Dopaminergic inhibition of secretin-stimulated choleresis byincreased PKC-� expression and decrease of PKA activity

Shannon Glaser,3 Domenico Alvaro,6 Tania Roskams,8 Jo Lynne Phinizy,3

George Stoica,7 Heather Francis,3 Yoshiyuki Ueno,5 Barbara Barbaro,2

Marco Marzioni,2 Jeremy Mauldin,2 Sobia Rashid,2 Maria Grazia Mancino,6

Gene LeSage,1 and Gianfranco Alpini1,2,4

Departments of 1Internal Medicine and 2Medical Physiology, 3Division of Research and Education, Scott &White Hospital, Texas A&M University System Health Sciences Center, College of Medicine, and 4Central TexasVeterans Health Care System, Temple, Texas 76504; 5Department of Internal Medicine, Tohoku UniversitySchool of Medicine, Aobaku, Sendai, Japan 980-8574; 6Division of Gastroenterology, University of Rome, LaSapienza, Rome, Italy 00100; 7Department of Veterinary Pathobiology, Texas A&M University, College Station,Texas, and 8Department of Morphology and Molecular Pathology, University of Leuven, 3000 Leuven, Belgium

Submitted 25 July 2002; accepted in final form 6 December 2002

Glaser, Shannon, Domenico Alvaro, Tania Roskams,Jo Lynne Phinizy, George Stoica, Heather Francis,Yoshiyuki Ueno, Barbara Barbaro, Marco Marzioni,Jeremy Mauldin, Sobia Rashid, Maria Grazia Mancino,Gene LeSage, and Gianfranco Alpin. Dopaminergic inhi-bition of secretin-stimulated choleresis by increasedPKC-� expression and decrease of PKA activity. Am JPhysiol Gastrointest Liver Physiol 284: G683–G694, 2003.First published December 27, 2002; 10.1152/ajpgi.00302.2002.—To determine the role and mechanisms of action bywhich dopaminergic innervation modulates ductal secretion inbile duct-ligated rats, we determined the expression of D1, D2,and D3 dopaminergic receptors in cholangiocytes. We evaluatedwhether D1, D2 (quinelorane), or D3 dopaminergic receptoragonists influence basal and secretin-stimulated choleresis andlumen expansion in intrahepatic bile duct units (IBDU) andcAMP levels in cholangiocytes in the absence or presence ofBAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), or rottlerin. We evaluated whether 1) quinelo-rane effects on ductal secretion were associated with increasedexpression of Ca2�-dependent PKC isoforms and 2) increasedexpression of PKC causes inhibition of PKA activity. Quineloraneinhibited secretin-stimulated choleresis in vivo and IBDU lumenspace, cAMP levels, and PKA activity in cholangiocytes. Theinhibitory effects of quinelorane on secretin-stimulated ductalsecretion and PKA activity were blocked by BAPTA-AM, chel-erythrine, and H7. Quinelorane effects on ductal secretion wereassociated with activation of the Ca2�-dependent PKC-� butnot other PKC isoforms. The dopaminergic nervous systemcounterregulates secretin-stimulated ductal secretion in exper-imental cholestasis.

intrahepatic biliary epithelium; bile flow; secretin receptor

DUCTAL SECRETION IS REGULATED by gastrointestinal hor-mones/peptides (3, 7, 19, 20, 33, 37, 64), bile salts (4),and nerves (12, 34, 38, 39). Although secretin does not

increase bile flow in normal rats (7, 29, 41), recentstudies (29) have shown that secretin increases biliarybicarbonate in a dose-dependent fashion and is muchmore effective when administered via the hepatic ar-tery. In addition to bile duct-ligated (BDL) rats (7, 24,43, 64), in which there is enhanced ductal mass (7),secretin has also been shown to stimulate bicarbonatesecretion in isolated intrahepatic bile duct units(IBDU) (12) and purified cholangiocytes (9, 41) of nor-mal rats. The stimulatory effect of secretin on ductalsecretion occurs by interaction with receptors ex-pressed only by cholangiocytes (11) through an in-crease in intracellular cAMP levels (3, 37, 38, 40, 41,43, 64), which leads to the opening of the cystic fibrosistransmembrane conductance regulator (CFTR) chan-nel (22) and activation of the Cl�/HCO3

� exchanger (3,12, 41) with subsequent secretion of bicarbonate intobile (7, 24, 38, 41, 43, 64). Bombesin and vasoactiveintestinal peptide stimulate ductal secretion (19, 20).Gastrin, somatostatin, and endothelin-1 inhibit secre-tin-stimulated choleresis in BDL rats (5, 18, 24, 64).

Cholangiocytes proliferate in response to liver in-jury/toxins including BDL (5, 7, 23, 43), partial hepa-tectomy (41), acute carbon tetrachloride administra-tion (43), or chronic feeding of �-naphthylisothiocya-nate (40) or bile salts (6). Cholangiocyte proliferation isassociated with enhanced basal and secretin-stimu-lated ductal secretion (5, 7, 11, 23, 24, 33, 38). The BDLrat model has been widely used for evaluating basaland secretin-stimulated ductal secretion (5, 7, 11, 23,24, 33, 38). In these studies, we used the BDL ratmodel, because it allows: 1) the isolation of a greaternumber of pure cholangiocytes compared with normalrats (7, 8) and 2) the in vivo evaluation of the stimula-tory effect of intravenous administration of secretin on

Address for reprint requests and other correspondence: G. Alpini,Associate Professor, Internal Medicine and Medical Physiology, TheTexas A&M University System, Health Sciences Center COM andCentral Texas Veterans Health Care System, 702 SW H. K. DodgenLoop, Temple, TX 76504 (E-mail: galpini@tamu.edu).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Gastrointest Liver Physiol 284: G683–G694, 2003.First published December 27, 2002; 10.1152/ajpgi.00302.2002.

http://www.ajpgi.org G683

bile flow (7, 8, 38, 43, 64), which is absent in normalrats (7, 8, 23, 41).

There is growing information regarding the role ofnerves in the regulation of ductal secretion (12, 34, 38,39). ACh induces intracellular Ca2� concentration([Ca2�]i) increases and oscillation in rat cholangiocytesdue to both influx of extracellular Ca2� and mobiliza-tion of thapsigargin-sensitive [Ca2�]i stores (49). Otherstudies (12) have shown that ACh, by interaction withM3 ACh receptor subtypes, increases secretin-stimu-lated Cl�/HCO3

� exchanger activity in cholangiocytesby Ca2�-calcineurin-mediated, PKC-independent mod-ulation of adenyl cyclase. Interruption of the cholin-ergic system of BDL rats by vagotomy decreasescholangiocyte cAMP levels and inhibits secretin-stim-ulated ductal secretion (38). The �2-adrenergic recep-tor agonist UK-14304 inhibits cholangiocarcinomagrowth through time course-dependent modulation ofRaf-1 and B-Raf activities (34). The �1-adrenergic ag-onist phenylephrine potentiates secretin-stimulatedductal secretion through a Ca2�- and PKC-dependentamplification of the adenylyl cyclase system (39).

In a number of cells, D1 and D2 dopaminergic recep-tors modulate adenylyl cyclase activity with a stimu-latory effect mediated by D1 dopaminergic receptorsand inhibitory effects regulated by D2 dopaminergicreceptors (26, 50, 59), the latter acting through D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and Ca2�-dependent intracellular pathways (28). Dopaminergicreceptors regulate secretory activity of several epithe-lia (15, 21, 56). The intrahepatic biliary epitheliumdisplays dopaminergic innervation (45, 46); however,no information exists regarding the role and mecha-nisms of action of the dopaminergic system in theregulation of ductal secretion and adenyl cyclase activ-ity in cholangiocytes. We addressed the following ques-tions. First, are functional D1, D2, and D3 dopaminer-gic receptors expressed by cholangiocytes? Second, doD1, D2, and D3 dopaminergic receptor agonists/antag-onists regulate basal and secretin-stimulated ductalsecretion? Third, are dopaminergic effects on ductalbile secretion associated with increased protein expres-sion of Ca2�-dependent (�, �I, �II, and �) and/or Ca2�-independent novel (� and �) and atypical (� and ) PKCisoforms? Fourth, are quinelorane effects on ductalsecretion associated with cytoskeleton-to-membranedistribution of PKC-�, whose protein expression is in-creased by quinelorane? Finally, is increased proteinexpression of PKC-� (after treatment with a dopami-nergic receptor agonist) associated with downregula-tion of PKA activity leading to inhibition of secretin-stimulated ductal secretion?

MATERIALS AND METHODS

Animal Model

Male Fischer 344 rats (150–175 g) were purchased fromCharles River (Wilmington, MA). The animals were kept in atemperature-controlled environment (22°C) with a 12:12-hlight-dark cycle and fed ad libitum with standard rat chow.The studies were performed in rats with cholangiocyte pro-

liferation induced by BDL (7) [for isolation of cells (5, 18, 23,24, 38, 43, 64) or IBDU (3, 47, 55)] or bile duct incannulation[BDI, for bile collection (7)] for 1 wk. BDL and BDI wereperformed as described (7). Before each procedure, animalswere anesthetized with pentobarbital sodium (50 mg/kgbody wt).

Materials

Reagents were purchased from Sigma (St. Louis, MO)unless otherwise indicated. The substrate for �-glutamyl-transpeptidase (�-GT), N-(�-L-glutamyl)-4-methoxy-2-naph-thylamide, was purchased from Polysciences (Warrington,PA). Rabbit antibodies reacting with the D1, D2, or D3dopaminergic receptors were purchased from Santa CruzBiotechnology (Santa Cruz, CA). Radioimmunoassay (RIA)kits for the determination of intracellular cAMP andIns(1,4,5)P3 levels were purchased from Amersham (Arling-ton Heights, IL). The antibodies (IgG) against the rat Ca2�-dependent PKC-�, PKC-�I, PKC-�II, PKC-�, and the Ca2�-independent novel (� and �) and atypical (� and ) PKCisoforms were purchased from Santa Cruz Biotechnology.The D1 dopaminergic receptor agonist, [()6-chloro-7,8-dihy-droxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrobromide 6-chloro-N-allyl-SKF-38393 hydrobromide] or()-SKF-82958 hydrobromide (48), the D2 dopaminergic re-ceptor agonist quinelorane (51), the D2 dopaminergic recep-tor antagonist eticlopride (31, 62), and the D3 dopaminergicreceptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin(7-OH-DPAT) (44) were obtained from Research Biochemi-cals International (Natick, MA).

Purification of Cholangiocytes

Pure cholangiocytes were obtained from BDL rats by im-munoaffinity separation (4–6, 18, 23, 24, 30, 37, 38, 40, 41,43, 64) by using a mouse monoclonal antibody against anunidentified membrane antigen expressed by all rat intrahe-patic cholangiocytes (30). Purity of isolated cholangiocytes wasevaluated by histochemistry for �-GT (58), a specific histochem-ical marker for cholangiocytes in rat liver (7, 41). Cell viability(�97%) was determined by trypan blue exclusion.

Expression of D1, D2, and D3 Dopaminergic Receptorsin Purified Cholangiocytes, Liver Sections, and Apicaland Basolateral Cholangiocyte Membranes

The expression of D1, D2, and D3 dopaminergic receptorswas evaluated by immunoblots in pure cholangiocytes fromboth normal and BDL rats. The expression of the D2 dopa-minergic receptor (the only receptor subtype expressed bycholangiocytes by immunoblots of whole cell lysate) was alsodetermined by immunohistochemistry in liver sections, cyto-spin smears of pure cholangiocytes, and immunoblots inapical and basolateral cholangiocyte membranes from BDLrats.

Immunoblots for D1, D2, or D3 dopaminergic receptors inpure cholangiocytes from BDL rats was performed as follows.Proteins (100 �g) were resolved by SDS-7.5% polyacrylamidegel electrophoresis and transferred onto a nitrocellulosemembrane (Bio-Rad, Hercules, CA). The membrane was in-cubated overnight with rotation at 4°C with a rabbit antibodyfor the D1, D2, or D3 dopaminergic receptor diluted 1:3,000with Tris-buffered saline (TBST; 50 mM Tris, 150 mM NaCl,and 0.05% Tween 20)-5% milk. The membrane was washedwith TBST and incubated for 1 h at room temperature witha secondary antibody, an anti-rabbit IgG peroxidase conju-gate [enhanced chemiluminescence (ECL) kit, Amersham

G684 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

Life Science, Little Chalfont, England] diluted 1:100,000with TBST. The membrane was washed with TBST, andproteins were visualized by using chemiluminescence (ECLPlus kit, Amersham Life Science). The intensity of the bandswas determined by scanning video densitometry by using theChemiImager 4000 low-light imaging system (Alpha Inno-tech, San Leandro, CA).

Immunohistochemistry for D2 dopaminergic receptor wasperformed in fresh frozen liver sections (5-�m thick) fromBDL rats. Sections were incubated for 30 min with primaryaffinity-purified goat polyclonal antibody against the D2 do-paminergic receptor (N-19, Santa Cruz Biotechnology) di-luted 1:50. The sections were incubated for 30 min withrabbit-anti-goat antibody (DAKO, Glostrup, Denmark), fol-lowed by incubation for 30 min with rabbit EnVision(DAKO). All reagents were diluted in PBS (pH 7.2). Eachincubation was followed by a wash in three changes of PBS,pH 7.2, for 15 min. The reaction product was developed withthe use of 3-amino-9-ethylcarbazole and H2O2. Sections thatwere not incubated with a primary antibody served as neg-ative controls. The sections were examined with a microscope(model BX 40; Olympus Optical).

Immunohistochemistry for D2 dopaminergic receptor wasperformed in cytospin smears of cholangiocytes. Cell smearswere fixed with ice-cold acetone for 5 min. After smears werewashed with PBS, endogenous peroxidase activity wasblocked with 70% methanol containing 3% H2O2. After beingblocked with normal rabbit serum, the cells were incubatedwith the primary antibody (dilution 1:100) at 4°C overnight.After washes with PBS, the conjugated antibody was de-tected by using a commercially available kit [Histofine SAB-PO(G) kit; Nichirei, Tokyo, Japan] according to the vendor’sinstructions. The reaction product was visualized with dia-minobenzidine. The negative control was performed by usingnormal goat serum instead of primary antibody.

To determine the subcellular distribution (apical vs. baso-lateral) of the D2 dopaminergic receptor in cholangiocytes,we evaluated by immunoblots (23) protein expression for theD2 dopaminergic receptor in membranes obtained from thebasolateral or apical domain of pure cholangiocytes fromBDL rats. Cholangiocyte apical and basolateral membraneswere prepared by isopycnic centrifugation on sucrose gradi-ents by using a technique previously used by Tietz et al. (63)in cholangiocytes from BDL rats.

We measured intracellular Ins(1,4,5)P3 and [Ca2�]i levels,second messenger system-activated by D2 dopaminergic in-nervation (17, 65). Cholangiocytes from BDL rats were incu-bated for 1 h at 37°C (37) and subsequently stimulated for 15min at 22°C with 0.2% BSA (basal value) or quinelorane (100�M) with 0.2% BSA. Intracellular Ins(1,4,5)P3 levels wereassessed by the [3H]Ins(1,4,5)P3 kit (Amersham) according tothe instructions provided by the manufacturer. Data wereexpressed as picomoles per million cells.

Calcium fluorescence measurements in purified cholangio-cytes from BDL rats were performed by using fluo 3-AM(Molecular Probes, Eugene, Oregon) and a microplate reader(Fluoroskan Ascent FL Thermolabsystems, Helsinki, Fin-land) equipped with three injectors (36). Isolated cholangio-cytes (4 104 per well) were loaded for 1 h at room temper-ature with 5 �M of fluo 3-AM in Tyrode’s salt solution (TSS)[(in mM): 137 NaCl, 2.7 KCl, 1 MgCl2, 0.2 NaH2PO4, 12NaHCO3, and 5.5 glucose] with 0.1% Pluronic F-127 (Molec-ular Probes). Loaded cells were washed two times with TSS.The loaded cells were subsequently resuspended in TSS andincubated for an additional 30 min at room temperature. Theloaded cells were then pelleted and resuspended at 4 104

cells per 100 �l of TSS. The 4 104 cells per well were added

to a 96-well black microplate. The baseline fluorescence wasmeasured 50 times at 2-s intervals. TSS alone or quinelorane(100 �M) dissolved in buffer were injected sequentially intoseparate wells, and the fluorescence intensity was measuredat 538 nm for 3 min at 1-s intervals. The excitation wave-length was 485 nm. [Ca2�]i was calculated as follows:[Ca2�]i � Kd(F � Fmin)/(Fmax � F). Fmax is fluorescenceintensity measured after permeabilization of the cells with1% NP-40. EGTA (0.1 M) was then added to chelate Ca2� andminimum fluorescence intensity (Fmin) was obtained. The Kd

of fluo 3-AM was 390 nm (35).

Effect of Dopaminergic Receptor Agonists onDuctal Secretion

Measurement of bile flow, bicarbonate concentration, andsecretion. BDI rats were surgically prepared for bile collec-tion (7). One jugular vein was incannulated with a polyeth-ylene-50 cannula to infuse Krebs-Ringer-Henseleit (KRH),secretin, or the selected dopaminergic receptor agonist/an-tagonist. Body temperature (37°C) was monitored with arectal thermometer. When steady-state bile flow wasachieved (60 min of KRH infusion from the beginning of bilecollection), we subsequently infused 1) secretin (100 nM) for30 min; 2) KRH until steady state; 3) quinelorane (100 �M)for 30 min; 4) KRH; or 5) quinelorane (100 �M) � secretin(100 nM) for 30 min, followed by a final infusion of KRH. Inseparate experiments, after steady-state bile flow wasreached, rats were subsequently infused with 1) secretin (100nM) for 30 min; 2) KRH until steady state; 3) eticlopride (100�M) for 30 min; 4) KRH until steady state; or 5) eticlopride (aD2 dopaminergic receptor antagonist; 100 �M) � quinelo-rane (100 �M) � secretin (100 nM) for 30 min, followed by afinal infusion of KRH. We evaluated the effects of a D1[()-SKF-82958 hydrobromide; 100 �M (48)] or a D3 (7-OH-DPAT; 100 nM) (44) dopaminergic receptor agonist on basaland secretin-stimulated bile flow and on bicarbonate concen-tration and secretion. Bile was collected every 10 min, andbile flow was determined by weight, assuming a density of 1.0g/ml. Biliary bicarbonate concentration (measured as CO2)was determined in bile by a Natelson microgasometer appa-ratus (Scientific Industries, Bohemia, NY).

Effect of Quinelorane on Ductal Lumen Expansion in IBDU

IBDU were isolated as described (3, 47, 55). IBDU wereincubated for 24 h at 37°C in minimum essential medium(GIBCO-BRL, Grand Island, NY) with 10% fetal calf serumto allow complete sealing of bile duct lumen (3, 47, 55).Ductal fluid secretion was estimated from the changes in thearea of IBDU lumen space, as previously described (3, 47,55), after perfusion with 1) 0.2% BSA (basal); 2) secretin(positive control, 100 nM) (3) with 0.2% BSA; 3) quinelorane(100 �M) in the absence or presence of secretin (100 nM) with0.2% BSA; or 4) BAPTA-AM (50 �M; an [Ca2�]i chelator) (12,23), chelerythrine (1 �M) or H7 (20 �M), two Ca2�-dependentPKC inhibitors (23, 32), or rottlerin (10 �M; a Ca2�-indepen-dent PKC-� inhibitor) (54) � quinelorane (100 �M) � secre-tin (100 nM) with 0.2% BSA.

Intracellular cAMP Levels in Purified Cholangiocytes

After isolation, pure cholangiocytes from BDL rats wereincubated for 1 h at 37°C (37) and subsequently incubated atroom temperature (4–6, 18, 23, 24, 37, 38, 40, 41, 43) with 1)0.2% BSA (basal) for 5 min; 2) secretin (100 nM) with 0.2%BSA for 5 min; 3) quinelorane (100 �M for 5 min) in theabsence or presence of secretin (100 nM for 5 min) with 0.2%

G685DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

BSA; 4) eticlopride (100 �M for 5 min) � quinelorane (100�M for 5 min) � secretin (100 nM for 5 min) with 0.2% BSA;or 5) eticlopride (100 �M for 5 min) in the absence or in thepresence of secretin (100 nM for 5 min) with 0.2% BSA. Weevaluated the effect of quinelorane (100 �M for 5 min) onsecretin-stimulated cAMP levels in the absence or presenceof BAPTA-AM (50 �M for 5 min), chelerythrine (1 �M for 5min), H7 (20 �M for 5 min), or rottlerin (10 �M for 5 min)with 0.2% BSA. We evaluated the effect of BAPTA-AM, H7,chelerythrine, or rottlerin on basal cAMP levels. Intracellu-lar cAMP levels were determined by RIA by using commer-cially available kits.

Are Quinelorane Effects on Secretin-Stimulated DuctalSecretion Associated with Increased Expression of Ca2�-dependent and Ca2�-independent PKC Isoforms?

We evaluated whether the inhibitory effects of quineloraneon secretin-stimulated ductal bile secretion are associatedwith increased protein expression of Ca2�-dependent (�, �I,�II, �) and/or Ca2�-independent novel (�, �, �) and atypical (�,) PKC isoforms, which regulate cell functions in severalepithelia including cholangiocytes (10, 14, 23, 32, 61, 68).

Cholangiocytes were stimulated for 90 min (14, 23) at 37°Cwith 0.2% BSA (basal) or quinelorane (100 �M) with 0.2%BSA and were subsequently analyzed for protein expressionfor PKC-�, -�I, -�II, -�, -�, -�, -�, -�, and - by immunoblots(23). The intensity of the bands was determined by scanningvideo densitometry by using the ChemiImager 4000.

Because quinelorane increases only the protein expressionfor the Ca2�-dependent PKC-�, we then determined whetherthe effects of quinelorane on secretin-stimulated ductal se-cretion were associated with intracellular redistribution ofPKC-�. To achieve this, we treated in vitro pure cholangio-cytes from 1-wk-old BDL rats at 37°C for 90 min (14, 23) with1) 0.2% BSA (basal) or 2) quinelorane (100 �M) in 0.2% BSA.Subsequently, we evaluated protein expression for PKC-� ina triton-soluble (containing cytoplasm and membrane) frac-tion and a triton-insoluble (containing cytoskeleton) fractionby immunoblots (23). Both triton-soluble and triton-insolublefractions were obtained from cholangiocytes as described(14, 23).

Is Quinelorane-Induced Increase in PKC-� ProteinExpression Associated with Downregulationof PKA Activity in Cholangiocytes?

We evaluated in pure cholangiocytes whether increasedexpression of PKC-� (induced by in vitro treatment with 100�M quinelorane) causes inhibition of secretin-stimulatedPKA activity, which regulates ductal secretion (13). PKAassay was performed according to the manufacturer’s in-structions by using PepTag assay protein kinase kits (Pro-mega, Madison, WI) for PKA. Purified cholangiocytes (5 106) from BDL rats were stimulated at 37°C with 1) 0.2%BSA (basal) for 30 min; 2) secretin (30 min at 100 nM) with0.2% BSA; 3) quinelorane (30 min at 100 �M) with 0.2% BSA;4) quinelorane (10 min at 100 �M) before stimulation withsecretin (30 min at 100 nM) with 0.2% BSA; or 5) cheleryth-rine, BAPTA-AM, H7, or rottlerin (10 min each) before treat-ment with quinelorane (100 �M; 10 min) followed by secretin(100 nM; 30 min) stimulation with 0.2% BSA. After stimula-tion of cholangiocytes with the appropriate agonist/antago-nist, samples were centrifuged at 1,400 rpm for 5 min. Sam-ples were resuspended in 1 ml of a lysis buffer (in mM: 20Tris �HCl, pH 7.4, 150 NaCl, 5 EDTA, pH 8.0, and 1 PMSFwith 10 �M leupeptin and 10 �M aprotinin). The sampleswere then sonicated with a cell membrane disrupter for three

30-s bursts. Samples were centrifuged at 2,000 rpm for 5 min,and the supernatant was saved. The supernatant was ana-lyzed with the PepTag assay for nonradioactive detection ofPKA activity from Promega. For PKA, the positive controlwas performed by using the same assay conditions as thesamples with the positive control containing 10 ng of PKAprovided with the kit. The negative controls (deionized wa-ter) were performed by using the same assay conditions asthe samples. Phosphorylated and nonphosphorylated peptidebands were visualized on a 0.8% agarose gel. Phosphorylatedpeptide bands were quantitated by scanning densitometry byusing the ChemiImager 4000. Densitometric values werenormalized on the basis of cell number.

Statistical Analysis

All data are expressed as means SE. Differences be-tween groups were analyzed by Student’s t-test when twogroups were analyzed or by ANOVA if more than two groupswere analyzed.

RESULTS

Cholangiocytes Express D2 Dopaminergic Receptors

Immunoblotting analysis shows that a band (migrat-ing at 55 kDa) (16) for the D2 dopaminergic receptorwas expressed by cholangiocytes from both normal andBDL rats (Fig. 1A), whereas D1 and D3 dopaminergicreceptors were absent (Fig. 1A). Immunohistochemis-try shows positive staining for D2 dopaminergic recep-tors in liver sections (Fig. 1B) and in cholangiocytesmears from BDL rats (Fig. 1C). Negative controls forD2 dopaminergic receptors for sections and cells fromBDL rats are shown in Fig. 1, B and C, respectively. Atthe subcellular level, we found that a band (migratingat 55 kDa) (16) for the D2 dopaminergic receptor waspresent in the basolateral but not the apical domain ofcholangiocytes (Fig. 1D).

Consistent with the concept that cholangiocytes con-tain functional D2 dopaminergic receptors, quinelo-rane increased intracellular Ins(1,4,5)P3 levels [0.37 0.059 vs. 0.18 0.018 (basal) pmol/1 106 cells, P �0.05] (Table 1). Parallel with changes in Ins(1,4,5)P3levels, quinelorane significantly (P � 0.05) increased[Ca2�]i levels of cholangiocytes compared with its cor-responding basal value (Table 1).

Quinelorane Inhibits Secretin-Stimulated Bileand Bicarbonate Secretion

Secretin increased bile flow and bicarbonate concen-tration and secretion compared with its correspondingbasal value (Table 2). When infused alone, the D2dopaminergic receptor agonist quinelorane did not al-ter basal bile flow and bicarbonate concentration andsecretion, but it decreased secretin-stimulated bile flowand bicarbonate concentration and secretion of BDLrats (Table 2). Consistent with the expression of D2dopaminergic receptors in cholangiocytes, the inhibi-tory effect of quinelorane on secretin-stimulated bicar-bonate-rich choleresis was abolished by eticlopride, aD2 dopaminergic receptor antagonist (Table 2). A po-tential shortcoming of these in vivo results is that theeffect of quinelorane on secretin-stimulated bile flow

G686 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

and bicarbonate secretion of BDL rats (Table 2) may beinfluenced by the in vivo vascular effects of the D2dopaminergic receptor agonist quinelorane (53, 66).Eticlopride alone did not alter basal bile flow andbicarbonate concentration and secretion (Table 2). Dueto the lack of expression of D1 and D3 dopaminergicreceptors, neither ()-SKF-82958 hydrobromide (a D1dopaminergic receptor agonist) nor 7-OH-DPAT (a D3dopaminergic receptor agonist) altered basal or secre-tin-stimulated bile flow and bicarbonate concentrationand secretion (Table 2).

Quinelorane Inhibition of Secretin-Stimulated IBDULumen Expansion Is Blocked by BAPTA-AM,Chelerythrine, and H7 but not Rottlerin

Secretin increased the area of IBDU luminal spacesin IBDU preparations from BDL rats (Fig. 2). Quinelo-rane alone did not alter the area of IBDU luminal

spaces (Fig. 2A). However, pretreatment of IBDU withquinelorane before the addition of secretin inhibitedthe stimulatory effect of secretin on the expansion ofIBDU luminal spaces (Fig. 2, B–F). The inhibitoryeffect of quinelorane on secretin-induced ductal lumenexpansion was abolished by pretreating purified IBDUwith BAPTA-AM (Fig. 2C), chelerythrine (Fig. 2D), andH7 (Fig. 2E) but not rottlerin (Fig. 2F). A possibleshortcoming of our studies is represented by the factthat the use of rottlerin excludes the role of PKC-� butnot of other calcium-independent PKC isoforms inquinelorane inhibition of secretin-stimulated ductal se-cretion.

Quinelorane Inhibition of Secretin-Stimulated cAMPLevels Is Blocked by BAPTA-AM, Chelerythrine,and H7 but not Rottlerin

Secretin increased intracellular cAMP levels ofcholangiocytes from BDL rats (Fig. 3). Quineloranealone did not alter basal cAMP levels but inhibitedsecretin-stimulated cAMP levels of cholangiocytesfrom BDL rats (Fig. 3). The inhibitory effect of quinelo-rane on secretin-stimulated cAMP levels was blockedby the D2 dopaminergic receptor antagonist eticlopride(Fig. 3). Consistent with the concept that the Ca2�-dependent PKC system regulates the effects of D2dopaminergic receptor agonists on cholangiocyte cAMPlevels, quinelorane inhibition of secretin-stimulatedcAMP synthesis was ablated by BAPTA-AM, chel-erythrine, and H7 but not rottlerin (Fig. 3). Due to thelack of expression of D1 and D3 dopaminergic recep-tors, neither ()-SKF-82958 hydrobromide (a D1 dopa-minergic receptor agonist) nor 7-OH-DPAT (a D3 do-

Fig. 1. A: Western blot analysis for D1,D2, and D3 dopaminergic receptors inpure cholangiocytes from both normaland bile duct-ligated (BDL) rats. A band(migrating at �55 kDa) for D2 dopa-minergic receptors was expressed bycholangiocytes from both normal andBDL rats. Cholangiocytes from normalor BDL rats did not express D1 and D3dopaminergic receptors. B: immunohis-tochemistry for D2 dopaminergic recep-tors in liver sections from 1-wk-old BDLrats. A specific reaction appears in bileducts (arrows). Original magnification 250. C: immunohistochemistry for D2dopaminergic receptors in cholangiocytesmears from 1-wk-old BDL rats. A spe-cific reaction appears in isolated cholan-giocytes. The negative controls for D2dopaminergic receptor for sections andcells are shown in B and C, respectively.Original magnification 250. D: distri-bution of the D2 dopaminergic receptorin apical and basolateral membranesfrom pure cholangiocytes from BDL rats.A band (migrating at 55 kDa) (16) for theD2 dopaminergic receptor was present inthe basolateral but not the apical domainof cholangiocytes. DR, dopaminergicreceptor.

Table 1 Effect of quinelorane on inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellularCa2� concentration ([Ca2�]i) levels in purifiedcholangiocytes from bile duct-ligated rats

Ins(1,4,5)P3 IR3 Levels,pmol/1 106 cells n [Ca2�]i Levels, nM n

Basal 0.180.018 5 189.096.017(n � 9)

9

Quinelorane 0.370.059* 10 242.9015.94*(n � 10)

10

Data are means SE; n � no. of rats. Consistent with the conceptthat cholangiocytes contain functional D2 dopaminergic receptors,quinelorane increased intracellular Ins(1,4,5) P3 and [Ca2�]i levels ofcholangiocytes compared to their corresponding basal values. *P �0.05 vs. its corresponding basal value.

G687DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

paminergic receptor agonist) altered basal or secretin-stimulated cAMP levels (results not shown).

Quinelorane Inhibition of Secretin-Stimulated DuctalSecretion Is Associated with Increased ProteinExpression and Membrane Translocation of PKC-�

Immunoblotting analysis shows that the protein forthe Ca2�-dependent PKC (�, �I, �II, �) and the Ca2�-independent novel (�, �, �) and atypical (�, ) PKCisoforms was expressed by cholangiocytes from BDLrats (Fig. 4A). Quinelorane did not enhance proteinexpression for the Ca2�-dependent PKC-�, -�I, and-�II but increased PKC-� protein expression (Fig. 4A).Quinelorane alone did not increase protein expressionof the Ca2�-independent novel (�, �, �) and atypical (�,) PKC isoforms (Fig. 4A).

Similar to that shown in other epithelia (14), PKC-�protein expression in cholangiocytes was significantlyhigher in the triton-soluble fraction compared with thetriton-insoluble fraction (Fig. 4B). Quinelorane inducescytoskeleton-to-membrane distribution of PKC-�,which was evidenced by increased PKC-� protein ex-pression only in the triton-soluble fraction (Fig. 4B).Consistent with PKC-� translocation to cholangiocytemembranes, there was a corresponding loss of PKC-�protein expression in the triton-insoluble fraction ofcholangiocytes stimulated with quinelorane (Fig. 4B).

Quinelorane-Induced Increase in PKC-� ExpressionIs Associated with Downregulation of PKA Activityin Cholangiocytes

We evaluated whether quinelorane-induced increasein PKC-� expression causes inhibition of secretin-stim-ulated PKA activity. We found that secretin increasesPKA activity and that quinelorane inhibits basal and

secretin-induced increases in PKA activity (Fig. 5). Theinhibitory effect of quinelorane on secretin-stimulatedPKA activity was ablated by eticlopride, BAPTA-AM,chelerythrine, and H7 but not rottlerin (Fig. 5).

DISCUSSION

The present study shows that cholangiocytes expressD2 (but not D1 and D3) dopaminergic receptors asdemonstrated by immunohistochemistry in liver sec-tions and both immunoblots and immunohistochemis-try in purified cholangiocytes. Immunoblots show thatthe D2 dopaminergic receptor was present in the baso-lateral but not the apical domain of cholangiocytemembranes. Stimulation of D2 dopaminergic receptorswith the D2/D3 dopaminergic receptor agonist quinelo-rane (27) induces 1) an increase in intracellularIns(1,4,5)P3 and Ca2� levels in purified cholangiocytes;2) inhibition of secretin-stimulated bicarbonate-richcholeresis in BDL rats and secretin-induced fluid se-cretion in IBDU; and 3) inhibition of secretin-inducedcAMP levels and secretin-stimulated PKA activity inpurified cholangiocytes. The inhibitory effects ofquinelorane on secretin-stimulated ductal secretionwere blocked by the selective D2 dopaminergic receptorantagonist eticlopride (27, 62), supporting the conceptthat quinelorane acts only through D2 dopaminergicreceptors in cholangiocytes. These findings indicatethat cholangiocytes express functionally active D2 do-paminergic receptors, which are involved in the coun-terregulation of secretin-stimulated, bicarbonate-richductal choleresis. We evaluated the signal transduc-tion pathways involved in quinelorane inhibition ofsecretin-stimulated ductal secretion by demonstratingthat 1) quinelorane effects are blocked by BAPTA-AM

Table 2 Effects of D1, D2, and D3 dopaminergic agonists on basal and secretin-stimulated bile flow,bicarbonate concentration, and secretion in 1-wk-old bile duct incannulated rats

TreatmentBile Flow

(�l/min/Kg)Bicarbonate Concentration

(mEq/Liter)Bicarbonate Secretion

(�Eq/min/Kg)

Basal before secretin 116.129.32 32.570.50 3.770.29Secretin 158.5016.50a 38.751.32b 6.050.64c

Basal before quinelorane 90.934.42 29.20.73 2.650.12Quinelorane 100.136.48 28.51.06 2.8402.9Basal before quinelorane � secretin 100.807.74 32.802.61 3.340.43Quinelorane � secretin 107.309.23 36.202.03 3.850.29Basal before eticlopride 80.277.20 27.501.15 2.260.26Eticlopride 90.817.19 25.500.99 2.340.12Basal before eticlopride � quinelorane � secretin 105.994.38 22.881.00 2.260.26Eticlopride � quinelorane � secretin 132.848.03a 26.970.72b 3.550.18c

Basal before SKF-82958 127.2732.96 22.481.44 2.840.67SKF-82958 130.9935.10 26.371.24 3.481.00Basal before SKF-82958 � secretin 128.8512.41 26.901.62 3.720.71SFK-82958 � secretin 180.0720.58a 34.752.41b 6.501.41c

Basal before 7-OH-DPAT 101.036.65 35.420.94 3.500.377-OH-DPAT 107.017.70 38.041.57 4.070.18Basal before 7-OH-DPAT � secretin 98.224.72 34.320.87 3.360.227-OH-DPAT � secretin 140.577.46a 41.061.91b 5.840.35c

Data are means SE of 5–18 experiments. aP � 0.05 vs. corresponding basal value of bile flow; bP � 0.05 vs. corresponding basal valueof bicarbonate concentration; cP � 0.05 vs. corresponding basal value of bicarbonate secretion; dP � 0.05 vs. corresponding value ofsecretin-stimulated bile flow, concentration, or secretion. Statistical analysis was performed by unpaired Student’s t-test. 7-OH-DPAT,7-hydroxy-N, N-di-n-propyl-2-aminotetralin.

G688 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

(a [Ca2�]i chelator), chelerythrine, and H7 (two PKCinhibitors) but not rottlerin (a PKC-� inhibitor); 2)quinelorane effects on secretin-stimulated ductal se-cretion are associated with increased protein expres-sion of the Ca2�-dependent PKC-� but not the Ca2�-

independent novel (�, �, �) and atypical (�, ) PKCisoforms; and 3) quinelorane-induced increase in pro-tein expression and cytoskeleton to membrane distri-bution of PKC-� was associated with inhibition of se-cretin-stimulated PKA activity.

Fig. 2. Measurement of basal and hormone-regulated ductal lumen expansion in intrahepatic bile duct units(IBDU) from BDL rats. Isolated IBDU were mounted in a perfusion chamber of a light microscope, and the lumensize was measured before or 10 min after addition of 1) 0.2% BSA; 2) 100 nM secretin with 0.2% BSA; 3) 100 �Mquinelorane with 0.2% BSA; or 4) quinelorane (100 �M) plus secretin (100 nM) in the absence or presence ofBAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfanyl)-2-methyl piperazine (H7), or rottlerin with 0.2% BSA.Secretin increased the lumen of isolated IBDU. Quinelorane did not alter basal IBDU lumen (A) but inhibitedsecretin-stimulated ductal lumen expansion (B–F). The inhibitory effect of quinelorane on secretin-induced ductallumen expansion was blocked by BAPTA-AM (C), chelerythrine (D), and H7 (E) but not rottlerin (F). *P � 0.05 vs.secretin-induced lumen expansion. Data are means SE of 6–22 experiments.

G689DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

A number of studies (12, 34, 38, 49) have shown thatthe nervous system is involved in the regulation ofcholangiocyte pathophysiology. We have demonstrated(12) that the cholinergic system regulates cholangio-cyte secretory functions. By acting on M3 ACh receptorsubtypes, ACh potentiates secretin-stimulated bicar-bonate secretion through an intracellular pathway in-volving Ca2�, calcineurin, and calmodulin but not PKC(12). The final target of this pathway is adenylyl cy-clase, whose activity is positively modulated, leading toamplification of secretin-induced cAMP levels (12).This results in marked stimulation of CFTR and Cl�/HCO3

� exchanger activity, which leads to maximal ex-cretion of bicarbonate in bile (12). Furthermore, sur-gical interruption of parasympathetic nerves (by vagot-omy) of BDL rats decreased cholangiocyte cAMP levelsand inhibits secretin-stimulated ductal secretion andcholangiocyte proliferation (38).

In this study, we demonstrated that dopaminergicinnervation is involved in the counterregulation of se-

cretin-stimulated choleresis. We first demonstrated inbile fistula BDL rats that quinelorane has no effect onbasal bile flow and biliary bicarbonate concentrationand secretion. However, quinelorane almost com-pletely abolished the stimulatory effect of secretin onbile flow and biliary bicarbonate concentration andsecretion. The inhibition of secretin-stimulatedcholeresis in bile fistula rats could be the consequenceof an indirect systemic effect of the dopaminergic sys-tem as well as effect on acetylcholine or prolactinrelease (1, 53, 66, 67). For these reasons, we directlyevaluated the effect of quinelorane on isolated IBDUfrom BDL rats and demonstrated that the expansion ofluminal space induced by secretin was completelyblocked by quinelorane, indicating a direct action oncholangiocytes by a specific interaction with D2 dopa-minergic receptors. This finding was also confirmed inpure cholangiocytes, in which we found that quinelo-rane (but not D1 or D3 dopaminergic receptor agonists)blocked secretin-stimulated cAMP synthesis.

In IBDU and cholangiocytes, we evaluated the intra-cellular mechanisms involved in quinelorane inhibi-tion of secretin-stimulated ductal secretion. We foundthat quinelorane increased intracellular cholangiocyteIns(1,4,5)P3 and Ca2� levels. In addition, by the use ofthe [Ca2�]i chelator BAPTA-AM, the PKC antagonistschelerythrine and H7, and the PKC-�-specific antago-nist rottlerin, we provided evidence that PKC-� plays arole in quinelorane inhibition of secretin-stimulatedductal secretion. This is supported by previous findingsshowing that, in many different cell types, D2 dopami-nergic receptors are coupled to Ca2�, Ins(1,4,5)P3, andPKC activation (17, 52, 60, 65). The intracellular mech-anisms by which quinelorane inhibits secretin-stimu-lated ductal secretion was demonstrated in isolatedcholangiocytes in which both cAMP levels and PKAactivity induced by secretin were abolished by pre-treatment with quinelorane. This is in keeping withthe known functional role of D2, which is a Gi-coupledreceptor whose activation or overexpression resultsin the inhibition of adenyl cyclase activity, as dem-onstrated in many different central and peripheralcells (25).

Although the involvement of the PKC pathway in theregulation of cell function is widely described in differ-ent cell types including cholangiocytes (10, 14, 23, 61,68), a novel aspect of our findings is the demonstrationthat the dopaminergic agonist inhibits secretin-stimu-lated ductal secretion by increasing protein expressionand inducing cytoskeleton-to-membrane distribution ofPKC-�. In support of the concept that different PKCisoforms can be activated differentially, we have shown(23) that gastrin inhibits cholangiocyte proliferationand secretin-stimulated ductal secretion through acti-vation of PKC-�. Similarly, insulin ursodeoxycholateor tauroursodeoxycholate inhibition of secretin-stimu-lated choleresis is associated with activation of PKC-�(2, 42). Feeding of taurocholate and taurolithocholateto normal rats stimulates cholangiocyte proliferationand secretin-stimulated secretion through activation ofPKC-� (10). The �1-adrenergic agonist phenylephrine

Fig. 3. Measurement of basal and hormone-regulated cAMP levels incholangiocytes from BDL rats. Cholangiocytes from BDL rats werestimulated for 5 min at 22°C with 1) 0.2% BSA (basal value); 2)secretin (100 nM) with 0.2% BSA; 3) quinelorane (100 �M) in theabsence or presence of secretin (100 nM); 4) eticlopride (100 �M) �quinelorane (100 �M) � secretin (100 nM) with 0.2% BSA; or 5)quinelorane (100 �M) � secretin (100 nM) in the presence ofBAPTA-AM (50 �M), chelerythrine (1 �M), H7 (20 �M), or rottlerin(10 �M) with 0.2% BSA. Intracellular cholangiocyte cAMP levelswere determined by radioimmunoassay. *P � 0.05 vs. basal values.#P � 0.05 vs. secretin-stimulated cAMP levels. Data are means SEof 7–71 experiments.

G690 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

increases secretin-stimulated ductal secretion througha Ca2�- and PKC-dependent amplification of adenylylcyclase (39). The different cross-talk between intracel-lular Ca2� and cAMP (which leads to stimulatory orinhibitory effects on secretin-stimulated ductal secre-tion) (2, 10, 12, 23, 38, 39, 42) likely depends on thetype of receptor [M3 acetylcholine (12), D2 dopaminer-gic, insulin (42), gastrin (23), or �1-adrenergic (39)] ortransporter (e.g., Na�-dependent apical bile acid trans-porter) (2, 10) that is up- or downregulated. For exam-ple, whereas there is activation of intracellular Ca2�

but not PKC (insensitivity to staurosporine) with ace-tylcholine (12), there is activation of intracellularIns(1,4,5)P3 and Ca2�- and specific isoforms of PKCwith quinelorane (PKC-�), gastrin (PKC-�) (23), insu-

lin (PKC-�) (42), taurocholate (PKC-�) (10), ursodeoxy-cholate (PKC-�) (2), or tauroursodeoxycholate (PKC-�)(2). Studies aimed to evaluate the type of PKC isoformactivated by the �1-adrenergic receptor agonist phen-ylephrine are ongoing. These interactions may resultin a different cross-talk between [Ca2�]i and specificadenylate cyclase isoforms, leading to inhibition orstimulation of adenylate cyclase and of secretin-stim-ulated ductal secretion.

Thus the dopaminergic system plays a role in thecomplex mechanisms regulating the secretory activi-ties of the intrahepatic biliary epithelium. By opposingthe cholinergic system (which potentiates secretin-stimulated ductal secretion and adenylate cyclase ac-tivity) (12), the dopaminergic system inhibits secretin-

Fig. 4. A: immunoblots for the Ca2�-dependent PKC-�, -�I, -�II, and -� andthe Ca2�-independent novel (�, �, �)and atypical (�, ) PKC isoforms inpurified cholangiocytes from 1-wk-oldBDL rats treated in vitro for 90 minwith 0.2% BSA (basal) or quinelorane(100 �M in 0.2% BSA). Quineloraneincreased protein expression of PKC-�but not PKC-�, -�I, -�II, -�, -�, -�, -�, or- in purified cholangiocytes. Data aremeans SE of 7 experiments. *P �0.05 vs. basal values. B: immunoblotsfor PKC-� in a triton-soluble fraction(containing cytoplasm � membrane)and a triton-insoluble fraction (con-taining cytoskeleton) obtained frompure cholangiocytes from BDL ratsstimulated with 0.2% BSA or quinelo-rane in 0.2% BSA. Quinelorane in-duces a membrane redistribution ofPKC-�, which was evidenced by in-creased PKC-� protein expression inthe triton-soluble but not in the triton-insoluble fraction of cholangiocytes.Data are means SE of 6–12 experi-ments. *P � 0.05 vs. its correspondingvalue of the triton-soluble fraction.#P � 0.05 vs. its corresponding basalvalue of the triton-soluble fraction.

G691DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

induced choleresis through inhibition of adenylatecyclase activity. Although both systems exert theirfunctions through increased intracellular Ins(1,4,5)P3and Ca2�, the cholinergic system acts via calmodulinand calcineurin but without recruitment of PKC (12),whereas the D2 dopaminergic system inhibits secretin-stimulated ductal secretion by increasing protein ex-pression and inducing cytoskeleton-to-membrane dis-tribution of PKC-�. However, a possible shortcoming ofour studies is that we would like to emphasize thatPKC-� may be not the only player in the regulation ofdopaminergic effects on secretin choleresis and thatother PKC isoforms (e.g., �, �1, �2, �, �, �, �, and ),although decreased or unchanged by quinelorane, mayhave a role in dopaminergic regulation of secretin-stimulated ductal secretion. Our studies have patho-physiological implications, because modulation of thedopaminergic system may play an important role in

the regulation of cholangiocyte proliferation and ductalsecretion in cholestatic liver diseases. This concept issupported by studies showing that 1) ACh potentiatessecretin-stimulated ductal bicarbonate secretion (12);2) interruption of the parasympathetic system (by va-gotomy) inhibits cholangiocyte proliferation and secre-tin-stimulated choleresis of BDL rats (38); and 3) pro-liferating cholangiocytes acquire phenotypic featuresof neuroendocrine tissue and expresses the neural ad-hesion molecule (57).

We thank S. Ambrus for technical assistance with immunohisto-chemistry.

This work was supported by a Scott & White Hospital grant andthe Texas A&M University System (to G. LeSage and G. Alpini), bya Scott & White Hospital grant (to S. Glaser), by National Instituteof Diabetes and Digestive and Kidney Diseases Grant DK-54208 (toG. LeSage), by a Veterans Affairs Merit Award and National Insti-tute of Diabetes and Digestive and Kidney Diseases Grant DK-58411

Fig. 5. Measurement of PKA activityin purified cholangiocytes (5 106)from BDL rats treated at 37°C with 1)0.2% BSA (basal) for 30 min; 2) secre-tin (30 min at 100 nM); 3) quinelorane(30 min at 100 �M); 4) quinelorane (10min at 100 �M) before stimulationwith secretin (30 min at 100 nM); or 5)BAPTA-AM, chelerythrine, H7, or rot-tlerin (10 min each) before treatmentwith quinelorane (10 min at 100 �M)followed by secretin (30 min at 100 nM)stimulation. PKA assay was performedaccording to the manufacturer’s in-structions by using PepTag assay pro-tein kinase kits. Phosphorylated pep-tide bands were quantitated byscanning densitometry by using theChemiImager 4000 low-light imagingsystem (Alpha Innotech). Data aremeans SE of 6 experiments; ns, notsignificant.

G692 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

(to G. Alpini), and by MURST Grant MM06215421/2 progetto nazio-nale 2000 (to D. Alvaro).

REFERENCES

1. Acquas E and Fibiger HC. Dopaminergic regulation of striatalacetylcholine release: the critical role of acetylcholinesteraseinhibition. J Neurochem 70: 1088–1093, 1998.

2. Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Fran-cis H, Phinizy JL, Angelico M, and LeSage G. Ursodeoxy-cholate and tauroursodeoxycholate inhibit cholangiocyte growthand secretion of BDL rats through activation of PKC alpha.Hepatology 35: 1041–1052, 2002.

3. Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL,Lasater J, and LeSage GD. Large but not small intrahepaticbile ducts are involved in secretin-regulated ductal bile secre-tion. Am J Physiol Gastrointest Liver Physiol 272: G1064–G1074, 1997.

4. Alpini G, Glaser SS, Rodgers R, Phinizy JL, RobertsonWE, Lasater J, Caligiuri A, Tretjak Z, and LeSage GD.Functional expression of the apical Na�-dependent bile acidtransporter in large but not small rat cholangiocytes. Gastroen-terology 113: 1734–1740, 1997.

5. Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, CaligiuriA, LeSage G, and LaRusso NF. Heterogeneity of the prolifer-ative capacity of rat cholangiocytes after bile duct ligation. Am JPhysiol Gastrointest Liver Physiol 274: G767–G775, 1998.

6. Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Fran-cis H, Baiocchi L, Holcomb LA, Caligiuri A, and LeSageGD. Bile acid feeding induces cholangiocyte proliferation andsecretion: evidence for bile acid-regulated ductal secretion. Gas-troenterology 116: 179–186, 1999.

7. Alpini G, Lenzi R, Sarkozi L, and Tavoloni N. Biliary phys-iology in rats with bile ductular cell hyperplasia. Evidence for asecretory function of proliferated bile ductules. J Clin Invest 81:569–578, 1988.

8. Alpini G, Lenzi R, Zhai WR, Slott PA, Liu MH, Sarkozi L,and Tavoloni N. Bile secretory function of intrahepatic biliaryepithelium in the rat. Am J Physiol Gastrointest Liver Physiol257: G124–G133, 1989.

9. Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, PodilaPV, LeSage G, and LaRusso NF. Morphological, molecular,and functional heterogeneity of cholangiocytes from normal ratliver. Gastroenterology 110: 1636–1643, 1996.

10. Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL,Francis H, and LeSage G. Bile acid feeding increased prolif-erative activity and apical bile acid transporter expression inboth small and large rat cholangiocytes. Hepatology 34: 868–876, 2001.

11. Alpini G, Ulrich CD II, Phillips JO, Pham LD, Miller LJ,and LaRusso NF. Upregulation of secretin receptor gene ex-pression in rat cholangiocytes after bile duct ligation. Am JPhysiol Gastrointest Liver Physiol 266: G922–G928, 1994.

12. Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C,Fraioli F, Romeo R, Marucci L, LeSage G, Glaser SS, andBenedetti A. Role and mechanisms of action of acetylcholine inthe regulation of rat cholangiocyte secretory functions. J ClinInvest 100: 1349–1362, 1997.

13. Alvaro D, Mennone A, and Boyer JL. Role of kinases andphosphatases in the regulation of fluid secretion and Cl�/HCO3

�

exchange in cholangiocytes. Am J Physiol Gastrointest LiverPhysiol 273: G303–G313, 1997.

14. Andre F, Rigot V, Remacle-Bonnet M, Luis J, Pommier G,and Marvaldi J. Protein kinase C-� and -� are involved ininsulin-like growth factor-I migration of colonic epithelial cells.Gastroenterology 116: 64–77, 1999.

15. Barton AC and Sibley DR. Agonist-induced desensitization ofD1-dopamine receptors linked to adenylyl cyclase activity incultured NS20Y neuroblastoma cells. Mol Pharmacol 38: 531–541, 1990.

16. Beischlag TV, Marchese A, Meador-Woodruff JH, DamaskSP, O’Dowd BF, Tyndale RF, van Tol HH, Seeman P, andNiznik HB. The human dopamine D5 receptor gene: cloning andcharacterization of the 5�-flanking and promoter region. Bio-chemistry 34: 5960–5970, 1995.

17. Bofill-Cardona E, Kudlacek O, Yang Q, Ahorn H, Freiss-muth M, and Nanoff C. Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the Gprotein activation switch. J Biol Chem 275: 32672–32680, 2000.

18. Caligiuri A, Glaser S, Rodgers RE, Phinizy JL, RobertsonW, Papa E, Pinzani M, and Alpini G. Endothelin-1 inhibitssecretin-stimulated ductal secretion by interacting with ETAreceptors on large cholangiocytes. Am J Physiol GastrointestLiver Physiol 275: G835–G846, 1998.

19. Cho WK and Boyer JL. Vasoactive intestinal polypeptide is apotent regulator of bile secretion from rat cholangiocytes. Gas-troenterology 117: 420–428, 1999.

20. Cho WK, Mennone A, Ryderg SA, and Boyer JL. Bombesinstimulates bicarbonate secretion from rat cholangiocytes: impli-cations for neural regulation of bile secretion. Gastroenterology113: 311–321, 1995.

21. Enjalbert A and Bockaert J. Pharmacological characteriza-tion of the D2 dopamine receptor negatively coupled with ade-nylate cyclase in rat anterior pituitary. Mol Pharmacol 23:576–584, 1983.

22. Fitz JG, Basavappa S, McGill J, Melhus O, and Cohn JA.Regulation of membrane chloride currents in rat bile duct epi-thelial cells. J Clin Invest 91: 319–328, 1993.

23. Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L,Kanno N, Caligiuri A, Phinizy JL, Chowdury U, Papa E,LeSage G, and Alpini G. Gastrin inhibits cholangiocyte growthin bile duct-ligated rats by interaction with cholecystokinin-B/gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2�)-,and protein kinase C alpha-dependent mechanisms. Hepatology 32:17–25, 2000.

24. Glaser SS, Rodgers R, Phinizy JL, Robertson WE, LasaterJ, Caligiuri A, Tretjak Z, LeSage G, and Alpini G. Gastrininhibits secretin-induced ductal secretion by interaction withspecific receptors on rat cholangiocytes. Am J Physiol Gastroin-test Liver Physiol 273: G1061–G1070, 1997.

25. Gordon AS, Yao L, Jiang Z, Fishburn CS, Fuchs S, andDiamond I. Ethanol acts synergistically with a D2 dopamineagonist to cause translocation of protein kinase C. Mol Pharma-col 59: 153–160, 2001.

26. Hayes G, Biden TJ, Selbie LA, and Shine J. Structuralsubtypes of the dopamine D2 receptor are functionally distinct:expression of the cloned D2A and D2B subtypes in a heterolo-gous cell line. Mol Endocrinol 6: 920–926, 1992.

27. Hemmati P, Shilliam CS, Hughes ZA, Shah AJ, RobertsJC, Atkins AR, Hunter AJ, and Heidbreder CA. In vivocharacterization of basal amino acid levels in subregions of therat nucleus accumbens: effect of a dopamine D(3)/D(2) agonist.Neurochem Int 39: 199–208, 2001.

28. Hernandez-Lopez S, Tkatch T, Perez-Garci E, GalarragaE, Bargas J, Hamm H, and Surmeier DJ. D2 dopaminereceptors in striatal medium spiny neurons reduce L-type Ca2�

currents and excitability via a novel PLC(beta)1-IP3-cal-cineurin-signaling cascade. J Neurosci 20: 8987–8995, 2000.

29. Hirata K and Nathanson MH. Bile duct epithelia regulatebiliary bicarbonate excretion in normal rat liver. Gastroenterol-ogy 121: 396–406, 2001.

30. Ishii M, Vroman B, and LaRusso NF. Isolation and morpho-logical characterization of bile duct epithelial cells from normalrat liver. Gastroenterology 97: 1236–1247, 1989.

31. Izquierdo-Claros RM, Boyano-Adanez MC, Larsson C,Gustavsson L, and Arilla E. Acute effects of D1- and D2-receptor agonist and antagonist drugs on somatostatin binding,inhibition of adenylyl cyclase activity and accumulation of ino-sitol 1,4,5-trisphosphate in the rat striatum. Brain Res MolBrain Res 47: 99–107, 1997.

32. Jones BA, Rao YP, Stravitz RT, and Gores GJ. Bile salt-induced apoptosis of hepatocytes involves activation of proteinkinase C. Am J Physiol Gastrointest Liver Physiol 272: G1109–G1115, 1997.

33. Kanno N, LeSage G, Glaser S, and Alpini G. Regulation ofcholangiocyte bicarbonate secretion. Am J Physiol GastrointestLiver Physiol 281: G612–G625, 2001.

34. Kanno N, LeSage G, Phinizy JL, Glaser S, Francis H, andAlpini G. Stimulation of alpha2-adrenergic receptor inhibits

G693DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org

cholangiocarcinoma growth through modulation of Raf-1 andB-Raf activities. Hepatology 35: 1329–1340, 2002.

35. Kao J. Practical aspects of measuring [Ca2�] with fluorescentindicators. Methods Cell Biol 40: 155–181, 1994.

36. Kassack M, Hofgen B, Lehmann J, Eckstein N, Quillan J,and Sadee W. Functional screening of G protein-coupled recep-tors by measuring intracellular calcium with a fluorescencemicroplate reader. J Biomolecular Screening 7: 233–246, 2002.

37. Kato A, Gores GJ, and LaRusso NF. Secretin stimulatesexocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J Biol Chem 267: 15523–15529, 1992.

38. LeSage EG, Alvaro D, Benedetti A, Glaser S, Marucci L,Baiocchi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R,Francis H, and Alpini G. Cholinergic system modulatesgrowth, apoptosis, and secretion of cholangiocytes from bileduct-ligated rats. Gastroenterology 117: 191–199, 1999.

39. LeSage G, Glaser S, Alvaro D, Marzioni M, Ueno Y, FrancisH, and Alpini G. Alpha-1 (but not beta-1) adrenergic agonistspotentiate secretin-stimulated ductal secretion in bile duct li-gated (BDL) rats through cross-talk between Ca2�-dependentPKC and adenylyl cyclase pathways (Abstract). Hepatology 34:A1219, 2001.

40. LeSage G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, KannoN, Phinizy JL, Francis H, and Alpini G. Regression of cholan-giocyte proliferation after cessation of ANIT feeding is coupledwith increased apoptosis. Am J Physiol Gastrointest LiverPhysiol 281: G182–G190, 2001.

41. LeSage G, Glaser SS, Gubba S, Robertson WE, Phinizy JL,Lasater J, Rodgers RE, and Alpini G. Regrowth of the ratbiliary tree after 70% partial hepatectomy is coupled to in-creased secretin-induced ductal secretion. Gastroenterology 111:1633–1644, 1996.

42. LeSage G, Marucci L, Alvaro D, Glaser S, Marzioni M,Patel T, Francis H, Phinizy JL, and Alpini G. Insulin inhib-its secretin-induced ductal secretion by activation of PKC alphaand inhibition of PKA activity. Hepatology 36: 641–651, 2002.

43. LeSage GD, Glaser SS, Marucci L, Benedetti A, PhinizyJL, Rodgers R, Caligiuri A, Papa E, Tretjak Z, JezequelAM, Holcomb LA, and Alpini G. Acute carbon tetrachloridefeeding induces damage of large but not small cholangiocytesfrom BDL rat liver. Am J Physiol Gastrointest Liver Physiol 276:G1289–G1301, 1999.

44. Maj J, Rogoz Z, and Skuza G. The anticataleptic effect of7-OH-DPAT: are dopamine D3 receptors involved? J NeuralTransm 106: 1063–1073, 1999.

45. Mann R, Bhathal PS, and Bell C. Aminergic innervation ofthe gall bladder in man and dog. Clin Auton Res 1: 205–213,1991.

46. Mann R, Bhathal PS, and Bell C. Sympathetic innervation ofthe liver in man and dog: an immunohistochemical study. ClinAuton Res 1: 141–145, 1991.

47. Mennone A, Alvaro D, Cho W, and Boyer JL. Isolation ofsmall polarized bile duct units. Proc Natl Acad Sci USA 92:6527–6531, 1995.

48. Murray AM and Waddington JL. The induction of groomingand vacuous chewing by a series of selective D-1 dopaminereceptor agonists: two directions of D1:D2 interaction. EurJ Pharmacol 160: 377–384, 1989.

49. Nathanson MH, Burgstahler AD, Mennone A, and BoyerJL. Characterization of cytosolic Ca2� signaling in rat bile ductepithelia. Am J Physiol Gastrointest Liver Physiol 271: G86–G96, 1996.

50. Ng GY, Trogadis J, Stevens J, Bouvier M, O’Dowd BF, andGeorge SR. Agonist-induced desensitization of dopamine D1receptor-stimulated adenylyl cyclase activity is temporally andbiochemically separated from D1 receptor internalization. ProcNatl Acad Sci USA 92: 10157–10161, 1995.

51. Noh JS and Gwag BJ. Attenuation of oxidative neuronalnecrosis by a dopamine D1 agonist in mouse cortical cell cul-tures. Exp Neurol 146: 604–608, 1997.

52. Nowicki S, Kruse MS, Brismar H, and Aperia A. Dopamine-induced translocation of protein kinase C isoforms visualized inrenal epithelial cells. Am J Physiol Cell Physiol 279: C1812–C1818, 2000.

53. Quevedo M, Prieto JC, and Perez-Olea J. The effect offenoldopam on the blood pressure of the rat. Gen Pharmacol 32:123–125, 1999.

54. Reyland ME, Anderson SM, Matassa AA, Barzen KA, andQuissell DO. Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells. J Biol Chem274: 19115–19123, 1999.

55. Roberts S, Kuntz S, Gores G, and LaRusso N. Regulation ofbicarbonate-dependent ductular secretion assessed by lumenalmicropuncture of isolated rodent intrahepatic bile ducts. ProcNatl Acad Sci USA 90: 9080–9084, 1993.

56. Roberts-Lewis JM, Roseboom PH, Iwaniec LM, and GnegyME. Differential downregulation of D1-stimulated adenylatecyclase activity in rat forebrain after in vivo amphetamine treat-ments. J Neurosci 6: 2245–2251, 1986.

57. Roskams T, De Vos R, and Desmet V. ‘Undifferentiatedprogenitor cells’ in focal nodular hyperplasia of the liver. Histo-pathology 28: 291–299, 1996.

58. Rutemburg AM, Kim H, Fischbein JW, Hanker JS, Was-serkrug HL, and Seligman AM. Histochemical and ultrastruc-tural demonstration of gamma-glutamyl transpeptidase activity.J Histochem Cytochem 17: 517–526, 1969.

59. Senogles SE. The D2 dopamine receptor isoforms signalthrough distinct Gi� proteins to inhibit adenylyl cyclase. J BiolChem 269: 23120–23127, 1994.

60. Senogles SE. The D2s dopamine receptor stimulates phospho-lipase D activity: a novel signaling pathway for dopamine. MolPharmacol 58: 455–462, 2000.

61. Shanmugam M, Krett NL, Maizels ET, Cutler REJ, PetersCA, Smith LM, O’Brien ML, Park-Sarge OK, Rosen ST, andHunzicker-Dunn M. Regulation of protein kinase C delta byestrogen in the MCF-7 human breast cancer cell line. Mol CellEndocrinol 148: 109–118, 1999.

62. Svenningsson P, Lindskog M, Ledent C, Parmentier M,Greengard P, Fredholm BB, and Fisone G. Regulation of thephosphorylation of the dopamine- and cAMP-regulated phospho-protein of 32 kDa in vivo by dopamine D1, dopamine D2, andadenosine A2A receptors. Proc Natl Acad Sci USA 97: 1856–1860, 2000.

63. Tietz P, Levine S, Holman R, Fretham C, and LaRusso NF.Characterization of apical and basolateral plasma membranedomains derived from cultured rat cholangiocytes. Anal Biochem254: 192–199, 1997.

64. Tietz PS, Alpini G, Pham LD, and LaRusso NF. Somatosta-tin inhibits secretin-induced ductal hypercholeresis and exocy-tosis by cholangiocytes. Am J Physiol Gastrointest Liver Physiol269: G110–G118, 1995.

65. Vallar L and Meldolesi J. Mechanisms of signal transductionat the dopamine D2 receptor. Trends Pharmacol Sci 10: 74–77,1989.

66. Vegh A, Papp JG, Semeraro C, Fatehi-Hasanabad Z, andParratt JR. The dopamine receptor agonist Z1046 reducesischaemia severity in a canine model of coronary artery occlu-sion. Eur J Pharmacol 344: 203–213, 1998.

67. Verbeeck WJ, Berk M, Paiker J, and Jersky B. The prolac-tin response to sulpiride in major depression: the role of the D2receptor in depression. Eur Neuropsychopharmacol 11: 215–220,2001.

68. Zhu GH, Wong BC, Slosberg ED, Eggo MC, Ching CK, YuenST, Lai KC, Soh JW, Weinstein IB, and Lam SK. Overex-pression of protein kinase C-beta1 isoenzyme suppresses indo-methacin-induced apoptosis in gastric epithelial cells. Gastroen-terology 118: 507–514, 2000.

G694 DOPAMINERGIC REGULATION OF CHOLANGIOCYTE SECRETION

AJP-Gastrointest Liver Physiol • VOL 284 • APRIL 2003 • www.ajpgi.org