Volume 130, Issue 6, Pages 1831-1847 (May 2006) 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, Antonio Benedetti  Gastroenterology  Volume 130, Issue 6, Pages 1831-1847 (May 2006) DOI: 10.1053/j.gastro.2006.02.021 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Expression of the δOR, μOR, and κOR 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 μOR and κOR expression are observed among small and large cholangiocytes, whereas the δOR displays a higher expression in large rather than small cholangiocytes. (C) Quantitative immunoblots: after 1-week BDL, only the expression of the δOR is down-regulated, whereas no modifications are observed for μOR and κOR (*P < .03 vs the other groups; data are expressed as mean ± SE of 3 experiments). Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Effect of OR activation on cholangiocyte proliferation in vitro, measured by PCNA protein expression and incorporation of BrdU. (A) Incubation with the δOR selective agonist (0.1 μmol/L) induced a marked reduction of the PCNA protein expression in BDL, but not normal, cholangiocytes. (B) Activation of the μOR by its selective agonist (0.1 μmol/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 κOR selective agonist. (D) Similar changes were also observed by the BrdU incorporation studies, which showed that δOR (top graphs) and μOR (middle graphs) ligands modify BDL cholangiocyte proliferation in a dose-dependent fashion. (E) EGF increases BDL cholangiocyte proliferation. Such an event is prevented by δOR activation, whereas it is further enhanced by μOR 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. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 (A) The inhibitory effect of δOR activation on BDL cholangiocyte proliferation (lane 2) is neutralized by preincubation with a Ca2+ chelator (lane 8), by a CamKII inhibitor (lane 7), and by a Ca2+-dependent PKC inhibitor (lane 6). (B) The increase observed in cholangiocyte proliferation after μOR 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 Ca2+ chelator (lane 8), with a CamKII inhibitor (lane 7), or with a Ca2+-dependent PKC inhibitor (lane 6). *P < .05 vs the other groups; data are expressed as mean ± SE of at least 3 experiments. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 (A) Incubation of BDL cholangiocytes with the δOR selective agonist (lane 2) significantly increases the intracellular IP3 levels (top) and the CamKIIα (middle) and PKCα (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 Ca2+ chelator (lane 8) neutralized both the δOR activation-induced CamKIIα (middle) and PKCα (bottom) phosphorylation but not the δOR activation-induced increase in intracellular IP3 levels. The CamKII inhibitor (lane 7) neutralized the δOR activation-induced increase in CamKIIα phosphorylation (middle) but only partially inhibited the increase in PKCα phosphorylation (bottom). Preincubation with the Ca2+-dependent PKC inhibitor (lane 6) only blocked the increase in PKCα phosphorylation (bottom). (B) The incubation of BDL cholangiocytes with the μOR selective agonist did not modify the intracellular IP3 levels (top) and the CamKIIα (middle) and PKCα (bottom) phosphorylation. *P < .05 vs the other groups; data are expressed as mean ± SE of at least 3 experiments. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 (A) Incubation with the δOR 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 Ca2+ chelator (lane 8), a CamKII inhibitor (lane 7), and a Ca2+-dependent PKC inhibitor (lane 6). (B) Incubation with the μOR 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 μOR 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. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 (A) The in vivo administration to BDL rats of the δOR 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), HCO3− excretion (bottom, middle), and intracellular cAMP synthesis (bottom, right); *P < .05 vs basal. (B) In contrast, in vivo treatment of BDL rats with the μOR 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. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

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 δOR (lane 3) and μOR (lane 4) activation on cell growth (*P < .04 vs basal). Data are expressed as mean ± SE of at least 4 experiments. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

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 δOR. This ligand/receptor interaction increases the IP3 levels and, as a consequence, the CamKIIα and PKCα 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 μOR. Gastroenterology 2006 130, 1831-1847DOI: (10.1053/j.gastro.2006.02.021) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions