NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis.

The endogenous metabolite of estradiol, estradiol 17β-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work.

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.

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.

Adaptive downregulation of Cl-/HCO3- exchange activity in rat hepatocytes under experimental obstructive cholestasis.

In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis.

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver.

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 β-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis.

Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis.

Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases.

Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-D-glucuronide-induced cholestasis in rats.

Estradiol-17β-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets.

EGFR participates downstream of ERα in estradiol-17β-D-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets.

Estradiol-17β-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα.

Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis.

At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17β-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.

G-protein-coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis.

Unlabelled: Estradiol-17ß-D-glucuronide (E17G) activates different signaling pathways (e.g., Ca(2+) -dependent protein kinase C, phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinases [MAPKs] p38 and extracellular signal-related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2).