Search

Uptake of xenobiotics by hepatocytes is mediated by specific proteins, including organic anion transporting polypeptides (OATPs), residing on the basolateral (sinusoidal) plasma membrane. Many of the OATPs have PDZ consensus binding sites, determined by their C-terminal 4 amino acids, while others do not. Mouse and rat OATP1A1 are associated with PDZK1, which is necessary for their trafficking to the plasma membrane. humanOATP1B1 (hOATP1B1) is a major drug transporter in human liver. Although localized to the plasma membrane, it was thought to lack a PDZ consensus motif, suggesting that the trafficking paradigm for murine OATPs is not applicable to human liver. The aim of the present study was to determine whether hOATP1B1 is a ligand for hPDZK1. hOATP1B1 immunoprecipitates with hPDZK1 following co-expression in 293T cells as well as in normal human liver. Co-expression with each of the 4 PDZ domains revealed interaction with domain 1 only. A truncated version of hOATP1B1 that lacks its terminal 4 amino acid PDZ binding motif as well as hOATP1B3, which does not contain a PDZ binding consensus motif, failed to interact with hPDZK1. Immunofluorescence microscopy of hOATP1B1 in stably transfected HeLa cells that endogenously express hPDZK1 showed that it distributes predominantly along the plasma membrane whereas hOATP1B1 lacking its terminal 4 amino acids distributes primarily intracellularly with little plasma membrane localization. Similar to findings in rats and mice, human OATP1B1 is a ligand for PDZK1 and requires interaction with PDZK1 for optimal trafficking to the hepatocyte plasma membrane. SIGNIFICANCE: Previous studies suggested that OATP1B1, a major xenobiotic transporter in human liver, does not have a PDZ binding consensus motif and does not follow the paradigm for subcellular trafficking and function that was established for OATP1A1 in murine liver. We now demonstrated that OATP1B1 but not OATP1B3 has a PDZ binding consensus motif that mediates binding to PDZK1 and is required for its trafficking to the plasma membrane. Such interaction could be an important previously unrecognized modulator of transport function., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is the causative agent of coronavirus disease 2019 (COVID-19). The presenting symptoms of this virus are variable, and there is an increasing body of literature on risk factors for mortality. The aim of this study was to evaluate the effect of initial aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and preexisting liver disease, including cirrhosis, in a cohort of patients admitted with COVID-19 infection at a tertiary care hospital network in the Bronx, New York. We reviewed 3,352 patients who had a positive SARS-CoV2 nasal swab, were over 18 years of age, and had an associated inpatient admission and discharge (or death) to the Montefiore Medical Center from February 28, 2020, to May 22, 2020. Of these, 39/86 (45%) patients died when the initial ALT was >5 times the upper limit of normal (ULN); 115/230 (50%) patients died when the initial AST was >3 times the ULN. The mortality of patients without preexisting liver disease was 26.6% compared to a mortality rate of 29.5% in patients with liver disease. Subgroup analysis showed a mortality of 36.1% in the patients with cirrhosis. Cirrhosis conferred a hazard ratio for mortality of 1.67 (95% confidence interval, 1.09, 2.55; P  = 0.019). The baseline Model for End-Stage Liver Disease score was not prognostic in the cirrhosis cohort. There was no statistical difference between mortality in patients with a history of compensated or decompensated cirrhosis. The most common cause of death in the cirrhosis cohort was respiratory failure. Conclusion : COVID-19 hepatitis may lead to poor outcomes in patients who are hospitalized for the disease. Patients with cirrhosis are at a higher risk of COVID-19-related mortality., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI-US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells., Competing Interests: The authors have declared that no competing interests exist.

The liver plays an essential role in removing endogenous and exogenous compounds from the circulation. This function is mediated by specific transporters, including members of the family of organic anion transport proteins (OATPs) and the Na + -taurocholate transporting polypeptide (NTCP). In the present study, transporter protein expression was determined in liver samples from patients with cirrhosis or controls without liver disease. Five transporters (OATP1A2, OATP1B1, OATP1B3, OATP2B1, and NTCP) were studied. Transporter content in homogenates of human liver was quantified on western blots probed with transporter-specific antibodies in which a calibrated green fluorescent protein-tagged transporter standard was included. Liver samples from 21 patients with cirrhosis (hepatitis C in 17 and alcohol abuse in 4) and 17 controls without liver disease were analyzed. Expression of each of the transporters had a large spread, varying by an order of magnitude in cirrhotic and control livers. OATP1B1 was the most abundant transporter in controls ( P  < 0.01) but was significantly lower in cirrhotic livers as was NTCP expression ( P  < 0.01). There was little difference in transporter expression with respect to age or sex. Despite the large variability in transporter expression within a group, analysis in individuals showed that those with high or low expression of one transporter had a similar magnitude in expression of the others. Conclusion: Differences in transporter expression could explain unanticipated heterogeneity of drug transport and metabolism in individuals with and without liver disease., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Accumulating somatic mutations have been implicated in age-related cellular degeneration and death. Because of their random nature and low abundance, somatic mutations are difficult to detect except in single cells or clonal cell lineages. Here, we show that in single hepatocytes from human liver, an organ exposed to high levels of genotoxic stress, somatic mutation frequencies are high and increase substantially with age. Considerably lower mutation frequencies were observed in liver stem cells (LSCs) and organoids derived from them. Mutational spectra in hepatocytes showed signatures of oxidative stress that were different in old age and in LSCs. A considerable number of mutations were found in functional parts of the liver genome, suggesting that somatic mutagenesis could causally contribute to the age-related functional decline and increased incidence of disease of human liver. These results underscore the importance of stem cells in maintaining genome sequence integrity in aging somatic tissues., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Organic anion transport proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density protein, drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n = 12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex., (© 2019 by the American Association for the Study of Liver Diseases.)

Background: Faecal microbiota transplantation (FMT) has shown promise in alleviating the symptoms of irritable bowel syndrome (IBS); however, controlled data on this technique are scarce. The aim of this clinical trial was to assess the efficacy of FMT in alleviating diarrhoea-predominant IBS (IBS-D)., Methods: We did a double-blind, randomised, placebo-controlled crossover trial in patients aged 18-65 years with moderate-to-severe IBS-D defined by an IBS-Symptom Severity Score (IBS-SSS) of more than 175, recruited from three US centres. Patients were randomly assigned (1:1) in blocks of four with a computer-generated randomisation sequence to receive FMT capsules followed by identical-appearing placebo capsules, or placebo capsules followed by FMT capsules. All participants and study team members were masked to randomisation. An independent staff member assigned the treatments according to consecutive numbers. Patients received either 75 FMT capsules (each capsule contained approximately 0·38 g of minimally processed donor stool) or 75 placebo capsules over 3 days (25 capsules per day). All patients crossed over to the alternate treatment at 12 weeks. The primary outcome was difference in IBS-SSS between the groups at 12 weeks. Intention-to-treat analyses were done and all patients who received study drug were included in an adverse events analysis. The trial was terminated during recruitment because results from an interim analysis revealed futility. The study is registered with ClinicalTrials.gov, number NCT02328547., Findings: From May 28, 2015, to April 21, 2017, 48 patients were randomly assigned to receive FMT first (n=25) or placebo first (n=23). Three participants were lost to follow-up in the FMT group. IBS-SSS did not differ between FMT recipients (mean 221 [SD 105]) and placebo recipients (236 [95]) at 12 weeks (p=0·65), after adjustment for baseline scores. The most common drug-related adverse events included abdominal pain (five [10%] of the 48 participants while receiving FMT capsules vs four [8%] while receiving placebo), nausea (four [8%] vs two [4%]), and exacerbation of diarrhoea (three [6%] vs eight [17%]). One serious adverse event that was unrelated to study drug (acute cholecystitis) was reported in a patient while receiving placebo capsules., Interpretation: FMT was safe, but did not induce symptom relief at 12 weeks compared with placebo. Additional studies are needed to determine the efficacy of FMT for IBS-D., Funding: National Institutes of Health., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule-based minus-end-directed motor proteins, the well-characterized dynein, and the less-studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti-aging purposes., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking.

The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumours is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. We studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events. We find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on our samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas. Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression. The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically reversible epigenetic field defect in HCV-infected liver.

Hyperbilirubinemia is a common finding in individuals with a history of substance abuse. Although this may indicate a serious disorder of liver function, this is not always the case. An understanding of bilirubin formation, metabolism, and transport can provide a helpful approach to dealing with these patients. This is typified by studies of patients treated with the antiretroviral drug atazanavir. Atazanavir has been associated with hyperbilirubinemia in as many as one-third of individuals for whom it has been prescribed, evoking concerns of hepatotoxicity. The studies in this report were designed to determine mechanisms by which this occurs. The data show that this drug inhibits the enzyme UDP-glucuronosyl transferase-1A1, responsible for conjugating bilirubin with glucuronic acid. This conjugation step is required for bilirubin excretion into bile, and when it is inhibited, bilirubin refluxes from the liver into the circulation, causing unconjugated hyperbilirubinemia. Other parameters of bilirubin formation, binding to albumin in the circulation, uptake into hepatocytes, and intracellular protein binding in hepatocytes were unaffected by atazanavir. The effect of atazanavir on serum bilirubin levels is reversible, consistent with lack of structural damage to the liver., (© 2017, The American College of Clinical Pharmacology.)

Na(+)-taurocholate cotransporting polypeptide (ntcp) mediates bile acid transport, also serving as the hepatitis B virus receptor. It traffics in vesicles along microtubules, requiring activity of protein kinase C (PKC)ζ for motility. We have now found that the epidermal growth factor receptor (EGFR) is the target of PKCζ activity and that EGFR and ntcp colocalize in vesicles. ntcp-containing vesicles that are not associated with EGFR have reduced microtubule-based motility, consistent with intracellular accumulation and reduced surface expression of ntcp in cells following EGFR knockdown., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We explored potential genetic risk factors implicated in nonalcoholic fatty liver disease (NAFLD) within a Caribbean-Hispanic population in New York City. A total of 316 individuals including 40 subjects with biopsy-proven NAFLD, 24 ethnically matched non-NAFLD controls, and a 252 ethnically mixed random sampling of Bronx County, New York were analyzed. Genotype analysis was performed to determine allelic frequencies of 74 known single-nucleotide polymorphisms (SNPs) associated with NAFLD risk based on previous genome-wide association study (GWAS) and candidate gene studies. Additionally, the entire coding region of PNPLA3, a gene showing the strongest association to NAFLD was subjected to Sanger sequencing. Results suggest that both rare and common DNA variations in PNPLA3 and SAMM50 may be correlated with NAFLD in this small population study, while common DNA variations in CHUK and ERLIN1, may have a protective interaction. Common SNPs in ENPP1 and ABCC2 have suggestive association with fatty liver, but with less compelling significance. In conclusion, Hispanic patients of Caribbean ancestry may have different interactions with NAFLD genetic modifiers; therefore, further investigation with a larger sample size, into this Caribbean-Hispanic population is warranted.

Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three-dimensional (3D) "sandwich" is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4-fold), carboxyfluorescein succinimidyl ester (4.8-fold), and fluorescein (30-fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L-FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12-18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long-term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Many of the compounds taken up by the liver are organic anions that circulate tightly bound to protein carriers such as albumin. The fenestrated sinusoidal endothelium of the liver permits these compounds to have access to hepatocytes. Studies to characterize hepatic uptake of organic anions through kinetic analyses, suggested that it was carrier-mediated. Attempts to identify specific transporters by biochemical approaches were largely unsuccessful and were replaced by studies that utilized expression cloning. These studies led to identification of the organic anion transport proteins (oatps), a family of 12 transmembrane domain glycoproteins that have broad and often overlapping substrate specificities. The oatps mediate Na(+)-independent organic anion uptake. Other studies identified a seven transmembrane domain glycoprotein, Na(+)/taurocholate transporting protein (ntcp) as mediating Na(+)-dependent uptake of bile acids as well as other organic anions. Although mutations or deficiencies of specific members of the oatp family have been associated with transport abnormalities, there have been no such reports for ntcp, and its physiologic role remains to be determined, although expression of ntcp in vitro recapitulates the characteristics of Na(+)-dependent bile acid transport that is seen in vivo. Both ntcp and oatps traffic between the cell surface and intracellular vesicular pools. These vesicles move through the cell on microtubules, using the microtubule based motors dynein and kinesins. Factors that regulate this motility are under study and may provide a unique mechanism that can alter the plasma membrane content of these transporters and consequently their accessibility to circulating ligands.

We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered.

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Previous studies identified a family of organic anion transport proteins (OATPs), many of which have C-terminal PDZ binding consensus sequences. In particular, the C-terminal four amino acids of Oatp1a1, a transporter on rat and mouse hepatocytes, comprise a consensus binding site for PDZK1. In PDZK1 knockout mice and in transfected cells where PDZK1 expression was knocked down, Oatp1a1 accumulates in intracellular vesicles. The present study tests the hypothesis that Oatp1a1 traffics to and from the cell surface in vesicles along microtubules, and that PDZK1 guides recruitment of specific motors to these vesicles. Oatp1a1-containing vesicles were prepared from wild-type and PDZK1 knockout mice. As seen by immunofluorescence, kinesin-1, a microtubule plus-end directed motor, was largely associated with vesicles from wild-type mouse liver, whereas dynein, a minus-end directed motor, was largely associated with vesicles from PDZK1 knockout mouse liver. Quantification of motility on directionally marked microtubules following addition of 50 µM ATP showed that wild-type vesicles moved equally toward the plus and minus ends whereas PDZK1 knockout vesicles moved predominantly toward the minus end, consistent with net movement toward the cell interior. These studies provide a novel mechanism by which PDZK1 regulates intracellular trafficking of Oatp1a1 by recruiting specific motors to Oatp1a1-containing vesicles. In the absence of PDZK1, Oatp1a1-containing vesicles cannot recruit kinesin-1 and associate with dynein as a predominant minus-end directed motor. Whether this is a result of direct interaction of the Oatp1a1 cytoplasmic domain with dynein or with a dynein-containing protein complex remains to be established.

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.

Texas-Red-asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.

Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1(EE)), phosphomimetic] or alanine [A634A635 (oatp1a1(AA)), nonphosphorylatable]. Distribution of oatp1a1(AA) and oatp1a1(EE) was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1(AA) was now expressed largely on the cell surface, while oatp1a1(EE) remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1(EE) internalizes quickly compared with oatp1a1(AA). To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study.

Randomized controlled trials of hepatitis C virus (HCV) therapy with pegylated interferon and ribavirin have demonstrated sustained viral response rates (SVRs) of 54%-63% (efficacy). Treatment results in clinical practice (effectiveness) may not be equivalent. The goal of this study was to assess the effectiveness of HCV treatment with pegylated interferon and ribavirin in a treatment-naïve, human immunodeficiency virus (HIV)-negative, United States urban population with many ethnic minority patients. We evaluated 2,370 outpatients for HCV therapy from 2001 to 2006 in the Faculty Practice of the Albert Einstein College of Medicine or the attending-supervised Montefiore Medical Center Liver Clinic. Care was supervised by one experienced physician under conditions of everyday clinical practice, and appropriate ancillary resources were made available to all patients. Two hundred fifty-five patients were treated with a mean age of 50 years (60% male, 40% female; 58% Hispanic, 20% African American, 9% Caucasian, 13% other; 68% genotype 1, the remainder genotypes 2 or 3). Patients had at least one liver biopsy. Intention-to-treat analysis (ITT) showed SVR in 14% of genotype 1 patients and 37% in genotype 2/3 patients (P < 0.001). SVR was significantly higher in faculty practice (27%) than in clinic patients (15%) by intention-to-treat (P = 0.01) but not per-protocol analysis (46% faculty practice, 34% clinic). 3.3% of 1,656 treatment-naïve, HIV antibody-negative individuals ultimately achieved SVR. Current hepatitis C therapies may sometimes be unavailable to, inappropriate for, and ineffective in United States urban patients. Treatment with pegylated interferon and ribavirin was less effective in this population than is implied by multinational phase III controlled trials. New strategies are needed to care for such patients.

Unlabelled: Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase-independent manner., Conclusion: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase-independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells.

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2-Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 alpha'' (CK2alpha'') which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of (125)I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%-60% in Trf2-Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on (125)I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2-Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa(488)-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein-protein or protein-lipid interactions that orchestrate specific steps in membrane protein trafficking.

Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5'-adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.

This chapter presents fluorescence microscope assays that can be used to study microtubule (MT)-based movement and receptor-ligand sorting in vitro. The strategy is to isolate endosomes in a concentrated active form and store them in frozen aliquots for single use. Glass microchambers are then constructed and coated with fluorescent MTs, and the endosomes are thawed and bound to the MTs. Proteins of interest are then detected and quantified by immunofluorescence. For motility experiments, time-lapse movies are captured using multichannel fluorescence microscopy, and motility is initiated by the addition of ATP. Movies are later categorized and quantified for MT-based motility and other associated events such as endocytic fission. These techniques were developed to assess the role of MTs and MT motor proteins in endocytic processing within liver cells, and we have streamlined a rapid procedure for isolating abundant, highly motile endosomes from rat liver. Cultured cells and other organelles can also be examined, and many important biological questions concerning intracellular traffic and organelle composition can be studied by creative adaptation of the protocols that are presented.

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.

In the asialoglycoprotein receptor (ASGPR) endocytic pathway, internalized receptors pass through early, recycling, and sorting endosomal compartments before returning to the cell surface. Sorting motifs in the cytoplasmic domain (CD) and protein interactions with these sequences presumably direct receptor trafficking. Previous studies have shown that association of a potential sorting heat shock protein (HSP) heterocomplex with the ASGPR-CD was regulated by casein kinase 2 (CK2)-mediated phosphorylation. Mass spectrometry and immunoblot analyses identified five of these ASGPR-CD-associated proteins as the molecular chaperones glycoprotein 96, HSP70, HSP90, cyclophilin A, and FK 506 binding protein. The present study was undertaken to determine whether any of the adaptor protein complexes (AP1, AP2, or AP3) were selectivity associated with the ASGPR-CD. In conjunction with molecular chaperones, AP2 and AP1 were recovered from a CK2 phosphorylated agarose-GSH-GST-ASGPR-CD matrix. Binding of AP3 was independent of the phosphorylation status of the CD matrix. Inhibition of CK2-mediated phosphorylation with tetrabromobenzotriazole prevented AP recovery within an immunoadsorbed ASGPR complex. Rapamycin, which dissociates the HSP heterocomplex from ASGPR-CD, thereby altering receptor trafficking also, inhibited AP association. Similar results were obtained with an inhibitor of HSP90 heterocomplex formation, geldanmycin. The data presented provide evidence that recruitment of AP1 and AP2, which is necessary for appropriate receptor trafficking, is mediated by the interaction of AP with the ASGPR-CD-bound HSP complex.

Flavonoids are a class of polyphenolic compounds widely present in the diet and herbal products. The interactions of flavonoids with some major efflux transporters [e.g., P-glycoprotein, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein] have been reported; however, their interactions with uptake transporters are largely unknown. Organic anion-transporting polypeptide OATP1B1 is a liver-specific uptake transporter important in hepatic drug disposition. Our objective was to evaluate the effects of 20 naturally occurring flavonoids, and some of their corresponding glycosides, on the uptake of [3H]dehydroepiandrosterone sulfate (DHEAS) in OATP1B1-expressing and OATP1B1-negative HeLa cells. Many of the tested flavonoids (including biochanin A, genistein, and epigallocatechin-3-gallate) significantly inhibited [3H]DHEAS uptake in a concentration-dependent manner in OATP1B1-expressing cells, with biochanin A being one of the most potent inhibitors with an IC50 of 11.3 +/- 3.22 microM. The flavonoids had negligible or small effects in OATP1B1-negative cells. Four of the eight pairs of tested flavonoids and their glycosides, namely, genistein/genistin, diosmetin/diosmin, epigallocatechin/epigallocatechin-3-gallate, and quercetin/rutin, exhibited distinct effects on [3H]DHEAS uptake. For example, genistin did not inhibit DHEAS uptake, whereas genistein did, and rutin stimulated uptake, whereas quercetin had no effect. [3H]Biochanin A uptake was similar in OATP1B1-expressing and OATP1B1-negative cells, suggesting that it is not a substrate for OATP1B1. A kinetic study revealed that biochanin A inhibited [3H]DHEAS uptake in a noncompetitive manner, with a Ki of 10.2 +/- 1.89 microM. Taken together, these results indicate that flavonoids are a novel class of OATP1B1 modulators, suggesting the potential for diet-drug interactions.

The roles of vascular binding, flow, transporters, and enzymes as determinants of the clearance of digoxin were examined in the rat liver. Digoxin is metabolized by Cyp3a and utilizes the organic anion transporting polypeptide 2 (Oatp2) and P-glycoprotein (Pgp) for influx and excretion, respectively. Uptake of digoxin was found to be similar among rat periportal (PP) and perivenous (PV) hepatocytes isolated by the digitonin-collagenase method. The Km values for uptake were 180 +/- 112 and 390 +/- 406 nM, Vmax values were 13 +/- 8 and 18 +/- 4.9 pmol/min/mg protein, and nonsaturable components were 9.2 +/- 1.3 and 10.7 +/- 2.5 microl/min/mg for PP and PV, respectively. The evenness of distribution of Oatp2 and Pgp was confirmed by Western blotting and confocal immunofluorescent microscopy. When digoxin was recirculated to the rat liver preparation in Krebs-Henseleit bicarbonate (KHB) for 3 h in absence or presence of 1% bovine serum albumin (BSA) and 20% red blood cell (rbc) at flow rates of 40 and 10 ml/min, respectively, biexponential decays were observed. Fitted results based on compartmental analyses revealed a higher clearance (0.244 +/- 0.082 ml/min/g) for KHB-perfused livers over the rbc-albumin-perfused livers (0.114 +/- 0.057 ml/min/g) (P < 0.05). We further found that binding of digoxin to 1% BSA was modest (unbound fraction = 0.64), whereas binding to rbc was associated with slow on (0.468 +/- 0.021 min(-1)) and off (1.81 +/- 0.12 min(-1)) rate constants. We then used a zonal, physiologically based pharmacokinetic model to show that the difference in digoxin clearance was attributed to binding to BSA and rbc and not to the difference in flow rate and that clearance was unaffected by transporter or enzyme heterogeneity.

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM&#95;017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule-based organelle motility. The protocols utilize small-volume, disposable "microchambers" designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluorescent secondary antibodies are added. Our work has focused on the isolation and use of endocytic vesicles from rat liver, and we present these protocols. However, the techniques can be adapted for other organelles or cell types. Multiple fluorescent probes, rapid image capture, and immunofluorescence under non-fixation conditions allow for measurements of the location and intensity changes of endogenous proteins upon addition of ATP or upon addition of other proteins or regulatory factors. We review measurements of microtubule-based motility as well as measurements for protein localization and protein segregation in vitro.

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.

After internalization, endocytic material is actively transported through the cytoplasm, predominantly by microtubule motor proteins. Microtubule-based endocytic transport facilitates sorting of endocytic contents, vesicle fusion and fission, delivery to lysosomes, cytosolic dispersal, as well as nuclear uptake and cytosolic egress of pathogens. Endosomes, like most organelles, move bidirectionally through the cytosol and regulate their cellular location by controlling the activity of motor proteins, and potentially by controlling microtubule and actin polymerization. Control of motor protein activity is manifest by increased microtubule "run lengths", and the binding of motor proteins to organelles can be regulated by motor protein receptors. A mechanistic understanding of how organelles control motor protein activity to allow for endocytic sorting presents an exciting avenue for future research.

Transport of a series of 3H-radiolabeled C23, C24, and C27 bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (alpha or beta) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Km similar to that of oatp1-mediated transport of [35S]sulfobromophthalein (Km = 3.7 vs. 3.3 muM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-gamma-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-gamma-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-gamma-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

A recent study (Cui, Y., Konig, J., Leier, I., Buchholz, U., and Keppler, D. (2001) J. Biol. Chem. 276, 9626-9630) suggests that human OATP2 (SLC21A6), also known as OATP-C and LST1, mediates hepatic bilirubin transport. Because of methodologic concerns, this study was designed to examine this issue using a bilirubin transport assay that was validated in overnight cultured rat hepatocytes. These studies showed that cultured rat hepatocytes transported bilirubin with kinetics virtually identical to the transport of sulfobromophthalein. This assay was then used to quantify bilirubin transport by HeLa cells that had been stably transfected with OATP2 under regulation of a metallothionein promoter. Immunoblot analysis revealed abundant expression of OATP2 after incubation of cells for 48 h in zinc, whereas uninduced cells had no expression of this protein. In OATP2-expressing (zinc-induced) HeLa cells at 37 degrees C, the uptake of [35S]sulfobromophthalein was substantial (51.6 +/- 16.5 pmol/15 min/mg protein, n = 5) with little cell-associated ligand in non-expressing (uninduced) cells (0.54 +/- 0.16 pmol/15 min/mg protein, n = 5, p < 0.002). In contrast, there was no difference (p > 0.2) in cell-associated [3H]bilirubin in induced (OATP2-expressing) as compared with uninduced cells (11.25 +/- 3.02 pmol/15 min/mg protein versus 9.15 +/- 1.68 pmol/min/mg protein, respectively, n = 5) We obtained similar results in OATP2-transfected HEK293 cells that were used in the original report. The existence of a bilirubin transporter has been an important field of investigation for many years. Although the current study indicates that a role for OATP2 in hepatocyte bilirubin transport is unlikely, it provides new and sensitive tools that can be adapted to examine the function of putative bilirubin transporters in the future.

Bile acids are cholesterol derivatives that serve as detergents in bile and the small intestine. Approximately 95% of bile acids secreted by hepatocytes into bile are absorbed from the distal ileum into the portal venous system. Extraction from the portal circulation by the hepatocyte followed by reexcretion into the bile canaliculus completes the enterohepatic circulation of these compounds. Over the past few years, candidate bile acid transport proteins of the sinusoidal and canalicular plasma membranes of the hepatocyte have been identified. The physiology of hepatocyte bile acid transport and its relationship to these transport proteins is the subject of this Themes article.

Rifampin, a member of the rifamycin class of antibiotics, is well known for its ability to induce drug-metabolizing enzymes and transporters, through activation of the pregnane X receptor. Available data suggest rifampin entry into hepatocytes may be transporter-mediated. Accordingly, it is therefore plausible that modulation of the achievable intracellular concentration of rifampin by drug uptake transporters would influence the degree of induction. In this study, we expressed an array of known hepatic uptake transporters to show the key hepatic rifampin uptake transporters are liver-specific members of the organic anion transporting polypeptide family (OATP). Indeed, both OATP-C and OATP8 seemed capable of mediating rifampin uptake into HeLa cells. OATP-C, however, seemed to have far greater affinity and capacity for rifampin transport. In addition, several allelic variants of OATP-C known to be present among European and African Americans were found to have markedly decreased rifampin transport activity. In cell-based, transactivation assays, OATP-C expression was associated with increased cellular rifampin retention as well as potentiation of PXR reporter gene activity. This is the first demonstration of an uptake transporter such as OATP-C, in modulating PXR function, and sheds important new insight into our understanding of the molecular determinants of PXR-mediated inductive processes.

Diabetes in subjects with hepatocyte nuclear factor (HNF)-1alpha gene mutations (maturity-onset diabetes of the young [MODY]-3) is characterized by impaired insulin secretion. Surprisingly, MODY3 patients exhibit hypersensitivity to the hypoglycemic actions of sulfonylurea therapy. To study the pharmacogenetic mechanism(s), we have investigated glibenclamide-induced insulin secretion, glibenclamide clearance from the blood, and glibenclamide metabolism in wild-type and Hnf-1alpha-deficient mice. We show that despite a profound defect in glucose-stimulated insulin secretion, diabetic Hnf-1alpha(-/-) mice have a robust glibenclamide-induced insulin secretory response. We demonstrate that the half-life (t(1/2)) of glibenclamide in the blood is increased in Hnf-1alpha(-/-) mice compared with wild-type littermates (3.9 +/- 1.3 vs. 1.5 +/- 1.8 min, P

A membrane protein trafficking mutant (Trf1) of HuH-7 alters the asialoglycoprotein (ASGPR) and transferrin receptor subcellular distribution. Expression cloning of a cDNA complementing the trf1 mutation led to the discovery of a novel casein Kinase 2 catalytic subunit (CK2alpha"). To purify potential CK2alpha" phosphorylation-dependent sorting proteins from cytosol, the ASGPR cytoplasmic domain was expressed as a GST fusion protein and immobilized on glutathione-agarose. In the absence of phosphorylation, only trace amounts of cytosol protein were bound and eluted. When the fusion protein was phosphorylated, a heterocomplex of potential sorting proteins was recovered. Mass spectrometer and immunoblot analysis identified five of these proteins as gp96, HSP70, HSP90, cyclophilin-A, and FKBP18. Treatment of HuH-7 with rapamycin to disrupt the heterocomplex reduced surface ASGPR binding activity by 65 +/- 5.7%. In Trf1 cells, surface-binding activity was 48 +/- 7% of that in HuH-7 and was not further reduced by rapamycin treatment. Immunoanalysis showed significantly fewer surface receptors on rapamycin-treated HuH7 cells than on nontreated cells, with no affect on the level of surface receptors in Trf1 cells. The data presented provide evidence that phosphorylation of the ASGPR cytoplasmic domain is required for the binding of specific molecular chaperones with the potential to regulate receptor trafficking.