Quantitative immunoferritin localization of [Na+,K+]ATPase on canine hepatocyte cell surface

Distribution of [Na+,K+]ATPase on the cell surface of canine hepatocytes was investigated quantitatively by incubating prefixed and dissociated liver cells with ferritin antibody conjugates against canine kidney holo[Na+,K+]ATPase. We found that [Na+,K+]-ATPase exists bilaterally both on the bile canalicular and sinusoid-lateral surfaces. The particle density on the bile canalicular surface was much higher (approximately 2.5 times) than that on the sinusoid-lateral surface. In the latter region, the enzyme was detected almost equally both on the sinusoidal and lateral surfaces. On all the surfaces, the distribution of the enzyme was homogeneous and no clustering of the enzyme was detected. Total number of the enzyme on the sinusoid-lateral surface was, however, approximately three times higher than that on the bile canalicular region, because the sinusoid-lateral surface represents approximately 87% of the total cell surface of a hepatocyte. We suggest that the [Na+, K+]ATPase on the bile canalicular surface is responsible for the bile acid-independent bile flow and the other transport processes on the bile canalicular cell surface, while that on the sinusoid-lateral surface is responsible not only for the active transport of Na+ but also for the secondary active transport of various substances in this region.     

Distribution of an asialoglycoprotein receptor on rat hepatocyte cell surface

Direct ferritin immunoelectron microscopy was applied to visualize the distribution of the hepatocyte cell surface of the asialoglycoprotein receptor which is responsible for the rapid clearance of serum glycoproteins and lysosomal catabolism. For this purpose, rabbit antibody against the purified hepatic binding protein specific for asialoglycoproteins was prepared and coupled to ferritin by glutaraldehyde. The specific antibody conjugates were incubated with the hepatocytes, which were isolated from rat liver homogenate after fixation by glutaraldehyde perfusion. These cells preserved well the original polygonal shape and polarity, and it was easy to identify the sinusoidal, lateral, and bile canalicular faces. The surface density of the ferritin particles bound to the sinusoidal face was about four times higher than that of particles bound to the lateral face, while the bile canalicular face was hardly labeled and almost at the control level. Using the surface area of hepatocyte measured by morphometrical analyses, it was estimated that approximately 90% of bound ferritin particles were at the sinusoidal face, approximately 10% at the lateral face, and approximately 1% at the bile canalicular face. Nonhepatic cells such as endothelial and Kupffer cells had no receptor specific for asialoglycoproteins.     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

Preservation of hepatocyte plasma membrane domains during cell division in situ in regenerating rat liver

We have utilized antibodies against five domain-specific integral proteins of the rat hepatocyte plasma membrane to examine the fates of the plasma membrane domains during hepatocyte division in the regenerating rat liver. The proteins were quantified on immunoblots of liver homogenates prepared during the peak of hepatocyte mitotic activity, 28-30 hr after two-thirds hepatectomy. Two sinusoidal/lateral proteins, CE 9 and the asialoglycoprotein receptor, and one bile canalicular protein, dipeptidylpeptidase IV, were not changed significantly in amount; whereas one sinusoidal/lateral protein, the epidermal growth factor receptor, and one bile canalicular protein, HA 4, were reduced to less than or equal to 50% of control levels. Light microscopic examination of plastic sections of regenerating liver tissue revealed that the mitotic hepatocytes generally appeared to retain normal contacts with neighboring interphase hepatocytes. Immunofluorescence was used to localize the domain-specific proteins on mitotic hepatocytes identified in 0.5-micron frozen sections of 28- to 30-hr regenerating liver tissue. Independent of mitotic stage, the hepatocytes retained mutually exclusive bile canalicular and sinusoidal/lateral domains, as defined at the molecular level by the distributions of specific proteins, such as HA 4 and CE 9, respectively.     

A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes

Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper).     

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.     

Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes

The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane- intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma; there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes.     

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.     

Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.     

Cell surface expression by adult rat hepatocytes of a non-collagen glycoprotein present in rat liver biomatrix

A rabbit antiserum raised against ACI rat liver biomatrix was used to identify components common to biomatrix and plasma membranes of adult hepatocytes. Biomatrix was isolated from intact rat livers by reverse perfusion via the inferior vena cava with sodium deoxycholate, nucleases and lipid extracting solvents. Immunoprecipitation analysis of detergent extracts of hepatocytes surface-labeled with 125I indicated that antibodies, purified from anti- biomatrix antiserum by adsorption and desorption from intact hepatocytes, showed reactivity with a single MW 105 kD component, designated Hep 105. Indirect immunofluorescence analysis showed that Hep 105 was expressed in some regions of the perisinusoidal space and in all three domains of the hepatocyte plasma membrane and was present on some but not all of the fibrous elements in frozen sections of biomatrix . The presence of Hep 105 on biomatrix was confirmed by immunoprecipitation analysis which showed that Hep 105 was present in components solubilized from biomatrix by sequential treatment with 0.5 M acetic acid, 0.05% collagenase and 4 M urea. Further characterization using immunoprecipitation analysis in combination with immobilized lectins and two-dimensional polyacrylamide gel electrophoresis (PAGE) indicated that Hep 105 was a non-collagen glycoprotein which showed charge heterogeneity and existed on the cell surface as a disulfide-linked heterodimer of apparent MW 125 kD. Two hybridomas, constructed by fusing P3 X 63Ag8 myeloma cells with spleen cells from mice immunized with intact hepatocytes, were shown by immunodepletion and two-dimensional gel electrophoretic analysis to be secreting monoclonal antibodies (Mab) against Hep 105. Examination of frozen sections of rat liver stained by indirect immunofluorescence showed that reactivity of both Mabs was concentrated in the bile canalicular domain of the hepatocyte plasma membrane, suggesting that the reactive epitopes were not accessible in the sinusoidal and intercellular membrane domains. Taken together, these results suggest that Hep 105 may play a role in the interactions between hepatocytes and extracellular matrix.     

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment.

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.     

Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.     

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive subcanalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per micron of plasma membrane were 7.86 vs 2.46 (P less than 0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P less than 0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.     

A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain

Indirect immunofluorescence was used to establish a domain-specific marker for hepatocyte plasma membranes. In frozen sections of fixed rat liver (0.5-4 microns), antibodies directed against rat intestinal leucine aminopeptidase (LAP) recognized an antigen that was restricted to the bile canalicular plasma membrane. Fluorescence was not observed on the sinusoidal or lateral membranes, and intracellular staining was not detected. The liver antigen was identified as LAP, based on its chemical similarity to intestinal LAP. First, immunoprecipitation experiments using trypsin-solubilized intestinal LAP (G-200 fraction, 91% pure) established a correlation between the loss of LAP enzyme activity from the soluble fraction and the appearance in the specific immunoprecipitates of polypeptides migrating on SDS PAGE between 110,000 and 130,000 daltons. The antigen precipitated from a detergent extract of liver plasma membranes had the same electrophoretic mobility. Second, the chymotryptic map of the major band in the liver immunoprecipitate was similar to that of purified intestinal LAP.     

Cell surface expression and bile acid transport function of one topological form of m-epoxide hydrolase

The bifunctional hepatic protein, microsomal epoxide hydrolase (mEH), plays a central role in the metabolism of many xenobiotics as well as mediating the Na(+)-dependent uptake of bile acids in parallel with the Na(+)-taurocholate co-transporting protein (ntcp). Previous studies have established that mEH is expressed in the endoplasmic reticulum with two topological orientations, where the type II form is targeted to the plasma membrane. In this report the topology and transport properties of mEH as a function of plasma membrane expression in cultured hepatocytes, transfected Madin-Darby canine kidney cells expressing mEH (MDCK[mEH]), and the human hepatoma cell line, HepG2, were studied using confocal fluorescence microscopy and substrate uptake measurements. Analysis of mEH localization with an anti-mEH monoclonal antibody demonstrated the expression of one topological form on the plasma membrane of hepatocytes and MDCK[mEH] cells where both systems exhibited Na(+)-dependent bile acid uptake. In contrast, Na(+)-dependent bile acid transport in HepG2 cells and hepatocytes in culture (72 h) was substantially reduced as was the expression of ntcp. Although the total mEH level was undiminished, the decrease of bile acid transport was associated with the loss of mEH surface expression possibly resulting from an alteration in mEH endoplasmic reticulum topology and/or the plasma membrane protein targeting system in these de-differentiated cells.     

Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical 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.     

High density lipoprotein (HDL) particle uptake mediated by scavenger receptor class B type 1 results in selective sorting of HDL cholesterol from protein and polarized cholesterol secretion

High density lipoprotein (HDL) mediates reverse transport of cholesterol from atheroma foam cells to the liver, but the mechanisms of hepatic uptake and trafficking of HDL particles are poorly understood. In contrast to its accepted role as a cell surface receptor, scavenger receptor class B type 1 (SR-BI) is shown to be an endocytic receptor that mediates HDL particle uptake and recycling, but not degradation, in both transfected Chinese hamster ovary cells and hepatocytes. Confocal microscopy of polarized primary hepatocytes shows that HDL particles enter both the endocytic recycling compartment and the apical canalicular region paralleling the movement of SR-BI. In polarized epithelial cells (Madin-Darby canine kidney) expressing SR-BI, HDL protein and cholesterol undergo selective sorting with recycling of HDL protein from the basolateral membrane and secretion of HDL-derived cholesterol through the apical membrane. Thus, HDL particles, internalized via SR-BI, undergo a novel process of selective transcytosis, leading to polarized cholesterol transport. A distinct process not mediated by SR-BI is involved in uptake and degradation of apoE-free HDL in hepatocytes.