Tyrosine Aminotransferase Inducibility Research Articles

Effects of dexamethasone and cAMP on tyrosine aminotransferase expression in cultured fetal rat hepatocytes

Fetal hepatocyte cultures were used to investigate tyrosine aminotransferase (TyrAT) expression during development. Previous studies showed that TyrAT is synthesized by hepatocytes isolated from 15-day-gestation fetuses maintained in culture for two or more day, then exposed to dexamethasone. TyrAT expression was essentially undetectable on the first day of culture of hepatocytes derived from 15-day-gestation, or less mature, fetuses. Dexamethasone and cAMP are potent inducers of TyrAT and they synergistically induce TyrAT to extremely high levels when added simultaneously to cultured fetal hepatocytes. The effects of dibutyryl-cAMP (Bt2cAMP) alone and in combination with dexamethasone on TyrAT expression are investigated. Hepatocytes isolated from 15-day-gestation fetuses exposed to both inducers possessed detectable levels of TyrAT activity and mRNA on day 1 of culture, and this increased by day 3. In contrast, hepatocytes exposed to either inducing agent alone were essentially negative on day 1, but positive on day 3. This was shown to be a consequence of transcription. When 13-day-gestation hepatocytes were maintained in culture under identical conditions detectable levels of TyrAT mRNA were evident on day 1, and this increased by day 3. Immunocytochemical studies revealed that the appearance and subsequent increase in TyrAT production elicited by dexamethasone and Bt2cAMP were due to changes in the proportion of hepatocytes expressing the enzyme. Therefore, in the presence of both dexamethasone and Bt2cAMP, TyrAT expression can be detected in some cells at an earlier stage of liver development than reported previously.

Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes

Dexamethasone can promote the differentiation of different tissues in vivo while dimethylsulfoxide is a commonly used inducer of differentiation in various tumor cell types in culture. In the present study, the effects of dexamethasone and dimethylsulfoxide on growth and functional activities of cultured differentiating suckling rat hepatocytes stimulated with various combinations of EGF, insulin, and glucagon were evaluated. Hepatocytes stimulated with EGF and either insulin or glucagon entered S phase and mitosis after a lag period of 24 h. These hormonal factors thus provide simple combinations of hepatocyte-growth regulators. Dexamethasone in the presence of EGF and glucagon inhibited the initiation of DNA synthesis and mitosis, but it had no effect on EGF-insulin stimulated cultures. Such a differential effect of dexamethasone was observed at concentrations ranging from 4 nM to 200 microM. alpha-Fetoprotein, albumin, and tyrosine aminotransferase were used as typical markers of hepatocyte differentiation status. Irrespective of the combinations of growth-promoting factors used, dexamethasone inhibited alpha 1-fetoprotein production and maintained albumin production and tyrosine aminotransferase inducibility. In contrast, dimethylsulfoxide at 2% inhibited hepatocyte growth and supported the maintenance of the production of both alpha 1-fetoprotein and albumin, independent of the hormonal growth regulators used. On this basis, dexamethasone and dimethylsulfoxide act as distinct modulators of growth and maturation of cultured differentiating suckling rat hepatocytes.

The inducibility of tyrosine aminotransferase in monolayer cultures of hepatocytes from neonatal rats.

Hepatocytes from neo- and postnatal rat liver were isolated, purified from non-hepatocytes (erythropoietic cells), and cultured in sufficient quantity to investigate enzyme inducibility. Tyrosine aminotransferase (TAT) in neo- and postnatal hepatocytes was induced by maximally responsive doses of glucagon, dexamethasone, DB-cAMP, theophylline, and combinations thereof. In cultures from newborn parenchymal cells TAT enzyme-specific activity showed only a moderate inducibility; however, responsiveness to the combination was fully developed 10 days after birth and did not differ from values found in adult liver cells. The results also show the existence of the "permissive" effect of glucocorticoid during postnatal age, and indicate that the development of a possibly involved receptor complex for the induction of TAT is largely completed 5-10 days after birth.

Primary monolayer culture of liver parenchymal cells and kidney cortical tubules as a useful new model for biochemical pharmacology and experimental toxicology. Studies in vitro on hepatic membrane transport, induction of liver enzymes, and adaptive changes in renal cortical enzymes.

Freshly isolated liver parenchymal cells were maintained in either short-term monolayer, suspension of long-term monolayer culture. Rapidly occurring processes through hepatocellular membrane, e.g., the enhanced amino acid transport and the concomitantly increased potassium influx following progressive starvation, were kinetically evaluated best in short-term monolayer culture. The inducibility of tyrosine aminotransferase by glucagon, dexamethasone, and a combination of both was compared in suspension and in monolayer culture. The induction of slowly inducible foreign compound-metabolizing enzymes, (e.g., ethoxycoumarin-O-dealkylase, p-nitroanisole-O-demethylase, and UDP-glucuronyltransferase) by phenobarbital, 3-methylcholanthrene, and dexamethasone were studied in long-term monolayer culture. The latter system was also used to maintain isolated kidney cortical tubules for the investigation of renal enzyme adaptation during progressive time in culture.

Reactivity of the liver to glucocorticoids during chemical hepatocarcinogenesis

Liver cells during chemical carcinogenesis (3′-methyldimethylaminoazobenzene) and cells of primary hepatomas retain the property of reacting to partial hepatectomy by increased incorporation of3H-thymidine into DNA, just as under normal conditions this process is inhibited by dexamethasone. The inducibility of tyrosine aminotransferase (EC 2.6.1.5) likewise remained unchanged, whereas induction of tryptophan pyrrolase (EC 1.13.11.11) in primary hepatomas was abolished. The adequacy of a model of chemical carcinogenesis of an organ if the heterogeneity of its cell populations is disregarded is discussed.

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.

The effect of hormones on glucocorticoid binding capacity of rat liver cytosol

The relationship between the glucocorticoid binding capacity of rat liver cytosol and the activity of tyrosine aminotransferase has been studied in adrenalectomized male rats. Bilateral adrenalectomy of male rats caused an increase within 3 days in the level of specific dexamethasone binding of liver cytosol accompanied by a rapid decrease in tyrosine aminotransferase activity. Known inducers of tyrosine aminotransferase were administered in vivo to test their effect on dexamethasone binding capacity, in order to determine whether the induction was by an indirect mechanism involving an increase in glucocorticoid binding capacity. Insulin, adrenalin, glucagon, dibutyryl cyclic AMP and oestradiol caused a significant increase in the activity of the enzyme, with no change in the specific dexamethasone binding. Tetracosactrin, a synthetic analogue of ACTH, had no effect on either parameter. It was concluded that the induction of tyrosine aminotransferase by the compounds tested was not mediated by an increase in glucocorticoid receptor activity.

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.

Human Regulatory Gene for Inducible Tyrosine Aminotransferase in Rat-Human Hybrids

The inducibility of tyrosine aminotransferase (EC 2.6.1.5) by corticosteroid hormones in rat-human hybrid clones was studied. The presence of human X chromosome activity in the cells was always associated with the suppression of tyrosine aminotransferase inducibility in all the clones examined. Negative correlation between the human X chromosome and inducibility of the enzyme was clearly established. Corticosteroid receptor was present to the same extent in hybrid cell clones that either contained or lost the human X chromosome. The human repressor for inducible tyrosine aminotransferase has a linkage relationship with glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and hypoxanthine-guanine-phosphoribosyltransferase (EC 2.4.2.8) and, therefore, can be assigned to the X chromosome.

Glucocorticoid receptors: Relations between steroid binding and biological effects

Steroid binding has been studied in cytoplasmic extracts of cultured rat hepatoma cells to investigate the mechanism of enzyme induction by glucocorticoids. The affinity of inducer steroids for the specific receptors contained in the extracts is directly related to the potency of these steroids as inducers of tyrosine amino-transferase. The ability of anti-inducer steroids to compete with inducers for binding is similar to their ability to inhibit induction. Furthermore, the affinity of an anti-inducer for the receptors can be predicted from its ability to inhibit inducer binding. These and other correlations allow distinction between the specific cytoplasmic receptors and a number of other molecules, includingplasma transcortin, which also bind glucocorticoid hormones. Further experiments were carried out to determine whether an allosteric model, proposed earlier, could explain the differential effects of inducer, suboptimal inducer and anti-inducer steroids. According to the model, steroids interact with either, or both, of two conformational states of the receptors; the uncomplexed receptors are predominantly in one (inactive) form and binding by inducers, but not by anti-inducers, increases the concentration of the other (active) conformation. Consistent with this idea, we find that receptors are less stable when they are free, or complexed by an anti-inducer, than when they are bound by an inducer. Furthermore, kinetic studies are in accordance with the proposal that binding by inducers, but not anti-inducers, is associated with conformational changes in the receptor molecules. Specific glucocorticoid receptors were also characterized in other tissues and found to be similar to those in hepatoma tissue culture cells.

Glucocorticoid and mineralocorticoid receptors for aldosterone

Specific binding of aldosterone and dexamethasone by rat kidney and hepatoma tissue culture (HTC) cell cytosol has been studied. In cytosol of HTC cells, aldosterone and dexamethasone bind to a single class of sites with affinities that correspond with their potencies as inducers of tyrosine aminotransferase. Kidney cytosol, however, contains two classes of specific aldosterone binding sites. The higher affinity sites bind aldosterone with an affinity (equilibrium, dissociation, constant) which is similar to the plasma concentration of aldosterone required for antinatriuresis. The lower affinity aldosterone-binding sites are present at a higher concentration than the higher affinity sites and also bind dexamethasone with a high affinity. We have tentatively identified these two classes of binding sites in renal cytosol as “mineraloeonicoid” and “glucocorticoid” receptors, respectively.

Induction of tyrosine aminotransferase with N 6 ,O 2 -dibutyryl adenosine 3'-5'monophosphate in rat-hepatoma cells grown in culture.

The dibutyryl analogue of adenosine 3′:5′ monophosphate (Bu2‐Ado‐3′:5′‐P) stimulates tyrosine‐aminotransferase synthesis in rat‐hepatoma cells cultured in Dulbecco medium supplemented with excess L‐tyrosine. This effect of Bu2‐Ado‐3′ :5′‐P is not observed in cells incubated in regular Dulbecco medium. It has been shown previously that tyrosine‐aminotransferase activity increases in hepatoma cells incubated in a glucose‐free medium that is also supplemented with excess L‐tyrosine. The increase in enzyme activity observed after administration of Bu2‐Ado‐3′:5′‐P and after glucose removal can be prevented by cycloheximide or actinomycin‐D. Bu2‐Ado‐3′ :5′‐P in the absence of glucose does not cause an additive increase in tyrosine‐amino transferase activity. The latter observation suggests that increased enzyme synthesis in each circumstance occurs via the same mechanism. Combination experiments, using Bu2‐Ado‐3′ :5′‐P and cortisol, indicate that each of these inducers of tyrosine aminotransferase acts independently. In light of recent observations concerning β‐galactosidase induction in bacteria, a mechanism of tyrosine‐aminotransferase induction in these mammalian‐derived cells is discussed.