Canalicular Membrane Research Articles

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.

Prostate-specific membrane antigen expression in hepatocellular carcinoma: potential use for prognosis and diagnostic imaging.

AimThe prostate-specific membrane antigen (PSMA) is currently being established as a potent diagnostic marker in many tumor types. So far, its evidence in hepatocellular carcinoma (HCC) is sparse. The aim of our study was a comprehensive evaluation of PSMA expression and its prognostic role in patients with hepatocellular carcinoma as well as feasibility test of PSMA as an agent for diagnostic imaging.MethodsThe cohort for immunohistochemistry consisted of 153 patients with HCC. For validation purposes the HCC cohort (n = 359) of The Cancer Genome Atlas was analyzed on transcript level as well.ResultsOn immunohistochemistry, non-tumorous liver tissue showed PSMA expression on canalicular membranes in all cases. In tumor tissue two patterns of expression, with a canalicular (41.1% of tumors) and a neovascular (89.9% of tumors) staining were seen. Completely negative for both two patterns were only 4.1% of tumors; conversely, 79.2% of the tumors showed high levels of PSMA protein expression at any location. At mRNA level higher FOLH1 (PSMA) expression rates were statistically significant and independently associated with longer overall survival times.Additionally, a case report of successful diagnostic 68Ga-PSMA-11 PET/CT in a patient with HCC progression on multiple therapy lines is provided.ConclusionsMajority of hepatocellular carcinomas show high levels of PSMA expression on tumor vessels and on canalicular membrane of the tumor cells. Putative diagnostic, prognostic and therapeutic value of PSMA in HCC warrants further clinically oriented investigations.

Regulation of Plasma Membrane Localization of the Na⁺-Taurocholate Co-Transporting Polypeptide by Glycochenodeoxycholate and Tauroursodeoxycholate.

Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.

Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models

Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.

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.

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. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (JOH-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 ± 0.3 vs. 3.1 ± 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 ± 0.3 vs. 3.5 ± 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.

Modeling the Effect of BSEP Inhibitors in Lipid Bilayers by Means of All-Atom Molecular Dynamics Simulation

The human bile salt export pump (BSEP) is a membrane protein expressed on the canalicular plasma membrane domain of hepatocytes, which mediates the active transport of unconjugated and conjugated bile salts from liver cells into bile. Genetically inherited defects in BSEP expression or activity causes cholestatic liver injury, and many drugs that cause cholestatic drug-induced liver injury (DILI) in humans have been shown to inhibit BSEP activity in vitro and in vivo, suggesting that this could be one of the mechanisms that initiates human DILI. The relationship between BSEP inhibition and molecular physicochemical properties has been previously investigated, identifying calculated lipophilicity and molecular weight to be significantly correlated with the BSEP inhibition. Predictive BSEP classification models, constructed through multiple quantitative structure–activity relationship modeling approaches, exhibit significant anomalies with differences in experimental IC50 values of three orders of magnitude...

ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine.

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts. Among these transporters, ABCB4 is essential for the translocation of phosphatidylcholine (PC) lipids from the inner to the outer leaflet of the canalicular membrane of hepatocytes. ABCB4 deficiency can result in altered PC to bile salt ratios, which led to intrahepatic cholestasis of pregnancy, low phospholipid associated cholelithiasis, drug induced liver injury or even progressive familial intrahepatic cholestasis type 3. Although PC lipids only account for 30-40% of the lipids in the canalicular membrane, 95% of all phospholipids in bile are PC lipids. We discuss this discrepancy in the light of PC synthesis and bile salts favoring certain lipids. Nevertheless, the in vivo extraction of PC lipids from the outer leaflet of the canalicular membrane by bile salts should be considered as a separate step in bile formation. Therefore, methods to characterize disease causing ABCB4 mutations should be considered carefully, but such an analysis represents a crucial point in understanding the currently unknown transport mechanism of this ABC transporter.

Impaired Hepatic Phosphatidylcholine Synthesis Leads to Cholestasis in Mice Challenged With a High-Fat Diet.

Phosphatidylethanolamine N‐methyltransferase (PEMT) is a hepatic integral membrane protein localized to the endoplasmic reticulum (ER). PEMT catalyzes approximately 30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt–/– mice fed a high‐fat diet (HFD) develop steatohepatitis. Interestingly, portions of the ER located close to the canaliculus are enriched in PEMT. Phospholipid balance and asymmetrical distribution by adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) on the canalicular membrane is required for membrane integrity and biliary processes. We hypothesized that PEMT is an important supplier of PC to the canaliculus and that PEMT activity is critical for the maintenance of canalicular membrane integrity and bile formation following HFD feeding when there is an increase in overall hepatic PC demand. Pemt+/+ and Pemt–/– mice were fed a chow diet, an HFD, or a choline‐supplemented HFD. Plasma and hepatic indices of liver function and parameters of bile formation were determined. Pemt–/– mice developed cholestasis, i.e, elevated plasma bile acid (BA) concentrations and decreased biliary secretion rates of BAs and PC, during HFD feeding. The maximal BA secretory rate was reduced more than 70% in HFD‐fed Pemt–/– mice. Hepatic ABCB11/bile salt export protein, responsible for BA secretion, was decreased in Pemt–/– mice and appeared to be retained intracellularly. Canalicular membranes of HFD‐fed Pemt–/– mice contained fewer invaginations and displayed a smaller surface area than Pemt+/+ mice. Choline supplementation (CS) prevented and reversed the development of HFD‐induced cholestasis. Conclusion: We propose that hepatic PC availability is critical for bile formation. Dietary CS might be a potential noninvasive therapy for a specific subset of patients with cholestasis.

Characterisation of a functional rat hepatocyte spheroid model

Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo-like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.

A Three-dimensional Cell Culture Method with a Micromesh Sheet and Its Application to Hepatic Cells.

In vitro 3D cultures of hepatocytes are increasingly being used to assess human hepatic metabolism and toxicity in drug development. Here, we developed an in vitro 3D cell culture method with a microstructured mesh sheet and applied it to culturing human hepatoma HepG2 cells. The micromesh sheet is constituted of fine mesh strands and apertures that are each much larger than a single cell in size. Proliferating on a micromesh sheet, HepG2 cells spread out in a planar manner and then formed a multilayered cell sheet, so that cell-cell adhesion was dominant over cell-substrate adhesion as being different from 2D cultures. In micromesh cultures, the increase rate in thickness of the cell mass was visually slower than that in spheroid cultures, enabling us to clearly observe inside cells of the cell population by microscopy. Micromesh-cultured HepG2 cells showed higher viability compared with spheroid-cultured cells. The multilayered HepG2 cell sheet increased expression of hepatic marker genes and induced cell polarization with bile canalicular membranes. Furthermore, a combination of micromesh cultures with medium perfusion further induced expression of hepatic marker genes in HepG2 cells; especially CYP1A1 and CYP1A2 mRNA increased 86-fold and 43-fold compared with 2D controls, respectively, which were much higher than those in spheroid cultures. Thus, this simple and versatile micromesh culture method holds some advantages over traditional spheroid cultures and is expected to be instrumental in culturing more differentiated hepatic cells such as HepaRG cells and primary hepatocytes for future preclinical testing.

ABCB4 Gene Aberrations in Human Liver Disease: An Evolving Spectrum.

ATP-binding cassette subfamily B member 4 (ABCB4) is a phospholipid translocator at the canalicular membrane of the hepatocyte, which "flops" phosphatidylcholine into bile. Dysfunction of this transporter due to ABCB4 gene variants can cause liver diseases and has been called ABCB4 deficiency. Several diseases including progressive familial intrahepatic cholestasis type 3 (PFIC3), low phospholipid-associated cholelithiasis (LPAC), a subgroup of patients developing intrahepatic cholestasis of pregnancy (ICP), drug-induced liver injury and chronic cholangiopathy with biliary fibrosis and cirrhosis were attributed to ABCB4 deficiency and characterized in the past decade. LPAC and ICP are usually caused by monoallelic variants, whereas patients affected by PFIC3 are homozygous or compound heterozygous carriers of ABCB4 variants. Treatment with ursodeoxycholic acid is often effective, but as the more severe forms of ABCB4 deficiency progress, nevertheless, new diagnostic and therapeutic approaches are warranted. Current functional classifications for ABCB4 deficiency-associated mutations can guide the development of novel genotype-based targeted pharmacotherapies for these conditions. Recently, increasing evidence from genome-wide association studies is emerging on associations of ABCB4 variants with hepatobiliary malignancies.

Relevance of Breast Cancer Resistance Protein to Brain Distribution and Central Acting Drugs: A Pharmacokinetic Perspective.

Breast Cancer Resistance Protein (BCRP, also known as ABCG2) is gaining momentum as a key transporter that restricts the permeability of a large number of therapeutic agents through the Blood-brain Barrier (BBB). BCRP is highly expressed in the apical membranes of epithelial cells of the small and large intestine, renal proximal tubules and canalicular membrane of hepatocytes, determining the gastrointestinal absorption and biodisposition of its substrates. It is also expressed in the luminal surface of endothelial cells of the BBB and Bloodspinal Cord Barrier (BSCB), where it undoubtedly limits the entry of a wide range of therapeutics into the CNS, potentially contributing to the therapeutic failure of CNS-acting drugs. As the U.S. Food and Drug Administration and the European Medicines Agency recommend pre-clinical evaluation and clinical assessment of BCRP-mediated drug-drug interactions, compounds that are currently recognized as BCRP substrates, inhibitors or inducers will be addressed, focusing on their pharmacokinetic behaviour in plasma and brain. Recent studies indicated a strong BCRP expression in the microvasculature of the BBB in brain tumors, hypothesizing that this phenomenon critically influences the penetration of drugs in these tumors and potentially contributes to the failure of antitumor therapy. BCRP expression in brain tissue from patients or animal models of neurological and neurodegenerative diseases has also been investigated, and the role of BCRP and its implications for novel therapeutic interventions was also herein demonstrated. The clinical significance of BCRP in drugs disposition is currently undeniable.

Thyroid hormone receptor β1 stimulates ABCB4 to increase biliary phosphatidylcholine excretion in mice

The ATP-binding cassette transporter ABCB4/MDR3 is critical for biliary phosphatidylcholine (PC) excretion at the canalicular membrane of hepatocytes. Defective ABCB4 gene expression and protein function result in various cholestatic liver and bile duct injuries. Thyroid hormone receptor (THR) is a major regulator of hepatic lipid metabolism; we explored its potential role in ABCB4 regulation. Thyroid hormone T3 stimulation to human hepatocyte models showed direct transcriptional activation of ABCB4 in a dose- and time-dependent manner. To determine whether THRβ1 (the main THR isoform of the liver) is involved in regulation, we tested THRβ1-specific agonists (e.g., GC-1, KB-141); these agonists resulted in greater stimulation than the native hormone. KB-141 activated hepatic ABCB44 expression in mice, which enhanced biliary PC secretion in vivo. We also identified THR response elements 6 kb upstream of the ABCB4 locus that were conserved in humans and mice. Thus, T3-via THRβ1 as a novel transcriptional activator regulates ABCB4 to increase ABCB4 protein levels at the canalicular membrane and promote PC secretion into bile. These findings may have important implications for understanding thyroid hormone function as a potential modifier of bile duct homeostasis and provide pharmacologic opportunities to improve liver function in hepatobiliary diseases caused by low ABCB4 expression.

Double Heterozygous Mutation Causing PFIC2 with Synchronic Hepatocellular Carcinomas before Two Years of Age

Introduction Bile salts are exclusively synthesized in the liver. The bile salt export pump (BSEP), an adenosine triphosphate (ATP)-dependent transporter, is exclusively located in the hepatic canalicular membrane, and exports the bile salts into the canaliculus against a concentration gradient. Mutations in the ABCB11 gene alter BSEP and cause progressive intrahepatic familial cholestasis (PFIC2) type 2. This disease is characterized by low GGT hepatitis and early onset of cholestasis and pruritus; these patients are at increased risk of developing hepatocellular carcinoma (HCC) or cholangiocarcinoma. Aim To communicate a case or PFIC 2 with early development of synchronic HCC, with two ABCB11 heterozygous mutations, one of them not described before. Case Report This is a female referred to this center at 3 months of age due to cholestasis and coagulopathy. Coagulation corrected after vitamin K administration; hepatocellular enzymes and bilirubin persisted elevated with normal GGT. Further workup was done, ruling out viral hepatitis, biliary atresia and cystic fibrosis. Alpha-fetoprotein and bile serum salts were markedly elevated. A percutaneous liver biopsy was remarkable for giant cell hepatitis with moderate activity, portal and periportal fibrosis with incomplete bridges. PFIC was suspected and a molecular analysis was requested. Two heterozygous mutations were identified: p.Glu297Gly already reported in association to PFIC2 and p.Arg303Met previously not identified as related with this disease. The patient remained clinically stable, with progression of cholestasis and pruritus that was managed medically. At 17 months of age she presented sustained increase of Alpha-fetoprotein. A liver ultrasound showed a 7 mm nodule that was confirmed by CT hepatic angiography. She was promptly evaluated for liver transplant; extra hepatic lesions were excluded. Three weeks later the patient was transplanted with a split liver. The transplant was uneventful and she was discharged home 10 days after the surgery. The explanted liver showed cirrhosis and 4 nodules of poorly differentiated HCC measuring less than 1 cm each. The Alpha-fetoprotein decreased to normal values three weeks after TX and it has remained normal. The patient is now 14 months post TX with a functioning graft and showing no lesions of HCC. Conclusion PFIC type 2 is one of the few entities that might present HCC at a very early age. It is important to perform an adequate follow up in order to make the appropriate diagnosis and to perform a timely adequate liver transplant. It is of great interest for the medical community that newly identified mutations are reported, in order to enrich the medical knowledge.

Therapeutic Doses of Eltrombopag do not Inhibit Hepatic BCRP in Healthy Volunteers: Intravenous Ceftriaxone as a Model.

Ceftriaxone elimination occurs through breast cancer resistance transporter (BCRP) and multidrug resistance-associated protein 2 (MRP-2) which are expressed on the canalicular membrane of hepatocytes. Eltrombopag, a thrombopoetin receptor agonist used in the treatment of immune thrombocytopenic purpura, is reported in in vitro studies as an inhibitor of intestinal BCRP but not an inhibitor of hepatic BCRP. Thus, the present study evaluates the effect of therapeutic doses of eltrombopag on the clinical pharmacokinetics of intravenous ceftriaxone. Healthy adult (n=12) were treated with oral doses of eltrombopag (0, 25 or 50 mg) 28 and 4 h prior to intravenous ceftriaxone administration (1g). Serial blood samples were collected up to 48 h after ceftriaxone administration and plasma samples were analysed by LC-MS/MS using 50 μL aliquots (total concentration) and 100 μL (unbound concentration). A method to analyze total and unbound ceftriaxone in plasma using LC-MS/MS was developed and validated with linearity from 1 to 200 μg/mL. Both methods are sensitive, precise and accurate with coefficients of variation less than 15% in the study of inter- and intra-assay precision and accuracy. Ceftriaxone pharmacokinetics in healthy adults were described using a bicompartmental model, with a mean clearance of 0.96 L/h (CI95% 0.71-1.20) and AUC0-∞of 1106 mg.h/mL (CI95% 811-1400) for volunteers that received only ceftriaxone; clearance of 0.95 L/h (CI95% 0.77-1.13) and AUC0-∞ of 1083 mg.h/mL (CI95% 876-1290) for volunteers that received ceftriaxone plus 25 mg of eltrombopag and clearance of 0.96 L/h (CI95% 0.74-1.19) and AUC0-∞ of 1072 mg.h/mL (CI95% 872-1273) for volunteers that received ceftriaxone plus 50 mg of eltrombopag. The results do not support the existence of a clinical pharmacokinetic drug interaction involving hepatic BCRP in human subjects receiving intravenous ceftriaxone and oral eltrombopag.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Manganese transport and toxicity in polarized WIF-B hepatocytes.

Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 μM MnCl2. However, the hepatocytes were resistant to 4-h exposures of up to 500 μM MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Subcellular Distribution of Cholesterol and Sphingolipids in Rat Hepatocytes

Hepatocytes are central in energy and lipid homeostasis of mammals and as such are key players in maintaining and regulating serum cholesterol levels. In addition, hepatocytes produce bile, which has detergent properties and is the main product of cholesterol metabolism. The canalicular membrane is the site of bile formation and needs protective mechanisms against the detergent properties of bile. One such mechanism could be a specific lipid composition. We therefore aimed to use a targeted lipidomics approach to determine simultaneously the cholesterol and sphingolipid composition of the organelles involved in lipid synthesis in hepatocytes. Subcellular organelles were isolated from rat liver with standard methodologies and lipids were analyzed by either electrospray ionization mass spectrometry (glycerophospholipids and sphingolipids) or gas chromatography coupled mass spectrometry (sterols). Our data indicate that there is a distinct difference in cholesterol and sphingolipid content in the organelle fractions. Highest cholesterol content is found in basolateral plasma membranes and in the Golgi fraction, while endoplasmic reticulum displays a low cholesterol content. A high proportion of sphingomyelin is present in basolateral plasma membranes, smooth endoplasmic reticulum and in the Golgi apparatus. Ceramide content is higher in basolateral membranes compared to organelles and the canalicular plasma membrane. Glucosylceramide levels are very low and are mostly found in Golgi fractions. Our preliminary data so far show a parallel distribution of cholesterol and sphingolipids in subcellular fractions of rat hepatocytes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The importance of membrane microdomains for bile salt-dependent biliary lipid secretion.

ABSTRACTAlternative models explaining the biliary lipid secretion at the canalicular membrane of hepatocytes exist: successive lipid extraction by preformed bile salt micelles, or budding of membrane fragments with formation of mixed micelles. To test the feasibility of the latter mechanism, we developed a mathematical model that describes the formation of lipid microdomains in the canalicular membrane. Bile salt monomers intercalate into the external hemileaflet of the canalicular membrane, to form a rim to liquid disordered domain patches that then pinch off to form nanometer-scale mixed micelles. Model simulations perfectly recapitulate the measured dependence of bile salt-dependent biliary lipid extraction rates upon modulation of the membrane cholesterol (lack or overexpression of the cholesterol transporter Abcg5–Abcg8) and phosphatidylcholine (lack of Mdr2, also known as Abcb4) content. The model reveals a strong dependence of the biliary secretion rate on the protein density of the membrane. Taken together, the proposed model is consistent with crucial experimental findings in the field and provides a consistent explanation of the central molecular processes involved in bile formation.