Williams Medium Research Articles

Maintenance of cytochrome P-450IA2, IIB1 and IIB2 mRNAs by metyrapone in rat hepatocyte culture

The primary culture of rat hepatocytes for 24 h in standard medium results in the loss of 50% o f their cytochrome 1’-450 (1’-450) content [ I ] . This loss can be prevented by culturing hcpatocytes with 0.5 mwmetyrapone which stimulates 1’450 synthesis 121. However, P 4 5 0 is a collective term for a superfamily of haemoproteins 131. In order to gain an insight into the molecular action of metyrapone we have compared its effects on the expression of the 1’-450IA and 1’-45011B subfamilies in hepatocyte culture with those of the classical inducers, phenobarbitone (PB) and P-naphthoflavone (BNF). Hepatocytes were prepared from 250-280 g male CD rats and cultured in Williams medium E as previously described [ 11. Total 1’-450 content was assayed [ 11 and total RNA isolated [4]. 1’-4501A2 and I’-45011B mRNAs were quantified after 24 and 72 h of culture by Northern blot hydridization to their respective cDNAs [ 5,6]. Rat hepatocytes cultured for 24 h contained 40 f 1 Soh of their initial 1’-450 content. Concomitant with this decrease, the abundance of the mRNAs encoding 1’-4501A2 and the P-4501IB subfamily declined to 37% and 33%, respectively. After 72 h of culture both total 1’-450 content and the levels of the mRNAs declined further (Table 1 ). Culturing hepatocytes in medium containing 0 . 5 mMmetyrapone prevented the loss of total 1’-450 content throughout the culture period. This was paralleled at 24 h by a higher abundance of both 1’-4501A2 and P-4501IB mRNAs compared with untreated cultures. However, at 72 h 1’-450IA2 mRNA had declined whereas P45011B mRNAs increased above the value found in freshly isolated hepatocytes. This observation suggests that metyrapone may

A functional and structural heterogeneity is formed among fetal rat hepatocytes during culture.

Fetal rat hepatocytes (22-day-old, full-term) in vivo were homogeneous in ultrastructure and glucose 6-phosphatase (G6Pase) distribution throughout the liver acinus. These cells were isolated and cultured for 17 days in Williams medium E containing 10% fetal calf serum, dexamethasone, insulin, and glucagon. Heterogeneity among hepatocytes appeared progressively during culture. There were variations in numbers of binucleated cells, cytoplasmic ultrastructure (e.g., in the distribution of glycogen and the quantity of ultrastructure (e.g., in the distribution of glycogen and the quantity of mitochondria), and intensity of histochemical reactivity for G6Pase. The cells could be classified generally into two types: type I cells had some characteristics of periportal hepatocytes and type II cells those of centrolobular hepatocytes in in vivo adult liver. The results show that a functional and structural heterogeneity can be formed among fetal hepatocytes in monolayer culture without differences in supply of oxygen, nutrients, and hormones. A hidden heterogeneity might already exist among hepatocytes in full-term fetuses even though it cannot be detected ultrastructurally or cytochemically.

Excretion process of copper from copper pre-loaded rat liver parenchymal cells

Excretion of copper from copper pre-loaded rat liver parenchymal cells was investigated. The copper pre-loaded cells were isolated from the liver of copper-administered rats and were maintained for 4 days in modified Williams medium E; sodium pyruvate was increased while calcium was reduced in concentration, and 10% fetal bovine serum and insulin were added. For the first 30 hr after plating, loss of copper was slow, with a half-life of more than 100 hr: subsequently the rate of loss was faster, with a half-life of 51 ± 9 hr. Copper pre-loaded in the cells was mostly bound to metallothionein and the amount of copper-metallothionein decreased with time of culture. Furthermore, the relative ratio of the two iso-metallothioneins changed with length of culture.

Arginase as one of the inhibitory principles in the density-dependent as well as plasma membrane-mediated inhibition of liver cell growth in vitro

Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2–3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 × 10 6 to 1.0 × 10 6 nuclei/dish (Falcon, Ø 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (<30 μg protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.

L-Proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.

Use of hepatocytes in primary culture for biochemical studies on liver functions.

Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its alpha-adrenergic action. The beta-action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland. These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.

Biochemical functions of adult rat hepatocytes in primary culture.

Liver parenchymal cells from adult rats were isolated by treatment with collagenase and cultured as monolayers in Williams medium E with 10% fetal or calf serum. The additions of dexamethasone and insulin to the medium were essential for maintaining liver functions of the cells. These cells synthesized and secreted various serum proteins into the medium. Gluconeogenesis and glycogenolysis were enhanced by glucagon, and lipogenesis was stimulated by insulin. Many enzymes were also induced by various hormones. These activities were very low in freshly isolated cells, but were restored when the cells were cultured for a few days. Markers of plasma membranes, such as 5'-nucleotidase and insulin receptors, were reduced to half the normal levels on freshly isolated cells, but they were restored to the normal levels during culture of the cells without added hormones. Analysis of the profile of amino acids in the medium showed that freshly isolated cells were in a catabolic state of protein turnover and released branched chain amino acids into the medium, but that cultured cells consumed amino acids, not only for protein synthesis, but also for other metabolic processes, such as gluconeogenesis. These findings show that freshly isolated cells have impaired functions and are unsuitable for use in studies of liver metabolism, but that after culture for a few days the cells regain the activity of normal liver and hance become useful for studies of liver functions. Studies with these cells are simpler and give clear results than studies in vivo.

DIFFERENTIATION OF A PRIMARY CHEMICALLY INDUCED RAT NEPHROBLASTOMA IN ORGAN CULTURE.

The differentiation in organ culture of a rat nephroblastoma is compared with differentiation of normal rat metanephric tissue under the same conditions. The nephroblastoma arose in a 19 week old female Fischer F344 rat given a single intraperitoneal injection of 4.0 μmole methyl(methoxymethy1)nitrosamine (DMN-OMe)/g body weight at one day of age. The tumor consisted almost entirely of spindle cells although a few well-differentiated tubules were scattered throughout the tumor mass. No primitive tubules were seen, but focal aggregates of tumor cells suggestive of nascent epithelial differentiation were frequent. Fragments of the nephroblastoma were cultured on gelfoam sponge in Williams Medium E supplemented with hydrocortisone, insulin, and fetal bovine serum. Within one day extensive tubulogenesis was observed. High mitotic activity resulted in a steady increase in the size of cultured explants over a period of 6 days. By day six, differentiating tubules filled the explant tissue. Cultured fragments were nearly indistinguishable histologically from normal F344 rat fetal kidney explanted to organ culture on day 15 of gestation and grown in vitro for the same period.

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis.

Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5 X 10(7) and 1.0 X 10(8) cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions or freshly isolated cells and cultured cells were compared in this medium. In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated. These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.

OXYGEN CONSUMPTION AND CARBON DIOXIDE PRODUCTION DURING THE GROWTH OF YEAST

111 the present experiments the oxygen consumption and carbon dioxide production of a pure strain of yeast have been investigated throughout the first and second cycles of its growth in a synthetic nutrient solution. Few measurements of the respiration of yeast in the literature are comparable to these observations, since other investigators have used different kinds of yeast under various experimental conditions which have not always been stated clearly. The measurements have usually been made only for short intervals of time. Copping (2) has published curves for the oxygen consumption, carbon dioxide production and cell count of Saccharomyces cerevisiae on different media for a period of about four days but readings were made only at daily intervals and no attempt was made to correlate the gaseous exchange of the yeast with its growTth. For these experiments a pure strain of Saccharomyces cerevisiae Hansen previously obtained from a single cell (Richards, 5)2 was grown in William 's medium.3 The seedings were made with 0.10 cc. of a suspension from yeast cultures of about the same age and the technique of measuring the multiplication of the yeast wras that of Richards (6). The oxygen consumption and carbon dioxide production of the yeast were determined with differential microrespirometers of the type devised by Thunberg (9) and more recently described by Fenn (3). For the measurements of oxygen consumption 0.5 cc. portions of 20 per cent, sodium hydroxide were introduced into small cups in the two chambers of the respirometer to absorb carbon dioxide. Into one chamber was placed 1 cc. of medium containing yeast and into the other enough water to make the air spaces equal. As oxygen was consumed the index drop in a horizontal capillary connecting the two chambers was drawn toward the chamber containing the yeast. The oxygen consumption was obtained by multiplying the movement of the drop in centimeters per minute by the volume of the capillary in cubic centimeters per centimeter. Twice this value was used