Endogenous Opioids Modulate the Growth of the Biliary Tree in the Course of Cholestasis

Transcription

1 GASTROENTEROLOGY 2006;130: Endogenous Opioids Modulate the Growth of the Biliary Tree in the Course of Cholestasis MARCO MARZIONI,* GIANFRANCO ALPINI,, STEFANIA SACCOMANNO,* SAMUELE DE MINICIS,* SHANNON GLASER,, HEATHER FRANCIS, LUCIANO TROZZI,* JULIET VENTER, FIORENZA ORLANDO,** GIAMMARCO FAVA,* CINZIA CANDELARESI,* GIAMPIERO MACARRI,* and ANTONIO BENEDETTI* *Department of Gastroenterology, Università Politecnica delle Marche, Ancona, Italy; Division of Research, Central Texas Veterans Health Care System, Department of Internal Medicine, Division of Research and Education, Scott & White Hospital and Texas A&M University System Health Science Center College of Medicine, Temple, Texas; and **Department of Research and Animal Care N. Masera, IRCCS, Ancona, Italy Background & Aims: There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. Methods: The presence of the opioid receptor (OR), OR, and OR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. Results: Cholangiocytes express all 3 receptors studied. OR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas OR resulted in a slight increase in cell growth. The OR signal is mediated by the IP3/CamKII /PKC pathway, which inhibits the camp/pka/erk1/2/akt cascade. In contrast, OR activation stimulates the camp/pka/erk1/2/akt cascade but does not affect the IP3/CamKII /PKC pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. Conclusions: The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the OR expressed by cholangiocytes. Cholangiopathies are chronic cholestatic diseases that affect cholangiocytes, the epithelial cells lining the intrahepatic biliary tree. 1,2 Such chronic cholestatic diseases represent a challenge for the clinician; because definitive medical treatments are not yet available, cholangiopathies often lead to liver failure. Indeed, 20% of liver transplants among adults and 50% of those among pediatric patients are due to these disorders. 3 Many of the problems that arise in the management of cholangiopathies can at least in part be ascribed to the poor knowledge about their pathophysiology. Cholangiopathies are indeed commonly characterized by an impaired proliferative response to duct injury. This, together with increased cell death, leads to vanishing of bile ducts. 2,3 However, very little is known about which factors are able to affect cholangiocyte growth, with particular regard to those endogenous molecules or systems that affect cholangiocyte proliferation either way. 2 In recent years, several experimental models have been used to investigate those issues. In particular, it has been shown that if cholangiocytes do not proliferate in a normal state, 2 marked growth of the biliary epithelium can be achieved, for example, by subjecting rodents to extrahepatic biliary obstruction by bile duct ligation (BDL). 1,2,4 Such studies helped to clarify that cholangiocyte functional activity is closely coupled with cholangiocyte proliferation. 1,5 Nerves, neuropeptides, and neuroendocrine hormones play a significant role in the regulation of cholangiocyte biology. 4,6 11 Interestingly, it has been shown that in the Abbreviations used in this paper: BAPTA, 2-bis(2-aminophenoxy)- ethane-n,n,n=,n=-tetraacetic acid; BDL, bile duct ligation; BrdU, bromodeoxyuridine; BSA, bovine serum albumin; CamkII, calcium-calmodulin kinase II ; DAMGO, [D-Ala 2, N-Me-Phe 4, Gly 5 -ol]-enkephalin; DPDPE, [D-pen2,5]-enkephalin; EGF, epidermal growth factor; ERK, extracellular signal regulated kinase; IP 3, inositol 1,4,5-triphosphate; OR, opioid receptor; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C by the American Gastroenterological Association Institute /06/$32.00 doi: /j.gastro

2 1832 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 course of cholangiopathies, the biliary epithelium acquires neuroendocrine features that are not present in the normal liver 12 ; however, the link of this event to cholangiocyte biology is still obscure. We have recently shown that after BDL, cholangiocytes overexpress the neuroendocrine hormone serotonin, which is secreted in an autocrine-paracrine fashion to limit the growth of these cells associated to cholestasis. 11 There is increasing evidence that endogenous opioid peptides have receptor-mediated roles as growth regulators not only in neuronal but also in nonneuronal cells and tissues. 13 Specifically, in the gastrointestinal tract, endogenous opioids have been shown to affect pancreatic, 14 colonic, 15 and esophageal 16 cell proliferation. They commonly exert their effects interacting with 3 classic receptors, the,, and opioid receptors (ORs), 17,18 all of which belong to the G protein coupled receptor superfamily. 18 Interestingly, it has been shown that in the course of cholestasis, the opioidergic neurotransmission and opioid peptide plasma and hepatic levels are markedly increased Such discoveries led to an understanding of the fundamental role played by opioid peptides in the genesis of the pruritus of cholestasis. 23 Interestingly, it has also been shown that in the course of experimental cholestasis, the messenger RNA for preproenkephalin, which codes for Met-enkephalin and Leuenkephalin and Met-enkephalin containing peptides, is expressed in the liver. In addition, Met-enkephalin immunoreactivity is expressed in the liver, particularly in cholangiocytes, of patients with primary biliary cirrhosis 19 (the most common of the cholangiopathies among adults) 2 and of rats with cholestasis. 24 Despite the abundant evidence strongly linking the opioidergic system to cholestasis, the meaning of that link is still obscure. Specifically, it is not known yet whether endogenous opioid peptides might affect cholangiocyte biology. Therefore, we developed a study that aimed to answer the following questions. (1) Do cholangiocytes express OR, OR, and OR? (2) Does the activation of those receptors affect cholangiocyte proliferation and functional activity? (3) Which are the intracellular pathways that mediate the OR signal in cholangiocytes? (4) Do endogenous opioids participate in the neuroendocrine peptide loop that regulates cholangiocyte proliferation in the course of cholestasis? Materials and Methods Materials Reagents were purchased from Sigma Chemical Co (St Louis, MO) unless otherwise indicated. Intracellular adenosine 3=,5=-cyclic monophosphate (camp) and inositol 1,4,5-triphosphate (IP 3 ) levels were determined with radioimmunoassay kits purchased from Amersham (Arlington Heights, IL). Antibodies for immunoblotting and immunohistochemistry were purchased from Santa Cruz Biotechnologies Inc (Santa Cruz, CA) unless otherwise indicated. The antibody anti cytokeratin-19 was purchased from Novocastra (Milan, Italy). Expression of ORs in Cholangiocytes The expression of ORs was evaluated by immunohistochemistry in formalin-fixed and paraffin-embedded liver sections. To inactivate endogenous biotin, after immersion in hydrogen peroxide diluted in methanol (3%) to reduce endogenous peroxidase activity, sections were incubated with avidin and afterward with biotin. Liver sections were then incubated with or without the OR primary antibodies (1:20 dilution in 1 phosphate-buffered saline) overnight. After exposure to biotinylated secondary antibody, reactive sites were detected using peroxidase-conjugated streptavidin and diaminobenzidine (0.06%) in 1 phosphate-buffered saline containing 0.03% H 2 O 2. Slides were counterstained with Mayer hematoxylin To evaluate the presence of ORs in the biliary epithelium in the course of human chronic cholestatic liver disease, the same procedure was performed in stored paraffin-embedded liver biopsy specimens randomly selected from a group of patients with primary biliary cirrhosis followed up in our center for 10 years. Rat brain was used as positive control. The expression of the receptors was also evaluated by immunoblots both in whole rat cholangiocyte lysates. 25,27 Rat brain and 0.2% bovine serum albumin (BSA) were used as positive and negative controls, respectively. Experimental Design In vitro studies. To evaluate the effects of OR activation on cholangiocyte proliferation, pure normal and (1 week) BDL rat cholangiocytes were subjected to long-term incubation (5 hours) at 37 C with 0.2% BSA (control), with either [D-pen2,5]-enkephalin (DPDPE; 0.1 mol/l, a OR selective agonist) 14 or [D-Ala 2, N-Me-Phe 4, Gly 5 -ol]-enkephalin (DAMGO; 0.1 mol/l, a OR selective agonist), 14 or with dynorphin A (0.1 mol/l, a OR selective agonist). 14 To study whether the changes observed in cholangiocytes after OR activation are mediated by the camp/protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) pathways, the experiments were also performed by preincubating the cells for 30 minutes at 37 C with either Rp-cAMP (100 mol/l, 28 a camp-dependent PKA inhibitor), PD98059 (50 mol/l, an MEK inhibitor), 29 or wortmannin (100 nmol/l, a PI3K inhibitor), 28 respectively, followed by the incubation with OR agonists or 0.2% BSA as described previously. Furthermore, to investigate whether ORs transduct the message through the activation of Ca 2 signaling, cells were also preincubated with either

3 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION bis(2-aminophenoxy)-ethane-N,N,N=,N=-tetraacetic acid (BAPTA)/AM (5 mol/l, an intracellular Ca 2 chelator), 8,11 KN62 (10 mol/l, a calcium-calmodulin kinase II inhibitor), 30 or Ro (0.5 mol/l, a Ca 2 -dependent protein kinase C [PKC] inhibitor), 31 respectively, followed afterward by incubation with OR agonists or 0.2% BSA as described previously. In the same fashion, to verify whether the activation of ORs may interfere with the effect played by the epidermal growth factor (EGF) on cholangiocyte proliferation, 32 cells were incubated with EGF (10 ng/ml) 33 for 5 hours (long-term incubation) after a 30-minute preincubation with either 0.2% BSA or with OR agonists as described previously. As a control of the ligand/receptor dependency of the effects of EGF, cells were also preincubated with AG1478 (10 mol/l, an EGF receptor inhibitor). 33 To show that the effects observed in these experiments are actually due to a specific agonist/receptor interaction, cells were incubated with the OR selective agonists after preincubation with naloxone (1 mol/l, a general OR antagonist) 34 for 30 minutes. Finally, to provide evidence that enkephalins might be secreted by cholangiocytes in an autocrine/paracrine fashion, BDL rat cholangiocytes were subjected to long-term incubation (5 hours) at 37 C with 0.2% BSA or naloxone. At the end of the incubation time, which was chosen as in previous studies 11,28 in light of the fact that the changes in proliferating cell nuclear antigen (PCNA) protein expression are very early events in the proliferative process, 35 cells were lysed to obtain proteins for immunoblotting, as described in the following text. In a second set of experiments, pure normal and BDL rat cholangiocytes were incubated at 37 C with increasing doses of DPDPE, DAMGO, or dynorphin A ( nmol/l and mol/l) for 12 hours. Such a longer incubation was chosen to assess the changes in cell proliferation on OR activation assaying the bromodeoxyuridine (BrdU) incorporation, an event that occurs later in the cell proliferation process. 36 In vivo studies. Male Fischer 344 rats ( g; INRCA, IRCCS, Ancona, Italy) were kept in a temperaturecontrolled environment (20 C 22 C) with a 12-hour lightdark cycle and free access to drinking water and standard rat chow. To study the effects of OR agonist administration on cholangiocyte proliferation and, as a consequence, on their functional activity, our studies were performed in (1) normal rats and rats with BDL 1 (for cholangiocyte purification or liver sections) or bile duct incannulation (BDI) (BDI for bile collection) for 1 week 1 and (2) normal, BDL, BDI (that immediately after BDL or BDI) rats treated with intraperitoneal injections of either DPDPE (10 mg kg 1 day 1 ) 37 or DAMGO (0.1 mg kg 1 day 1 ) 38 for 1 week. To verify the actual in vivo effects of endogenous opioids on the development of chronic cholestasis, BDL rats were also treated with daily intraperitoneal injections of naloxone (0.4 mg kg 1 day 1 ). 39 The animals were fasted overnight before each experiment. 11 Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). Study protocols were performed in compliance with the institution guidelines. Isolation of Pure Cholangiocytes and Assessment of Cell Viability Purification 40 of cholangiocytes from normal or 1-week BDL rats was performed using a monoclonal antibody (immunoglobulin M, kindly provided by Dr R. Faris, Brown University, Providence, RI) against an unidentified membrane antigen expressed by all rat intrahepatic cholangiocytes. 40 Separation of small and large cholangiocyte subpopulations was obtained by counterflow elutriation as previously reported. 41 At the end of each procedure, purity of cholangiocytes was assessed by cytochemistry for -glutamyltransferase, 4,9,11 a specific marker for cholangiocytes. 1 Cell viability at the end of the purification procedure was determined by trypan blue exclusion and found to be 97%. Cell viability was also assessed at the end of in vitro experiments by both trypan blue exclusion and the cytotoxicity detection kit (Roche, Monza, Italy). The latter, based on the measurement of the activity of lactate dehydrogenase released in the culture medium by damaged cells, was performed according to the instructions supplied by the vendor. Evaluation of Cholangiocyte Proliferation The changes in cholangiocyte proliferation were assayed by both immunoblots for PCNA protein expression 4 and measurement of BrdU incorporation 33 for the in vitro studies. The assessment of the bile duct mass by the quantitative immunohistochemistry Cytokeratin (CK)-19, a specific marker of cholangiocytes, 11,42 was used for the in vivo studies. Immunoblots for the evaluation of PCNA protein expression were performed as previously described, 4,11 comparing the amount of loaded proteins with immunoblots for -actin. 11 Incorporation of BrdU was measured using the cell proliferation enzyme-linked immunosorbent BrdU assay according to the instructions provided by the vendor (Roche), as previously described. 33 Immunohistochemistry for cytokeratin-19 was performed in frozen liver sections as previously shown by us. 11,42 After examination of the stainings with a microscope, photographs of 7 different fields per group (selected in a blinded, random fashion) were taken. Then, by computerized analysis, the volume percent of liver occupied by ducts was calculated from the total number of points over hepatic tissue and the number of points over cytokeratin-19 positive ducts, as previously reported. 11,42 The quantitative analysis was performed using an Olympus microscope (Olympus Vanox AHBT3; Olympus Optical Co Ltd, Tokyo, Japan) equipped with a computerized image analysis system (CUE-3 Image Analyzer; Galai Production Ltd, Migdal Haemek, Israel).

4 1834 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Evaluation of Cholangiocyte Functional Activity The functional activity of cholangiocytes was determined measuring (1) the basal and secretin-stimulated bile and bicarbonate secretion in BDI rats and (2) basal and secretinstimulated camp levels in pure cholangiocytes. Following anesthesia, animals were surgically prepared for bile collection as described by us. 1 After steady spontaneous bile flow was reached (60 70 minutes from the infusion of Krebs Ringer-Henseleit buffer), the animal was infused for 30 minutes with secretin (100 nmol/l) followed by a final infusion of Krebs Ringer-Henseleit buffer for 30 minutes. Bile was collected every 10 minutes and immediately stored at 70 C in preweighed tubes. Bicarbonate concentration (measured as total CO 2 ) was determined using a Natelson microgasometer apparatus (Scientific Industries, Bohemia, NY). Intracellular camp levels were evaluated as previously described 4,5,41 ; following incubation at 37 C for 1 hour, 4 cells ( ) were incubated for 5 minutes at room temperature 4,8,11 with 0.2% BSA (basal) or 100 nmol/l secretin with 0.2% BSA. Intracellular camp levels were then determined using commercially available radioimmunoassay kits (Amersham) according to the vendor s instructions. Biliary Met-enkephalin Excretion The detection and quantification of Met-enkephalin immunoreactivity in bile was assayed using the Met-Enkephalin Radioimmunoassay Kit (Peninsula Laboratories, San Carlos, CA) according to the vendor s instructions. Briefly, Metenkephalin was extracted as follows. Bile samples were eluted with 1% trifluoroacetic acid (buffer A; 100 ml) and centrifuged for 20 minutes at 17,000g. Sep columns (treated with 200 mg of C18) were equilibrated and the samples loaded onto the column. After 2 washes with buffer A, the peptide was eluted with 60% acetonitrile (buffer B) and dried using a centrifugal concentrator. After Met-enkephalin extraction from bile aliquots, samples were dissolved in radioimmunoassay buffer (100 L). Samples were then incubated overnight at 4 C with 100 L of rabbit antiserum. At the end, 125 I-peptide was added and a second overnight incubation followed. After the addition of goat anti-rabbit immunoglobulin G serum (100 L) and normal rabbit serum (100 L), samples were mixed and incubated at room temperature for 90 minutes. Finally, 100 ml of radioimmunoassay buffer was added, and samples were vortexed and centrifuged at 1700g for 20 minutes. Supernatant was aspirated, and samples were counted on a gamma counter. Data were obtained by measuring the amount of 125 I-peptide bound as a function of the concentration of the unlabeled peptide in a standard reaction mixture. A standard curve was constructed from which the concentration of the peptide in unknown samples was determined. Bile samples were obtained from 6 different animals under the infusion of Krebs Ringer Henseleit buffer or secretin as previously described. Measurement of PKA Activity PKA activity was evaluated using the PepTag Assay Protein Kinase Kit (Promega, Milan, Italy) according to the instructions of the manufacturer as previously described by us. 8,11 Evaluation of Calcium-Calmodulin Kinase II, PKC, Extracellular Signal Regulated Kinase 1/2, and AKT Phosphorylation Cells were resuspended in lysis buffer (20 mmol/l Tris-HCl, ph 7.4, 150 mmol/l NaCl, 5 mmol/l EDTA, 1% Nonidet P-40, 1 mmol/l aprotinin, 1 mmol/l phenylmethylsulfonyl fluoride, and 1 mmol/l leupeptin) and sonicated 3 times (10-second bursts). Proteins (30 g/lane) were resolved by sodium dodecyl sulfate/7.5% polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated overnight at 4 C with either anti phospho-calcium-calmodulin kinase II (CamKII ), 43 anti-camkii, 43 anti phospho-pkc, 44 anti- PKC, 9 anti phospho-extracellular signal regulated kinase (ERK) 1/2, 11 anti ERK 1, 11 anti ERK 2, 11 anti phospho- AKT, 28 and anti-akt, 28 followed by incubation with the corresponding secondary antibody. After several washes, proteins were visualized using chemiluminescence (ECL Plus Kit; Amersham). The intensity of the bands was determined by scanning video densitometry using the Chemi Doc imaging system (BioRad, Milan, Italy). Measurement of IP 3 Levels Intracellular IP 3 levels were measured from cholangiocytes ( ) by the IP 3 [3H] assay kit according to the instructions supplied by the manufacturer (Amersham). 8,11 Statistical Analysis All data are expressed as mean SE. For the in vitro experiments, data are expressed as percentage of basal value. The differences between groups were analyzed by Student t test if 2 groups were analyzed, or analysis of variance if more than 2 variables were considered. Results Heterogenic Expression of ORs in Cholangiocytes in Normality and Cholestasis Both immunohistochemistry (Figure 1A) and immunoblots (Figure 1B) showed that normal rat cholangiocytes express OR, OR, and OR. In particular, immunoblots showed a heterogenic expression along the biliary tree for OR that was found to be more abundant in large rather than in small cholangiocytes (Figure 1B). In contrast, no major differences among the 2 cell subpopulations for OR and OR (Figure 1B) were observed. After 1 week BDL, expression of OR in cholangiocytes was significantly

5 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1835 Figure 1. Expression of the OR, OR, and OR in cholangiocytes. (A) Immunohistochemical detection of OR (arrows) in rat brain (top row), rat liver (middle row), and primary biliary cirrhosis (bottom row) (original magnification 50 ). (B) Immunoblots show that normal rat cholangiocytes express the 3 receptor subtypes. No major differences in OR and OR expression are observed among small and large cholangiocytes, whereas the OR displays a higher expression in large rather than small cholangiocytes. (C) Quantitative immunoblots: after 1-week BDL, only the expression of the OR is down-regulated, whereas no modifications are observed for OR and OR (*P.03 vs the other groups; data are expressed as mean SE of 3 experiments). diminished compared with normal, whereas OR and OR expression was not modified (Figure 1C). In primary biliary cirrhosis sections, cholangiocytes were homogeneously positive for the OR and OR stainings. In contrast, even if evident, the OR staining was less intense and scattered (Figure 1A). Biliary Excretion of Met-enkephalin in Normality and Cholestasis In normal rats, a minimal amount of Met-enkephalin was found in bile. Interestingly, biliary Metenkephalin immunoreactivity was almost tripled in BDL

6 1836 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Table 1. Biliary Met-enkephalin Excretion Assessed by the Specific Met-Enkephalin Radioimmunoassay Kit Basal (pg min 1 kg Secretin (pg min 1 kg body wt 1 ) body wt 1 ) Normal rat BDL rat a a NOTE. In rats subjected to 1-week BDL, a significant increase of the amount of Met-enkephalin in bile is observed compared with normal rats. The biliary Met-enkephalin excretion further increased after the infusion of secretin to BDL rats. Biliary Met-enkephalin excretion was quantified as picograms per minute per kilogram of body weight. Data are expressed as mean SE of at least 6 experiments. a P.05 vs normal rat basal. rats, and when cholangiocyte choleresis was triggered by the infusion of secretin, biliary Met-enkephalin immunoreactivity was found to be further (even if not statistically) increased (Table 1). Effect of OR Activation on Cholangiocyte Proliferation In Vitro In vitro, the long-term activation of OR by incubation with the selective agonist DPDPE markedly reduced the proliferation of BDL but not of normal rat cholangiocytes (Figure 2A). In contrast, when BDL cholangiocytes were incubated with DAMGO, the selective OR agonist, PCNA protein expression was slightly but significantly increased without, however, modifying normal rat cholangiocytes (Figure 2B). Surprisingly, incubation with the OR selective agonist was not associated with changes in any sense of normal or BDL rat cholangiocytes (Figure 2C). Similar results were obtained when the changes in cell proliferation were assayed by the measurement of BrdU, which also showed that both DPDPE and DAMGO exerted their effects on cell growth in a dose-dependent manner (Figure 2D). DPDPE was also found able to counteract the enhancement of BDL cholangiocyte proliferation induced by EGF; in contrast, the presence of DAMGO further increased the effect of EGF, even if without reaching statistical significance (Figure 2E). All of those findings were associated with no changes in cell viability (not shown). Characterization of Intracellular OR Signaling The inhibition of BDL cholangiocyte proliferation induced by OR activation was neutralized by preincubation with the intracellular Ca 2 chelator BAPTA/ AM, the CamKII inhibitor KN62, and the Ca 2 - dependent PKC inhibitor Ro but not by the camp-dependent PKA inhibitor Rp-cAMP, the MEK inhibitor PD98056, or the PI3K inhibitor wortmannin (Figure 3A). As a confirmation of the relevance of Ca 2 signaling in transducing the OR message, OR activation determined a significant increase in IP 3 intracellular levels and in CamKII and PKC phosphorylation. The specificity of such findings was confirmed by the fact that intracellular Ca 2 chelation with BAPTA/AM prevented the OR-induced increase in CamKII and PKC phosphorylation but did not modify the OR-induced increase in IP 3 levels. Furthermore, preincubation with KN62 blocked the OR-induced increase in CamKII phosphorylation in an only partial blockage of the ORinduced enhancement of PKC phosphorylation and in no changes in IP 3 levels. With a similar meaning, the presence of Ro neutralized the increase in PKC phosphorylation due to OR activation, without affecting IP 3 levels and CamKII phosphorylation (Figure 4A). The OR activation determined a marked reduction of both PKA activity and ERK1/2 and AKT phosphorylation. Such changes were mediated by the activation of Ca 2 signaling, because they were no longer evident when cells were preincubated with BAPTA/AM, KN62, or Ro (Figure 5A). All of those findings were associated with no changes in cell viability (not shown). Characterization of the Intracellular OR Signaling The increase in BDL cholangiocyte proliferation determined by OR activation was prevented by preincubation with Rp-cAMP, PD98056, and wortmannin, whereas such an effect was not observed when cells were pretreated with BAPTA/AM, KN62, or Ro (Figure 3B). In confirmation of that, OR activation did not increase IP 3 levels or CamKII and PKC phosphorylation (Figure 4B). In contrast, cell stimulation with the OR selective agonist resulted in a significant enhancement of both PKA activity and ERK1/2 and AKT phosphorylation. The increase in PKA activity associated with OR activation was only neutralized by preincubation with the PKA-specific inhibitor RpcAMP, which was also able to prevent the eliciting of ERK1/2 and AKT phosphorylation. Furthermore, if the MEK inhibitor PD98056 blocked ERK1/2 phosphorylation induced by OR activation, it did not exert any effect on AKT phosphorylation. In an opposite fashion, the PI3K inhibitor wortmannin significantly reduced AKT phosphorylation associated with OR activation without, however, modifying ERK1/2

7 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1837 Figure 2. Effect of OR activation on cholangiocyte proliferation in vitro, measured by PCNA protein expression and incorporation of BrdU. (A) Incubation with the OR selective agonist (0.1 mol/l) induced a marked reduction of the PCNA protein expression in BDL, but not normal, cholangiocytes. (B) Activation of the OR by its selective agonist (0.1 mol/l) determined a slight, although significant, increase of PCNA protein expression in BDL, but not normal, cholangiocytes. (C) There were no changes of PCNA protein expression in normal or BDL cholangiocytes when cells were incubated with the OR selective agonist. (D) Similar changes were also observed by the BrdU incorporation studies, which showed that OR (top graphs) and OR (middle graphs) ligands modify BDL cholangiocyte proliferation in a dose-dependent fashion. (E) EGF increases BDL cholangiocyte proliferation. Such an event is prevented by OR activation, whereas it is further enhanced by OR activation. Data are expressed as mean SE of at least 3 experiments. *P.04 vs basal; # P.05 vs the other groups; P.05 vs the other groups.

8 1838 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Figure 3. (A) The inhibitory effect of OR activation on BDL cholangiocyte proliferation (lane 2) is neutralized by preincubation with a Ca 2 chelator (lane 8), by a CamKII inhibitor (lane 7), and by a Ca 2 -dependent PKC inhibitor (lane 6). (B) The increase observed in cholangiocyte proliferation after OR activation (lane 2) is abolished by preincubation with a camp-dependent PKA inhibitor (lane 5), with an MEK (lane 3), and with a PI3K inhibitor (lane 4), whereas no changes were observed when cells were preincubated with a Ca 2 chelator (lane 8), with a CamKII inhibitor (lane 7), or with a Ca 2 -dependent PKC inhibitor (lane 6). *P.05 vs the other groups; data are expressed as mean SE of at least 3 experiments. phosphorylation. The blockage of the Ca 2 signaling by BAPTA/AM, KN62, or Ro did not change the effects of OR activation on PKA activity and ERK1/2 and AKT phosphorylation (Figure 5B). All of those findings were associated with no changes in cell viability (not shown). Effect of In Vivo OR and OR Activation on the Proliferative and Functional Response of the Biliary Tree in the Course of Cholestasis The in vivo administration of the selective OR agonist significantly diminished the bile duct mass of the BDL-treated compared with untreated animals (Figure 6A, top). Such a reduction was also coupled with a marked reduction of cholangiocyte functional activity, as deduced by the blunted response to secretin of bile flow, bicarbonate secretion, and intracellular camp synthesis (Figure 6A, bottom). In contrast to the effect of in vivo OR activation, but similar to what is observed in vitro, the treatment of BDL rats with the OR selective agonist slightly enhanced bile duct mass and maintained the response to secretin of bile flow, bicarbonate secretion, and intracellular camp synthesis (Figure 6B). Role of OR Blockage on the Growth of the Biliary Tree in the Course of Cholestasis In vivo administration of naloxone to BDL rats further enhanced the growth of the biliary tree in response to cholestasis, as shown by the increased bile duct mass of naloxone-treated compared with untreated animals (Figure 7A). Similarly, when pure BDL cholangiocytes were incubated in vitro in the presence of naloxone, PCNA protein expression was significantly augmented (Figure 7B). Moreover, the previously described effects of OR and OR activation on BDL cholangiocyte proliferation were receptor/ligand dependent, because they were no longer evident when cells were preincubated with the general receptor antagonist naloxone (Figure 7B). Discussion The current investigation provides evidence that endogenous opioid peptides are major determinants of the proliferative response of the biliary tree to cholestasis. In particular, this study shows the following: (1) Cholangiocytes express the OR, OR, and OR, with OR the only one among those that is down-regulated in the course of cholestasis. (2) Although normal rat cholangiocyte proliferation is not modified by OR agonists, activation by the OR agonist markedly diminishes BDL cholangiocyte pro-

9 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1839 Figure 4. (A) Incubation of BDL cholangiocytes with the OR selective agonist (lane 2) significantly increases the intracellular IP 3 levels (top) and the CamKII (middle) and PKC (bottom) phosphorylation. None of these were modified by preincubation with the camp-dependent PKA inhibitor (lane 5), the MEK (lane 3), and the PI3K inhibitor (lane 4). The Ca 2 chelator (lane 8) neutralized both the OR activation-induced CamKII (middle) and PKC (bottom) phosphorylation but not the OR activation-induced increase in intracellular IP 3 levels. The CamKII inhibitor (lane 7) neutralized the OR activation-induced increase in CamKII phosphorylation (middle) but only partially inhibited the increase in PKC phosphorylation (bottom). Preincubation with the Ca 2 -dependent PKC inhibitor (lane 6) only blocked the increase in PKC phosphorylation (bottom). (B) The incubation of BDL cholangiocytes with the OR selective agonist did not modify the intracellular IP 3 levels (top) and the CamKII (middle) and PKC (bottom) phosphorylation. *P.05 vs the other groups; data are expressed as mean SE of at least 3 experiments. liferation, OR activation slightly enhances the effect of BDL on cell growth, and OR activation does not affect cholangiocyte proliferation at all. (3) Those changes in cell growth are mediated by OR- and OR-induced parallel changes in the camp/pka/erk1/2/pi3k cascades; OR inhibits such a pathway by activating the Ca 2 /CamKII / PKC cascade, whereas OR activation does not result in any modification of such a cascade. (4) The general OR antagonist naloxone further enhances cholangiocyte growth after BDL.

10 1840 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Figure 5. (A) Incubation with the OR selective agonist (lane 2) markedly reduced PKA activity (top) and ERK1/2 (middle) and AKT phosphorylation (bottom), an effect that was neutralized by preincubation with a Ca 2 chelator (lane 8), a CamKII inhibitor (lane 7), and a Ca 2 -dependent PKC inhibitor (lane 6). (B) Incubation with the OR selective agonist (lane 2) enhanced both PKA activity (top) and ERK1/2 (middle) and AKT phosphorylation (bottom), effects that were all neutralized by preincubation with a camp-dependent PKA inhibitor (lane 5). In contrast, preincubation with the MEK (lane 3) and the PI3K (lane 4) inhibitors was only associated with blockage of the effect of OR activation on EKR1/2 phosphorylation and AKT phosphorylation, respectively. *P.05 vs the other groups; P.05 vs perk1 of the other groups; # P.05 vs perk2 of the other groups; data are expressed as mean SE of at least 3 experiments. Cholangiopathies are among the most frequent causes of liver transplantation in the United States. 3 They are a broad spectrum of diseases sharing the feature of being chronic conditions inducing loss of bile ducts and chronic cholestasis, leading to liver failure. 2 The vanishing of bile ducts observed in these diseases is at least in part ascribed to the impairment of the proliferative response of cholangiocytes to liver injury. 2 However, little is still known about the factors that regulate cholangiocyte proliferation in normal and diseased states. Recent studies have discovered a close connection between endogenous opioid peptides and chronic cholestasis. In particular, it has been shown that opioid peptide plasma levels and activity, opioid peptide hepatic concentration, and central opioidergic neurotransmission are increased in the course of both human and experimental cholestasis ,45 Those studies resulted in the discovery that endogenous opioids play a fundamental role in the genesis of pruritus typical of cholestasis. 23 Interestingly, endogenous opioid peptides have also been 3 Figure 6. (A) The in vivo administration to BDL rats of the OR selective agonist markedly reduced bile duct mass (top, P.05 vs BDL 0.9% NaCl) and cholangiocyte functional activity, given the loss of the response to secretin of bile flow (bottom, left), HCO 3 excretion (bottom, middle), and intracellular camp synthesis (bottom, right); *P.05 vs basal. (B) In contrast, in vivo treatment of BDL rats with the OR selective agonist slightly but still significantly increased bile duct mass (top, P 05 vs BDL 0.9% NaCl) and cholangiocyte functional activity was maintained (bottom, *P.05 vs basal). Data are expressed as mean SE of at least 3 experiments.

11 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1841

12 1842 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Figure 7. (A) In vivo administration of the OR antagonist naloxone to BDL rats markedly enhanced bile duct mass ( P.05 vs BDL 0.9% NaCl). (B) Similarly, in vitro incubation of BDL cholangiocytes with naloxone (lane 2) significantly increased cholangiocyte proliferation, also neutralizing the effect of both OR (lane 3) and OR (lane 4) activation on cell growth (*P.04 vs basal). Data are expressed as mean SE of at least 4 experiments. shown to act as growth regulators in neural 13 and nonneural cells ,46 In light of that, we wondered whether endogenous opioid peptides might also have a role in the regulation of cholangiocyte proliferation. Such a hypothesis (ie, that peptides of nervous origin such as opioids might govern the growth of the biliary epithelium) was not completely irregular, in our opinion, given the fact that cholangiocyte growth and functional activity are known to be under the direct control of nerves and neuroendocrine hormones. 4,6 11 Moreover, cholangiocytes themselves, in the course of cholestasis, express a neuroendocrine phenotype that is not typical of these cells in normal states. 12 To begin to address the general aim of the current study, we verified if cholangiocytes express the classic OR, OR, OR, and OR. 17,18 We found that although cholangiocytes express all 3 receptors, only OR displayed a higher expression in large cholangiocytes compared with small cholangiocytes, for example, in those of the biliary epithelial cells that proliferate in response to BDL (Figure 1). 41 In addition, among the 3 receptors, OR was the only one in which expression was downregulated after BDL (Figure 1). Given the presence of the 3 receptors in cholangiocytes, the following step was to study if their long-term activation determined changes in cell proliferation. Cell incubation with the OR selective agonist resulted in a marked, dose-dependent reduction of BDL (eg, hyperplastic) 1,4,41 but not normal (eg, quiescent) 2,47 rat cholangiocyte proliferation (Figure 2). Interestingly, OR activation did not exert such an antiproliferative effect but rather determined a slight but significant increase of the growth of BDL (but not normal) rat cholangiocytes, as if OR might be able to enhance but not to trigger cell proliferation. 48 Those dual effects of OR and OR activation were also reproduced when BDL cholangiocyte proliferation was further elicited by EGF. In contrast to both OR and OR, OR activation did not determine any modification of either normal or BDL cholangiocyte proliferation. Such differential distribution and effects of OR on cholangiocytes were surprising. However, features similar to those we found in our cells were observed in neural cells. ORs are heterogeneously expressed in cells of the central nervous system, a condition that is believed to reflect the proliferative state of those cells. 49,50 In particular, a higher expression of the OR was observed

13 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1843 in more mature cells compared with immature ones. 51 This parallels the higher expression of OR that we found in large cholangiocytes, for example, those cells considered more mature and sensible to injury, rather than in small cholangiocytes, which are considered less mature and more resistant to duct injury. 47 Furthermore, the presence in the cell membrane of different OR subtypes is often associated with a differential effect of their activation on cell growth 49,52 or function, 53 again in a fashion similar to what was observed in our experiments. Moreover, the ability of ORs to interfere with the proproliferative effect of EGF in cholangiocytes 32 is a feature analogous to what is seen in other cells. 54,55 To provide additional confirmation and a mechanistic significance to the findings regarding cell proliferation, we then wanted to investigate the intracellular messengers that mediate the effects of OR and OR activation. This is of particular interest, because there is still poor knowledge on the intracellular signals governing cholangiocyte proliferation in the course of chronic cholestasis. We observed that incubation with the OR selective agonist, which reduces BDL cholangiocyte proliferation, inhibited camp/pka activity and ERK1/2 (one of the downstream MAPK) 56 and AKT (an immediate downstream of PI3K) 28 phosphorylation (Figure 5). The camp/pka cascade, the MAPK and PI3K pathways, are a major determinant of cholangiocyte growth 6,28 and are modulated by neuroendocrine hormones that affect cholangiocyte proliferation. 11 In contrast, OR activation increased the activity/phosphorylation of such pathways; as confirmation, the OR effect of cell growth was abolished by preincubation with the inhibitors of those signals (Figure 3). These results are in accordance with the previously shown ability of OR activation to interfere with such pathways. 17,54 Recently, many studies have agreed in defining Ca 2 signaling as one of the most significant determinants of the growth of the biliary epithelium 8,11,57 60 and that dysregulation of Ca 2 signaling is a unifying feature of late-stage cholangiopathies. 59 Because ORs have been shown to be able to modulate cell growth by affecting Ca 2 signaling, 54,61 we tested the hypothesis that the differential changes in cell proliferation associated with OR activation were mediated by such a pathway. We found that the OR but not OR selective agonist significantly increased the intracellular IP 3 levels and the phosphorylation of the Ca 2 -dependent PKC and of CamKII, a molecule known to act as an intracellular Ca 2 sensor 54 (Figure 4). As confirmation, the blockage of Ca 2 signaling by a Ca 2 chelator, a CamKII, and a Ca 2 -dependent PKC inhibitor neutralized the inhibitory effect of OR activation on cholangiocyte proliferation (Figure 3). Similarly, the blockage of the Ca 2 signal also neutralized the inhibitory effect of OR activation on camp/pka activity and ERK1/2 and AKT phosphorylation (Figure 5). The CamKII inhibitor did not completely abolish the OR-induced increase of PKC phosphorylation (Figure 4), thus suggesting that CamKII inhibits the camp/pka cascade probably by both a direct effect and also indirectly through PKC (Figure 8). These data are in accordance with what has been previously shown 11,60 and propose the concept that Ca 2 signaling might be the common pathway triggered by those inhibitory factors that counterbalance the overgrowth of the biliary epithelium. In light of the in vitro findings, we wanted to verify whether OR and OR are functionally active in vivo as well. Interestingly, we found that in vivo treatment of BDL rats with the OR selective agonist resulted in a significant reduction of bile duct mass (Figure 6). In a fashion similar to what was observed in vitro, chronic in vivo administration of the OR selective agonist did not diminish but rather increased the bile duct mass of BDL rats, maintaining the functional response to cholestasis as well (Figure 6). Both our in vitro and in vivo studies showed that endogenous opioid peptides have, in cholangiocytes, 2 receptors available to interact with and to affect cell proliferation: the OR, which exerts an evident antiproliferative effect, and the OR, which in contrast sustains the growth of the biliary epithelium. The last step of our investigation was thus to verify which one is mostly activated by endogenous opioid peptides in the course of chronic cholestasis. To answer this last question, we administered the general receptor antagonist naloxone to BDL rats. 39 This resulted in a further increase of the growth of the biliary tree in response to cholestasis. Similarly, the in vitro incubation of cholangiocytes purified from BDL rats with naloxone significantly increased cell proliferation (Figure 7). These findings suggest that in the course of cholestasis, endogenous opioid peptides likely interact mostly with the OR, thus determining an antiproliferative effect, because when the action of endogenous opioid peptides is blocked by a receptor antagonist, cell proliferation is strongly increased. This hypothesis is also indirectly confirmed by the changes of OR expression after BDL (Figure 1). After BDL, OR is the only one of the 3 receptors in which cholangiocyte expression is down-regulated. Such a condition, which typically happens to ORs on interaction with ligands (as a sort of negative feedback), 62 has been shown to occur in the central nervous system in the course of cholestasis. 20 In vivo, the previously described changes in the growth of the biliary tree were not asso-

14 1844 MARZIONI ET AL GASTROENTEROLOGY Vol. 130, No. 6 Figure 8. (Top) Schematic working model of the effect of endogenous opioid peptides on cholangiocytes. In the course of cholestasis, cholangiocytes express opioid peptides; these peptides (that might also derive from other cell types), once secreted, mostly interact with the OR. This ligand/receptor interaction increases the IP3 levels and, as a consequence, the CamKII and PKC phosphorylation. The activation of such a pathway inhibits the camp/pka, ERK1/2, AKT pathway, an event that is then directly responsible for the reduction of cell proliferation. (Bottom) Depiction of the pathway that mediates the increase of cholangiocyte proliferation due to the pharmacologic activation of OR. ciated with significant modifications of collagen deposition (not shown). Additional, specifically designed investigations will thus be needed to ascertain the possible effects of endogenous opioid peptides as modulators of liver fibrogenesis. It is indeed necessary to rule out that such a lack of major differences might be influenced by the fact that collagen deposition after 1-week BDL is small if compared with longer times of biliary obstruction, which are commonly used as a model of liver fibrosis.63,64 In previous investigations, it has been shown that Met-enkephalin immunoreactivity is expressed by the biliary epithelium in experimental24 and human cholestasis19 and that preproenkephalin messenger RNA, which codes for Met-enkephalin, is detected in cholestatic livers.19 These findings explain the marked increase in biliary Met-enkephalin excretion that we have observed in our study. Previous experiments19,24 also suggested that cholangiocytes themselves are able to synthesize such peptides, an hypothesis that is confirmed by the current investigation, because Met-enkephalin biliary excretion was found to be markedly increased after BDL and by the observation that the OR antagonist naloxone increased the proliferation of virtually pure colonies of BDL cholangiocytes40,41 (Figure 7), a condition in which the contribution of opioid peptides can originate almost uniquely from cholangiocytes themselves. If the in vivo study does not rule out the possible role of endogenous opioid peptides of different origin (eg, paracrine release by other cells such as hepatocytes or nerves),19,24,65 overall our findings provide evidence for the first time of what was previously hypothesized,21 for example, the participation of endogenous opioids in the autocrine/ paracrine loop of neuropeptides/neuroendocrine peptides released by cholangiocytes in the course of cholestasis to limit the overgrowth of the biliary tree.11 This parallels what was previously shown in other epithelial cells, where Met-enkephalin has been recognized as a very strong endogenous inhibitor of cell growth Naturally, species differences, implying that it is uncertain whether endogenous opioid peptides exert the same effect in human livers, should be considered when interpreting our findings. However, the fact that we observed the presence of ORs on the biliary epithelium in liver sections from patients with primary biliary cirrhosis might also explain those previous reports showing that

15 May 2006 ENDOGENOUS OPIOIDS AND CHOLANGIOCYTE PROLIFERATION 1845 the administration of OR antagonists to patients with primary biliary cirrhosis was associated with amelioration of plasma bilirubin levels. 45 In summary, we have shown that cholangiocytes express OR, OR, and OR, the activation of which is associated with heterogenic effects on cholangiocyte proliferation. In particular, we found that endogenous opioid peptides are likely secreted in an autocrine/paracrine fashion to inhibit, by interacting with the OR, the excessive growth of cholangiocytes in response to chronic cholestasis. The findings of the current investigation open novel perspectives for the understanding of the pathophysiology of chronic cholestasis and may open additional options for the medical therapy or prevention of cholangiopathies and cholangiocarcinoma. References 1. Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 1988;81: Lazaridis KN, Strazzabosco M, LaRusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 2004;127: Annual Report of the U. S. Organ Procurement and Transplantation Network and the Scientific Registry for Transplant Recipients: transplant data Rockville, MD: Department of Health and Human Services, Health Resources and Services Administration, Office of Special Programs, Division of Transplantation, Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G, Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-b/gastrin receptors via D-myoinositol 1,4,5-triphosphate-, Ca(2 )-, and protein kinase C alphadependent mechanisms. Hepatology 2000;32: Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 2001;281:G612 G Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD, Glaser SS, Francis H, Mancino MG, Ueno Y, Barbaro B, Benedetti A, Attili AF, Alvaro D. Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis. Gastroenterology 2004;127: LeSage G, Alvaro D, Benedetti A, Glaser S, Marucci L, Eisel W, Caligiuri A, Baiocchi L, Rodgers R, Phinizy JL, Francis H, Alpini G. Cholinergic system modulates growth, apoptosis and secretion of cholangiocytes from bile duct ligated rats. Gastroenterology 1999;117: LeSage GD, Marucci L, Alvaro D, Glaser S, Benedetti A, Marzioni M, Patel T, Francis H, Phinizy JL, Alpini G. Insulin inhibits secretininduced ductal secretion by activation of PKC alpha and inhibition of PKA activity. Hepatology 2002;36: LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L, Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2 )- and PKC-dependent stimulation of camp. Hepatology 2004;40: Tietz PS, Alpini G, Pham LD, LaRusso NF. Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. Am J Physiol 1995;269:G110 G Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J, Fava G, LeSage GD, Alpini G. Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin. Gastroenterology 2005;128: Roskams T, van den Oord JJ, De Vos R, Desmet VJ. Neuroendocrine features of reactive bile ductules in cholestatic liver disease. Am J Pathol 1990;137: Glasel JA. The effects of morphine on cell proliferation. Prog Drug Res 2000;55: Zagon IS, Smith JP, McLaughlin PJ. Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor. Int J Oncol 1999;14: Zagon IS, Hytrek SD, McLaughlin PJ. Opioid growth factor tonically inhibits human colon cancer cell proliferation in tissue culture. Am J Physiol 1996;271:R511 R Zagon IS, Wu Y, McLaughlin PJ. Opioid growth factor is present in human and mouse gastrointestinal tract and inhibits DNA synthesis. Am J Physiol 1997;272:R1094 R Simon EJ. Opioid receptors and endogenous opioid peptides. Med Res Rev 1991;11: Wollemann M, Benyhe S. Non-opioid actions of opioid peptides. Life Sci 2004;75: Bergasa NV, Liau S, Homel P, Ghali V. Hepatic Met-enkephalin immunoreactivity is enhanced in primary biliary cirrhosis. Liver 2002;22: Bergasa NV, Rothman RB, Vergalla J, Xu H, Swain MG, Jones EA. Central mu-opioid receptors are down-regulated in a rat model of cholestasis. J Hepatol 1992;15: Bergasa NV, Vergalla J, Swain MG, Jones EA. Hepatic concentrations of proenkephalin-derived opioids are increased in a rat model of cholestasis. Liver 1996;16: Swain MG, Rothman RB, Xu H, Vergalla J, Bergasa NV, Jones EA. Endogenous opioids accumulate in plasma in a rat model of acute cholestasis. Gastroenterology 1992;103: Jones EA, Bergasa NV. The pruritus of cholestasis. Hepatology 1999;29: Bergasa NV, Sabol SL, Young WS III, Kleiner DE, Jones EA. Cholestasis is associated with preproenkephalin mrna expression in the adult rat liver. Am J Physiol 1995;268:G346 G Bao L, Jin SX, Zhang C, Wang LH, Xu ZZ, Zhang FX, Wang LC, Ning FS, Cai HJ, Guan JS, Xiao HS, Xu ZQ, He C, Hokfelt T, Zhou Z, Zhang X. Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion. Neuron 2003;37: Melone M, Brecha NC, Sternini C, Evans C, Conti F. Etorphine increases the number of mu-opioid receptor-positive cells in the cerebral cortex. Neuroscience 2000;100: Zhang N, Rogers TJ, Caterina M, Oppenheim JJ. Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize muopioid receptors on dorsal root ganglia neurons. J Immunol 2004;173: Marzioni M, LeSage G, Glaser S, Patel T, Marienfeld C, Ueno Y, Francis H, Alvaro D, Phinizy JL, Tadlock L, Benedetti A, Marucci L, Baiocchi L, Alpini G. Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct ligated rats. Am J Physiol 2003;284:G837 G Qiao L, Yacoub A, Studer E, Gupta S, Pei XY, Grant S, Hylemon PB, Dent P. Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes. Hepatology 2002;35: Wright DC, Hucker KA, Holloszy JO, Han DH. Ca 2 and AMPK both mediate stimulation of glucose transport by muscle contractions. Diabetes 2004;53: Tian Y, Laychock SG. Protein kinase C and calcium regulation of adenylyl cyclase in isolated rat pancreatic islets. Diabetes 2001; 50: