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

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

París

Congreso; The International Liver Congress. 2018; 2018

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/sunknown. 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 itinteracts with bile salts and potentially activates AC.Isolated rat hepatocyte coupletswere preincubated with the S1PR2 antagonist JTE-013 (JTE,10 μM), AC inhibitor MDL12330 (MDL, 20 μM) or PKA inhibitor KT5720 (KT, 250 nM) and then exposed toTLC (2.5 μM). Coupletswere also co-preincubated with JTE and either MDL, KTor PI3K inhibitor wortmannin (W,100 nM). Changes in Mrp2 activity were evaluated by assessing the canalicular vacuolaraccumulation (cVA) of glutathione methylfluorescein (GMF), an Mrp2 substrate. Mrp2 localization was studied by immunofluorescence, followed by confocal microscopy. PKA and AKTactivationweredetermined 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 JTEor DDA (AC inhibitor) were administered 10 min before TLC. Finally, biliary flow was measured for 30 min in 5 min-periods. (% 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 thatS1PR2, 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 thedelocalization of Mrp2 induced by TLC. TLC diminished bile flow (see figure). JTE or DDA only increased recovery after the initial drop. a: p < 0.05 vs control, b: p < 0.05 vs TLC