Adrenergic receptor agonists prevent bile duct injury induced by adrenergicdenervation by increased cAMP levels and activation of Akt

Shannon Glaser,1,2 Domenico Alvaro,6 Heather Francis,1 Yoshiyuki Ueno,5 Luca Marucci,7

Antonio Benedetti,7 Sharon De Morrow,1 Marco Marzioni,7 Maria Grazia Mancino,6 Jo Lynne Phinizy,1

Ramona Reichenbach,4 Giammarco Fava,3 Ryun Summers,3 Julie Venter,3 and Gianfranco Alpini2,3,4

1Division of Research and Education, Departments of 2Internal Medicine and 3Medical Physiology, 4Central TexasVeterans Health Care System, College of Medicine, Scott and White Hospital and The Texas A & M UniversitySystem Health Science Center, Temple, Texas; 5Division of Gastroenterology, Tohoku University School of Medicine,Aobaku, Sendai, Japan; 6Department of Clinical Medicine, University of Rome, “La Sapienza,” Polo Pontino, Latina;and 7Department of Gastroenterology, Polytechnic University of Marche, Ancona, Italy

Submitted 4 July 2005; accepted in final form 20 November 2005

Glaser, Shannon, Domenico Alvaro, Heather Francis, Yo-shiyuki Ueno, Luca Marucci, Antonio Benedetti, Sharon De Mor-row, Marco Marzioni, Maria Grazia Mancino, Jo Lynne Phinizy,Ramona Reichenbach, Giammarco Fava, Ryun Summers, JulieVenter, and Gianfranco Alpini. Adrenergic receptor agonists pre-vent bile duct injury induced by adrenergic denervation by increasedcAMP levels and activation of Akt. Am J Physiol Gastrointest LiverPhysiol 290: G813–G826, 2006. First published December 8, 2005;doi:10.1152/ajpgi.00306.2005.—Loss of parasympathetic innervationafter vagotomy impairs cholangiocyte proliferation, which is associ-ated with depressed cAMP levels, impaired ductal secretion, andenhanced apoptosis. Agonists that elevate cAMP levels preventcholangiocyte apoptosis and restore cholangiocyte proliferation andductal secretion. No information exists regarding the role of adrener-gic innervation in the regulation of cholangiocyte function. In thepresent studies, we investigated the role of adrenergic innervation oncholangiocyte proliferative and secretory responses to bile duct liga-tion (BDL). Adrenergic denervation by treatment with 6-hydroxydo-pamine (6-OHDA) during BDL decreased cholangiocyte proliferationand secretin-stimulated ductal secretion with concomitant increasedapoptosis, which was associated with depressed cholangiocyte cAMPlevels. Chronic administration of forskolin (an adenylyl cyclase acti-vator) or �1- and �2-adrenergic receptor agonists (clenbuterol ordobutamine) prevented the decrease in cholangiocyte cAMP levels,maintained cholangiocyte secretory and proliferative activities, anddecreased cholangiocyte apoptosis resulting from adrenergic dener-vation. This was associated with enhanced phosphorylation of Akt.The protective effects of clenbuterol, dobutamine, and forskolin on6-OHDA-induced changes in cholangiocyte apoptosis and prolifera-tion were partially blocked by chronic in vivo administration ofwortmannin. In conclusion, we propose that adrenergic innervationplays a role in the regulation of biliary mass and cholangiocytefunctions during BDL by modulating intracellular cAMP levels.

apoptosis; bile ducts; growth; nerves; secretin

CHOLANGIOCYTES ARE THE TARGET cells of cholangiopathies (7),which are characterized by dysregulation of the balance be-tween cholangiocyte proliferation/apoptosis (7). In experimen-tal models, cholangiocyte proliferation is achieved by a num-ber of pathological maneuvers, including bile duct ligation(BDL; see Refs. 6, 7, 20, 31, 34), whereas cholangiocyte

damage by apoptosis occurs after acute administration ofcarbon tetrachloride (CCl4; see Ref. 35) or chronic feeding ofthe toxin �-naphthylisothiocyanate (33). Although cholangio-cyte proliferation is associated with increased basal and secre-tin-stimulated ductal secretion (6, 20, 31, 34), apoptosis iscoupled with decreased basal and secretin-stimulated ductalsecretory activity (35).

There is growing information regarding the mechanismsregulating the balance between cholangiocyte proliferation/apoptosis (4, 11, 19, 24). Cholangiocyte proliferation is differ-entially regulated by a number of hormones and neuropeptides(4, 11, 19, 24). Although somatostatin inhibits cholangiocyteproliferation of BDL rats by a decrease in the synthesis of theintracellular cAMP system (4), gastrin inhibits both hyperplas-tic and neoplastic cholangiocyte proliferation by an increase inCa2�/protein kinase C (PKC)-regulated cholangiocyte apopto-sis (19, 24). Interruption of the cholinergic innervation byvagotomy impairs cholangiocyte proliferation and enhancesapoptosis by a decrease in intracellular cAMP levels, thusleading to a decrease in the number of intrahepatic cholangio-cytes in response to BDL (25, 31). Maintenance of cAMPlevels, by forskolin administration, prevents the effects ofvagotomy on cholangiocyte proliferation and apoptosis (31).

In many different cell types, activation of the cAMP/proteinkinase A (PKA)/mitogen/extracellular signal-regulated kinase(MEK)/mitogen-activated protein kinase (MAPK) intracellularpathway is associated with a wide range of biological re-sponses, including differentiation, survival, inhibition ofgrowth, and apoptosis (13, 29, 39, 56). Recent evidence indi-cates that the cAMP/PKA/MEK/MAPK cascade is involved inthe modulation of cholangiocyte functions by different agents(3, 5, 17, 34). We have shown that elevation of cAMP: 1)protects cholangiocytes from vagotomy-induced apoptosis and2) stimulates the proliferation of normal rat cholangiocytes(17). cAMP has been demonstrated to promote the activationof cell survival factors such as protein kinase B (Akt; see Refs.12 and 28) by cAMP-dependent phosphorylation of Akt inneurons. cAMP has also been implicated in the activation ofphosphatidylinositol 3-kinase (PI3-kinase) to modulate bileacid secretion in WIF-B9 cells (23).

Address for reprint requests and other correspondence: G. Alpini, CentralTexas Veterans Health Care System and The Texas A & M Univ. SystemHealth Science Center College of Medicine, Temple, TX 76504 (e-mail:galpini@tamu.edu).

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

Am J Physiol Gastrointest Liver Physiol 290: G813–G826, 2006.First published December 8, 2005; doi:10.1152/ajpgi.00306.2005.

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The intrahepatic biliary epithelium displays adrenergic in-nervation (45, 54); however, no information exists regardingthe role and mechanisms of action by which adrenergic nervesregulate intracellular cAMP levels and the balance betweencholangiocyte proliferation/apoptosis in rats with cholangio-cyte hyperplasia induced by BDL. We addressed the followingquestions: 1) Do cholangiocytes from normal and BDL ratsexpress �1- and �2-adrenergic receptors? 2) Does administra-tion of 6-hydroxydopamine [6-OHDA, which causes degener-ation of adrenergic terminal fibers (14)] alter the expression of�1- and �2-adrenergic receptors in cholangiocytes from BDL

rats; 3) Does 6-OHDA induce bile duct damage and cholan-giocyte apoptosis with subsequent inhibition of cholangiocyteproliferation and ductal functional activity? 4) Does chronicadministration of dobutamine (a specific �1-adrenergic recep-tor agonist; see Ref. 55), clenbuterol (a specific �2-adrenergicreceptor agonist; see Ref. 15), or forskolin (an adenylyl cyclaseactivator; see Ref. 27) (which all increase intracellular cAMPlevels; see Refs. 1, 17, 41) prevent 6-OHDA activation ofcholangiocyte apoptosis, and inhibition of cholangiocyte pro-liferation and ductal bile secretion? 5) Are 6-OHDA effects oncholangiocyte proliferative and secretory capacity associated

a

b

Fig. 1. a: Immunohistochemistry for �1- and�2-adrenergic receptors in liver sections fromnormal and 1-wk bile duct ligation (BDL)rats. A specific reaction appears in bile ductsof normal and 1-wk BDL rats. Original mag-nification �250. b: Immunofluorescent stain-ing for �1 (A and B)- and �2 (C and D)-adrenergic receptors. Cytospin smears weremade from freshly isolated cholangiocytesfrom normal (A and C) or BDL (B and D)rats. Specific immunoreactivity is shown inred, and the smears were counterstained withDAPI (blue). No staining could be seen whenprimary antibodies were omitted (E). Immu-noblotting analysis for �1- and �2-adrenergicreceptors in protein (10 �g) from whole celllysate from BDL rats and BDL rats treatedwith 6-hydroxydopamine (6-OHDA; c) andapical and basolateral membranes (d) frompurified cholangiocytes from BDL rats. c: Byimmunoblots, we detected the protein for �1-and �2-adrenergic receptors in purifiedcholangiocytes from BDL rats and BDL ratstreated with 6-OHDA. Cholangiocyte proteinexpression for �1- and �2-adrenergic recep-tors was not altered in BDL rats comparedwith BDL � 6-OHDA-treated rats comparedwith cholangiocytes from BDL rats. AR �adrenergic receptor. Data are means � SE of7 experiments. d: These receptor subtypeswere found mostly in the basolateral mem-branes of BDL cholangiocytes.

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with changes in the cAMP/PKA and Akt cell survival path-ways? 6) Does chronic administration of the cAMP-stimulatingagonists (clenbuterol, dobutamine, or forskolin) prevent6-OHDA-induced alterations in the cAMP/PKA and Akt path-ways? and 7) Does administration of wortmannin, a PI3-kinaseinhibitor (40), block the protective effects of clenbuterol,dobutamine, and forskolin on 6-OHDA-induced changes incholangiocyte apoptosis, proliferation, and secretion?

MATERIALS AND METHODS

Animal models. Male Fischer 344 rats (150 to 175 g) were pur-chased from Charles River Laboratories (Wilmington, MA), main-tained in a temperature-controlled environment (20–22°C) with a12:12-h light-dark cycle, and fed ad libitum standard rat chow.Animals had free access to drinking water. The studies were per-formed in: 1) rats with BDL (for preparation of liver blocks andisolated cholangiocytes) or bile duct incannulation (BDI) for bile

c

d

Fig. 1—Continued

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collection (6) for 1 wk and 2) rats that (immediately after BDL orBDI) received a single intraportal injection of 6-OHDA [whichinduces degeneration of adrenergic terminal fibers (14), 50 mg/kgbody wt in 0.9% NaCl plus 0.3% ascorbic acid] followed by dailyintraperitoneal injections of 0.9% NaCl, dobutamine [a �1-adrenergicreceptor agonist (55), 0.5 �g/g body wt], clenbuterol [a �2-adrenergicreceptor agonist (15), 0.5 �g/g body wt], or forskolin [an adenylylcyclase activator (27), 0.04 mg/100 g body wt, 2 times/day] for 7days. BDL or BDI was performed as previously described (6). Toevaluate if Akt plays a role in the modulation of cholangiocyteproliferation/apoptosis by adrenergic agonists, we evaluated cholan-giocyte apoptosis and proliferation in liver sections from rats that(immediately after BDL) received a single intraportal injection of6-OHDA followed by daily intraperitoneal injections of NaCl, dobut-amine, clenbuterol, or forskolin in the presence of daily injections ofwortmannin [a PI3-kinase inhibitor (40), 0.7 mg/kg body wt (43)] inDMSO for 7 days. We also evaluated the effects of administration ofwortmannin alone on cholangiocyte growth and apoptosis in liversections from 1-wk BDL rats and rats that (immediately after BDL)received a single intraportal injection of 6-OHDA followed by dailyintraperitoneal injections of 0.9% NaCl. Because we have previouslyshown (31) that chronic intraperitoneal injections of DMSO do notalter cholangiocyte apoptosis and proliferation of BDL rats, we didnot include this group in our study. In all animals, body weight, wetliver weight, and liver weight-to-body weight ratio were determined.Study protocols were performed in compliance with institutionalguidelines (Institutional Animal Care and Use Committee).

Materials. Reagents were purchased from Sigma Chemical (St.Louis, MO) unless otherwise indicated. Porcine secretin was pur-chased from Peninsula (Belmont, CA). The mouse anti-cytokeratin 19(CK-19) antibody was purchased from Amersham (Arlington Heights,IL). The substrate for �-glutamyltranspeptidase (�-GT), N-(�-L-glu-tamyl)-4-methoxy-2-naphthylamide was purchased from Polysciences(Warrington, PA). RIA kits for the determination of intracellularcAMP levels were purchased from Amersham. The antibodies for the�1- and �2-adrenergic receptors, proliferating cell nuclear antigen(PCNA), total and phosphorylated PKA, and total and phosphorylatedAkt were purchased from Santa Cruz Biotechnology (Santa Cruz,CA). Receptor expression in cholangiocytes was confirmed with�1-adrenergic receptor antibody (PA1–049 from ABR-AffinityBioReagents, Golden, CO) and �2-adrenergic receptor (M-20 fromSanta Cruz). The antibody for �-actin (AC-74) was purchased fromSigma-Aldrich (St. Louis, MO). �1-adrenergic receptor is an affinity-purified rabbit polyclonal antibody raised against a peptide mapping atthe COOH-terminus of �1-adrenergic of mouse origin. �2-adrenergicreceptor is a rabbit polyclonal antibody raised against amino acids338–413 mapping at the COOH-terminus of �2-adrenergic receptorof human origin. Phosphorylated PKA-� (Ser96) is a rabbit polyclonalaffinity-purified antibody raised against a short amino acid sequencecontaining phosphorylated Ser96 of PKA-� of human origin. TotalPKA-� cat is an affinity-purified rabbit polyclonal antibody raisedagainst a peptide mapping at the COOH-terminus of PKA-� cat of

human origin. Phosphorylated Akt1/2/3 (Ser473)-R is a rabbit poly-clonal antibody raised against a short amino acid sequence containingphosphorylated Ser473 of Akt1/2/3 of human origin. Total Akt1/2/3 isa goat polyclonal IgG (sc-1618) raised against the COOH-terminus ofAkt1, which recognizes Akt1 and to a lesser extent Akt2/3. Water-soluble forskolin {forskolin, 7-deacetyl-7-[O-(N-methylpiperazino)-�-butyryl], dihydrochloride; see Refs. 17 and 21}, an adenylyl cyclaseactivator (27), was purchased from Calbiochem-Nova Biochem (SanDiego, CA).

Purification of cholangiocytes. Purified cholangiocytes [97–98%pure (4, 5, 19, 20, 22, 26) by �-GT histochemistry (47)] from theselected groups of animals were obtained by immunoaffinity beadseparation (22) using a mouse monoclonal OC-2 antibody (IgM,kindly provided by Dr. R. Faris, Brown University, Providence, RI)against an unidentified membrane antigen expressed by all rat intra-hepatic cholangiocytes (22). Cell viability assessed by trypan blueexclusion was 97%.

Expression of �1- and �2-adrenergic receptors in liver sections andpurified cholangiocytes. Immunohistochemistry for �1- and �2-adren-ergic receptors was performed in paraffin-embedded liver sections (5�m thick) from normal and 1-wk BDL rats. The endogenous perox-idase activity of the sections was blocked by treatment with 3%hydrogen peroxide for 15 min. Sections were processed by the SignetUSA Ultrastreptavidin Detection System-Alkaline Phosphatase kit(Signet Laboratories, Dedham, MA) as follows. Sections were incu-bated for 30 min with normal serum blocking reagent, washed for 5min in Tris �HCl (pH 7.4) buffer (Tris buffer), and subsequentlyincubated for 15 min with avidin and biotin blocking solution. Afterwashes, sections were incubated for 40 min with primary antibody

Table 1. Liver weight, body weight, and liver-to-body weight ratio in 1-wk BDL rats and BDL rats that (immediately after BDL)received a single intraportal injection of 6-OHDA (50 �g/kg body wt) followed by ip injections of 0.9% NaCl, clenbuterol(0.5 �g/g body wt), dobutamine (0.5 �g/g body wt), or forskolin (0.04 mg/100 g body wt) two times/day for 7 days

Treatment Liver Wt, g Body Wt, g Liver-to-Body Wt Ratio, %

BDL 10.01�0.40 164.85�5.61 5.59�0.34BDL � 6-OHDA � NaCl 6.85�0.91* 150.91�6.47 4.45�0.42*BDL � 6-OHDA � clenbuterol 8.80�0.43 150.93�3.34 5.81�0.22BDL � 6-OHDA � dobutamine 8.85�0.50 167.78�3.34 5.31�0.32BDL � 6-OHDA � forskolin 8.08�1.00 152.88�13.32 5.22�0.20

Values are means � SE of 14 values. BDL, bile duct ligation; 6-OHDA, 6-hydroxydopamine. *P 0.05 vs. corresponding values of 1-wk BDL rats and BDLrats that (immediately after BDL) received a single intraportal injection of 6-OHDA followed by ip injections of clenbuterol, dobutamine, or forskolin twotimes/day for 7 days.

Table 2. H&E staining of liver sections from 1-wk BDL ratsand BDL rats that (immediately after BDL) received a singleintraportal injection of 6-OHDA (50 mg/kg body wt)followed by ip injections of NaCl, clenbuterol, dobutamine,or forskolin for 7 days

Treatment Inflammation Necrosis Lobular Damage

BDL � � �BDL � 6-OHDA � NaCl �/� �/� �/�BDL � 6-OHDA � clenbuterol � �/� �BDL � 6-OHDA � dobutamine � �/� �BDL � 6-OHDA � forskolin � �/� �

We evaluated, by hemotoxylin and eosin (H&E) staining of paraffin-embedded liver sections (3 slides evaluated/group, 5 �m thick), the degree ofportal inflammation, necrosis, and lobular morphology (disarrangement ofhepatocytes). At least 10 different portal areas were evaluated for inflamma-tion, apoptosis, and lobular damage. Results were semiquantified into 4degrees (�, �, ��, ���) in comparison with the BDL samples (BDLserved as internal controls and were judged as �). There were no significantdifferences in the extent of portal inflammation, necrosis, and lobular damagebetween the different groups of animals.

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against the �1- or �2-adrenergic receptor diluted 1:200. After washeswith Tris buffer, the sections were incubated for 30 min with biotin-ylated secondary anti-goat antibody at a dilution of 1:200. Afterwashes with Tris buffer, sections were incubated for 30 min with UltraStreptavidin-alkaline phosphatase in conjugate buffer. After washeswith Tris buffer, the sections were then developed with HistomarkRED (KPL Laboratories, Gaithersburg, MD) and counterstained withmethyl green. Finally, the sections were dehydrated, mounted withPermount (Fisher Scientific), coverslipped, and examined with amicroscope (model BX 40; Olympus Optical, Tokyo, Japan). Sectionsthat were not incubated with a primary antibody served as negativecontrols.

The expression of �1- and �2-adrenergic receptors was evaluatedby immunofluorescence in cytospin smears of purified cholangiocytesfrom normal and 1-wk BDL rats. The cells were permeabilized in 1�PBS containing 0.2% Triton X-100 (PBST) and blocked in 4% BSA(in PBST) for 1 h at room temperature. Antibodies directed against�1- and �2-adrenergic receptors were diluted (1:100 and 1:10, respec-tively) in 1% BSA/PBST and were added to the slides for 2 h at roomtemperature. Cells were then washed 3 � 10 min in PBST, and a 1:50dilution (in 1% BSA/PBST) of cy3-conjugated anti-rabbit antibody(Jackson Immunochemicals) was added for 1 h at room temperature.Cells were washed again for 3 � 10 min in PBST and mounted onmicroscope slides with Antifade gold containing DAPI as a counter-stain (Molecular Probes). Images were taken on an Olympus IX71

fluorescence microscope with a DP70 digital camera. For the mergedpictures, images from each channel were overlayed electronicallyusing Adobe Photoshop software.

The expression of �1- and �2-adrenergic receptors was measuredby immunoblots (20) in protein (10 �g) from whole lysate from ratheart (positive control) and purified cholangiocytes from 1-wk BDLrats and rats that (immediately after BDL) were treated with a singleinjection of 6-OHDA. Purified cholangiocytes (3.0 � 106) wereresuspended in lysis buffer [20 mM Tris �HCl (pH 7.4), 150 mMNaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM aprotinin, 1 mMphenylmethylsulfonyl fluoride, and 1 mM leupeptin] and sonicated sixtimes (30-s bursts). Proteins (10 �g/lane) were resolved by SDS-7.5%PAGE and transferred to a nitrocellulose filter. After blocking, thefilter was incubated overnight at 4°C with a rabbit anti-�1- or -�2-adrenergic receptor antibody (1:200) followed by incubation with agoat anti-rabbit IgG horseradish peroxidase antibody [diluted 1:2,500with Tris-buffered saline-Tween 20 (TBST)]. After several washes,the filter was visualized using chemiluminescence (ECL Plus kit,Amersham Life Science, Little Chalfont, Buckinghamshire, UK).

To determine the subcellular distribution of the �1- and �2-adren-ergic receptors in cholangiocytes, we evaluated by immunoblots (20)the protein expression for these receptor subtypes in protein (10 �g)from membranes from the basolateral or apical domain (18, 32, 52) ofpurified cholangiocytes from 1-wk BDL rats. The amount of cholan-giocyte apical and basolateral membrane protein was determined

Fig. 2. Measurement of the no. of cholangiocytes undergoing apoptosis in liver sections from 1) 1-wk BDL rats; 2) BDL rats that (immediately after BDL)received a single intraportal injection of 6-OHDA followed by ip injections of NaCl, clenbuterol, dobutamine, or forskolin for 7 days; and 3) rats that(immediately after BDL) received a single intraportal injection of 6-OHDA followed by daily ip injections of NaCl, dobutamine, clenbuterol, or forskolin in thepresence of daily injections of wortmannin for 7 days. A single intraportal injection of 6-OHDA increased the no. of apoptotic cholangiocytes compared withBDL rats. Chronic administration of clenbuterol, dobutamine, or forskolin to rats prevented the stimulatory effects of 6-OHDA on the number of apoptoticcholangiocytes. Chronic administration of wortmannin partially blocks the protective effects of clenbuterol, dobutamine, and forskolin on 6-OHDA-inducedincreases in cholangiocyte apoptosis. *P 0.05 vs. cholangiocyte apoptosis of BDL rats. Data are means � SE of 6 experiments. Original magnification �40.

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using a Pierce BSA Protein Assay Kit from Pierce Biotechnology(Rockford, IL). Cholangiocyte apical and basolateral membranes wereprepared by isopycnic centrifugation on a three-step sucrose gradient(38, 34, and 31% wt/wt) as described by us and others (18, 33, 52).We and others have characterized the purity of these membranes usingspecific markers for the basolateral (i.e., Na�-K�-ATPase) and apical(i.e., alkaline phosphatase) domain of cholangiocyte membranes asdescribed (33, 52).

Evaluation of inflammation, necrosis, and lobular damage. In theselected group of animals, we evaluated, by hematoxylin and eosin(H&E) staining of paraffin-embedded liver sections (3 slides evalu-ated/animal, 4–5 �m thick), the degree of portal inflammation, ne-crosis, and lobular morphology (disarrangement of hepatocytes). Atleast 10 different portal areas were evaluated for inflammation, apop-tosis, and lobular damage. Results were semiquantified into fourdegrees (�, �, ��, ���) in comparison with the BDL samples(BDL served as internal controls and were judged as �). After theselected staining, liver sections were examined in a coded fashion bylight microscopy with an Olympus BX-40 microscope equipped witha camera. After being stained, sections were evaluated in blindedfashion with a microscope (Olympus Optical U-PMTVC). One hun-dred fifty cells per slide were counted in a coded fashion in 10nonoverlapping fields.

Evaluation of cholangiocyte apoptosis. Cholangiocyte apoptosiswas evaluated by TUNEL analysis in liver sections from the selectedgroups of animals. TUNEL analysis (the number obtained, n � 6,derives from the analysis of 3 slides/animal) was performed using a

commercially available kit (Wako Chemicals, Tokyo, Japan) as de-scribed by us (33). After counterstaining with hematoxylin solution,sections were examined by light microscopy with an Olympus BX-40microscope equipped with a camera. At least 100 cells/slide werecounted in a coded fashion in 10 nonoverlapping fields.

Cholangiocyte proliferative capacity. Cholangiocyte proliferationwas evaluated by measurement of 1) PCNA- and CK-19-positivecholangiocytes (33) in liver sections and 2) PCNA protein expression(20, 33) in purified cholangiocytes from the selected group of animals.Immunohistochemistry for PCNA (in paraffin-embedded liver sec-tions, 5 �m) and CK-19 (in frozen liver sections, 5 �M) wasperformed as previously described (33, 34). The number obtained,n � 6, derives from the analysis of 3 slides/animal. After the selectedstaining, sections were counterstained with hematoxylin and exam-ined in a random, blinded fashion with an Olympus BX 40 microscope(Olympus Optical). Data were expressed as number of PCNA- orCK-19-positive cholangiocytes per each 100 cholangiocytes in 7different fields.

Cholangiocyte proliferation was also assessed by quantitative im-munoblotting measurement of protein expression for PCNA (a markerof cell proliferation; see Refs. 19, 20, 24, 25, 33), as previouslydescribed (33) in protein (10 �g) from whole lysate samples frompurified cholangiocytes from the selected groups of animals. Ratspleen and BSA were the positive and negative controls, respectively.The amount of protein loaded was normalized by immunoblots for�-actin, the internal control (8). The intensity of the bands wasdetermined by scanning video densitometry using the ChemiImager4000 low light imaging system (Alpha Innotech, San Leandro, CA).

Cholangiocyte secretory activity. Ductal secretion was evaluatedby assessment of basal and secretin-stimulated bile flow and bicar-bonate concentration and secretion in vivo from the selected groups ofanimals. After anesthesia, rats were surgically prepared for bilecollection as described (6). One jugular vein was incannulated with aPE-50 cannula (Intramedic, Clay-Adams brand; Becton-Dickinson,Sparks, MD) to infuse either Krebs-Ringer-Henseleit (KRH) or se-cretin (100 nM). The rate of fluid infusion was adjusted according toboth the rate of bile flow and the value of the arterial hematocrit. Bodytemperature was monitored with a rectal thermometer and maintainedat 37°C by using a heating pad (Harvard Homeothermic BlanketControl Unit; Harvard Apparatus, Kent, England). Immediately afterthe bile duct was incannulated, the biliary fistula tubing was connectedto another tube of larger diameter (6) to initiate the collection of bile.When steady-state bile flow was achieved (60–70 min from thebeginning of bile collection), secretin (100 nM) was infused for 30min followed by a final infusion of KRH for 30 min. Bicarbonateconcentration (measured as total CO2) in bile from the selected groupof animals was determined by an ABL 520 Blood Gas System(Radiometer Medical, Copenhagen, Denmark).

Analysis of intracellular signaling mechanisms. IntracellularcAMP levels (a functional index of cholangiocyte proliferation/loss;see Refs. 2, 4, 19, 20, 31, 33, 35) were measured as follows. Afterpurification, pure cholangiocytes from the selected group of animals

Fig. 3—Continued

Fig. 3. Measurement of proliferating cell nuclear antigen (PCNA; A) or cytokeratin 19 (CK-19)-positive cholangiocytes (C) in liver sections from 1) 1-wk BDLrats; 2) BDL rats that (immediately after BDL) received a single intraportal injection of 6-OHDA followed by ip injections of NaCl, clenbuterol, dobutamine,or forskolin for 7 days; and 3) rats that (immediately after BDL) received a single intraportal injection of 6-OHDA followed by daily ip injections of NaCl,dobutamine, clenbuterol, or forskolin in the presence of daily injections of wortmannin for 7 days. A single intraportal injection of 6-OHDA significantly reducedthe no. of PCNA (A)- and CK-19 (B)-positive cholangiocytes compared with BDL rats. Administration of clenbuterol, dobutamine, or forskolin to rats preventedthe inhibitory effects of 6-OHDA on the no. of PCNA- or CK-19-positive cholangiocytes. Chronic administration of wortmannin partially blocks the protectiveeffects of clenbuterol, dobutamine, and forskolin on 6-OHDA-induced decreases in cholangiocyte proliferation of BDL rats. Data are means � SE of 6experiments derived from the analysis of 3 slides/animal. *P 0.05 vs. the corresponding value of BDL rats. The no. of PCNA- and CK-19-positivecholangiocytes derives from the analysis of different fields of 3 slides/group. Original magnification �40 (PCNA) and �20 (CK-19). C: measurement of PCNAprotein expression in cholangiocytes from 1-wk BDL rats and rats that (immediately after BDL) received a single portal injection of 6-OHDA followed by ipinjections of NaCl, clenbuterol, dobutamine, or forskolin for 7 days. A single intraportal injection of 6-OHDA significantly reduced PCNA protein expressionin purified cholangiocytes compared with cholangiocytes from 1-wk BDL rats. Chronic administration of clenbuterol, dobutamine, or forskolin to rats preventedthe inhibitory effects of 6-OHDA on PCNA protein expression. *P 0.05 vs. PCNA protein expression of cholangiocytes from all the other groups of animals.Data are means � SE of 4 experiments.

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were incubated for 1 h at 37°C (to regenerate membrane proteinsdamaged by proteolytic enzymes during cell isolation; see Ref. 26)and subsequently incubated at room temperature for 5 min (20, 31, 33,34) with 0.2% BSA (basal) or secretin (100 nM) with 0.2% BSA.Intracellular basal and secretin-stimulated cAMP levels were deter-mined by RIA using a commercially available kit. The proteinexpression of phosphorylated PKA and Akt (Ser473) was evaluated byimmunoblots (20). After stripping of the membrane, the expression oftotal PKA and Akt (Ser473) was evaluated by immunoblots (20). Theintensity of the bands was determined by scanning video densitometryusing the ChemiImager 4000 low-light imaging system (Alpha Innotech).

Statistical analysis. All data are expressed as means � SE. Thedifferences between groups were analyzed by Student’s t-test whentwo groups were analyzed or ANOVA if more than two groups wereanalyzed.

RESULTS

�1- and �2-Adrenergic receptors are expressed by cholan-giocytes predominately in the basolateral membrane. Both �1-and �2-adrenergic receptors were found by immunohistochem-istry in bile ducts of liver sections (Fig. 1a) and by immuno-fluorescence in purified cholangiocytes (Fig. 1b) from normaland 1-wk BDL rats. By immunoblots, we detected the proteinfor �1- and �2-adrenergic receptors (63 and 68 kDa, respec-tively) in purified cholangiocytes from BDL and BDL �6-OHDA rats (Fig. 1c). Previous studies have indicated that�1- and �2-adrenergic receptors can form homodimers andheterodimers and that �-adrenergic receptors have complexglycosylation resulting in a smeared band when analyzed bySDS-PAGE (48, 49, 51). The heterogeneity of band sizespresent most likely results from different glycosylation statesand dimerization. The presence of nonglycosylated monomeric�1- and �2-adrenergic receptors were also detected at 50 and46 kDa, respectively. Protein expression for �1- and �2-adrenergic receptors was unchanged between cholangiocytesfrom BDL rats and BDL rats treated with a single dose of6-OHDA (Fig. 1c). �1- and �2-Adrenergic receptor subtypeswere found mostly in the basolateral membranes of BDLcholangiocytes (Fig. 1d).

6-OHDA-induced reduction of the number of CK-19-posi-tive cholangiocytes and liver to body weight ratio was pre-vented by cAMP activators. No significant differences in bodyweight were observed between 1-wk BDL rats and BDL ratsthat (immediately after BDL) received a single intraportalinjection of 6-OHDA (50 mg/kg body wt) followed by intra-peritoneal injections of NaCl, clenbuterol, dobutamine, orforskolin for 7 days (Table 1). Consistent with the findings that6-OHDA induces a decrease in the number of bile ducts, asingle intraportal injection of 6-OHDA to BDL rats signifi-cantly decreased both liver weight and liver-to-body weightratio (an index of liver growth, including cholangiocytes; seeRefs. 6 and 17) compared with BDL control rats (Table 1).Chronic administration of clenbuterol, dobutamine, or forsko-lin prevented the 6-OHDA-induced decrease in liver weightand the liver-to-body weight ratio, which was similar to that of1-wk BDL rats (Table 1). Light microscopy of liver sections(stained with H&E) showed that there were no significantdifferences in the degree of portal inflammation, necrosis,and lobular damage among the selected groups of animals(Table 2).

6-OHDA-induced increases in cholangiocyte apoptosis,which was prevented by chronic administration of clenbuterol,dobutamine, or forskolin. A single intraportal injection of6-OHDA did not alter cholangiocyte apoptosis in liver sectionsof normal rats [0.4 � 0.25 of apoptotic cholangiocytes (normalrats) vs. 0.8 � 0.38 of apoptotic cholangiocytes (normal rats �6-OHDA), not significant difference]. 6-OHDA induced anincrease in cholangiocyte apoptosis compared with 1-wk BDLrats (Fig. 2). Chronic administration of clenbuterol, dobut-amine, or forskolin prevented 6-OHDA-induced increases incholangiocyte apoptosis, which remained similar to that of1-wk BDL rats (Fig. 2). Consistent with the concept that Aktplays a role in the modulation of cholangiocyte apoptosis byadrenergic agonists, chronic administration of wortmannin par-tially blocks the protective effects of clenbuterol, dobut-amine, and forskolin on the 6-OHDA-induced increase incholangiocyte apoptosis of BDL rats (Fig. 2). Administra-tion of wortmannin alone did not change cholangiocyteapoptosis in BDL rats and BDL � 6-OHDA saline-treated rats(not shown).

6-OHDA-induced inhibition of cholangiocyte proliferation,which was prevented by chronic administration of clenbuterol,dobutamine, or forskolin. A single intraportal injection of6-OHDA did not alter cholangiocyte proliferation of normalrats [0.2 � 0.2 PCNA-positive cholangiocytes (normal rats) vs.0.6 � 0.25 of PCNA-positive cholangiocytes (normal rats �6-OHDA), not a significant difference]. Similarly, administra-tion of 6-OHDA to normal rats did not alter the number ofCK-19-positive cholangiocytes [32.2 � 2.35 CK-19-positivecholangiocytes (normal rats) vs. 31.2 � 2.70 of CK-19-posi-tive cholangiocytes (normal rats � 6-OHDA), not significantdifference]. 6-OHDA induced a decrease in the number ofPCNA- and CK-19-positive cholangiocytes compared with1-wk BDL rats (Fig. 3, a-b). Chronic administration of clen-buterol, dobutamine, or forskolin prevented 6-OHDA-induceddecreases in the number of PCNA- and CK-19-positive cholan-giocytes, the values of which were similar to that of 1-wk BDLrats (Fig. 3, a and b). Consistent with the concept that Aktplays a role in the modulation of cholangiocyte proliferation byadrenergic agonists, chronic administration of wortmannin par-tially blocks the protective effects of clenbuterol, dobutamine,and forskolin on the 6-OHDA-induced decrease in the numberof PCNA- and CK-19-positive cholangiocytes of BDL rats(Fig. 3, a and b). Administration of wortmannin alone did notchange cholangiocyte proliferation in BDL rats and BDL �6-OHDA saline-treated rats (not shown). Chronic administra-tion of clenbuterol, dobutamine, or forskolin also prevented the6-OHDA-induced decrease in the PCNA protein expression incholangiocytes, the levels of which were similar to that of 1-wkBDL rats (Fig. 3c).

6-OHDA induces inhibition of secretin-stimulated bile andbicarbonate secretion, which was prevented by chronic admin-istration of clenbuterol, dobutamine, or forskolin. Secretinincreased bile flow and bicarbonate concentration and secretioncompared with the corresponding basal value in 1-wk BDL rats(Table 3). A single intraportal injection of 6-OHDA ablated theincrease in secretin-stimulated bile flow and bicarbonate con-centration and secretion compared with the correspondingbasal value (Table 3). Chronic administration of clenbuterol,dobutamine, or forskolin after intraportal injection of 6-OHDAprevented the 6-OHDA-induced reduction in secretin-stimu-

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lated bile flow and bicarbonate concentration and secretion(Table 3).

6-OHDA inhibits the cAMP/PKA pathway, which was pre-vented by chronic administration of clenbuterol, dobutamine,or forskolin. We next evaluated if 6-OHDA administrationinduces a decrease in basal and secretin-stimulated cAMPsynthesis (50), a functional index of cholangiocyte prolifera-tion and secretion (20, 26, 31, 33, 34). A single intraportal

injection of 6-OHDA significantly decreased basal cAMP lev-els in purified cholangiocytes compared with basal cAMPlevels of cholangiocytes from 1-wk BDL rats (Fig. 4). Secretinincreased intracellular cAMP levels in purified cholangiocytesfrom 1-wk BDL rats compared with the corresponding basalvalue (Fig. 4). A single intraportal injection of 6-OHDAablated the increase in secretin-stimulated intracellular cAMPlevels compared with the corresponding basal value (Fig. 4).Chronic administration of clenbuterol, dobutamine, or forsko-lin, after intraportal injection of 6-OHDA, prevented the6-OHDA-induced decrease in both basal and secretin-stimu-lated cAMP levels (Fig. 4).

We next determined if 1) 6-OHDA effects on cholangiocyteproliferative and secretory capacity are associated withchanges of the cAMP-dependent PKA pathway and 2) chronicadministration of clenbuterol, dobutamine, or forskolin prevent6-OHDA effects on the cAMP-dependent PKA pathway. Thedata show that a single intraportal injection of 6-OHDA to ratsdecreased the expression of the phosphorylated PKA protein(Fig. 5). Chronic administration of clenbuterol, dobutamine, orforskolin, following intraportal injection of 6-OHDA, pre-vented the 6-OHDA-induced changes in the phosphorylation ofPKA (Fig. 5).

6-OHDA inhibition of Akt phosphorylation was prevented bychronic administration of clenbuterol, dobutamine, or forsko-lin. Treatment with 6-OHDA in BDL rats induced a decrease inAkt1/2/3 (Ser473) phosphorylation (Fig. 6) in comparison withcontrol BDL rats. However, chronic administration of forsko-lin, clenbuterol, or dobutamine prevented the impaired Aktphosphorylation induced by 6-OHDA (Fig. 6).

DISCUSSION

The data demonstrate that normal and BDL cholangiocytesexpress �1- and �2-adrenergic receptors. 1) The expression of�1- and �2-adrenergic receptors was similar between cholan-giocytes from BDL rats and BDL rats treated with 6-OHDA; 2)chemical sympathetic denervation of the liver by 6-OHDAadministration increased cholangiocyte apoptosis and de-creased cholangiocyte proliferation and secretin-stimulatedductal secretion in BDL rats; 3) the effects of 6-OHDA wereassociated with decreased cAMP levels and decreased phos-

Fig. 4. Measurement of basal and secretin-stimulated cAMP levels in cholan-giocytes from 1-wk BDL rats and rats that (immediately after BDL) receiveda single portal injection of 6-OHDA (50 mg/kg body wt) followed by ipinjections of NaCl, clenbuterol, dobutamine, or forskolin for 7 days. A singleintraportal injection of 6-OHDA significantly inhibited both basal and secretin-stimulated cAMP levels compared with cholangiocytes from 1-wk BDL rats.Chronic administration of clenbuterol or dobutamine prevented the inhibitoryeffects of 6-OHDA on both basal and secretin-stimulated cAMP levels. *P 0.05 vs. the corresponding basal value. #P 0.05 vs. secretin-inducedcholangiocyte cAMP levels. Data are means � SE. No. above bars indicate no.of experiments.

Table 3. Effect of secretin on bile flow and bicarbonate concentration and secretion in BDI rats that (immediately after BDI)received a single intraportal injection of 6-OHDA (50 mg/kg body wt) followed by daily ip injections of 0.9% NaCl, clenbuterol(0.5 �g/g body wt), dobutamine (0.5 �g/g body wt), or forskolin (0.04 mg/100 g body wt) two times/day for 7 days

Treatment

Bile Flow, �l�min�1�kg body wt�1 Bicarbonate Concentration, mEq/lBicarbonate Secretion, �eq�min�1�kg body

wt�1

Basal Secretin Basal Secretin Basal Secretin

BDI 88.75�8.59 (12) 133.74�5.76a (12) 44.91�1.20 (12) 58.36�2.24b (12) 3.95�0.20 (12) 7.68�0.40c (12)BDI � 6-OHDA �

NaCl 86.93�6.38 (13) 96.92�6.67 (13) 34.12�1.87e (13) 35.40�2.03 (13) 2.95�0.21f (13) 3.30�0.29 (13)BDI � 6-OHDA �

clenbuterol 80.59�4.42 (11) 118.26�4.85a (11) 46.94�4.62 (11) 62.34�4.94b (11) 3.70�0.24 (11) 7.50�0.75c (11)BDI � 6-OHDA �

dobutamine 84.27�4.77 (19) 118.26�4.85a (19) 37.50�1.20 (19) 47.53�2.03b (19) 3.14�0.18 (19) 5.70�0.30c (19)BDI � 6-OHDA �

forskolin 113.97�10.11d (8) 154.46�16.67a (8) 67.28�12.69e (8) 135.30�26.94b (8) 6.78�1.33f (8) 16.72�2.37c (8)

Data are means � SE; no. of rats in parentheses. BDI, bile duct incannulation; NS, not significant. P 0.05 vs. corresponding basal value of bile flow (a), vs.corresponding basal value of bicarbonate secretion (b), vs. corresponding basal value of bicarbonate secretion (c), vs. basal bile flow of BDI rats (d), vs. basal bicarbonateconcentration of BDI rats (e), and vs. basal bicarbonate secretion of BDI rats (f). Statistical analysis was performed by both unpaired Student’s t-test and ANOVA.

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phorylation of PKA and Akt; 4) 6-OHDA-induced changes incholangiocyte proliferative and secretory capacity were pre-vented by the �1-adrenergic receptor agonist dobutamine andthe �2-adrenergic receptor agonist clenbuterol (adrenergic re-ceptors that both couple to Gs-proteins to activate adenylylcyclase; see Ref. 57) and by forskolin (adenylyl cyclase ago-nist; see Ref. 27) administration; and 5) consistent with theconcept that Akt plays a role in the modulation of cholangio-cyte apoptosis by adrenergic agonists, chronic administrationof wortmannin to rats partially blocks the protective effects ofclenbuterol, dobutamine, and forskolin on 6-OHDA effects oncholangiocyte apoptosis and proliferation.

Recent studies have demonstrated how the nervous systemplays a key role in the modulation of apoptotic, proliferative,and secretory activities of the intrahepatic biliary epithelium(9, 25, 31, 42). The cholinergic system positively modulatesthe proliferative and secretory activities of cholangiocytes byregulating adenylate cyclase activity by an intracellular path-way involving Ca2� and calcineurin but not PKC (9, 31). Theserotoninergic system exerts an antiproliferative effect by in-hibiting the cAMP/PKA/Src/MAPK pathway via inositoltrisphosphate/Ca2�/PKC signaling (37). The dopaminergicsystem inhibits secretin-stimulated ductal secretion of BDLrats through activation of PKC-� and downregulation ofcAMP-dependent PKA activity (18). No information exists onthe role of the adrenergic system in the modulation of cholan-giocyte pathophysiology, although the adrenergic innervationof the intrahepatic biliary system has been described (45). Inthe present study, we evaluated the effect of chemical sympa-

thetic denervation of the liver on the enhanced proliferative andsecretory activities of the intrahepatic biliary epithelium of theBDL rat model of experimental cholestasis (2, 4, 6, 7, 19, 20).Ligation of the common bile duct induces ductal hyperplasia(4, 6, 7, 20), which is associated with enhanced secretoryactivity and responsiveness to the choleretic stimulus of secre-tin (6). Immediately after BDL, rats were submitted to oneintraportal single injection of 6-OHDA, a procedure describedto induce a selective chemical adrenergic denervation of theliver (14). After BDL � 6-OHDA (1 wk), we evaluatedintrahepatic bile duct mass and the proliferative and secretoryactivities of cholangiocytes in comparison with control BDLrats submitted to one intraportal injection of saline instead of6-OHDA. We next evaluated whether the effects of chemicaladrenergic denervation of the liver could be reversed by ad-renergic agonists or by the adenylate cyclase stimulator fors-kolin (27). For this purpose, BDL � 6-OHDA rats were treatedby daily intraperitoneal injections of dobutamine (a �1-adren-ergic receptor agonist; see Ref. 55), clenbuterol (a �2-adren-ergic receptor agonist; see Ref. 15), forskolin (adenylate cy-clase stimulator; see Ref. 27), or NaCl. Subsequently, theproliferative and secretory activities of the intrahepatic biliaryepithelium were evaluated compared with BDL rats treatedwith 6-OHDA.

The first finding of our study is the expression of both the�1- and �2-adrenergic receptors in normal and BDL cholan-giocytes with preferential basolateral localization. This hasbeen demonstrated by immunoblotting of apical and basolat-eral membranes in purified cholangiocytes from 1-wk BDL rats

Fig. 5. Evaluation of the effects of 1)6-OHDA on the phosphorylation (p) of theprotein kinase A (PKA) pathway and 2)chronic administration of clenbuterol, do-butamine, or forskolin on 6-OHDA-in-duced changes in the phosphorylation ofPKA. A single intraportal injection of6-OHDA to rats decreased the phosphor-ylation of PKA. Chronic administration ofclenbuterol, dobutamine, or forskolin, af-ter an intraportal injection of 6-OHDA,prevented the 6-OHDA-induced changesin the expression of the phosphorylation ofPKA (total PKA � tPKA). *P 0.05 vs.all the other groups. Data are means � SEof 7 experiments.

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(Fig. 1d). 6-OHDA causes degeneration of adrenergic terminalfibers (14) but does not destroy the �1- and �2-adrenergicreceptors, since the protein for these two receptors was presentat similar levels in cholangiocytes from BDL rats and BDL ratstreated with a single intraportal injection of 6-OHDA. Thus the�1- and �2-adrenergic receptor agonists are able to interactwith their own receptor, preventing 6-OHDA-induced ductdamage. The finding that �-adrenergic receptors are involvedin the modulation of the proliferative response of cholangio-cytes to BDL is also supported by the fact that the chemicaldestruction of hepatic adrenergic fibers by 6-OHDA is fol-lowed by decreased bile duct mass, which in turn is caused bydepression of cholangiocyte proliferation (evaluated by PCNAimmunoblots) and by the activation of apoptosis (evaluated byTUNEL analysis in liver sections).

The specificity of 6-OHDA effects was demonstrated by thecapability of �-adrenergic agonists (clenbuterol and dobut-amine) to completely reverse the effects of 6-OHDA on bileduct mass, cholangiocyte proliferation, and apoptosis. In addi-tion, this is consistent with decreased liver weight and liver-to-body weight ratio after 6-OHDA and with the prevention ofthese effects by administration of dobutamine or clenbuterol.We next evaluated the intracellular transduction pathways bywhich �-adrenergic receptors modulate cholangiocyte prolif-eration.

The �1- and �2-adrenergic receptors (rhodopsin/�-adrener-gic receptors) are prototypes of class I G protein-coupled

receptors, since they induce cAMP increase via adenylyl cy-clase activation and then PKA phosphorylation in a number ofcells (46, 53). Intracellular cAMP plays an important role in theregulation of cholangiocyte proliferation (4, 20, 31, 33–35). Insupport of this concept, experimental maneuvers causing inhi-bition of duct proliferation are associated with decreasedcAMP levels in cholangiocytes (31, 35), whereas induction ofproliferation is associated with enhanced cholangiocyte cAMPlevels (4, 20, 31, 34). Maintenance of cAMP levels, by fors-kolin administration, prevents the activation of cholangiocyteapoptosis and inhibition of duct proliferation/secretion inducedby vagotomy (31). Furthermore, in vivo and in vitro activationof cholangiocyte cAMP levels alone is sufficient to increaseduct proliferation and secretion through changes in the PKA/Src/MAPK pathway (17). A number of hormones (4, 10, 19,20), neuropeptides (31, 37, 38), and bile salts (2, 8, 36, 38)modulate cholangiocyte proliferation by acting on cAMP andPKA activity, which, in turn, induce changes in the Ras/Raf/Shc/Src/extracellular/signal-regulated kinase cascade. Inagreement, we found that reduction of cholangiocyte prolifer-ation by 6-OHDA administration is associated with decreasedcAMP levels and PKA phosphorylation and that prevention of6-OHDA effects by clenbuterol or dobutamine is linked withmaintenance of these intracellular mediators. Most impor-tantly, the inhibitory effects of 6-OHDA on cholangiocyteproliferation were maintained by forskolin, and this is consis-tent with a central role of cAMP in mediating the �-adrenergic

Fig. 6. Evaluation of the effects of 1)6-OHDA on the phosphorylation of proteinkinase B (Akt; Ser473) and 2) chronic admin-istration of clenbuterol, dobutamine, or for-skolin on 6-OHDA-induced changes in thephosphorylation of Akt (Ser473). A singleintraportal injection of 6-OHDA decreasedthe phosphorylation of Akt. Chronic admin-istration of clenbuterol, dobutamine, or for-skolin, after an intraportal injection of6-OHDA, prevented the 6-OHDA-inducedchanges in the phosphorylation of Akt (tAkt �total Akt). *P 0.05 vs. all the othergroups. Data are means � SE of 3 experi-ments.

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modulation of cholangiocyte proliferation. Several hypothesescan explain our findings. The first possibility is that prolifer-ating cholangiocytes respond directly to manipulation of ad-renergic nerves, and to this regard a sparse adrenergic inner-vation of the rat intrahepatic biliary epithelium has beendocumented (45, 54). A second, most likely possibility is thatcholangiocytes respond to extracellular catecholamines. Theexpression of �-adrenergic receptors in cholangiocytes andchanges of the signaling pathways associated with these ad-renergic receptors during treatment with 6-OHDA or �-adren-ergic agonists are all consistent with this explanation. Al-though, we have shown that �1 (phenylephrine; see Ref. 32)-and �2 (UK14,304; see Ref. 16)-adrenergic receptor agonistsregulate biliary functions by increases in cholangiocyte intra-cellular Ca2� concentration and decreases in cholangiocytecAMP levels, respectively, we cannot exclude that changes in�-adrenergic vascular tone after 6-OHDA treatment mighthave influenced our findings. Indeed, a number of effects of the�-adrenergic system have been documented in the normal andinjured rat liver, including fibrogenesis, regulation of vasculartone, proliferation of the oval cell compartment, and release ofosmolites and prostaglandins (30, 44). However, the fact thatall the effects of the chemical sympathectomy on cholangio-cyte pathophysiology have been prevented by the administra-tion of selective �1- and �2-agonists indicates a major roleplayed by these receptors in the balance between cholangiocyteproliferation/loss.

We also demonstrated that 6-OHDA administration resultedin a decrease in the phosphorylation of Akt (Ser473) and that thechronic administration of the cAMP-stimulating agonists clen-buterol, dobutamine, and forskolin activated the phosphoryla-tion of Akt. Previous reports have demonstrated that cAMP canpromote the activation of cell survival factors such as AKT(12, 28) via cAMP-dependent phosphorylation of AKT inneurons. Also, cAMP can activate PI3-kinase-dependent bileacid secretion in WIF-B9 (23). Our data suggest a link betweencAMP and activation of cell survival pathways via an Akt-dependent mechanisms in cholangiocytes. Our studies showthat clenbuterol and dobutamine have very similar effects oncAMP but that dobutamine may influence Akt phosphorylationto a lesser degree compared with clenbuterol (Fig. 6). This maybe because of differences in cholangiocyte �1- and �2-receptorsignaling that influence cholangiocytes or the doses of the �1-and �2-adrenergic receptor agonists used. Because wortmannin(a PI3-kinase inhibitor; see Ref. 40) only partially blocks theprotective effects of clenbuterol, dobutamine, and forskolin on6-OHDA-induced changes in cholangiocyte apoptosis, prolif-eration, and secretion, further studies are warranted to evaluatethe other possible intracellular mechanisms that (in addition tocAMP and PI3-kinase) may regulate adrenergic modulation ofcholangiocyte function. Further evaluation of the link betweencAMP and Akt-dependent cell survival mechanisms is beingconducted.

Regarding ductal secretion, we found that 6-OHDA admin-istration abolished secretin-induced choleresis typical of BDLrats (6) and that the administration of clenbuterol, dobutamine,and forskolin prevented 6-OHDA inhibition of ductal secre-tion. The enhanced response to the choleretic effect of secretinis a typical feature of BDL rats (6), which is associated withamplification of the cAMP/PKA response to this hormone (4,18, 20). A number of previous studies have shown that changes

in proliferation are associated with parallel changes in basaland secretin-stimulated cholangiocyte secretory activities (2, 6,8, 19, 20, 31, 33–35, 38). In fact, maneuvers inducing inhibi-tion of cholangiocyte proliferation in BDL rats (i.e., by vagot-omy, acute CCl4 treatment, or chronic administration of gastrinor the bile salts ursodeoxycholate or tauroursodeoxycholate)are associated with impairment of secretin-stimulated cholere-sis (2, 19, 20, 31, 35), whereas, on the contrary, stimulation ofcholangiocyte proliferation (i.e., after partial hepatectomy, byfeeding of the bile acids taurocholate and taurolithocholate; seeRefs. 5 and 34) induces amplification of secretin-stimulatedcAMP levels and secretin-induced choleresis. The findings ofthe present study further confirm this general concept that 1)inhibition of proliferation by 6-OHDA is associated with abol-ishment of secretin-stimulated choleresis and secretin-inducedcAMP levels and 2) 6-OHDA effects on cholangiocyte func-tions are prevented by maintaining proliferation through theadministration of �-adrenergic agonists or forskolin.

In conclusion, the role of the �-adrenergic system in themodulation of cholangiocyte apoptotic, proliferative, and se-cretory activities, shown in this study, adds a new piece in thecomplex puzzle of the regulation of cholangiocyte pathophys-iology by the nervous system. Understanding the mechanismsby which nerves regulate the balance between cholangiocyteproliferation/loss may be important in patients with denervatedlivers after liver transplantation.

GRANTS

The study was supported by a Veterans Affairs Research Scholar award anda Veterans Affairs Merit Award, a grant award to G. Alpini from Scott &White Hospital and Texas A&M University, National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-58411 and DK-062975 to G.Alpini, partially by a grant award from Scott & White Hospital to S. Glaser andH. Francis, by MIUR Grant Cofin 2003 (no. 2003060498_002) to D. Alvaro,by Health and Labour Sciences Research Grants for the Research on Measuresfor Intractable Disease (from the Ministry of Health, Labor, and Welfare ofJapan), and by a Grant-in-Aid for Scientific Research C (16590573) fromJapan Society for the Promotion of Science to Y. Ueno.

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