Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

Pro-inflammatory cytokines enhance dilatation of bile canaliculi caused by cholestatic antibiotics.

Many drugs can induce liver injury, characterized by hepatocellular, cholestatic or mixed hepatocellular-cholestatic lesions. While an inflammatory stress is known to aggravate hepatocellular injury caused by some drugs much less evidence exists for cholestatic features. In this study, the influence of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α), either individually or combined, on cytotoxic and cholestatic properties of antibiotics was evaluated using differentiated HepaRG cells. Six antibiotics of various chemical structures and known to cause cholestasis and/or hepatocellular injury in clinic were investigated. Caspase-3 activity was increased with all these tested hepatotoxic drugs and except with erythromycin, was further augmented in presence of cytokines mainly when these were co-added as a mixture. TNF-α and IL-1ß aggravated cytotoxicity of TVX more than IL-6. Bile canaliculi (BC) dilatation induced by cholestatic drugs was increased by co-treatment with IL-6 and IL-1ß but not with TNF-α. Reduced accumulation of carboxy-dichlorofluorescein, a substrate of the multi-drug resistance-associated protein 2, in antibiotic-induced dilatated BC, was further extended in presence of individual or mixed cytokines. In conclusion, our data demonstrate that pro-inflammatory cytokines either individually or in mixture, can modulate cholestatic and/or cytotoxic responses to antibiotics and that the extent of these effects is dependent on the cytokine and the cholestatic antibiotic.

A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule.

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs.

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

UNLABELLED: The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. CONCLUSION: These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery.

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas-permeable membranes.

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%-O2 (+)] or physiological oxygen concentrations [10%-O2 (+), 5%-O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas-impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS-O2 (-)]. The results indicated that the hepatocytes under 10%-O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS-O2 (-) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug-metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long-term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen-permeable membrane system to provide a simple method for in vitro studies.

A physiologically based model of hepatic ICG clearance: interplay between sinusoidal uptake and biliary excretion.

Although indocyanine green (ICG) has long been used for the assessment of liver function, the respective roles of sinusoidal uptake and canalicular excretion in determining hepatic ICG clearance remain unclear. Here this issue was addressed by incorporating a liver model into a minimal physiological model of ICG disposition that accounts of the early distribution phase after bolus injection. Arterial ICG concentration-time data from awake dogs under control conditions and from the same dogs while anesthetized with 3.5% isoflurane were subjected to population analysis. The results suggest that ICG elimination in dogs is uptake limited since it depends on hepatocellular uptake capacity and on biliary excretion but not on hepatic blood flow. Isoflurane caused a 63% reduction in cardiac output and a 33% decrease in the ICG biliary excretion rate constant (resulting in a 26% reduction in elimination clearance) while leaving unchanged the sinusoidal uptake rate. The terminal slope of the concentration-time curve, K, correlated significantly with elimination clearance. The model could be useful for assessing the functions of sinusoidal and canalicular ICG transporters.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice.

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.

Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C.

UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.

The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease.

The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.

Bile canalicular secretion - tales from Vienna and Yale.

The isolated rat hepatocyte couplet was developed to facilitate studies of canalicular bile formation in a primary bile secretory unit in culture. This preparation enabled the first determination of electrical driving forces across the canalicular membrane in an intact hepatocyte. The basic electrical properties of the hepatocyte were able to be described using this model which also facilitated functional studies of canalicular bile formation using video and confocal imaging techniques. More recently isolated hepatocytes in collagen sandwich cultures have been adapted for the studies of bile formation in polarized hepatocytes requiring longer term experiments where proteins involved in the bile secretory process can be specifically knocked down using adenoviral siRNA techniques. This presentation reviews the historical development of the hepatocyte couplet preparations and the seminal contributions of Jürg Graf to this field.

Transcellular water transport in hepatobiliary secretion and role of aquaporins in liver.

In the context of the osmotic model of bile formation, we used isolated rat hepatocyte couplets and performed volume measurements by video image analysis to analyze the transport of water between the bile canalicular lumen, liver cells and the surrounding bathing medium. Increasing bath osmolarity by the addition of sucrose led to shrinkage of cells that preceded shrinkage of the canalicular lumen by approx. 1 sec. Thermodynamic modeling of water transport across the basolateral and apical cell membranes and across a paracellular pathway (tight junctions) revealed high hydraulic water permeabilities of both cell membranes of approx. 3*10(-4) cm*sec(-1)*(osmol/kg)(-1) indicating transcellular water flux between bathing medium and bile. Tight junctions exhibited low water permeability but allowed for electrolyte permeation that enables canalicular spaces to shrink below van't Hoff equilibrium during the osmotic maneuver. The results are discussed with respect to the role of different types of membrane aquaporins being expressed in hepatocytes.

Osmoregulation of bile formation.

Bile secretion by liver parenchymal cells is the result of vectorial transcellular transport of solutes and involves the coordinated action of transport proteins at the basolateral (sinusoidal) and apical (canalicular) membranes of the hepatocyte. A complex network of signals controls uptake and efflux transporters on a long- and a short-term timescale, including regulation at the level of gene transcription, protein translation and maturation, covalent modification, and dynamic localization of transporter proteins, as well as substrate availability. Evidence has shown that the hepatocellular hydration state exerts powerful control on the transcellular transport of solutes, such as conjugated bile acids and glucuronide and glutathione conjugates. This is of physiological significance because liver cell hydration is a dynamic parameter, which changes within minutes under the influence of hormones, nutrients, and oxidative stress. Thus, osmoregulation of bile formation is of physiological and pathophysiological interest.

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions.

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

The bile salt export pump.

Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver disease.

Cytochrome P450 expression-induction profile and chemically mediated alterations of the WIF-B9 cell line.

BACKGROUND INFORMATION: WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF-B9 cell line as a model for analysing drug-induced hepatic effects. The drug metabolism potential of WIF-B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized. RESULTS: Competitive reverse transcriptase-PCR revealed that four rat (1A1, 2B1/2, 2E1 and 4A1) and four human (1A1, 2Cs, 2D6 and 2E1) CYP mRNA isoforms were constitutively expressed in WIF-B9 cells. The rat CYP forms were expressed at levels 2-4 orders of magnitude higher than the human forms. Exposure for 20-72 h to increasing concentrations of CYP reference inducers (beta-naphthoflavone, 3-methyl cholanthrene, dexamethasone, phenobarbital, clofibrate and pregnenolone 16alpha-carbonitrile) revealed that the rat CYP 1A1, 1A2, 3A1, 3A2 and 4A1 and human CYP 1A1 and 2Cs mRNAs were inducible. Rat CYP 1A1 and 1A2 were the most inducible isoforms since they were overexpressed up to 100-fold after 20-48 h of treatment with beta-naphthoflavone. Human CYP 1A1 and 2Cs mRNAs were induced 3-fold after 48 h of treatment with phenobarbital. Other mechanisms involved in hepatotoxicity were explored using microscopy and immunofluorescence. The WIF-B9 cell line exhibited fragmentation and dilatation of bile canaliculi upon exposure to erythromycin, and to isoniazid and cytochalasins, respectively. Monensin promoted cell depolarization and cytoplasmic granulation. Ethionine promoted cytoplasmic vacuolation and dilatation of the Golgi structures. CONCLUSIONS: These results indicate that the CYP expression and induction profiles and the morphological features of WIF-B9 cells allow prediction in vitro of the induction and hepatotoxicity profiles of chemical entities.

Coordinated movement of bile canalicular networks reconstructed by rat small hepatocytes.

Hepatocytes in vivo have a potential for liver regeneration, but it has been very difficult to reconstruct hepatic organoids in vitro. Recent studies have shown that small hepatocytes (SHs) can reconstruct hepatic organoids including functional bile canaliculi (BC). In the present study we analyzed the movement of BC formed in the hepatic organoids, focusing on the coordination of contraction and dilation among cells and the mechanism producing the coordination. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Time-lapse images of BC movements were taken and analyzed in cells treated with or without cytochalasin B (CB). Time-lapse images revealed that all BC, regardless of region contracted in a coordinated manner. Actin filaments were observed along the BC even after the BC networks treated with CB dilated markedly. Microinjection of dye was also carried out to investigate the flow thorough BC. Secreted fluorescein from the injected cell flowed along BC, and gap junctional protein connexin 32 was expressed along BC networks, suggesting cell-to-cell communication. Thus, groups of hepatocytes in the hepatic organoids act in a coordinated manner through intercellular communication.

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity.

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.

Long-term effect of treating pregnant rats with ursodeoxycholic acid on the congenital impairment of bile secretion induced in the pups by maternal cholestasis.

Transient latent cholestasis in young rats born from mothers with obstructive cholestasis during pregnancy (OCP) has been reported. The cause of this congenital impairment and the long-term effect on the pups of treating their mothers with ursodeoxycholic acid (UDCA) during pregnancy were investigated. Complete biliary obstruction was imposed on day 14 of pregnancy and UDCA treatment was begun on day 15. Serum bile acids (BAs) concentrations were elevated in 4-week-old pups born from OCP, but not OCP + UDCA, mothers. However, gas chromatographic/mass spectrometric analysis of BA species in basal bile indicated the presence of significant differences among all experimental groups (control, OCP, and OCP + UDCA). Canalicular plasma membrane fluidity was reduced in OCP, but not in OCP + UDCA, pups. Screening by reverse transcription followed by real-time quantitative polymerase chain reaction of the steady-state levels of mRNA of genes related to hepatobiliary function revealed changes (upregulation of Cyp7a1 and Mrp1 and down-regulation of Abcg5 and Abcg8) in OCP group, which were prevented by UDCA treatment. Electron microscopy examination showed multilamellar bodies occupying part of the canalicular lumen in OCP pups. Their number and size were reduced in animals born from OCP + UDCA mothers. In OCP, but not OCP + UDCA, the stimulation of bile flow and BA output induced by taurocholate administration were reduced and cholesterol/BA output ratio was increased, whereas phospholipid/BA output ratio was enhanced in both groups (OCP > OCP + UDCA). In conclusion, UDCA treatment of rats with cholestasis during pregnancy has long-term beneficial effects on their offspring by preventing in part the congenital impairment in hepatobiliary function of the pups that affects their biliary lipid secretion.