Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies.

Abstract

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-/zm 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 Iocalizations 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.Epithelial cells exhibit a striking polarity that reflects the different functions carried out at the different surfaces of the cell. The assumption that the functional differences among surface domains of such cells are matched by compositional differences has been generally confirmed (see references 8, 32, and 4 l). However, the extent of molecular restriction is still not known, because so few endogenous membrane molecules have been identified and definitively localized in epithelial cells. Furthermore, there is little information on the steadystate intracellular distributions of plasma membrane (PM) ~ molecules in this type of cell or on the cellular mechanisms of their surface expression.1 Abbreviations used in this paper: BC, bile canalicular; LS, lateral; Mabs, monoclonal antibodies; PBS, 0.15 M NaC1, 0.02 M NaP~ (pH 7.4); PBSa, PBS containing 0.02% (wt/vol) NAN3; PFA, paraformaldehyde; PLP, 0.01 M Na-metaperiodate, 0.075 M lysine, 2% (wt/vol) paraformaldehyde; PM, plasma membrane; SF, sinusoidal; WGA, wheat germ agglutinin.The principal epithelial cell of the liver, the hepatocyte, has three functionally and morphologically distinct surface domains. The sinusoidal (SF) domain, specialized for exchange of metabolites with the blood, is characterized by irregular microvilli that extend into the space of Disse and by numerous coated pits. The ...

EfficientIn Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells

“…2E-G), similar to those formed by hepatocytes after several days in primary cultures 27,28 and to the in vivo canalicular structures. 24 Lateral and basolateral markers such as cadherins, HA 321 and CE9, 22 were excluded from membrane of WIF 12-6 tubular and round BC, whereas other apical proteins, such as dipeptidyl peptidase IV and 5Ј-nucleotidase 14,22 and the tight junction-associated protein 578 BRAVO, BENDER, AND CASSIO ZO-1 were found concentrated in these structures or lining them, respectively (results not shown). In view of their hepatic polarized phenotype, the capacity of these four clones to transport CGamF was examined.…”

“…To confirm the membrane polarity of the four cell lines used in this study, we investigated the distribution of an apical plasma membrane protein, HA4. 22 This marker protein was chosen because it has been reported to be a possible canalicular bile acid carrier. 23 As shown in Fig.…”

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex

“…4,5 The function of CEACAM1 is unknown, however, it has been suggested that CEACAM1 may act as a cell adhesion molecule. [6][7][8][9][10] CEACAM1 is highly expressed in apical domains of hepatocytes 11,12 and secreted into bile. Its concentration in hepatic bile was estimated to be about 10 mg/L, 13 whereas serum concentrations were 10 to 20 times lower in healthy subjects 14 ; hence CEACAM1 may be considered to be a bile-specific protein.…”

Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic‐like stem cells

“…45 Liver biliary cells were identified with OC2, OC3, OC4, OC5, OC10, DPP-IV, and OV6 (Hixson) for biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells Walborg et al, 1985;Faris et al, 1991;Gordon et al, 2000). 46 Liver canalicular cells were identified with antibodies H4Ac19 (DSHB), DPP-IV, OV6, and LAP (Hixson) for bile canalicular cells, liver progenitor cells, biliary epithelial cells, and canalicular cell surface protein Hubbard et al, 1985;Walborg et al, 1985;Faris et al, 1991;Gordon et al, 2000). 47 Pancreatic progenitor cells were tentatively identified as three-dimensional structures void of chondrogenic or osteogenic phenotypic markers.…”