No abstract

The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (Wi) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.Past efforts to produce long-term proliferation of adult hepatocytes in culture have met with limited success (1-8 Substrata. Cells were plated on one of the following substrata: (i) Tissue culture plastic. Sixty-millimeter tissue culture dishes (Falcon). (ii) Type I collagen gels. Sixty-millimeter tissue culture dishes (Falcon) were coated with type I collagen gels prepared from rat tail tendons (10). (ifi) Normal rat liver biomatrix. Livers from normal Sprague-Dawley rats were utilized to prepare biomatrix by the methods of Reid (12). The biomatrix was pulverized into a fine powder with a Spex-Mill liquid nitrogen pulverizer (Spex-Mills, Metuchen, NJ), thickly smeared onto 60-mm Petri dishes (Falcon) and then sterilized by 'y irradiation (cesium-135) at 10,000 rads (100 grays). Just before use, the plates were rinsed with serum-free RPMI 1640 medium supplemented with penicillin at 200 units/ml and streptomycin at 200 pug/ml. (iv) Rat regenerating liver biomatrix. Five days after partial hepatectomy (18), livers were used for biomatrix preparation (12). (v) Normal rat liver biomatrix prepared by low-salt extraction. Biomatrix from normal rat liver was prepared by extracting the tissue with 1.0 M NaCl and omitting the initial wash in distilled water, prepared as described (11).Morphological Studies. Cultures of hepatocytes were plated under various conditions and maintained for 1-2 weeks. They were examined by using a Nikon inverted phase-contrast microscope. Cultures were evaluated by a number of investigators independently. Representative cultures were selected and photographed with phase-contrast microscopy.[3H]Thymidine Incorporation. At 1411The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody pioduced against ligandin, bear entirely sinlilar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was showh to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.ligandin is a cytoplasmic protein found in abundance in the liver of rat, man, and other species. This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1, 2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3-7). Fractionation of the protein on the basis of any one of its binding activities has resulted in apparently identical, highly purified preparations (7-9); Thus, the term, ligandin (2), is synonymous with that of azo-dye carcinogenbinding protein (8), corticosteroid binding I protein (9), and Y protein (7)$, §.The glutathione S-transferases (EC 2.5.1.18) from rat liver (iO-i3) have sevieral physical properties in common with rat liver ligandin (2). When crude liver extracts were subjected to filtration on Sephadex G-75, the fraction containing ligandin also served as a source of enzymatic activity for the conjugation of glutathione (GSH) with 1,2-dichloro-4-nitrobenzene (14). However, subsequent fractionation resulted in removal of alriost all GSH transferase activity with this substrate despite virtually complete recovery of ligandin (t, 15).Since four of the GSH transferases of rat liver (transferases A, B; C, and E) have been purified to homogeneity (11-13), (w/v) and the precipitate was removed by centrifugation after an additional 4 hr at 4°. The superratant fluid was used directly for enzyme assays with 1,2-ehloro-4-nitrobenzene. The precipitate was washed twice with phosphate-buffered saline at pH 7.4, containing 2%o polyethylene glycol. The residue was dissolved in 0.5 M NaOH, and absorbance at 280 nm was determined.

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance) . Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans) . Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, longterm survival (months), and retention of some hepatocyte-specific functions.Despite numerous advances in cell culture procedures (2, 3,14,16,18,24,35), the culture of differentiated epithelial cells, particularly from normal tissues, has remained especially difficult. We have proposed that the shortcomings of cell culture techniques are primarily that cells are isolated from the extracellular matrix and from association with other cell types with which they may be interdependent (30). Culture methods, such as organ culture or the culture oftissue fragments, which retain tissue architecture, preserve the differentiative state of the cells, whereas clonal cell cultures usually undergo a dedifferentiative process (16,40). Thus, to culture differentiated cells it seems logical to ascertain the critical variables of the tissue matrix and to evolve culture procedures dependent upon them.In a previous paper, we presented techniques that we refer to as "socio-cell culture techniques" (30) and that are, in essence, attempts to simulate some of the cell-cell relationships of the tissue matrix relevant to epithelial cells . The techniques we described involve the culturing of epithelial cells on substrates of reconstituted basement membrane and in medium THE JOURNAL OF CELL BIOLOGY " VOLUME 87 OCTOBER 1980 255-263 © The Rockefeller University Press -0021-9525/80/10/0255/09 $1 .00 supplemented with hormones, serum, and with conditioned medium from feeder layers of primary cultures of fibroblasts . In this article, we present new techniques that have superseded the earlier ones. They include the isolation of connective tissue fibers called biomatrix, representing a significant portion ofthe in vivo extracellular matrix (basement membranes and ground substance), and techniques using these fibers as substrates for cultures of differentiated cells . Our lon...

Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking.We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATPdependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions.Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs.These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.Bile acids are the predominant organic solutes in bile. Only 5% of the total bile acid pool is produced daily as a result of 7 ␣ hydroxylation of cholesterol in the liver; the remaining 95% of bile acids undergoes enterohepatic circulation and is transferred into the bile from plasma by mechanisms that are only partially understood (1, 2). Three bile acid transporters have been identified in the basolateral plasma membrane of hepatocytes, but none are known to associate with vesicular trafficking of bile acids to the canaliculus (3-5). Although bile acids bind with varied affinity to several hepatocellular cytoplasmic proteins (6), the role of protein binding in intracellular movement is uncertain. Because administration of microtubular inhibitors, such as colchicine, reduces bile acid secretion but not uptake from plasma, a microtubule-based vesicular mechanism of transcellular bile acid transport has been proposed (7,8).In recent years, several ATP-dependent transporters have been functionally identified in CMVs 1 for bile acids, organic cations, phosphatidylcholine, and nonbile acid organic anions (9 -13). Cloning identified the respons...

The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. Taurocholate is the major bile acid in humans and rats. Its uptake from plasma by hepatocytes utilizes a Na' cotransport system that requires Na',K+-ATPase for generation of a Na' gradient (1, 2). Two pathways for intracellular bile acid transport have been demonstrated. One involves microtubule-dependent vesicular transport from the Golgi apparatus to the bile canaliculus (3, 4). The other is the major physiologic pathway and involves direct transport to the canaliculus, possibly by cytosolic binding proteins (5). The secretion of bile acids into the canaliculus is a major determinant ofbile flow. Although the contents ofthe bile canaliculus have never been measured due to its inaccessibility, studies ofductal bile suggest that there is a large bile acid concentration gradient between the hepatocyte and canalicular contents (6, 7). Previous studies using vesicles selectively derived from the canalicular domain of hepatocyte plasma membrane revealed carrier-mediated transport of taurocholate and other bile acids that is driven by an outside-positive membrane potential (8, 9). However, the membrane potential is insufficient to explain the concentration gradients of bile acids that are postulated to occur across the canalicular membrane (6).We have described (10, 11) two distinct ATP-dependent transport systems in the canalicular membrane. One system utilizes P-glycoprotein, the product of the multidrugresistance gene, and transports mainly hydrophobic cations, such as daunomycin (10). The carrier protein for the second system has not been purified but its substrates are nonbile acid organic anions, such as bilirubin diglucuronide, oxidized glutathione (GSSG), and glutathione S-conjugates (11-13).The latter transport system is defective in TR-mutant rats, which have a phenotype that resembles the defect in patients with Dubin-Johnson syndrome (11,14). While studying these transport processes, an ATP-dependent transport of bile acids in canalicular membrane vesicles (CMVs) was detected and has been now characterized. (GSBSP) and dinitrophenyl glutathione (GSDNP) were synthesized nonenzymatically (16,17). Rabbit polyclonal antibody against the extracellular domain of -glutamyltransferase (y-GT) was kindly provided by Masayasu Inoue (Kumamoto University Medical School, Kumamoto, Japan). Bilirubin diglucuronide, which was purified by HPLC (18), was kindly provided by N. Roy-Cho...

The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.

Multiple-indicator dilution studies of labeled bilirubin uptake were carried out on isolated perfused rat livers with variable ligandin concentrations (from normal and thyroidectomized animals with and without phenobarbital pretreatment). Ligandin concentrations, measured immunologically, increased 25% after thyroidectomy and approximately doubled after phenobarbital pretreatment but decreased to normal during perfusion in the thyroidectomized nonpretreated group. A distributed two-compartment model was fitted to the dilution data and estimates of influx, efflux, and sequestration coefficients were obtained. Influx and sequestration coefficients did not vary significantly between the groups. Efflux coefficients were significantly smaller (P less than 0.001), and hepatic ligandin concentrations were significantly larger (p less than 0.001) in phenobarbital-treated rats than in other groups. The efflux coefficient varied inversely with ligandin concentration and the volume of distribution in tissue, as perceived from the plasma space, increased in proportion to the concentration of ligandin. The increased net uptake of tracer bilirubin by the liver of phenobarbital-pretreated animals is due to decreased tracer efflux secondary to the increase in intracellular binding of bilirubin by ligandin.