Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies

The hepatocyte plasma membrane consists of three morphologically and functionally distinct domains, the sinusoidal, the lateral and the canalicular. To study the distribution of antigenic determinants among these domains, we prepared monoclonal antibodies by immunizing mice with a crude, plasma membrane-enriched liver fraction. Four monoclonal antibodies were obtained that recognized various parts of the rat hepatocyte plasma membrane when tested by indirect immunofluorescence and immunoperoxidase assay performed on formaldehyde-fixed liver tissue. Each antibody gave a different staining pattern when analyzed by light and electron microscopy. A59 exclusively labelled the part of the sinusoidal membrane facing the sinusoids. A39 mainly labelled the sinusoidal membrane. B1 mainly labelled the lateral membrane, while the labelling by B10 was almost completely limited to the canalicular membrane. Immunoblotting showed that the antibody B1 recognized an antigen of approximately 100 kilodaltons and that B10 recognized an antigen of approximately 125 to 130 kilodaltons. These antibodies allow us to distinguish the three domains of the hepatocyte plasma membrane.     

Analysis of hepatocyte plasma membrane domains during rat development using monoclonal antibodies

The plasma membrane of adult rat hepatocyte consists of three domains, which have been identified by the monoclonal antibodies A39 and A59 as markers of the sinusoidal domain, B1 of the lateral, and B10 of the canalicular domains (Eur J Cell Biol 39:122, 1985). These monoclonal antibodies were used to study, by indirect immunocytochemistry, formation of the hepatocyte plasma membrane domains during development, from day 15 of gestation to day 35 post partum. The antigens defined by A39, B1, and B10 were detected, from day 15, over the major part of the hepatocyte plasma membrane except for the membranes of newly formed bile canaliculi, which were not labeled by B1 and only poorly labeled, if at all, by A39 and B10. As soon as fetuses were 16 days old, B1 labeled predominantly the lateral domain, as in the adult. Labeling with B10 progressively intensified on the membranes of bile canaliculi, but localization was not exclusively canalicular until day 21 post partum. A39 intensely labeled the canalicular membranes at 19-21 days of gestation, while at 35 days post partum it exhibited the predominantly sinusoidal labeling observed in adult hepatocytes. The antigen defined by A59 was not detected before birth and was found exclusively on the sinusoidal domain, as in the adult. These results show that the patterns of antigen distribution on different plasma membrane domains establish themselves at different rates. The marked differences observed between fetal or neonatal and adult hepatocytes might be responsible for immaturity of liver functions in the neonate.     

Hepatocyte polarity and the peroxisomal compartment: a comparative study

In search of factors that regulate the phenotype of the peroxisomal compartment in wild-type liver parenchymal cells, we compared hepatocyte polarity to peroxisome differentiation, using adult liver as the standard. Differentiation parameters were evaluated in a three-dimensional culture model (spheroid), in 'sandwich' and monolayer primary hepatocyte cultures, and in 15.5 and 18.5-day-old foetal rat liver.Peroxisomes, studied by immunohistochemistry, enzyme histochemistry, and catalase specific activity, were better differentiated depending on foetal age (day 18.5 > day 15.5) and culture type (spheroid > sandwich > monolayer). The hepatocyte polarity markers ATP-, ADP-, and AMP-hydrolysing activities were, in all models, mislocalized at the lateral plasma membrane, whereas in contrast the multidrug resistance-associated protein 2 (mrp2) antigen was always correctly immunolocalized at the apical membrane domain. In cultures, the correct secretion of fluorescein (mrp2-mediated) into bile canaliculi was observed. Bile canaliculi (branching, ultrastructure and immunolocalization of the tight-junction associated protein ZO-1), were better differentiated in 18.5 than in 15.5-day-old foetal liver and in spheroid > sandwich > monolayer cultures.Our results show a parallelism between changes of the peroxisomal compartment and bile canalicular structure together with mrp2-mediated secretory function. Distinct polarization characteristics do not necessarily change simultaneously, suggesting different regulatory mechanisms.     

Analysis by flow cytometry of rat hepatocytes from different acinar zones

Many functional, morphological and biochemical differences among hepatocytes from different acinar zones have been described. Therefore, it will facilitate studies on liver metabolism rapid, non-destructive procedures to isolate hepatocytes from these zones. Flow cytometry is a new powerful tool which, however, has not been used thus far to accomplish the separation of hepatocytes from different acinar zones. We describe here various cytometric parameters which characterize hepatocyte populations, separated by isopycnic centrifugation in Percoll gradients. The intraacinar origin of the different hepatocytes was assessed by enzymatic and morphological measurements.     

Impact of neonatal cryptosporidial gastroenteritis on epigenetic programming of rat hepatocytes

Inflammation, malnutrition and growth retardation during critical time-windows of development play a powerful role in ontogenetic programming of the life-long risk to many adult diseases (including metabolic syndrome, obesity and diabetes). Cellular mechanisms and the accurate timing and duration of critical periods for the liver remain obscure. To resolve this problem, we developed a postnatal suckling-weanling rat model of mild, moderate, and acute gastroenteritis challenged by a protozoan parasitic spread throughout the whole world, namely Cryptosporidium parvum. The physiological state of the liver was evaluated by hepatocyte ploidy and protein content that were measured by cytophotometry and image analysis on isolated cells. Hepatocyte ploidy is known to irreversibly increase after stress and is associated with the decrease in liver physiological capacity. Hepatocyte hypertrophy reflects cell functional loading. From our results, cryptosporidiosis is able to provoke a burst in premature hepatocyte polyploidization and hypertrophy (in proportion to parasitic load), and thus plays an important role in epigenetic programming of hepatocyte structure and function. We revealed two sensitive periods in liver growth. The first period (the less sensitive) covers the time before the establishment of homoiothermy, i.e. 6-9 days after birth. The second period (the more sensitive) covers the time of weaning when the change of type of nutrition and the peak of hepatocyte polyploidization and differentiation occurs. Thus, our data provide direct evidence that phenomenon of ontogenetic programming is reflected at the cellular level.     

Etude de l'evolution des activites des enzymes mitochondriaux de l'hepatocyte au cours du developpement du rat

Study on the evolution of mitochondrial enzyme activities in hepatocyte during rat development

Some constitutive enzymes of the three isolated fractions of mitochondria outer membrane, inner membrane and matrix, have been investigated in rat hepatocyte during a period varying from the foetal state to the 15th day after birth.

In the three mitochondrial fractions, activities of the studied enzymes present different evolutions. In the matrix, the tricarboxylic enzyme activities have already reached their normal values before birth. In the outer membrane, the NADH-cytochrome c reductase activity increases regularly, in the same way as that of the endoplasmic reticulum NADH-cytochrome c reductase. In the inner membrane, the oxygen consumption is very low before birth, then increases suddenly from the 5th to the 8th day after birth, when it reaches the normal values. The limiting factor of the respiratory chain activities is neither cytochrome oxidase nor the first dehydrogenases.     

Expression of rat hepatocyte plasma membrane antigens in hybrid clones: assignment of genes coding for two antigens of the basolateral domain to chromosomes 11 and 13

Expression of three rat hepatocyte plasma membrane antigens defined by monoclonal antibodies (mAbs) was examined by immunofluorescence in mouse hepatoma x rat hepatocyte hybrid clones segregating rat chromosomes. The antigen defined by mAb B1, a marker of the lateral domain of the hepatocyte plasma membrane in vivo, was expressed in hybrids retaining the rat chromosome 11. The antigen defined by mAb A39, mainly located on the sinusoidal (basal) domain of the plasma membrane in vivo, was expressed when chromosome 13 was present. The genes coding for these two antigens were thus assigned to chromosomes 11 and 13, respectively. The antigen defined by mAb B10, exclusively located on the canalicular (apical) domain of the plasma membrane in vivo, was not expressed in most hybrid clones, and the chromosome location of the gene could not be determined.     

Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.     

QSG‐7701 human hepatocytes form polarized acini in three‐dimensional culture

Hepatocytes are polarized and fulfill a variety of liver‐specific functions in vivo; but the polarized tissue structure and many of these functions are lost when the cells are cultured on plastic. To recapitulate the polarized structure and tissue‐specific function of liver cells in culture, we established a three‐dimensional (3D) culture assay with the human hepatocyte line QSG‐7701. In 3D Matrigel culture, QSG‐7701 cells formed polarized spheroids with a center lumen, which is reminiscent of bile canaliculi in the liver. Immunofluoresence analysis showed that F‐actin bundles and radixin were mainly located at the apical membrane and that α6 and β1 integrins were localized basally in 3D culture. Lumen formation was associated with the selective apoptosis of centrally located cells and was accompanied by proliferative suppression during acinar development. Compared to QSG‐7701 cells in 2D or agarose gel cultures, the cells in 3D Matrigel culture maintained a given direction of biliary excretion and acquired higher levels of cytochrome P450 and albumin expression. Our study shows that the immortal human hepatocytes, QSG‐7701, in 3D Matrigel culture reacquire cardinal features of glandular epithelium in vivo, providing an ex vivo model to study liver‐specific function and tumorigenesis. J. Cell. Biochem. 110: 1175–1186, 2010. Published 2010 Wiley‐Liss, Inc.     

The ultrastructural localization of antigen B10 of the hepatocyte plasma membrane in mouse hepatomas

The ultrastructure of the murine hepatocyte plasma membrane antigen (Ag B10) was studied by immunoelectron microscopy in 5 spontaneous and 3 chemical-induced hepatomas. Ag B10 was associated with plasmalemma of bile canaliculi and membrane of microvilli as in normal liver. Sometimes it was connected with plasmalemma of lateral domain of tumor cells. The availability of Ag B10 in the matrix of bile canaliculi and within microvilli was shown.     

The establishment of hepatocyte cell surface polarity during fetal liver development

Antibodies to six glycoproteins present in different domains of the hepatocyte plasma membrane were used to study the establishment of cell surface polarity during rat fetal liver development. The proteins were immunoprecipitated from fetal liver homogenates between 14 and 21 days of gestation and quantified by immunoblotting. Aminopeptidase N, CE 9, and HA 321, which reside in the apical, basolateral, and lateral plasma membrane in the adult hepatocyte, respectively, were present in high concentrations at 14 days of gestation and remained high until birth. In contrast, two apical proteins (HA 4 and dipeptidyl peptidase IV) and two basolateral proteins (ASGP receptor and EGF receptor) were first detected between 16 and 18 days of gestation and increased linearly until birth. HA 4 was the only molecule for which the fetal and adult forms differed, with the former having a faster mobility on SDS-PAGE, due to differences in N-linked oligosaccharides. With two exceptions, the localization of the molecules from earliest detection was restricted to the same domain as that in the adult. At 15 days of gestation, HA 321 and a small portion of aminopeptidase were detected on the basolateral membrane. By 21 days both molecules had assumed their adult localization pattern. Our results indicate that the biogenesis of cell surface polarity is an early event, implying that the mechanisms for sorting plasma membrane molecules are functional very early in development. Furthermore, the different patterns of appearance of the six molecules, irrespective of domain, indicate that the biochemical composition of the cell surface changes dramatically during fetal liver development.     

Dynamics of structural changes in rat liver after single dose of gamma-irradiation

Histological changes and alterations in biophysical and biochemical parameters in liver of gamma-irradiate rats have been investigated. The gamma-irradiation of the whole body of rats with a single dose of 1 Gy did not cause any impairments of beam structure of rat liver, but resulted in the lymphocytic infiltrations of portal tracts which were not accompanied by formation of spotty areas of necrosis in adjacent areas of lever parenchyma. gamma-Irradiation stimulated proliferation of the hepatocytes and induced time-dependent mitochondrial structure lesions. Post-irradiation changes in cell cytoplasm appeared as disordering in reticulum-endothelial system, among them enlarging and fragmentation of its cisterns, cytoplasmic vacuolization, enhancement of the number of lysosomes and of the lipid inclusion contents. These facts revealed the mobilization of the additional energy resources for recovery of metabolic processes in rat liver. Post-irradiation increase of the level of the hepatocyte membrane lipid peroxidation products preceded liver morphological alterations. The membrane lipid microviscosity decreased in 1 and 3 days after irradiation. As a result of damages of hepatocyte membrane, the activity of the alanin- and asparagin-aminotransferases in blood serum increased 6 hours after. We can conclude that the whole body single gamma-irradiation with a dose of 1 Gy leads to the reversible but significant damages to the rat liver cell membrane structures. These damages might be the reason of radiation-induced liver morphological alterations.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.     

Human hepatocytes in mice receiving pre-immune injection with human cord blood cells

It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34(+) (hCBCD34(+)) cells during pre-immune stages of development in immunocompetent mice might also lead to human-mouse liver chimerism. Freshly isolated hCBCD34(+) cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human alpha-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34(+) cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Effects of keratin filament disruption on exocrine pancreas-stimulated secretion and susceptibility to injury

Disruption or absence of hepatocyte keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic keratin disruption in transgenic mice that express keratin 18 Arg89 --> Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type keratin 18 and harbor normal extended keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic keratin filament disruption, the apicolateral keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their (125)I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical keratins may function differently in different tissues.     

Liver scarring induced by polychlorinated biphenyl administration to mice previously treated with diethylnitrosamine

Exposure of mice for 8 weeks to drinking water containing diethylnitrosamine (DEN) was accompanied by alterations in hepatocyte structure and varying degrees of liver nonparenchymal cell (NPC) proliferation. Eighteen and a half weeks after cessation of DEN exposure, there was a 47% incidence of hepatocellular nodules. Centrilobular hepatocyte hypertrophy occurred consistently in mice given intraperitoneal injections of the polychlorinated biphenyl (PCB) mixture Aroclor 1254. PCB administration to mice previously treated with DEN was not accompanied by increases in gross liver nodule incidence above that induced by DEN, but many more developing microscopic nodules within the liver were observed in DEN-treated mice given Aroclor 1254 than in mice treated only with DEN. Aroclor 1254 administration over a 16-week period to mice previously treated with DEN was accompanied by an 83% incidence of severe distortion of liver structure resulting from nodule formation, uneven patterns of hepatocyte growth, and extensive deposition of scar tissue containing proliferating bile ducts. Morphological evidence of intestinal metaplasia was observed in proliferating bile duct-like structures during an early stage of liver adenofibrosis.     

Effect of narrow fractions of chromatin non-histone proteins on the expression of membrane tumor-associated antigen MA-50 and phosphorylation of proteins of cultured rat hepatocytes

As earlier reported, the main component of narrow fractions of chromosomal non-histone proteins (NHP) of kidney and of Zaidel hepatoma cells has its own protein kinase activity, and is identified as a heteroorgan NHP-antigen, which is intrinsic to the definite renal tissue and absent in the liver. Effects of narrow fractions of kidney and Zaidel hepatoma NHP on biosynthetic processes and sizes of hepatocytes were studied in vitro. It has been shown that as a result of a 5 h incubation of rat hepatocytes with a narrow fraction of renal NHP the proportion of small hepatocytes increases approximately by 12% as compared with that of cells cultivated without NHP. Besides, binding of organ-specific anti-kidney immune serum with a small hepatocyte population rises by more than 20%, which results from the expression of tumor-associated heteroorgan kidney-specific antigen on the hepatocyte surface. According to immunoprecipitation and subsequent electrophoresis, the molecular mass of a membrane heteroorgan antigen on the surface of hepatocytes amounts approximately to 65 kDa, and an active phosphorylation of cellular proteins takes place. The same effect on hepatocytes is produced by a narrow NHP fraction of chromatin of Zaidel hepatoma cells, whereas no phosphorylation is observed in the presence of liver NHP as well as in the absence of NHP. It is suggested that the heteroorgan NHP-antigen induces biosynthetic processes including synthesis of membrane tumorassociated antigen on the surface of hepatocytes cultivated in vitro by activation of cellular protein phosphorylation, which can lead to changes in size of cultivated cells.