Liver Of Adrenalectomized Rats Research Articles

The adaptive response to 3‐methylcholanthrene is altered in the liver of adrenalectomized rats

The aryl hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that mediates most effects of aromatic hydrocarbons, such as 3‐methylcholanthrene (MC). The in vivo regulation of hepatic AHR and whether changes in AHR levels affect the response to agonists are not fully understood. We discovered that the level of hepatic AHR protein was decreased in male adrenalectomized (ADX) rats 4 days after surgery. We studied the consequences of this AHR depletion by measuring expression of aromatic hydrocarbon‐responsive genes in the liver of MC‐treated ADX and SHAM‐operated rats at 6 and 54 h post‐MC. Hepatic AHR protein was depleted in ADX rats at 4 days, but not 6 days, post‐surgery. At this later time point, MC increased hepatic AHR protein in ADX but not SHAM rats. Hepatic cytochrome P450 1A1 (CYP1A1), AHR and P450 reductase (CYPRED) mRNA was induced by MC to a greater extent in SHAM versus ADX rats at 6 h. At 54 h post‐MC, induction by MC was greater in ADX versus SHAM rats for CYP1A1 and CYPRED mRNA. CYP1A1 protein levels corresponded to its mRNA. These results suggest that the adaptive response to MC in liver of ADX rats is impaired at 6 h but sustained and enhanced at 54 h relative to SHAM. Changes in the level of hepatic AHR caused by ADX affect the magnitude and duration of the response to aromatic hydrocarbons in vivo in a gene‐dependent manner. [Support: CIHR; OGS; U of Toronto; Peterborough K.M. Hunter Studentship]

Purification and primary structure of a macromolecular-translocation inhibitor II of glucocorticoid-receptor binding to nuclei from rat liver. Inhibitor II is the 11.5-kDa Zn2+-binding protein (parathymosin).

The nuclear binding of the activated glucocorticoid-receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI-I, MTI-II and MTI-III). In this study, we purified the most potent of the three species (MTI-II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two-dimensional PAGE. Purified MTI-II inhibits GR binding to DNA containing glucocorticoid-response elements. To obtain the amino acid sequence of MTI-II, we digested the MTI-II with endopeptidases. The N-terminal amino acid sequences of the four digested fragments indicated that MTI-II is an 11.5-kDa Zn2+-binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI-II (ZnBP, parathymosin) and that GR binds to the MTI-II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.

Cloning of Rat Dihydropyrimidine Dehydrogenase and Correlation of Its mRNA Increase in the Rat Liver with Age.

Overlapping cDNAs encoding dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of pyrimidine degradation, were isolated from a rat liver cDNA library, and a 4,353 bp cDNA sequence with a 3,075 bp open reading frame encoding a polypeptide of 1,025 residues with a molecular mass of 111,458 Da was determined. Western blot analysis of COS-7 cells that were transfected with an RT-PCR-derived DPD cDNA showed that the product from the cDNA binds to an anti-serum for DPD. Northern blot analysis was performed by using a partial fragment of the cloned cDNA as a probe. The mRNA level, the protein level, and the activity of DPD in the rat liver increased to the adult level by 3 mo of age. The levels of DPD in the liver of adrenalectomized rats increased 48 h after glucocorticoid administration. The increase of DPD after birth may be caused by the increase in glucocorticoid level.

Increased Glucagon Action on Lactate Gluconeogenesis in Perfused Liver of Dexamethasone-Treated Rats

To clearly understand the hyperglycemic action of glucocorticoids, we studied the action of glucagon on lactate gluconeogenesis in the liver of rats 7 days after adrenalectomy and after treatment with 1 mg/kg dexamethasone for 7 days. The liver was isolated and cyclically perfused at 20 ml/min with 25 ml of perfusion medium containing 5 mM lactate, [U-14C]lactate, and 0–100 ng/ml glucagon. In the absence of glucagon, incorporation of [14C]lactate into glucose carbon 1 did not change significantly in the adrenalectomized rat liver (1.66 ± 0.12% of total radioactivity for 5 min) and increased in the dexamethasone-treated rat liver (3.61 ± 0.54%,P< 0.01) compared to the normal rat liver (1.99 ± 0.28%). The response of lactate gluconeogenesis to glucagon was extremely blunted in the adrenalectomized rat liver and was much larger in the dexamethasone-treated rat than in the normal rat liver (at a glucagon concentration of 100 ng/ml, 2.13 ± 0.33, 8.55 ± 1.06, and 4.61 ± 0.53% for 5 min, respectively). Glucagon binding to liver plasma membrane was not changed by adrenalectomy and was decreased by dexamethasone treatment. These results suggest that glucocorticoids induce hyperglycemia by increasing the response to glucagon, together with the high basal activity of hepatic gluconeogenesis. In addition, these effects do not occur through changes in glucagon binding to receptors.

Purification and characterization of a macromolecular‐translocation inhibitor III of activated glucocorticoid‐receptor‐complex binding to nuclei from rat liver

Macromolecular-translocation inhibitors (MTI) of binding of the activated glucocorticoid-receptor complex (GRC) to nuclei from rat liver are separated into at least three components (MTI-I-III) by DEAE-cellulose column chromatography [Okamoto, K., Isohashi, F., Horiuchi, M. & Sakamoto, Y. (1982) Biochem. Biophys. Res. Commun. 108, 1655-1660]. In this study, we have purified MTI-III from the livers of adrenalectomized rats to apparent homogeneity, as determined by SDS/PAGE. The purification procedure consisted of DEAE-cellulose chromatography, acid treatment and sequential chromatographies using gel filtration, S-Sepharose and Mono S columns. The purified protein had a molecular mass of approximately 69 kDa, as estimated by SDS/PAGE, and the molecular mass of the inhibitor was approximately 68 kDa, as estimated by gel filtration. Thus, MTI-III exerts its inhibitory activity as a monomer. The sedimentation coefficient of MTI-III was approximately 3.7 S. Purified MTI-III was fairly stable at 4 degrees C but at higher temperatures, especially above 25 degrees C, it was rapidly inactivated. Under low-salt conditions, MTI-III was associated with activated GRC (4.2 S) and the resulting complex was detected on sucrose density gradients as a larger species (6.8 S). Initial treatment of nuclei or DNA-cellulose with MTI-III did not alter their abilities to bind activated GRC. These results indicate that MTI-III acts through an interaction with GRC.

A new approach to localize glucocorticoid receptor using DNA probe containing glucocorticoid responsive element DNA consensus sequences.

A method to localize glucocorticoid receptor (GR) by utilizing the specific interaction between GR and glucocorticoid responsive element (GRE) is described. In this study, pBR 322 DNA was thymine-thymine (T-T) dimerized by UV-irradiation and used as a probe, since pBR 322 DNA harbors GRE DNA consensus sequences and it is known that GR binds to pBR 322 DNA specifically. T-T dimerized pBR 322 DNA was incubated with fresh frozen sections of adrenalectomized rat liver, which were fixed in 4% paraformaldehyde in phosphate buffered saline, and the sites of reaction were visualized by a successive use of rabbit anti-T-T antibody and horseradish-peroxidase labeled goat anti-rabbit IgG antibody. As a result, GR was localized to the nuclei and nuclear membranes as well as cytoplasm of hepatocytes, and most of the staining was lost in the liver sections from rats, which were intraperitoneally injected with hydrocortisone. On the other hand, the staining for T-T dimerized DNA probe with cyclic AMP responsive element consensus sequence was localized solely to the nuclei and the staining pattern was not altered by an injection of the steroid, indicating that the staining for pBR 322 DNA is sequence-specific. Finally, this method should provide useful information on the localization of steroid hormone receptors with DNA-binding activity.

Purification and characterization of an adenosine triphosphate-stimulated factor that enhances the nuclear binding of activated glucocorticoid-receptor complex from rat liver.

The ATP-stimulated translocation promoter (ASTP), a protein that increases the nuclear binding of partially purified activated glucocorticoid-receptor complexes (GRC) in the presence of ATP, was purified from the liver of adrenalectomized rats to apparent homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification procedure consisted of DEAE-cellulose chromatography, heat treatment (60 C; 3.5 min), and sequential chromatographies on agarose gel filtration, hydroxylapatite, Mono Q, and S-Sepharose. ASTP had a mol wt of 93,000, as determined by gel filtration on Bio-Gel A-0.5 m, and was composed of two apparently identical subunits with mol wt of 48,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sedimentation coefficient of ASTP was 6.5s, and its isoelectric point was 4.5. ASTP activity was dependent on the physiological concentration of ATP, although ASTP did not bind to ATP-agarose. Neither ADP nor AMP affected its activity. An analysis of the nuclear binding behavior of GRC suggested that the affinity of GRC for nuclei in the presence of ASTP is much greater than that in the absence of ASTP. In the presence of ATP, ASTP increased the binding of GRC to chromatin, but not to DNA-cellulose, suggesting that ASTP acts as a regulatory component, altering the chromosomal binding of GRC in rat liver.

In vivo effects of dexamethasone and cycloheximide on the phosphorylation of 110-kDa proteins and the protein kinase activities of rat liver nucleoli.

In vivo effects of dexamethasone and cycloheximide on the phosphorylation of nucleolar proteins and the activities of nucleolar protein kinases of rat livers were studied. Phosphorylation of nucleolar proteins was accomplished by incubation of isolated nucleoli with [gamma-32P]ATP at 37 degrees C for 10 min followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Of several nucleolar phosphoproteins observed in the liver of adrenalectomized rats, the incorporation of 32P into nucleolar 110-kDa proteins was rapidly increased, reaching about 1.8-fold of the adrenalectomized level at 12 h after administration of dexamethasone. In addition, the activities of liver nucleolar protein kinases which were measured using casein or phosvitin as substrates were stimulated similarly by the administration of dexamethasone. It was found by DEAE-Sephadex column chromatography that nucleolar protein kinases were separated into NI and NII, the activities being increased about 35 and 80% by dexamethasone treatment (12 h), respectively, and that the enhancements were completely abolished by 1-h treatment of cycloheximide. The injection of cycloheximide also suppressed the hormone-induced enhancement of 32P incorporation into 110-kDa proteins but did not reduce the amount of the proteins at least until 2 h. Evidence was obtained indicating that the phosphorylation of the nucleolar 110-kDa proteins is accomplished primarily by protein kinase NII. These results suggest that continuous synthesis of protein(s) is necessary for glucocorticoid-induced enhancement of phosphorylation of 110-kDa proteins of liver nucleoli and that the nucleolar protein kinase NII is involved in the glucocorticoid-induced short-lived protein(s).

Increased rate of purine biosynthesis in rat liver after bilateral adrenalectomy

In bilaterally adrenalectomized rat liver the increased rate of de novo purine synthesis was shown by the increased [14C]glycine incorporation into hepatic acid-soluble purines with unchanged rapidly miscible glycine pool size and its turnover rate and by the increased rate of chasing of radiolabeled purines. At 24 h after adrenalectomy, the rate of de novo purine synthesis increased by 70%, 5-phosphoribosyl-1-pyrophosphate (PRPP) content increased by 200%, the specific activity of amidophosphoribosyltransferase (EC 2.4.2. 14; ATase) did not change, ATP and GTP showed a 33 and 24% decrease, and AMP and ADP showed a 245 and 38% increase. Combined, the metabolic pool size data reflected an unchanged total inhibitory potential on ATase. Replacement with corticosterone acetate for 24 h partially restored some of these abnormalities. These results suggest that the increase in the rate of de novo purine synthesis in adrenalectomized rat liver is secondary to increased catabolism of purine ribonucleotides and mediated by increased PRPP concentrations.

Effects of glucocorticoid and cycloheximide on the activity and amount of RNA polymerase I in nuclei of rat liver.

The activity of the template-engaged form of RNA polymerase I from livers of adrenalectomized rats was about 50-60% of that of normal control rats, and increased about 2-fold at 6 h after the administration of dexamethasone. However, no change was found in the activity of the 'free' form of RNA polymerase I or the template-engaged form of RNA polymerase II. Immunochemical studies using guinea-pig anti-(RNA polymerase I) serum disclosed that the total number of RNA polymerase I molecules did not vary during the treatment with dexamethasone. Cycloheximide caused a rapid decrease in the template-engaged form of RNA polymerase I activity in normal rats and in dexamethasone-treated (6 h) adrenalectomized rats, to the value in adrenalectomized rats, but affected it only slightly in adrenalectomized rats. The elongation rate of rRNA-precursor synthesis in liver nuclei was not affected by a change in the concentration of circulating dexamethasone. From these results, it is concluded that about half the rRNA-precursor synthesis in rat liver is regulated by glucocorticoids, probably through the synthesis of short-lived protein(s) which may play a role in conversion of the 'dormant' form of RNA polymerase I into the 'engaged' form.

MECHANISM OF INCREASED RATE OF DE NOVO PURINE BIOSYNTHESIS IN RAT LIVER AFTER BILATERAL ADRENALECTOMY: 92

In rat liver after bilateral adrenalectomy de novo purine biosynthesis was studied 24, 48 and 72 hours in comparison to sham-operated control. At these time points the rate of de novo purine biosynthesis assayed by [14C]glycine incorporation to hepatic purines significantly increased by 1.61, 1.95 and 2.01 folds respectively associated with comparable free glycine concentration in plasma or liver tissue. Hepatic concentration of 5-phosphoribosyl 1-pyrophosphate(PRPP) increased by 2.91, 1.50 and 1.75 folds at these 3 time points. 24 hours after bilateral adrenalectomy ATP and GTP concentration decreased by 33 and 24% respectively which were counterbalanced by the increased concentration of AMP(245%) and GMP(38%) associated with 18% decrease of adenylate energy charge. The replacement of corticosterone acetate to adrenal-ectomized animals for 24 hours partially reversed these changes. The results suggest that the increased rate of de novo purine biosynthesis in adrenal-ectomized rat liver is mediated by the increased concentration of PRPP and that it is compensatory against increased catabolism of purine ribonucleotides as a result of increased AMP concentration.

The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat.

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat-stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor-1 and modulator), did not affect the activity of synthase phosphatase in gel-filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D. Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G-component of synthase phosphatase for efficient conversion to the alpha-form. No G-component could be isolated from livers of adrenalectomized starved rats. Cross-combination of subcellular fractions from control and deficient livers revealed an almost total loss of G-component, with little loss of S-component. This specific loss of functional G-component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan-diabetes.

Ribosomal RNA synthesis in liver of adrenalectomized rats after partial hepatectomy

The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized — partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.

Partially purified "activated" receptor-glucocorticoid complex from rat liver: regulation of nuclear, chromatin, and DNA-cellulose binding of "activated" complex by pyrophosphate and ATP.

The effects of PP1 and ATP on nuclear binding of the "activated" receptor-[3H]-triamcinolone acetonide (TA) complex purified about 3,000-fold from adrenalectomized rat liver were investigated. ATP at up to 5 mM did not affect nuclear binding of the "activated" complex, but PP1 at 2-7 mM greatly enhanced it. However, ATP in the presence of PP1 decreased nuclear binding dose-dependently. Similar results were obtained in the case of chromatin binding, but PP1 alone did not alter DNA-cellulose binding of the "activated" complex, suggesting that the binding sites for chromatin and DNA on the "activated" complex are different. Furthermore, PP1 enhanced ATP-agarose binding of the "activated" complex, indicating that the PP1 binding site(s) on the receptor is different from the ATP binding site(s). The physicochemical properties of the "activated" receptor-glucocorticoid complex bound with ATP and/or PP1 were examined by sucrose density gradient centrifugation and Sephadex G-150 gel filtration. There was no detectable change in the sedimentation coefficient or molecular weight (about 4.2S; Mr approximately equal to 98,000) on binding with ATP and/or PP1. These results suggest that the binding of PP1 and PP1 plus ATP to the "activated" complex caused some allosteric change of the acceptor binding sites of the receptor, resulting in increase or decrease in its binding to nuclei, chromatin, or DNA.

Observations on the role of smooth endoplasmic reticulum in glucocorticoid-induced hepatic glycogen deposition

We have studied by quantitative electron microscopy the relationship of specific hepatic cellular organelles to glycogen synthesis using dexamethasone, a potent synthetic glucocorticoid, to induce glycogen deposition in livers of adrenalectomized rats. Chemical and ultrastructural glycogen determinations revealed that the livers of fasted adrenalectomized rats had very low glycogen levels. Dexamethasone caused a time-related increase in hepatic glycogen which was the result of increases in the number of hepatocytes depositing glycogen and the amount of glycogen in each cell. The surface density of smooth endoplasmic reticulum (SER) in centrilobular and periportal hepatocytes also increased after treatment with dexamethasone; this increase preceded glycogen deposition. The newly deposited glycogen was spatially associated with membranes of SER, and a continued increase in SER surface density was correlated temporally with the increasing glycogen volume density. In both centrilobular and periportal hepatocytes, the suface density of rough endoplasmic reticulum (RER) initially decreased after dexamethasone administration but later increased. These data support the hypothesis that dexamethasone-induced enhancement of SER is functionally associated with the increase in glycogen, and that although the initial increase in SER may occur through transformation of RER to SER, later increases in SER require synthesis of new membranes.

Characteristics of glucocorticoid-binding sites of rat liver: different effects of adrenalectomy on the binding.

Hydrocortisone (HC) in rat liver cytoplasmic fraction was bound to three different binding sites with high, medium and low affinity. Dissociation constants (Kd) were approx. 2.1, 22 and 208 nM; and the densities of these binding sites were about 40, 50 and 10% of total number of binding sites, respectively. The binding site for dexamethasone (DM) of the cytoplasmic fraction was the medium affinity one among these three components. The maximum number of binding sites (Bmax) of HC and DM was significantly increased by adrenalectomy. The Bmax of HC was about twice as great as that of DM in normal and adrenalectomized rat liver. DM inhibited 3H-HC binding in a dose-dependent manner but inhibition did not exceed 50% in either normal or adrenalectomized rats. Following adrenalectomy, the Bmax of the medium affinity-site for HC was significantly increased, while the high affinity component disappeared. By adding DM to the cytoplasmic fraction of adrenalectomized rat liver in vitro and in vivo, the Bmax of the medium affinity-site was significantly decreased, and a high affinity component of HC was revealed with a significant increase in the number of binding sites. These results indicate that the binding site for DM is one component of the HC binding site; and following adrenalectomy, the number of each type of binding site for glucocorticoids increases differently from the others.

CHARACTERISTICS OF GLUCOCORTICOID-BINDING SITES OF RAT LIVER: DIFFERENT EFFECTS OF ADRENALECTOMY ON THE BINDING

Hydrocortisone (HC) in rat liver cytoplasmic fraction was bound to three different binding sites with high, medium and low affinity. Dissociation constants (Kd) were approx. 2.1, 22 and 208 nM; and the densities of these binding sites were about 40, 50 and 10% of total number of binding sites, respectively. The binding site for dexamethasone (DM) of the cytoplasmic fraction was the medium affinity one among these three components. The maximum number of binding sites (Bmax) of HC and DM was significantly increased by adrenalectomy. The Bmax of HC was about twice as great as that of DM in normal and adrenalectomized rat liver. DM inhibited 3H-HC binding in a dose-dependent manner but inhibition did not exceed 50% in either normal or adrenalectomized rats. Following adrenalectomy, the Bmax of the medium affinity-site for HC was significantly increased, while the high affinity component disappeared. By adding DM to the cytoplasmic fraction of adrenalectomized rat liver in vitro and in vivo, the Bmax of the medium affinity-site was significantly decreased, and a high affinity component of HC was revealed with a significant increase in the number of binding sites. These results indicate that the binding site for DM is one component of the HC binding site; and following adrenalectomy, the number of each type of binding site for glucocorticoids increases differently from the others.

Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute s.c. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [3H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose-dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.

Influence of dl-ethionine on the nad content and on the activity of the poly(adpr)synthetase in the rat liver

1. 1. Ethionine, an analogue of methionine, is known to cause a number of serious changes. In this study, its influence on the NAD + NADH 2 content and on the activity of the poly(ADPR)synthetase in rat liver has been investigated. 2. 2. dl-Ethionine decreases the content of NAD + NADH 2 in adrenalectomized, but not in normal animals. 3. 3. After injection of nicotinamide the NAD + NADH 2 content in the liver of adrenalectomized rats is much higher than in that of normal ones. 4. 4. DL-Ethionine diminishes the NAD synthesis caused by nicotinamide in adrenalectomized animals and increases it in normal ones. dl-Tryptophan induces only a slight NAD synthesis, which is reduced in adrenalectomized animals by ethionine. 5. 5. 1-Methylnicotinamide slightly decreases the NAD concentration in normal animals, but increases it considerably in adrenalectomized ones. dl-ethionine reduces the NAD + NADH content in adrenalectomized animals in the presence of 1-methylnicotinamide. 6. 6. The activity of poly(ADPR)synthetase of in-vitro tested nuclei is slightly increased after treatment with dl-ethionine.

Spironolactone: a glucocorticoid agonist or antagonist?

Spironolactone displaces 3H-dexamethasone from HTC cell nuclei and inhibits the induction of tyrosine aminotransferase (TAT) activity by dexamethasone in HTC cells. Spironolactone alone fails to increase TAT activity in HTC cells or in the liver of adrenalectomized rats. These actions indicate that spironolactone lacks glucocorticoid agonist activity but can be an antagonistic to the actions of glucocorticoid as well as mineralocorticoid steroid hormones.