Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon.

The 7- to 10-fold increase in the rat liver serine:pyruvate aminotransferase activity after glucagon administration was shown to occur mainly in the mitochondrial matrix of parenchymal cells. The enzyme was purified from glucagon-treated rat liver mitochondria to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A specific rabbit antibody was prepared against the purified enzyme. Upon Ouchterlony double diffusion analysis, the mitochondrial extracts of glucagon-treated rat liver produced a single and fused precipitin line between the purified enzyme against the antibody. The supernatant fraction of glucagon-treated rat liver and the mitochondrial extracts of normal liver were also shown to make a single and fused precipitin line with the purified enzyme, when applied in large quantities. The quantitative immunotitration demonstrated that the glucagon-induced increase in the activity of liver serine:pyruvate aminotransferase were accompanied by the parallel increase in the amount of the enzyme antigen. Isotopic leucine incorporation studies showed that the relative rate of synthesis of the enzyme was increased approximately 10-fold by glucagon administration under the conditions employed. The rate of the degradation of the aminotransferase in the normal rat liver was a relatively slow process with a half-life of approximately 30 h. Thus the accumulation of serine:pyruvate aminotransferase in rat liver mitochondria by glucagon treatment can be ascribed mainly to the rise in the rate of enzyme synthesis.     

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.     

Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.     

Regulation of liver lipase. I. Evidence for several regulatory sites, studied in corticotrophin-treated rats

The activity of liver lipase, an enzyme that can be released from the liver by heparin, varies under several hormonal conditions. The site(s) at which regulation of the enzyme activity may occur was investigated in vitro. As a model, rats were used which had been treated with a corticotrophin analogue, to induce hypercortisolism, a condition in which liver lipase activity is lowered. Lipases isolated from heparin-containing perfusates of livers from ACTH or control rats were identical with respect to heat stability and specific activity as determined by immunotitration and binding to isolated non-parenchymal liver cells, indicating that the enzyme structure was not affected by the treatment. The secretion of liver lipase by isolated parenchymal liver cells was studied. During incubation of parenchymal cells derived from ACTH rats, less enzyme activity was found to be secreted when compared with hepatocytes isolated from control rats (ACTH rats, 2.30 +/- 0.2 mU/10(6) cells; control rats, 3.3 +/- 0.3 mU/10(6) cells). Liver lipase partially purified from control rats could be bound specifically to saturation by non-parenchymal cells, isolated from ACTH or control rats. Non-parenchymal cells from ACTH rats bound less lipase activity (29 mU/mg cell protein) than cells from control rats (50 mU/mg cell protein). This reduction in binding capacity seems to be due to a diminished number of binding sites, since the affinity based on Scatchard analysis and half-maximal binding was not different. These results suggest that the lowered liver lipase activity found during hypercortisolism may be due to an impaired synthesis and/or secretion of the enzyme by the parenchymal cells and to a reduced binding capacity of the non-parenchymal cells for liver lipase.     

Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues

An antibody against acetoacetyl-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of antibody-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immunosorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (microgram immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestyramine plus 0.2% ML-236B for 4 days, respectively, and decreased about 1.8-fold by fasting the animals or 1.3-fold by feeding them a diet containing 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the physiological role of this enzyme in cholesterol biosynthesis. Immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of immunologically crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.     

Localization of collagen prolyl hydroxylase to the hepatocyte : Studies in primary monolayer cultures of parenchymal cells from adult rat liver

Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocytes prepared from normal or regenerated adult rat liver and primary non-proliferating monolayer cultures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47% of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore, prolyl hydroxylase was localized by immunofluorescence uniformly to the hepatocytes in culture. Cultured hepatocytes converted [¹⁴C]proline to [¹⁴C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.     

Polymerized albumin receptor on rat liver cells.

The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of rat liver S-adenosyl-l-methionine decarboxylase (Short Communication)

The amount of S-adenosyl-l-methionine decarboxylase present in rat liver was enhanced 17-fold by administration of methylglyoxal bis(guanylhydrazone),* a specific inhibitor of the enzyme. The enzyme was purified 1400-fold in 50% yield from such liver extracts by chromatography on columns of the inhibitor bound to Sepharose. The purified enzyme had no spermidine synthetase activity.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

Effect of lipoprotein on 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity in rat liver cell culture: special suppressant effect of a lipoprotein isolated from hypercholesterolemic rat plasma.

A significant advance in cell culture methodology has permitted an analysis of the regulation of liver cell cholesterol metabolism by lipoproteins. The effect of rat lipoproteins on the activity of the rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase, was studied in monolayer cultures of adult rat hepatic parenchymal cells. Lipoproteins isolated from normocholesterolemic rat plasma, including low density lipoprotein, did not suppress HMG CoA reductase activity. However, enzyme activity was profoundly suppressed by a cholesterol rich d < 1.063 lipoprotein(s) isolated from hyperlipemic rat plasma. This lipoprotein may regulate the suppression of endogenous hepatic cholesterol biosynthesis which occurs after cholesterol feeding.     

Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A synthase and the chromosomal localization of the human gene

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was purified to homogeneity from rat liver cytoplasm. The active enzyme is a dimer composed of identical subunits of Mr = 53,000. The amino acid composition and the NH2-terminal sequence are presented. Partial cDNA clones for the enzyme were isolated by screening of a rat liver lambda gt11 expression library with antibodies raised against the purified protein. The identity of the clones was confirmed by hybrid selection and translation. When rats were fed diets supplemented with cholesterol, cholestyramine, or cholestyramine plus mevinolin, the hepatic protein mass of cytoplasmic synthase, as determined by immunoblotting, was 25, 160, and 1100%, respectively, of the mass observed in rats fed normal chow. Comparable changes in enzyme activity were observed. Approximately 9-fold increases in both HMG-CoA synthase mRNA mass and synthase mRNA activity were observed when control diets were supplemented with cholestyramine and mevinolin. When rats were fed these two drugs and then given mevalonolactone by stomach intubation, there was a 5-fold decrease of synthase mRNA within 3 h. These results indicate that cytoplasmic synthase regulation occurs primarily at the level of mRNA. This regulation is rapid and coordinate with that observed for HMG-CoA reductase. The chromosomal localization of human HMG-CoA synthase was determined by examining a panel of human-mouse somatic cell hybrids with the rat cDNA probe. Interestingly, the synthase gene resides on human chromosome 5, which has previously been shown to contain the gene for HMG-CoA reductase. Regional mapping, performed by examination of a series of chromosome 5 deletion mutants and by in situ hybridization to human chromosomes indicates that the two genes are not tightly clustered.     

Cell turnover in the repopulated rat liver: distinct lineages for hepatocytes and the biliary epithelium

The dynamics of cell renewal in the normal adult liver remains an unresolved issue. We investigate the possible contribution of a common biliary precursor cell pool to hepatocyte turnover in the chimeric long-term repopulated rat liver. The retrorsine (RS)-based model of massive liver repopulation was used. Animals not expressing the CD26 marker (CD26^-) were injected with RS, followed by transplantation of 2 million syngeneic hepatocytes isolated from a normal CD26-expressing donor. Extensive (80-90 %) replacement of resident parenchymal cells was observed at 1 year post-transplantation and persisted at 2 years, as expected. A panel of specific markers, including cytokeratin 7, OV6, EpCAM, claudin 7 and α-fetoprotein, was employed to locate the in situ putative progenitor and/or biliary epithelial cells in the stably repopulated liver. No overlap was observed between any of these markers and the CD26 tag identifying transplanted cells. Exposure to RS was not inhibitory to the putative progenitor and/or biliary epithelial cells, nor did we observe any evidence of cell fusion between these cells and the transplanted cell population. Given the long-term (>2 years) stability of the donor cell phenotype in this model of liver repopulation, the present findings suggest that hepatocyte turnover in the repopulated liver is fuelled by a cell lineage distinct from that of the biliary epithelium and relies largely on the differentiated parenchymal cell population. These results support the solid biological foundation of liver repopulation strategies based on the transplantation of isolated hepatocytes.     

Some kinetic properties of Mucor rouxii phosphofructokinase. Effect of cyclic adenosine 3', 5'-monophosphate.

Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O₂^? is an important intermediate in H₂O₂ formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O₂^t-, but might also help to regulate the level of the important antimicrobial agent, H₂O₂.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Influence of insulin on lysosomal activity and urea production in isolated parenchymal cells from rat liver.

Non-latent (free) activities of two lysosomal enzymes (acid phosphatase and beta-glucuronidase) and urea production were measured in purified rat liver parenchymal cells incubated in the presence and absence of insulin. Non-latent enzyme activity was measured by including 0.25M sucrose in the assay mixtures to provide osmotic protection to the lysosomes. Total enzyme activity was estimated with Triton X-100 in the homogenates. Insulin was found to inhibit ureogenesis and to reduce non-latent lysosomal enzyme activity in the hepatocytes in vitro. Our data support the idea that insulin inhibits autophagy in rat liver parenchymal cells. Such an effect of insulin may also explain the inhibitory action of insulin on urea production in the rat liver.     

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.     

Mitochondria contain a distinct DNA topoisomerase.

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.     

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 μg, was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo⁻¹ s⁻¹. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK ± CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4–5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.     

Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture

Summary The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, andα-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.