Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

Expression and induction of drug-metabolizing enzymes in cultured fetal rat hepatocytes

Anin vitro experimental model, fetal rat hepatocytes in culture, was metabolically characterized. Several enzymatic activities were expressed in these hepatocytes, namely, testosterone hydroxylations. Hepatocytes cultured up to 3 weeks in the presence of dexamethasone and phenobarbital still expressed some drug-metabolizing enzyme activities (e.g., ECOD). The enzymatic activities were measured both directly on monolayers during culture and on the corresponding harvested and homogenized cells. The results correlate perfectly with each other. The on cell procedure allows us to repeat the assay or to measure several activities on the same cells at different time intervals. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes, namely, those hydroxylating testosterone. This makes the model particularly attractive for induction experiments as well as for metabolic or toxicological studies needing longer treatments.Abbreviations BA benzanthracene - CLO clofibric acid - DEXA dexamethasone - DMSO dimethylsulfoxide - ECOD ethoxycoumarin-O-dethylase - PB phenobarbital - RER rough endoplasmic reticulum     

Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

Sialyltransferase activities in cultured rat hepatocytes

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.     

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0- 10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.     

Lipid synthesis in permeabilized cultured rat hepatocytes

Hepatic lipid synthesis was verified and studied in lysolecithin-permeabilized cultured rat hepatocytes and compared to that of intact liver cells. Triacylglycerol synthesis in permeabilized cells incubated in the presence of glycerol 3-phosphate and long chain fatty acids approached that of intact hepatocytes. Similarly, phosphatidylcholine synthesis in permeable cells incubated in the presence of exogenous CDP-choline was similar to that of intact hepatocytes and at the expense of microsomal neutral lipid synthesis. Phosphatidic acid accumulation in lysolecithin-permeabilized liver cells was remarkably increased as compared to that of intact cells, and its synthesis was mostly accounted for by the activity of mitochondrial glycerol-3-phosphate acyltransferase. Mitochondrial-generated phosphatidate was found to migrate to the endoplasmic reticulum, thus establishing a novel lipid esterification pathway which begins in mitochondrial glycerol 3-phosphate acylation and results in microsomal triacylglycerol and phospholipid synthesis. The free access of permeabilized liver cells to substrates and modulators of lipid synthesis, while maintaining an overall synthetic pattern similar to that of intact hepatocytes, makes them a system of choice for studying hepatic lipid synthesis in general and the microsomal/mitochondrial distribution of fluxes in particular.     

Oxygen uptake rates in cultured rat hepatocytes

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: lack of involvement in cytotoxicity

Galactosamine hepatotoxicity in vivo has long been associated with rapid and extensive depletion of hepatic uridine nucleotides. Depletion of uridine nucleotides is considered to be causal in the toxicity, as evidenced by the protective effect of uridine administration. However, the exact mechanism of galactosamine-induced hepatic necrosis is still unclear. We have previously shown that the addition of galactosamine to rat primary hepatocyte cultures dramatically decreases production of nitric oxide, as measured in the 24 hour culture medium. The present study investigates whether decreased nitric oxide production contributes to the toxicity of galactosamine in primary hepatocyte cultures. Similar concentration-response curves were observed for the decrease in nitric oxide production and galactosamine cytotoxicity, raising the possibility that there is a similar mechanism for these effects. Suppression of NO synthesis was a direct effect of galactosamine, rather than an indirect effect due to loss of cells from the cultures. Both cytotoxicity and the decrease in nitric oxide production were attenuated by coaddition of 3 mM uridine. However, galactosamine cytotoxicity was not enhanced by prior inhibition of hepatocellular NO synthesis nor was it attenuated by maintenance of culture NO levels with molsidomine or diethylamine NONOate. These data do not support a role for decreased hepatocyte nitric oxide production in galactosamine hepatocyte toxicity.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Amiloride blocks cell-free protein synthesis at levels attained inside cultured rat hepatocytes

Amiloride, a passive Na⁺ influx inhibitor, lowers initial rates and plateau levels of [³⁵S]met uptake into proteins in cell-free rabbit reticulocyte lysates (ID₅₀∽0.4 mM). Isolated hepatocytes take up amiloride through a saturable (K_m∽0.02 mM; V_max∽1.43 nmol/ 10⁶ cells/min) Na⁺-dependent process. Similar temperature dependent uptake occurs in cultured hepatocyte monolayers. In chemically defined media, under growth reinitiation conditions, amiloride lowers overall rates of cellular protein and albumin synthesis (ID₅₀∽0.4 and ∽0.028 mM, respectively). Amiloride concentrations (0.02 mM) that half-maximally inhibit reinitiation of hepatocyte DNA synthesis reach, within 30 min, cellular levels (∽0.14 mM) that block reticulocyte lysate protein synthesis by 25%. These findings complicate interpretations, from studies in many eukaryotic systems, of cause and effect between mitogen-activated membrane Na⁺ influxes and the reinitiation of DNA synthesis.     

Early membrane depressions in hepatocytes cultured on Primaria supports

The topography of spreading hepatocytes on positively charged Primaria plates was examined using scanning electron microscopy. The cells acquired a uniform morphology within 2 h. The spreading was rapid and the surface of the cells showed early prominent depressions or dips. The hepatocytes had either one or two of these structures which corresponded to the frequency of mononuclear and binuclear cells, respectively. The nuclear origin of the dips was strengthened after 6 h. They contained solid structures, whose number, size and shape were the same as nucleoli. The membrane dipping was independent of cell density and took place under conditions where phenotypic changes can occur. Kidney proximal tubule cells had no dips. Co-cultures of hepatocytes and kidney proximal tubule cells showed that the cell types behave differently.     

Secretion of alpha-tocopherol from cultured rat hepatocytes

Primary cultures of rat hepatocytes and rat liver perfusions were used to study hepatic secretion of alpha-tocopherol. The secretion of alpha-tocopherol from hepatocytes in culture was linear with time for 4 h. Ultracentrifugation of the medium revealed that 89.4 +/- 2.1% of alpha-tocopherol secreted during 4 h incubation was associated with the very-low density lipoprotein fraction (VLDL, d less than 1.006 g/ml). Oleic acid had no significant effect on the secretory rate of alpha-tocopherol, whereas eicosapentaenoic acid reduced the amount of alpha-tocopherol secreted to 48.4 +/- 12.7% of the control value after 20 h incubation (P less than 0.01). Monensin, a known inhibitor of VLDL secretion, reduced the secretion of alpha-tocopherol to 14.1 +/- 4.3% of the control value (P less than 0.02). Colchicine and chloroquine inhibited the secretion of alpha-tocopherol in the same order of magnitude as monensin. Hepatic perfusion after intravenous injection of in vivo labeled alpha-[3H]tocopherol lymph, showed that about 75% of the secreted radioactivity was in the VLDL fraction. From these results we conclude that most alpha-tocopherol is secreted from the liver associated with nascent VLDL in rats.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Receptor-mediated formation of multilayer aggregates of primary cultured adult rat hepatocytes on lactose-substituted polystyrene.

We used a lactose-substituted polystyrene, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLA-coated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates.