Silibinin (SIL) prevents estradiol 17ß-glucuronide (E217G)-induced bile salt excretory failure and internalisation of the canalicular bile salt export pump, Bsep, in the isolated rat hepatocyte couplet (IRHC) model: Possible involvement of cAMP

CROCENZI, FERNANDO A.; PORTESIO, MARÍA SOLEDAD; BASIGLIO, CECILIA L; COLEMAN, ROGER; ROMA, MARCELO G

Congreso; 38th Annual Meeting of the European Association for the Study of the Liver (EASL).; 2003

Sylimarin has beneficial effects in estrogen-induced cholestasis in vivo. Since E217G induces internalisation of Bsep, we studied, in IRHCs, whether Sil, the active component of silymarin, prevents changes in Bsep localisation/function. E217G (50 µM) diminished the % of IRHCs accumulating apically the fluorescent bile salt, cholyl-lysylfluorescein (CLF), and Sil (2.5 µM, 30 min) fully prevented this effect. Sil beneficial effect was abolished by the intracellular-calcium quelating agent, BAPTA/AM, but not by the PKA inhibitor, KT5720 (% of IRHCs accumulating CLF, referred to controls: E217G: 43±2*; E217G±Sil: 93±1; E217G±Sil±BAPTA/AM: 50±4*; E217G±Sil±KT5720: 92±3#; *p<0.05 vs control, #p<0.05 vs E217G). E217G induced internalisation of Bsep into intracellular vesicles, visualized by immunofluorescence, and this effect was prevented by Sil (% of canalicular Bsep content: Control: 69±2; E217G: 38±3*; Sil±E217G: 59±3*#; *p<0.05 vs control; #p<0.05 vs E217G). Since Sil is a potent inhibitor of cAMP phosphodiesterase, and cAMP protects against E217G-induced internalisation of another major canalicular pump, Mrp2, we analysed whether Sil increases cAMP in hepatocytes, and whether the cAMP analogue, DBcAMP, is able to mimic Sil effects. Sil increased hepatocellular cAMP levels by 36±6% (p<0.05). DBcAMP fully mimicked Sil ability to prevent E217G-induced impairment in CLF apical accumulation and Bsep internalisation. Furthermore, the beneficial effect of DBcAMP wal also calcium, but not PKA-dependent. Taken together, these results suggest that Sil prevents E217G-induced bile salt excretory failure by counteracting internalisation of Bsep, probably by a mechanism involving cAMP-induced cytosolic calcium elevations.