Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis

Administration of phalloidin, one of the toxic peptides of the mushroom Ama nita phalloides, leads to rapid and sustained cholestasis in rats. Although attributed to the interaction of phalloidin with microfilaments, the event s leading to cholestasis are incompletely understood. The adenosine triphos phate (ATP)-dependent, apical conjugate export pump, termed multidrug resis tance protein 2 (Mrp2) or canalicular multispecific organic anion transport er, is the major driving force for bile salt-independent bile flow. We inve stigated the role of Mrp2 in phalloidin-induced cholestasis, Bile flow decr eased to 53% and 31% of control at 15 and 30 minutes after phalloidin (0.5 mg/kg), respectively. Mrp2-mediated [H-3]leukotriene excretion into bile du ring the initial 45 minutes was reduced to 44% of control when [H-3]LTC4 wa s injected 15 minutes after phalloidin treatment. Mrp2 was progressively lo st from the hepatocyte canalicular membrane and detected predominantly on i ntracellular membrane structures together with other canalicular proteins i ncluding P-glycoproteins, ecto-ATPase, and dipeptidyl-peptidase IV. By cont rast, structures involved in intercellular adhesion (zonula occludens, zonu la adhaerens, and desmosomes) as well as intermediate filaments of the cyto keratin type appeared largely unaffected within 30 minutes after phalloidin , In line with the immunofluorescence analysis, immunoblots indicated a los s of Mrp2 and P-glycoproteins from the canalicular membrane and a 3- and 4. 6-fold increase of these transport proteins in the microsomal fraction, res pectively. Our results indicate that phalloidin induces marked alterations of the hepatocyte canalicular architecture and a loss of Mrp2 together with other proteins from the canalicular membrane. The resulting cholestasis ca n therefore be explained in part by the loss of export pumps, including Mrp 2, from the canalicular membrane.