Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration.

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Pranoprofen quantification in ex vivo corneal and scleral permeation samples: Analytical validation.

The investigation of the ocular permeability and/or distribution of pranoprofen (PF), a non-steroidal antiinflamatory drug, demands for the selective analysis of its transit through specific ocular membranes. Therefore, customised ex vivo permeation experiments through external ocular tissues (cornea and sclera) have been validated for this drug in addition to its HPLC-UV quantification following standard bioanalytical guidelines. Chromatographic conditions consist of an isocratic system to elute the drug with a C18 column with UV detection at 245 nm. Precision, expressed as the relative standard deviation (% RSD), ranged between 4.89 and 0.79% (intra-day) and between 9.02 and 2.14% (interday). Accuracy ranged between 5.15 and -1.92% in intra-day experiments and between 6.25 and -4.89% in inter-day experiments. Drug recovery from tissue samples was reproducible around 90% and considered satisfactory to adequately assess drug levels in target tissues. Results indicate that the procedure is valid for the quantitation of PF in those ophthalmic samples in the range of 6.5 µg/mL to 100 µg/ml. As a proof of concept, PF permeation profiles through porcine cornea and sclera with vertical diffusion cells have been generated and analyzed. Pilot experiments demonstrate its applicability to investigate permeation levels of PF from 22.31 µg/cm2 (about a 20% of the dose) until 500 µg/cm2 if required. Additionally, real tissue-retention samples were also generated to verify the goodness of this experimental setup.

Changes in sinusoidal plasma membrane enzyme activities during the pre-replicative phase of liver regeneration.

Changes in a range of plasma membrane enzyme activities during the early period of liver regeneration are thought to be related to the initiation of DNA synthesis and the triggering of cellular activation. The sinusoidal plasma membrane was isolated from control and partially hepatectomized animals at various intervals during the pre-replicative phase. The specific activities of 5'-nucleotidase, (Na+ + K+)-ATPase, Ca2+-ATPase, Mg2+-ATPase showed that after partial hepatectomy changes in the enzyme activities at the sinusoidal plasma membrane region occur. These changes are probably related to the remodeling of the cell-surface that occurs before the division of hepatocytes.

Reduced levels of sialic acid in the plasma membrane during hepatocellular proliferation.

When rats were infused with a solution containing triiodothyronine, amino acids, glucagon and heparin (solution A) the hepatocytes increased DNA synthesis and decreased plasma membrane sialic acid. In order to study whether the reduced levels of sialic acid in the plasma membrane were associated with hepatocyte proliferation, different mixtures of three components of solution A were infused into rats and the DNA synthetic activity as well as the sialic acid content measured. Results reported here show a correlation between DNA synthetic activity and sialic acid reduction suggesting that the decrease in the plasma membrane sialic acid can be a pre-replicative step associated to cell proliferation.

EGF triggers caveolin redistribution from the plasma membrane to the early/sorting endocytic compartment of hepatocytes.

In this study, we demonstrate that, in rat liver, epidermal growth factor (EGF) is responsible for the partial redistribution of caveolin-1 from the plasma membrane into the early/sorting endocytic compartment. Highly purified endosomes and plasma membrane fractions were isolated from control rat liver and from rats injected with EGF or pIgA for different times. Whereas in subcellular fractions from control hepatocytes most of caveolin was concentrated in the plasma membrane and the receptor-recycling fractions, after EGF injection there was a significant redistribution of caveolin toward the early/sorting (CURL) endocytic fractions. The recruitment of caveolin into the endocytic compartment was not induced by pIgA.

Antibodies to hepatic endosomes. Identification of two endosome antigens.

Endosome fractions were prepared from rat liver homogenates, and antibodies were raised in rabbits against the integral membrane proteins. Immunofluorescent studies showed that these antibodies identified primarily intracellular structures in liver sections, isolated hepatocytes and HepG-2 cells. Immunoelectron microscopy using protein A-gold confirmed that endocytic multivesicular structures, especially those located at the biliary pole of the hepatocyte, were labeled. Biochemical analysis showed that approximately 12 endosome antigens were present. A major 43 kDa glycosylated antigen corresponded to the asialoglycoprotein receptor subunit. A further antigen identified in endosomes was a 115 kDa polypeptide pI 4.3 previously identified as a major calmodulin-binding protein. The antigens identified in rat liver endosomes were different to those previously shown by other studies to be present in the Golgi apparatus and lysosomes.

A 220 kDa polypeptide, immunolocalized to epithelial tight junctions, is associated with brain clathrin preparations.

Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated endosomes from quiescent rat liver contain the signal transduction machinery. Differential distribution of activated Raf-1 and Mek in the endocytic compartment.

In this study we identify the molecules involved in the MAPK signal transduction pathway (Ras, Raf-1, Mek, Mek-P and MAPK) in highly purified endosomal fractions isolated from rat liver. Biochemical analysis shows that only the early-sorting endocytic compartment contains activated Raf-1 and Mek. Finally, the exogenous administration of EGF led to redistribution of Raf-1 from the caveolin-enriched plasma membrane into the endosomes.

Pre-replicative changes of the rat sinusoidal plasma membrane glycoproteins during hepatic regeneration.

Cell-surface glycoproteins of rat liver sinusoidal plasma membranes from control and regenerating livers were studied. The glycoproteins were labeled using specific methods for sialic acid (NAIO4/NaB3H4) and galactosyl/N-acetyl galactosaminyl residues (galactose oxidase/NaB3H4 and neuraminidase-galactose oxidase (NaB3H4) and the solubilized proteins were analyzed by gel electrophoresis. The patterns obtained with regenerating livers were quantitatively different from controls. This shows that cell surface glycoproteins change during liver regeneration.

Fibronectin isoforms in plasma membrane domains of normal and regenerating rat liver.

Plasma membrane fractions were obtained from the three surface domains of normal and regenerating adult rat livers. It was shown by immunoblotting that sinusoidal plasma membranes contained the characteristic 220 and 210 kDa fibronectin doublet, whereas bile canalicular plasma membranes contained a 220 kDa component. In lateral plasma membranes, 180, 190 and 220 kDa fibronectin isoforms were present. Fibronectin in the sinusoidal and canalicular plasma membranes was shown, by detergent/aqueous phase partitioning, to be more hydrophilic than isoforms in lateral plasma membranes. Changes in the distribution of fibronectin between plasma membrane domains occurred during liver regeneration and their significance, especially in relation to cell division, is discussed.

Calmodulin may decrease cell surface sialic acid and be involved in the expression of fibronectin during liver regeneration.

The decrease of sialic acid in plasma membrane glycoproteins and the expression of cell surface fibronectin were studied during the pre-replicative phase of liver regeneration. The aim of this study was to correlate these cell-surface events to the intracellular surge of calmodulin observed a few hours after partial hepatectomy. The fact that calmodulin decreased the specific activity of UDP-N-acetyl-D-glucosamine 2'-epimerase, a key regulatory enzyme in the biosynthesis of glycoprotein sialic acids, and that trifluoperazine prevented the desialylation indicates that the membrane desialylation is a calmodulin-dependent process. On the other hand, Western blotting using anti-rat fibronectin antibody in trifluoperazine-treated animals suggests that calmodulin may also be involved in the surface expression of fibronectin in regenerating hepatocytes.

Activation of Raf-1 is defective in annexin 6 overexpressing Chinese hamster ovary cells.

Annexin 6 is a Ca2+-dependent phospholipid-binding protein involved in membrane trafficking. In this study we demonstrate the association of Raf-1 with recombinant rat annexin 6. Raf-annexin 6 interaction was shown to be independent of cell activation by epidermal growth factor (EGF) or phorbol esters (12-O-tetradecanoyl-phorbol-13-acetate (TPA)). A stable Chinese hamster ovary (CHO)-anx6 cell line overexpressing annexin 6 was established to examine the function of annexin 6. In these cells, no increase of Ras-GTP levels, induced by EGF or TPA, was detected. In addition, the activity of Raf was completely inhibited, whereas the mitogen-activated protein kinase-P was unaffected.

Early induction of Na(+)-dependent uridine uptake in the regenerating rat liver.

Na(+)-dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham-operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased Vmax of uridine uptake (3.7 vs. 1.4 pmol/mg prot/3 s) without any change in Km (6 microM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na+ gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.

[Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes]. / Localización inmunocitoquímica de la anexina VI en el compartimiento endocítico de hepatocitos de hígado de rata.

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver.

We isolated and characterized a subcellular fraction derived from the blood-sinusoidal plasma membrane of hepatocytes enriched in caveolin and containing several of the molecular components described to be present in caveolae isolated from other cell types. A morphological study by electron microscopy revealed that it was composed of caveolae-attached membrane profiles. Immunoelectron microscopy of isolated fraction showed the specific labeling of internal caveolae membranes with anti-caveolin antibody. Finally, one- and two-dimensional electrophoresis and Western blotting were used for the biochemical analysis of this new rat liver plasma membrane fraction. From the biochemical and the morphological characterization, we conclude that the caveolae-enriched plasma membrane fraction is a plasma membrane fraction, which originates from specialized regions of the sinusoidal plasma membrane, enriched in caveolae.

Membrane transport in rat liver endocytic pathways: preparation, biochemical properties and functional roles of hepatic endosomes.

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol-treated rats, were analyzed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One- and two-dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.