title: Silibinin prevents cholestasis-associated retrieval of the bile salt export pump, Bsep, in isolated rat hepatocyte couplets: possible involvement of cAMP.
abstract: Estradiol-17beta-d-glucuronide (E(2)17G) and taurolithocholate (TLC) induce acute cholestasis-associated with retrieval of the bile salt export pump (Bsep), which parallels with alteration in transport activity. cAMP stimulates the apically directed vesicular trafficking of transporters, partially preventing these alterations. The hepatoprotector, silymarin, which inhibits cAMP-phosphodiesterase, prevents the cholestasis induced in vivo by both estrogens and TLC. We aimed to assess the ability of silibinin (Sil), the silymarin active component, to prevent the retrieval of Bsep induced by TLC and E(2)17G, and the associated alteration in its transport function. The possible involvement of cAMP as a second messenger and the intracellular signalling pathways implicated were also evaluated. Functional studies were performed analysing the proportion of isolated rat hepatocyte couplets (IRHC) accumulating the fluorescent bile salt analogue, cholyl-lysylfluorescein (CLF), into their sealed canalicular vacuoles. Cellular localisation of Bsep was assessed by immunofluorescent staining. Intracellular levels of cAMP were measured by ELISA. Sil (2.5microM) elevated by 40+/-3% intracellular cAMP, and mimicked the ability of dibutyryl-cAMP (10microM) to prevent internalisation of Bsep and the TLC (2.5microM)- and E(2)17G (50microM)-induced impairment in the capacity of IRHC to accumulate CLF apically. Preventive effects of Sil and dibutyryl-cAMP were not abolished by the specific protein kinase A inhibitors, KT5720 and H89. Contrarily, the intracellular Ca(2+) chelator, BAPTA/AM, significantly blocked the protective effect of both compounds. We conclude that Sil prevented TLC- and E(2)17G-induced bile salt secretory failure, at least in part, by avoiding redistribution of Bsep, by a mechanism probably involving cAMP-induced cytosolic Ca(2+) elevations.
authors.affiliation: Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas (Conicet-U.N.R.), S2002LRL Rosario, Argentina.
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Crocenzi
authors.forename: Cecilia L
authors.initials: CL
authors.lastname: Basiglio
authors.forename: Leonardo M
authors.initials: LM
authors.lastname: Pérez
authors.forename: María S
authors.initials: MS
authors.lastname: Portesio
authors.forename: Enrique J Sánchez
authors.initials: EJ
authors.lastname: Pozzi
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Roma
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D018929:Cell Culture Techniques; D002779:Cholestasis / Q000503:physiopathology* / Q000517:prevention & control; D000242:Cyclic AMP / Q000502:physiology*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000633:toxicity; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D008297:Male; D020944:Silybum marianum; D051381:Rats; D017208:Rats, Wistar; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D013658:Taurolithocholic Acid / Q000633:toxicity
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D012838:Silymarin; C025483:estradiol-17 beta-glucuronide; D000077385:Silybin; D004958:Estradiol; D013658:Taurolithocholic Acid; D003994:Bucladesine; D000242:Cyclic AMP