Apical Domain Of Hepatocytes Research Articles

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Microanatomy of the digestive system of Supachai's caecilian,Ichthyophis supachaiiTaylor, 1960 (Amphibia: Gymnophiona)

AbstractThe gross anatomy and microanatomy of the digestive system ofIchthyophis supachaiiwere investigated. The microscopic structures of the digestive system are similar to those in other caecilians. Functional and developing teeth are present in adults. The tongue contains the genioglossus muscle. The digestive tract is elongated and consists of the mucosa, submucosa, muscularis and adventitia/serosa. The oesophagus contains longitudinal folds and lacks oesophageal glands. We report for the first time the caecilian gastric rugae and specific localization of oxynticopeptic cells in the anterior gastric region. The intestinal folds are exclusively present in the anterior intestinal region. The liver comprises 30–40 incomplete hepatic lobes, lying in an imbricate manner. Each lobe is enveloped by haematopoietic tissue that produces and delivers blood cells into sinusoids. Hepatic parenchyma is organized into anastomosing, two‐cell‐thick plates, having sinusoids at the basal domain and bile canaliculi at the apical domain of hepatocytes. Pigment cells are scattered inside sinusoids. The pancreas contains pancreatic acini interspersed with islets of Langerhans. The gallbladder proper is thin and continuous with the cystic duct wall. Neutral and carboxylated acid mucosubstances are secreted along the digestive tract, while sulphated mucosubstances are not produced by the stomach and anterior intestinal regions.

Upregulation of Brain Expression of P‐Glycoprotein in MRP2‐deficient TR‐ Rats Resembles Seizure‐induced Up‐regulation of This Drug Efflux Transporter in Normal Rats

The multidrug resistance protein 2 (MRP2) is a drug efflux transporter that is expressed predominantly at the apical domain of hepatocytes but seems also to be expressed at the apical membrane of brain capillary endothelial cells that form the blood-brain barrier (BBB). MRP2 is absent in the transport-deficient (TR(-)) Wistar rat mutant, so that this rat strain was very helpful in defining substrates of MRP2 by comparing tissue concentrations or functional activities of compounds in MRP2-deficient rats with those in transport-competent Wistar rats. By using this strategy to study the involvement of MRP2 in brain access of antiepileptic drugs (AEDs), we recently reported that phenytoin is a substrate for MRP2 in the BBB. However, one drawback of such studies in genetically deficient rats is the fact that compensatory changes with upregulation of other transporters can occur. This prompted us to study the brain expression of P-glycoprotein (Pgp), a major drug efflux transporter in many tissues, including the BBB, in TR(-) rats compared with nonmutant (wild-type) Wistar rats. The expression of MRP2 and Pgp in brain and liver sections of TR(-) rats and normal Wistar rats was determined with immunohistochemistry, by using a novel, highly selective monoclonal MRP2 antibody and the monoclonal Pgp antibody C219, respectively. Immunofluorescence staining with the MRP2 antibody was found to label a high number of microvessels throughout the brain in normal Wistar rats, whereas such labeling was absent in TR(-) rats. TR(-) rats exhibited a significant up-regulation of Pgp in brain capillary endothelial cells compared with wild-type controls. No such obvious upregulation of Pgp was observed in liver sections. A comparable overexpression of Pgp in the BBB was obtained after pilocarpine-induced seizures in wild-type Wistar rats. Experiments with systemic administration of the Pgp substrate phenobarbital and the selective Pgp inhibitor tariquidar in TR(-) rats substantiated that Pgp is functional and compensates for the lack of MRP2 in the BBB. The data on TR(-) rats indicate that Pgp plays an important role in the compensation of MRP2 deficiency in the BBB. Because such a compensatory mechanism most likely occurs to reduce injury to the brain from cytotoxic compounds, the present data substantiate the concept that MRP2 performs a protective role in the BBB. Furthermore, our data suggest that TR(-) rats are an interesting tool to study consequences of overexpression of Pgp in the BBB on access of drugs in the brain, without the need of inducing seizures or other Pgp-enhancing events for this purpose.

Expression of the multidrug transporter MRP2 in the blood–brain barrier after pilocarpine-induced seizures in rats

Multidrug resistance proteins (MRPs; symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of a wide range of substrates from cells and thereby affect the bioavailability and disposition of many drugs. MRP2 (ABCC2) is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, but its location in the brain is a matter of debate. Most previous studies failed to determine MRP2 mRNA or protein in the brain or cell preparations from the brain of different species including humans. Based on our previous experience with the drug efflux transporter P-glycoprotein, we evaluated whether the immunohistochemical determination of MRP2 expression is sensitive to fixation and staining variables. Furthermore, we examined whether the MRP2 protein is overexpressed after experimentally induced seizures in rats, using the pilocarpine model of temporal lobe epilepsy. The MRP2 expression in the liver was used as positive control. MRP2 deficient TR − rats were used as negative controls. Despite various modifications in tissue fixation and immunohistochemical staining as well as use of different commercially available MRP2 antibodies, we never observed any unequivocal MRP2 staining in the brain of normal rats. However, after a pilocarpine-induced convulsive status epilepticus, clear MRP2 staining became visible in brain capillary endothelial cells and, less frequently, perivascular astroglia and neurons in various brain regions. In view of our recent data on brain access of antiepileptic drugs in MRP2 deficient TR − rats, seizure-induced over-expression of MRP2 in the blood–brain barrier is likely to impair drug penetration into the brain, thereby contributing to drug resistance in epilepsy.

Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.

Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.

An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics-based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA. We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes). (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face. The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.

Regulation of expression of the multidrug resistance-associated protein 2 (MRP2) and its role in drug disposition.

The multidrug resistance protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter accepting a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many endo- and xenobiotics. MRP2 generally performs excretory or protective roles, and it is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, as well as in the brain and the placenta. MRP2 is regulated at several levels, including membrane retrieval and reinsertion, translation, and transcription. In addition to transport of conjugates, MRP2 transports cancer chemotherapeutics, uricosurics, antibiotics, leukotrienes, glutathione, toxins, and heavy metals. Several mutagenesis studies have described critical residues for substrate binding and various naturally occurring mutations that eliminate MRP2 expression or function. MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by certain drugs and disease states.

Annexin VI defines an apical endocytic compartment in rat liver hepatocytes.

Annexin VI has been demonstrated previously to be a marker for hepatic endosomes. By western blotting with an affinity purified anti-annexin VI antibody it was shown that annexin VI was present in the three morphologically and functionally different endosomal fractions from rat liver. We have quantified the gold-labeled endosomes by immunoelectron microscopy in ultrathin Lowicryl sections of rat liver and now demonstrate that 80% of the total labeling with anti-annexin VI was associated with endocytic structures surrounding the bile canaliculus, the apical domain of hepatocytes, whereas only 20% was found in the subsinusoidal endosomes. In double immuno-gold labeling experiments 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization with antibodies to the polymeric immunoglobulin receptor. Finally, we demonstrate that 50% of endosomes containing internalized gold-labeled transferrin were double labeled with anti-annexin VI antibodies. Thus, annexin VI becomes the first known structural protein at the apical 'early' endocytic compartment of the hepatocyte that may be involved in the receptor recycling and transport to late endocytic/lysosomal compartment pathways.