Involvement of sphingosine-1P receptor 2 (S1PR2) in taurolithocholate-induced impairment of multidrug resistence associated protein 2 (Mrp2) in rat

ANDERMATTEN, ROMINA; CIRIACI, NADIA; RAZORI, MARIA VALERIA; MISZCZUK, GISEL S.; BAROSSO, ISMAEL R.; SANCHEZ POZZI, ENRIQUE J.

Paris

Congreso; International Liver Congress; 2018

EALS

Background and Aims: Taurolithocholate (TLC), a cholestatic bile salt, induces internalization of Mrp2. Signaling proteins,such as PI3K/ AKT, participate in this cholestasis but the initial receptor/s remain/s unknown. We focused our study in adenylyl cyclase (AC)/PKA, recently involved in estrogen-induced cholestasis, and in potential TLC receptors. S1PR2 arose as a possible initial TLC receptor since it interacts with bile salts and potentiallyactivates AC. Method: Isolated rat hepatocyte couplets were preincubated with the S1PR2 antagonist JTE-013 (JTE,10 μM), AC inhibitor MDL12330 (MDL, 20μM) or PKA inhibitor KT5720 (KT, 250 nM) and then exposed to TLC(2.5 μM). Couplets were also co-preincubated with JTE and either MDL, KT or PI3K inhibitor wortmannin (W,100nM). Changes in Mrp2 activity were evaluated by assessing the canalicular vacuolar accumulation (cVA) of glutathione methylfluorescein (GMF), an Mrp2 substrate. Mrp2 localization was studied by immunofluorescence, followed by confocal microscopy. PKA and AKT activation were determined by western blot using an antibody against phospho PKA substrate and phospho AKT. In isolated rat liver, cholestasis was induced by intraportal injection of TLC(4.5 μmol/liver).Inhibitors JTE or DDA (AC inhibitor) were administered 10min before TLC. Finally, biliary flow was measured for 30 min in 5min-periods. Results: (% of control±SEM; n=3-9) Treatment with JTE, MDL, KT and W partially prevented TLC-induced impairment in Mrp2 activity: TLC (55±2a), TLC+JTE (79±2a,b), TLC+MDL (80±9a,b), TLC+KT (74±10a,b), TLC+W (77±6a,b). JTE preventive effects did not improve the actions of MDL, KT or W: TLC+JTE+MDL (84±6a,b), TLC+JTE+KT(76±3a,b), TLC+JTE+W (79±6a,b), suggesting that S1PR2, AC, PKA and PI3K share the same pathway. Activation of PKA induced by TLC decreased in presence of JTE: TLC (248±15a), TLC+ JTE (138±33b). The same phenomenon occurs with AKT phosphorylation: TLC (584±23a), TLC+JTE (161±27a,b). Localization studies showed that JTE, MDL, KT and W prevented the delocalization of Mrp2 induced by TLC. TLC diminished bile flow (seefigure). JTE or DDA only increased recovery after the initial drop. a: p