Cultures Of Liver Parenchymal Cells Research Articles

Differential regulation of multidrug resistance-associated protein 2 (MRP2) and cytochromes P450 2B1/2 and 3A1/2 in phenobarbital-treated hepatocytes

Multidrug resistance-associated protein 2 (MRP2) is a drug efflux pump found at the biliary pole of hepatocytes. In the present study, we have investigated its expression in response to phenobarbital, a liver tumor promoter known to up-regulate hepatic cytochromes P450 (CYPs), such as CYP2B1/2 and CYP3A1/2. MRP2 mRNA and protein levels were found to be markedly increased in both primary rat and human hepatocytes exposed to phenobarbital. However, features of this up-regulation, especially the dose-response, were different from those of the induction of CYP2B1/2 and CYP3A1/2. In addition, hepatic MRP2 expression remained unaltered in rats treated by phenobarbital that, by contrast, increased CYP2B1/2 and CYP3A1/2 gene expression in the liver. Therefore, MRP2 and CYPs appeared differently regulated in response to phenobarbital in both in vivo and in vitro situations, suggesting that cellular and molecular mechanisms underlying up-regulation of MRP2 are, at least in part, unrelated to those operating for CYPs. Phenobarbital-related MRP2 induction in primary rat hepatocytes was associated with some phenotypic effects of the barbiturate, such as prolonged cell survival and inhibition of cell proliferation. Phenobarbital also inhibited growth of human hepatoma HepG 2 cells and increased their level of MRP2 gene expression. Such results may favor a putative relationship between phenobarbital-mediated MRP2 regulation in cultured liver parenchymal cells and alteration of cell cycle and survival.

Betaine as an osmolyte in rat liver: metabolism and cell-to-cell interactions.

Betaine was recently identified as an osmolyte in rat liver macrophages (Kupffer cells [KCs]) and sinusoidal endothelial cells (SECs). Betaine interferes with KC functions, such as phagocytosis, cytokine, and prostaglandin syntheses. As betaine is derived from choline, the present study was undertaken to evaluate osmosensitivity and cell heterogeneity of choline metabolism in rat liver. In the perfused rat liver after in vivo prelabeling with [14C]-choline, hypoosmotic stress induced a radioactivity release into the perfusate which was identified as [14C]-betaine by high-performance liquid chromatography (HPLC) analysis and which was inhibited by the anion exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Choline metabolism was studied in cultured liver parenchymal cells, (PCs), KCs, and SECs. Choline was taken up by all but betaine formation from choline was only detectable in PCs and not in KCs and SECs. Betaine formation in PCs was not stimulated by hyperosmolarity; rather, betaine has a role as an osmolyte in KCs and SECs but is of minor importance in PCs, as evidenced by only minor hyperosmolarity-induced betaine uptake. Thus, liver PCs can produce and release betaine derived from choline, and, thereby, possibly supply the osmolyte important for KC and SEC cell function. This may be another example for cell-to-cell interaction in the liver.

Deficiency of bile acid transport and synthesis in oval cells from carcinogen-fed rats

Freshly isolated oval cells, which we obtained from the livers of rats fed a choline-deficient/DL-ethionine-supplemented diet, did not transport bile acids. Compared with freshly isolated rat hepatocytes they took up only negligible amounts of [3H]taurocholate or [14C]cholate. The cells bound small amounts of radioactive bile acids. This portion of the total cell-associated radioactivity was enhanced on membrane permeabilization. In contrast to cultured liver parenchymal cells from untreated rats, no bile acid synthesis was detected in cultured oval cells. Cultured oval cells also lost the ability to conjugate exogenously added cholate (100 mumol/L) with taurine or glycine. However, when liver parenchymal cells were isolated from carcinogen-fed rats, bile acid uptake was diminished compared with that in hepatocytes from control animals. In particular, the maximum values of taurocholate and cholate uptake were decreased by 75% and 50%, respectively, whereas the Michaelis-Menten constant values were not altered. The study demonstrates that (a) oval cells lack typical liver parenchymal cell-specific properties such as bile acid uptake, bile acid synthesis and conjugation of bile acids with taurine/glycine and therefore do not contribute to bile acid dependent bile formation (b) proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet are part of the bile duct epithelial cell compartment); and (c) bile acid uptake is reduced in liver parenchymal cells of rats fed a choline deficient/DL-ethionine-supplemented diet, and this effect is due to a decrease in transport capacity without a decrease in transport affinity.

Copper Uptake and Retention in Liver Parenchymal Cells Isolated from Nutritionally Copper-Deficient Rats

Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (less than 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver, anemia, low plasma ceruloplasmin oxidase activity and increased 64Cu whole-body retention. Freshly isolated liver parenchymal cells from copper-deficient rats showed a higher 64Cu influx, which was associated with a higher apparent Vmax of 45 +/- 4 pmol Cu.mg protein-1.min-1 as compared with 30 +/- 3 pmol Cu.mg protein-1.min-1 for cells isolated from copper-sufficient rats. No significant difference in the apparent Km (approximately 30 mumol/L) was observed. Relative 64Cu efflux from cells from copper-deficient rats was significantly smaller than the efflux from cells from copper-sufficient rats after prelabeling as determined by 2-h efflux experiments. Analysis of the medium after efflux from cells from copper-deficient rats showed elevated protein-associated 64Cu, suggesting a higher incorporation of radioactive copper during metalloprotein synthesis. Effects of copper deficiency persist in primary cultures of parenchymal cells derived from copper-deficient rats, and short-term cultures of these cells offer a prospect for the study of cell biological aspects of the metabolic adaptation of the liver to copper deficiency.

Visualization of the interaction of native and modified lipoproteins with parenchymal, endothelial and Kupffer cells from human liver.

The interaction of low density lipoprotein, acetylated low density lipoprotein and apolipoprotein E-free high density lipoprotein with parenchymal, endothelial and Kupffer cells of human liver was visualized. For this purpose, the fluorescent phospholipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate was used to label the lipoproteins. The involvement of both parenchymal and nonparenchymal cells in the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-labeled low density lipoprotein and acetylated low density lipoprotein was studied using in vitro perfusion of human liver tissue blocks. In addition, primary hepatocyte cultures were used to visualize the interaction with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-labeled apolipoprotein E-free high density lipoprotein and (modified) low density lipoprotein. 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-low density lipoprotein showed a time-dependent and concentration-dependent interaction with both hepatocytes and Kupffer cells, although the intensity of the interaction with parenchymal cells varied strongly among the liver donors. Uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-low density lipoprotein by both cell types was strongly inhibited by the presence of excess unlabeled low density lipoprotein in the (perfusion) medium. Methylation and hydroxyacetaldehyde treatment of low density lipoprotein prevented the uptake of low density lipoprotein. This indicated that the uptake of low density lipoprotein by Kupffer and parenchymal cells was mediated by the low density lipoprotein receptor. 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-acetylated low density lipoprotein was mainly taken up in situ by liver endothelial cells and by a minor population of Kupffer cells. Polyinosinic acid, a known inhibitor of the scavenger receptor, prevented the uptake by liver endothelial cells. Therefore human liver endothelial cells express active scavenger receptors on their surface. Apolipoprotein E-free 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-high density lipoprotein was found to be associated with the membrane of cultured liver parenchymal cells but was not taken up intracellularly, indicating a cholesterol exchange process occurring extracellularly at the plasma membrane. The cellular localization of lipoprotein receptors and uptake of the various classes of lipoproteins are comparable with the situation in rats.

Effect of dietary restriction on the degradation of proteins in senescent mouse liver parenchymal cells in culture

We previously reported that the half-life of protein degradation in cells from old mice is about 50% longer than that in cells from young or middle-aged ones. In the present study we investigated the degradation rate of microinjected proteins (horseradish peroxidase (HRP) and ovalbumin (OVA)) and pulse-labeled proteins in hepatocytes from dietary-restricted old mice. Dietary restriction was initiated when mice were 23 months of age and performed in two steps (first 80% and then 60% of the ad libitum intake), the total period being 70 days. Hepatocytes were isolated from mice fed a restricted diet and fed ad libitum. The half-lives of HRP, OVA, and pulse-labelled proteins in the hepatocytes from mice fed a restricted diet were about 40% shorter than those in the cells from mice fed ad libitum. These values were close to those in the cells of young animals. These results are discussed in relation to our previous findings that a similar regimen reduces the percentage of heat-labile enzymes accumulated in tissues of aged animals.

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37°C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 μg/ml 125 I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 μg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4°C and specific cell-associated uptake at 37°C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of 125I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4°C as a function of ligand concentration, the response tended to be higher at 5 to 15 μg HDL/ml for the Cu-deficient treatment. However, at 37°C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.

Inactivation kinetics of horseradish peroxidase microinjected into hepatocytes from mice of various ages

We have studied degradation of proteins in liver parenchymal cells in culture obtained from mice of various ages. Horseradish peroxidase (HRP) was microinjected into the cells by the hypertonic-hypotonic method of Okada and Rechsteiner ( Cell, 29 (1982) 33–41). Rate of inactivation of HRP was examined biochemically. Average functional half-lives of the enzyme were 50 h, 53 h and 75 h in the cells of young (3–8 months old), middle-aged (18–20 months old) and old (28–30 months old) mice, respectively. Thus, half-life of the inactivation of the enzyme in “old” cells was about 50% longer than that in “young” or “middle-aged” cells, suggesting that rate of protein degradation is lowered in senescent animals.

Studies on the effect of the hepatocyte-stimulating factor on galactose- β1 → 4- N-acetylglucosamine α2 → 6-sialyltransferase in cultured hepatocytes

Rat hepatic Galβ1 → 4GlcNAcα2 → 6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Galβ1 → 4GlcNAcα2 → 6 isozyme.

Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone

Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin. Specific hormone requirements are needed by hepatocytes to retain the biochemical pattern of mature cells. Insulin supplementation of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence of dexamethasone amplified the insulin-induced glycogen synthesis. Cytophotometric analysis showed differences in the way that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in culture are functionally heterogeneous.

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17.BETA..

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.

Investigation for the mechanism of cadmium toxicity at cellular level

With quantitative techniques at electron microscopical level chromatin condensation and emptying of the interchromatin space have been established in the nuclei of the endothelial cells of small uterine vessels. The nuclear and cytoplasmic changes after cadmium administration show much similarity between endothelial cells of small uterine vessels and cultured liver parenchymal cells. Cytoplasmic changes in both cell types after cadmium administration are suggestive of a disturbance in ribosomal RNA synthesis as the main cause leading to ultimate cell lysis.

Native collagen formation by liver parenchymal cells in culture.

IN normal supporting tissue and repair tissue of animals collagen is usually considered to be produced by cells of mesenchymal origin. Little is known, however, about the type of cell which synthesises collagen in organs which undergo diffuse fibrosis without known physical trauma, such as in cirrhosis of the liver. Tissue culture has shown, however, that epithelial cells1, including cancer cells2 and smooth muscle cells3, can synthesise collagen. We report here that an epithelial clone, originating from the liver cell line of rat forms a considerable amount of native collagen in culture.

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B 12-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B 12 uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B 12. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.