Sphingosine 1-phosphate receptor 2/adenylyl cyclase/protein kinase A pathway is involved in taurolithocholate-induced internalization of Abcc2 in rats.

Taurolithocholate (TLC) is a cholestatic bile salt that induces disinsertion of the canalicular transporter Abcc2 (Mrp2, multidrug resistance-associated protein 2). This internalization is mediated by different intracellular signaling proteins such as PI3K, PKCε and MARCK but the initial receptor of TLC remains unknown. A few G protein-coupled receptors interact with bile salts in hepatocytes. Among them, sphingosine-1 phosphate receptor 2 (S1PR2) represents a potential initial receptor for TLC. The aim of this study was to evaluate the role of this receptor and its downstream effectors in the impairment of Abcc2 function induced by TLC. In vitro, S1PR2 inhibition by JTE-013 or its knockdown by small interfering RNA partially prevented the decrease in Abcc2 activity induced by TLC. Moreover, adenylyl cyclase (AC)/PKA and PI3K/Akt inhibition partially prevented TLC effect on canalicular transporter function. TLC produced PKA and Akt activation, which were blocked by JTE-013 and AC inhibitors, connecting S1PR2/AC/PKA and PI3K/Akt in a same pathway. In isolated perfused rat liver, injection of TLC triggered endocytosis of Abcc2 that was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Abcc2 substrate dinitrophenyl-glutathione until the end of the perfusion period. S1PR2 or AC inhibition did not prevent the initial decay, but they accelerated the recovery of these parameters and the reinsertion of Abcc2 into the canalicular membrane. In conclusion, S1PR2 and the subsequent activation of AC, PKA, PI3K and Akt is partially responsible for the cholestatic effects of TLC through sustained internalization of Abcc2.

Role of ERK1/2 in TNFα-induced internalization of Abcc2 in rat hepatocyte couplets.

TNFα is a cytokine whose levels are increased in inflammatory pathologies that are associated with cholestasis. Endocytic internalization of Abcc2 (multidrug resistance-associated protein 2), a canalicular transporter of organic anions that is implicated in the clearance of clinically important drugs, is a phenomenon that occurs in inflammatory liver diseases, and it has been established that cytokines act as mediators. However, the intracellular mechanism involved in this effect remains unknown. The aim of the present work was to characterize the internalization of Abcc2 induced by TNFα and to study the role of ERK1/2 and reactive oxygen species as signaling mediators of transporter internalization. Using rat hepatocyte couplets, we found that TNFα (6.25 pg/ml) induced a decrease in Abcc2 activity estimated by the accumulation of the Abcc2 substrate glutathione methylfluorescein in the canalicular vacuole that was accompanied by internalization of Abcc2 from the canalicular membrane. Inhibition of MEK1/2 (upstream of ERK1/2) partially prevented TNFα effects on Abcc2 internalization and activity impairment. Reactive oxygen species (ROS) scavengers such as vitamin C and mannitol partially prevented both TNFα-induced decrease in Abcc2 activity and ERK1/2 phosphorylation. Apocynin, a NADPH oxidase inhibitor, prevented the increase in ROS and the phosphorylation of ERK1/2 produced by TNFα. Taken together, these results indicate that TNFα activates a pathway involving NADPH oxidase, ROS and MEK1/2-ERK1/2 that is partially responsible for the internalization of Abcc2. This internalization leads to an altered transport activity of Abcc2 that could impair drug disposal, enhancing drug toxicity in patients suffering from inflammatory liver diseases.