cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway Heather Francis 3 , Shannon Glaser 3 , Yoshiyuki Ueno 5 , Gene LeSage 1,† , Luca Marucci 6 , Antonio Benedetti 6 , Silvia Taffetani 3 , Marco Marzioni 2,‡ , Domenico Alvaro 7 , Julie Venter 2 , Ramona Reichenbach 4 , Giammarco Fava 2 , Jo Lynne Phinizy 3 , Gianfranco Alpini 1,2,4, * 1 Department of Internal Medicine, Scott and White Hospital, The Texas A&M University System Health Science Center, College of Medicine, Temple, TX 76504, USA 2 Department of Medical Physiology, Scott and White Hospital, The Texas A&M University System Health Science Center, College of Medicine, Temple, TX 76504, USA 3 Division of Research and Education, Scott and White Hospital, The Texas A&M University System Health Science Center, College of Medicine, Temple, TX 76504, USA 4 Central Texas Veterans Health Care System, Temple, TX 76504, USA 5 Division of Gastroenterology, Tohoku University, School of Medicine, Aobaku, Sendai, Japan 6 Department of Gastroenterology, University of Ancona, Ancona, Italy 7 Division of Gastroenterology, La Sapienza University, Rome, Italy Background/Aims: To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion. Methods: Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway. Results: Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2. Conclusions: Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Bicarbonate secretion; Biliary epithelium; Proliferation; Secretin receptor Journal of Hepatology 41 (2004) 528–537 www.elsevier.com/locate/jhep 0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.009 Received 18 December 2003; received in revised form 6 May 2004; accepted 9 June 2004; available online 2 July 2004 † Present address: The University of Texas Houston Medical School, Houston, TX, USA. ‡ Present address: Department of Gastroenterology, University of Ancona, Ancona, Italy. Abbreviations: cAMP, cyclic adenosine 3 0 ,5 0 -monophosphate; BSA, bovine serum albumin; CK-19, cytokeratin-19; ERK, extracellular signal-regulated kinase; g-GT, g-glutamyltranspeptidase; PCNA, proliferating cellular nuclear antigen; PKA, protein kinase A. * Corresponding author. Tel.: þ 1-254-742-7044; fax: þ 1-254-742-7185. E-mail address: galpini@medicine.tamu.edu (G. Alpini).