and 4, at low pHi a smallpercentage of H/ extrusion occurred even in the absence ofCl0. When the experiment was repeated in Cl0-depleted cellsthat had been pretreated with 0.5 mmol/L DIDS (n Å 6), pHirecovery after Na/ readdition was similar (2.95 { 0.93) atpH 6.5 but was significantly lower at pHi 6.4 (4.34 { 2.02),suggesting that an Na/-dependent, DIDS-inhibitable Cl0-in-dependent transporter contributes to acid extrusion at lowpHi (Fig. 4 and Table 2).

Na-dependent Cl0/HCO03 exchange is expressed also in the

human biliary cell lines MZ-Cha-1 and SK-ChA-1 (Table 3).In control conditions, acidified cells recovered to baseline pHiat a rate of 10.5 { 1.41 mmol/L min and 14.5 { 4.0 mmol/L/min at pHi 6.6 in SK-ChA-1 and MZ-ChA-1 cells, respectively.After removal of external Na/, pHi recovery was greatly in-hibited (2.69 { 0.9 mmol/L/min at pHi 6.55; n Å 4 in SK-Cha-1 cells and 1.36 { 1.34 at pHi 6.42 in MZ-Cha-1 cells, Põ .01), and fully recovered to baseline after Na/ readmission.In the presence of amiloride (1 mmol/L), pHi recovery wasonly partly reduced (5.93 { 1.83 at pHi 6.6, n Å 5 in SK-Cha-1 cells, and 8.58 { 2.84 at pHi 6.6 n Å 12 in MZ-Cha-1 cells,P õ .01). Consistent with data from primary cholangiocyteFIG. 1. Representative pHi tracings showing the effects of Na/ removal

(B) and amiloride (C) on pHi recovery from an intracellular acid load induced cultures, recovery from an acid load was inhibited by preincu-by pulse-withdrawal of 20 mmol/L NH4Cl in the nominal absence of HCO0

3 bation of cells with 0.5 mmol/L DIDS for 40 minutes (0.03 {(Hepes). (A) Control pulse. In cholangiocytes perfused with Hepes (a-b), pHi0.01 n Å 3 and 2.25 { 1.65 n Å 5 for SK-Cha-1 and MZ-rises upon exposure to NH4Cl (b-c) because of the entry of the permeant weakCha-1 cells, respectively, P õ .001). While in MZ-ChA-1 cellsbase NH3 and its protonation to the impermeant NH/

4 . After withdrawal ofexternal NH4Cl (c), NH/

4 releases a proton and leaves the cell as NH3, thus absence of Na/ completely inhibited pHi recovery, an addi-acidifying the cytoplasm. Human cholangiocytes rapidly recovered to baseline tional organic anion/HCO0

3 exchange mechanism could be op-by extruding protons (nÅ 8 from one preparation) (c-d). (B) In this experiment,erative in SK-ChA-1 cells, that show a small Na/-dependent,at the time of NH4Cl withdrawal (c), external Na/ was substituted by cholineDIDS-inhibitable acid extrusion. To evaluate the Cl0-de-and pHi recovery was completely blocked until Na/ was readmitted (n Å 12

from two preparations) (d). (C) In this experiment, at the time of NH4Cl with- pendance of pHi recovery, cells were depleted of intracellulardrawal (c) cells were exposed to 1 mmol/L amiloride (c-d), an inhibitor of Na// Cl0, by incubation in Cl0-free KRB for 40 minutes and pHiH/ exchange; pHi recovery was greatly inhibited (n Å 7 from one preparation).

recovery was evaluated following a protocol similar to Fig.3C. In Cl0-depleted cells pHi recovery in the presence of Na/

and amiloride was 1.04 { 0.32 at pHi 6.6; n Å 4 in SK-Cha-



FIG. 3. Representative pHi tracings showing the effects of Na/ removal(A), amiloride (B), and Cl0 depletion (C) on pHi recovery from an intracellular

FIG. 2. Representative pHi tracings showing the effects of amiloride (B) acid load induced by pulse-withdrawal of 20 mmol/L NH4Cl in HCO03 /CO2

and amiloride / DIDS (C) as respect to controls (A) on pHi recovery from an buffered media (KRB). (A) Effects of Na/ removal; a-b: KRB; b-c: NH4; c-d:intracellular acid load induced by pulse-withdrawal of 20 mmol/L NH4Cl in choline (Na/-free); d-e: KRB (Na/ readmission); pHi recovery was completelyHCO0

3 /CO2 buffered media (KRB). (A) Control pulse. a-b: KRB; b-c: NH4Cl; c- blocked until Na/ was readmitted (n Å 4 from one preparation). (B) In thisd: KRB (n Å 7 from one preparation). (B) In this experiment, at the time of experiment, at the time of Na/ readmission (d-e), cells were exposed to 1NH4Cl withdrawal (c) cells were exposed to 1 mmol/L amiloride, that only mmol/L amiloride; pHi recovery was only partly inhibited (n Å 13 from threepartly inhibited pHi recovery (c-d) (Fig. 2b) (n Å 10 from one preparation). (C) preparations). (C) In this experiment, cells were previously depleted of intracel-This experiment shows the effect of DIDS on pHi recovery; cells were pre- lular Cl0 by preincubation in Cl0-free media for 40 minutes; a-b: Cl0-freetreated with 0.5 mmol/L DIDS for 40 minutes before the experiment and were media; b-c: exposure to NH4/NH3; c-d: exposure to Na/-free, Cl0-free media;then acidified by pulsing with NH4Cl as described (b-c); amiloride 1 mmol/L d-e: readmission of Na/, in the presence of 1 mmol/L amiloride, still in thewas added after NH4Cl withdrawal to inhibit Na//H/-dependent pHi recovery; absence of Cl0. Comparison with panel B clearly shows that pHi recovery isDIDS pretreatment clearly inhibited pHi recovery (c-d) (n Å 6 from one prepa- greatly inhibited by prior Cl0 depletion (n Å 30 from three preparations).ration).

AID Hepa 0023 / 5p1e$$0023 03-12-97 09:42:41 hpta WBS: Hepatology


pHi increased by about 0.22 { 0.05 pH units because of CO2

exit from the cells. Human cholangiocytes recovered to basalpHi by extruding HCO0

3 at a rate JOH0 Å 2.16 { 0.97 mmol/L/min, n Å 33. In the presence of KRB containing 25 mmol/L HCO0

3 /5% CO2 but without Cl0, alkalinization was higher(0.26 { 0.06 pH units; n Å 5) and pHi did not recover tobaseline until Cl0 was readministered (Fig. 5B). If the sameexperiment was repeated in cells pretreated with 1 mmol/LDIDS for 30 minutes, base extrusion was strongly inhibited(JOH0 Å 1.07 { 0.8, n Å 13, P õ .02) (Fig. 5C). These dataclearly indicate that human cholangiocytes possess a DIDS-inhibitable Cl0/HCO0

3 exchange, that mediates HCO03 extru-

sion after an intracellular alkaline pulse.Effect of cAMP on Bicarbonate Transport in Human Cholan-

giocytes. To evaluate the effects of cAMP on HCO03 transport

in human cholangiocytes, Cl0/HCO03 exchange activity was

measured during the alkalinization phase following extracel-lular Cl0 removal in the presence or the absence of agentsFIG. 4. Comparison between pHi recovery from an intracellular acid loadincreasing intracellular cAMP concentration.22,35-39 After ain HCO0

3 /CO2 buffered media. In the first part of the experiments (not shown)cholangiocytes were acid-loaded and then exposed to a Na/-free medium. Trac- first control removal of Cl0 cells recovered to basal pHi anding A shows pHi recovery after Na/ readmission; in tracing B, at the moment were perfused with IBMX (100 mmol/L), forskolin (3 mmol/L),of Na/ readmission amiloride 1 mmol/L was added; in tracing C cells were and DBcAMP (100 mmol/L) for 10 minutes.22,35-39 pHi beforedepleted in intracellular Cl0 by prior incubation in Cl0-free media (see Fig.

the second Cl0 removal pulse (7.05 { 0.18) was not signifi-3C) and Na/ was readmitted in the presence of amiloride (1 mmol/L) but stillin the absence of Cl0; in tracing D cells were exposed to the same protocol as cantly different from that measured before the first pulse.in tracing C, but were pretreated with DIDS 0.5 mmol/L (n Å 6 from one Cl0 was again removed and the activity of the exchangerpreparation). From these tracings it is evident that 1) H/ extrusion is mostly reassessed. In these conditions (n Å 33 from five prepara-Cl0-dependent; 2) a small percentage of H/ extrusion occurs in the absence of

tions) Cl0/HCO03 exchange activity increased from 1.30{ 1.31Cl0 at low pHi and is partly inhibited by DIDS (see also Table 2).

up to 2.36 { 2.41 (P õ .05) during the alkalinization phaseafter Cl-removal, and from 4.28 { 4.75 to 6.51 { 7.10 during

1 cells and 1.54 { 0.90 at pHi 6.6; n Å 4 in MZ-Cha-1 cells the acidification phase following Cl-readmission. Cl0/HCO03

versus 6.23 { 3.56 at pHi 6.6; n Å 4 and 9.86 { 2.52 at pHi exchange activity was also measured by evaluating the recov-6.6; n Å 5 in SK-Cha-1 and MZ-Cha-1 cells, respectively in ery rate from an acute intracellular alkalinization inducedthe presence of Cl0 and amiloride. These data indicate that by isohydric changes in CO2/HCO0

3 concentration in the per-also in human biliary cell lines the amiloride-insensitive, H/ fusion medium, as described above (Fig. 5). In experimentsextrusion mechanism activated in the presence of HCO0

3 is in which the alkaline pulse was performed after perfusionNa/ and Cl0-dependent and DIDS-inhibitable, as expected with IBMX, forskolin and DBcAMP for 10 minutes (samefor an Na/-dependent Cl0/HCO0

3 antiporter. doses), Cl0/HCO03 exchange activity was 2.92 { 0.88 (n Å 21

Demonstration of Cl0/HCO03 Exchange. Immunoreactivity from five preparations) compared with 1.80 { 0.49 (n Å 16

for the AE-2 anion exchanger (an Na/-independent Cl0/ from five preparations, P õ .02) measured in paired controlHCO0

3 exchange) has been shown at the apical membrane of pulses. These data are thus consistent with activation of chol-cholangiocyte in histological sections.33 To obtain functional angiocyte Cl0/HCO0

3 exchange by cAMP. To evaluate the con-evidence of its presence in cultured human cholangiocytes, tribution of Cl0 conductances to the stimulation of Cl0/pHi changes were monitored during acute removal of extra- HCO0

3 exchange activity induced by cAMP, cells were exposedcellular Cl0.11,12 In the presence of a Cl0/HCO0

3 exchange, this both to cAMP and to NPPB (10 mmol/L)12 and subjected tomaneuver causes intracellular alkalinization, because the alkali load procedure. JH/ in these conditions was 3.21HCO0

3 enters the cell in exchange with intracellular Cl0 that { 0.46 (n Å 6 from two preparations), indicating that NPPBis forced to exit.31,32 Indeed, following this protocol cholangio- was unable to prevent the cAMP-mediated changes in Cl0/cyte intracellular pH alkalinized by 0.18 { 0.06 pH units, HCO0

3 exchange activity.and cells extruded protons at a rate of JH/ Å 1.53 { 1.49 As discussed above, in human cholangiocytes Na/-depen-mmol/L/min (n Å 38 from three preparations). In cells prein- dent Cl0/HCO0

3 exchange is responsible for amiloride-insensi-cubated for 30 minutes with 1 mmol/L DIDS no pHi alkalin- tive, Na/- and HCO0

3 -dependent acid extrusion. Its activityization was recorded, consistent with the presence of a Cl0/ can be estimated by acidifying the cells and then measuringHCO0

3 exchange in cholangiocytes. H/ extrusion in the presence of amiloride in cells perfusedBecause in epithelial cells the AE-2 anion exchanger works with KRB. To study the effects of cAMP on Na/-dependent

as an acid loader, opposing acute changes of pHi in the alka- Cl0/HCO03 exchange activity, amiloride-independent H/ ex-

line direction, we also evaluated rates of recovery to basal trusion rates were measured before and after administrationpHi from an intracellular alkaline pulse induced by isohydric of IBMX, forskolin and DBcAMP (Fig. 6). JH/ at pH 6.6 in-variation of HCO0

3 /CO2 concentration in the perfusion me-dium (Fig. 5A).31,34 This maneuver increased intracellularHCO0

3 concentration without changing Cl0 concentration,TABLE 2. Effect of Cl0 Depletion on H/ Extrusion Ratesand thus enabled us to measure Cl0/HCO0

3 exchange in itsusual operation mode (i.e., as an alkali extruder) avoiding JH/ at pHi 6.4 JH/ at pHi 6.5 JH/ at pHi 6.6changes in volume and membrane potential induced by the

Amiloride† 14.51 { 5.57 9.40 { 3.73 5.19 { 3.44Cl0 depletion. Because the Na/-dependent Cl0/HCO03 ex-

0 Cl0 / amiloride‡ 6.56 { 3.63*

3.36 { 1.98 1.86 { 1.43change only functions as an acid extruder and it is not active0 Cl0 / amiloride / DIDS§ 4.34 { 2.02 2.95 { 0.93 2.03 { 0.93at alkaline pHi30 recovery rates from the alkaline pulse are

a representative measure of Cl0/HCO03 exchange. Human * P õ .05.

cholangiocytes perfused with KRB containing 50 mmol/L † See tracing B in Fig. 4.HCO0

3 /10% CO2, (pHo 7.4) were re-exposed to normal KRB ‡ See tracing C in Fig. 4.§ See tracing D in Fig. 4.containing 25 mmol/L HCO0

3 /5% CO2 (pH 7.4). At this point



TABLE 3. Comparison Between hBDE and Other Cell Lines in HCO3/CO2 Containing Media

hBDE MZ-ChA-1 SK-ChA-1

JH/ At pHi n JH/ At pHi n JH/ At pHi n

Control 15.8 { 3.50 6.6 7 14.5 { 4.00 6.6 8 10.5 { 1.41 6.6 90 Na/ 0.40 { 0.30 6.3 4 1.36 { 1.34 6.42 4 2.69 { 0.90 6.55 4%Amiloride 8.65 { 3.86 6.6 10 8.58 { 2.84 6.6 12 5.93 { 1.83 6.6 5%DIDS%amiloride 2.45 { 0.86 6.6 6 2.25 { 1.65 6.42 5 0.03 { 0.01 6.7 3

Recovery from Na-free%Na/ 17.6 { 9.90 6.7 4 17.4 { 7.85 6.7 9 14.97 { 8.42 6.7 4%Na/%amiloride 5.19 { 3.44 6.6 13 9.86 { 2.52 6.6 5 6.23 { 3.56 6.6 40 Cl%Na/%amiloride 1.86 { 1.43 6.6 30 1.54 { 0.90 6.6 3 1.04 { 0.32 6.6 40 Cl%Na/%amiloride%DIDS 2.03 { 0.93 6.6 6 0.94 { 0.31 6.6 4 1.55 { 0.61 6.65 5

creased from 8.24 { 3.62 mmol/L/min to 11.98 { 2.46 mmol/ The presence of Na//H/ exchanger is suggested by the fol-lowing observations: 1) in HCO0

3 -free media basal pHi de-L/min after stimulation (n Å 6, P õ .05), suggesting that anincrease in cAMP levels may stimulate HCO0

3 influx through creased after removal of external Na/ and during administra-tion of amiloride; 2) in the absence of HCO0

3 , recovery fromNa/-dependent Cl0/HCO03 exchange.

A role for a vesicular-type H/-ATPase in secretin-stimu- an intracellular acid load was Na/-dependent and amiloride-inhibitable. Sensitivity to amiloride suggests that cholangio-lated biliary HCO0

3 secretion has been suggested by the obser-vation that in microdissected pig intrahepatic ductules cAMP cyte Na//H/ exchanger may be functionally similar to the

growth factor-inducible isoform NHE-1, that in addition toanalogues and secretin stimulate Na/-independent and amil-oride-insensitive, baphilomycin A1-inhibitable H/ extrusion pHi homeostasis is involved in cell volume and mitosis regu-

lation. Figure 4 and Tables 1 and 2 show that a component offrom the cell.40 However, when human cholangiocytes wereacid-loaded and exposed to IBMX 100 mmol/L, forskolin 3 H/ extrusion is Na/-dependent, Cl0-independent, amiloride-,

and DIDS-insensitive. Also in HCO03 /CO2-free media a smallmmol/L, and DBcAMP 100 mmol/L in the absence of Na/ and

HCO03 no significant increase in H/ extrusion was recorded component of H/ extrusion appears to be amiloride-insensi-

tive. A similar observation has been reported also in rat hepa-(n Å 6) (Fig. 7). The absence of cAMP-dependent stimulationof Na-independent H/ extrusion is not consistent with the tocytes43 and cholangiocytes11,12; in both cases it has been

explained with incomplete inhibition of Na//H/ by amiloride.presence of a H/-ATPase in human cholangiocytes.However, in a recent report Marti et al.44 showed that, in

DISCUSSION addition to the Na//H/ exchange isoform 1 (NHE-1), themRNA for the amiloride-insensitive isoform 2 (NHE-2) couldIn this study we have used continuous cell lines and pri-also be amplified from rat cholangiocyte preparations (albeigtmary cultures of intrahepatic cholangiocytes to assess pHiof low purity). Moreover, Singh et al.45 have reported prelimi-regulation in the human biliary epithelium. Human cholangio-nary evidence for an apical Na//H/ exchange in microper-cytes were isolated from normal liver tissue not used for liverfused rat bile ductules; this carrier was active only at verytransplant, following recently described procedures.13,14,17,18

low pHi, but was inhibited by amiloride at the doses used inSome functional differences between cholangiocytes isolatedthis study. Therefore, at this time, the presence of an amilo-from ducts of different size have been described in rats.41

ride-insensitive Na//H/ isoform in our preparations of hu-Because the antibody used for the immunomagnetic separa-man cholangiocytes, although appealing, cannot be showntion of human cholangiocytes (HEA 125) recognizes an anti-nor dismissed and will have to await experiments on micro-gen expressed in all branches of the intrahepatic biliary tree,perfused bile ductules.the initial cell isolates could theoretically represent a mixture

When cells were studied in the presence of HCO03 , basalof cholangiocytes from ductules of different sizes; however

pHi decreased following extracellular Na/ removal, but notrecent studies employing a panel of phenotypic and func-during amiloride administration, and was lower in cells pre-tional markers suggest that the isolation and culture proce-incubated for 40 minutes with 0.5 mmol/L DIDS, an inhibitordure favors the isolation of cholangiocytes from intermediateof HCO0

3 -dependent transport systems. In the presence ofand medium size intrahepatic ducts.42 Cultured cells are posi-HCO0

3 , pHi recovery from an intracellular acid load was in-tive for cholangiocyte phenotypic markers such as cytokera-hibited by Na/ removal and DIDS administration, but wastin 19 and g-GT. With time in culture HEA 125 positivitypartly insensitive to amiloride. The amiloride-insensitivefades, but cells remain positive for cytokeratin 19 and g-component was inhibited by depletion of intracellular Cl0.GT and negative for mesenchymal (desmin) and endothelialThus the acid extrusion mechanism active in the presence(FactorVIII-related antigen) markers.of HCO0

3 /CO2 is Na/-, HCO03 -, and Cl0-dependent, indicatingOur results show that human cholangiocytes possess a

the presence of a Na/-dependent Cl0/HCO03 exchange. ThisNa//H/ exchange and both Na/-dependent and -independent

electroneutral acid extruder has an absolute requirement forCl0/HCO03 exchange, whereas no evidence was found for a

intracellular Cl0 and exchanges extracellular Na/ andvesicular-type H/-ATPase. The main base influx mechanismHCO0

3 with intracellular Cl0 and H/,30 but it has not yet beenis Na/-dependent Cl0/HCO03 exchange whereas Na/:HCO0

3cloned or characterized biochemically. Its presence has beencotransport is active only at very low pHi. The activity of bothreported in invertebrates, in mammalian mesangial cells,26anion exchangers is stimulated by cAMP (secretin’s secondsmooth muscle cells,46 and epithelial cells from rabbit esopha-messenger), suggesting that increased influx of HCO0

3 andgeal mucosa,47 rabbit kidney proximal tubule, and kidney b-efflux of HCO0

3 by Na-dependent and Na-independent Cl0intercalated cells from alkali loaded rabbits.48 The presence/HCO0

3 exchange, respectively, represents the molecularin human cholangiocytes of electroneutral Na/-dependentmechanism by which human cholangiocytes secrete HCO0

3

Cl0/HCO03 exchange is thus the main difference from rat chol-into the bile, both in basal conditions and after hormonal

angiocytes where HCO03 influx mechanism is mediated by anstimulation. Finally, lack of effect of NPPB suggests that

electrogenic Na/:HCO03 cotransport,11,12 that is engineered tostimulation of anion exchanger by cAMP is not mediated by

opening of Cl0 conductances. maintain pHi homeostasis following changes in membrane



FIG. 6. Representative pHi tracing showing the effects of agents raisingcAMP levels on pHi recovery from an acid load in the presence of HCO0

3 . Cellswere acid loaded as described above (NH4Cl pulsing). At the time of NH4Clwithdrawal and during recovery phase amiloride 1 mmol/L was present (toavoid the influence of Na//H/ exchange). As described above in these conditionspHi recovery is mediated by Na/-dependent Cl0/HCO0

3 exchange. The firstpart of the plot represents a control pulse: a-b: KRB; b-c: NH4Cl; c-d: KRB /amiloride; at point d amiloride was withdrawn; at point e the tracing waspaused, cells were allowed to stabilize baseline pHi and a 10-minute perfusionwith DBcAMP (100 mmol/L), forskolin (3 mmol/L), and IBMX (100 mmol/L) wasstarted and continued throughout the rest of the experiment; from f to g NH4Clwas administered, from g to h cells were perfused with KRB / amiloride(n Å 6 from 1 preparation). It is evident that pHi recovery is steeper afteradministration of cAMP agonists. Preliminary experiments showed no effectsof this double pulse procedure in the absence of cAMP agonists.

potential. At low pHi (6.4) a small component of H/ extrusionwas Cl0-independent. Although this phenomenon may be be-cause of incomplete intracellular Cl0 depletion the acid extru-sion that remains in Cl0-free solutions between pHi 6.4 and6.5 is partly inhibited by DIDS and by the removal of externalNa/ and is thus reminiscent of the Na/:HCO0

3 cotransportpresent in rat cholangiocytes. Presence of both Na-dependentCl0/HCO0

3 exchange and Na/:HCO03 cotransport has also

been reported in other mammalian cells.46 Signals regulatingNa/-dependent Cl0/HCO0

3 exchange activity have not beensystematically evaluated. Available information suggeststhat activity of this carrier is increased by stable expressionof cHras oncogene in NIH3T3 fibroblasts,49 by vasopressin inmesangial cells,34 by osmotic shrinkage in Chinese hamster

FIG. 5. Representative tracings showing pHi recovery from an alkaline load (A)and the effects of external Cl0 removal (B) and DIDS pretreatment (C). Intracellularalkaline load was induced as described in references 34 and 37 by applying andwithdrawing a medium containing 50 mmol/L NaHCO3 equilibrated with 10% CO2

to maintain external pH at 7.4. After exposure to this medium cells acutely acidifybecause of CO2 entry into the cell and then recover pHi toward baseline in about10 minutes (this part of the experiment is not shown in the plots). After pHi hadequilibrated giving a stable baseline cells are returned to the original KRB con-taining 5% CO2 and 25 mmol/L NaHCO3. Exit of CO2 out of the cells creates anintracellular alkali load from which cells recover thanks to a Cl0-dependent, DIDS- FIG. 7. Effects of cAMP on H/ extrusion in the absence of external Na/.inhibitable mechanism (nÅ 33 from five preparations). In plot B, point b, perfusion Cells were perfused with DBcAMP (100 mmol/L), forskolin (3 mmol/L), andmedium was changed from 50 mmol/L NaHCO3/10% CO2 KRB to a Cl0-free KRB IBMX (100 mmol/L) for 10 minutes before and throughout the experiment. a-buffered with 25 mmol/L NaHCO3/5% CO2; cells alkalinized, but pHi did not b: Hepes; b-c: NH4Cl; c-d: choline; d-e: Hepes. Part c-d of the tracing clearlyrecover until Cl0 was readmitted (c) (n Å 5 from three preparations). In plot C shows that cAMP does not stimulate Na/- and HCO0

3 -independent H/ extru-cells were preincubated for 30 minutes with 1 mmol/L DIDS. It is clear that the sion, as it could be expected if vacuolar-type H/ ATPase contributed to pHirecovery phase after returning to normal KRB is much slower in cells pretreated regulation in human cholangiocytes. Tracing is representative of six similarwith the inhibitor (n Å 13 from three preparations). experiments from one preparation.



ovary cells,50 and by cAMP in kidney b-intercalated cells.48 ATPase. Our data indicate that regulation of HCO03 secretion

in the human bile duct epithelium is likely different fromOur data show that in human cholangiocytes, HCO03 influx

through electroneutral Na/-dependent Cl0/HCO03 exchange rodents and pigs. In fact, Cl0/HCO0

3 exchange activity is in-creased by cAMP independently from changes in Cl0 conduc-is increased by cAMP. In rat cholangiocytes cAMP does not

directly stimulate Na:HCO03 cotransport activity that is acti- tances. Activation of Na/-dependent Cl0/HCO0

3 exchange bycAMP48,55 increases HCO0

3 influx and stimulates Cl0 efflux;vated only secondarily to the depolarization induced by open-ing of Cl0 channels.12 these changes in HCO0

3 and Cl0 electrochemical gradientsfacilitate the activity of the apical Cl0/HCO0

3 exchange. It isIn the pig, secretin promotes the clearance of vesicles lo-cated underneath the basolateral membrane of cholangio- tempting to speculate that the above differences in intracellu-

lar acid/base regulation underlay the higher choleretic effectcytes.51 In rat cholangiocytes secretin induces the release ofacridin orange (a fluorescent dye that distributes in acidic induced by secretin in humans.vesicles) in the extracellular medium,52 suggesting that an

Acknowledgment: The authors thank Elena Ossi, M.D. forexocytic process is involved in secretin action. In short-timehelpful discussion, P. McMaster, J.A.C. Buckels, and D.cultured pig bile ductules cAMP stimulates Na/- andMayer for having provided the liver samples. We also grate-HCO0

3 -independent baphylomycin A1-inhibitable recoveryfully acknowledge the collaboration of Drs. Alastair J. Strainfrom an intracellular acid load,40 suggesting that a vesicular-and Joanne Wilton.type H/-ATPase is inserted at the basolateral membrane fol-

lowing secretin administration. This mechanism seems to beREFERENCESrestricted to pig cholangiocytes because in our cultured chol-

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19. Momberg F, Moldenhauer G, Hammerling GH. Immunohistochemicaltions, opening of Cl0 conductances is not required.study of the surface expression of an Mr 34000 human epithelium specificIn rat cholangiocytes the Cl0 channel blocker NPPB12 in-glycoprotein in normal and malignant tissues. Cancer Res 1987;47:2883-hibits secretin- and cAMP-induced increase in Cl0/HCO0

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Na+-dependent and -independent Cl?/HCO3? exchange mediate cellular HCO3? transport in cultured human...

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