A new glucocorticoid receptor detected in host rat liver but not in various hepatomas.

Previously a new glucocorticoid receptor (Peak C), which eluted with 0.12 to 0.14 M NaCl from DEAE-cellulose column, was identified in addition to another receptor (Peak B), a classic type of glucocorticoid receptor, which eluted with 0.05 to 0.08 M NaCl. Peak C appeared after stress or injection of a high dose (20 micrograms/100 g body weight) of dexamethasone into rats. Peak C was also detected in the liver of rats bearing various tumors, but it was not found in malignant tumors (Yoshida sarcoma and Yoshida ascites hepatoma AH 130), a less malignant Yoshida ascites hepatoma (LY-5), or in minimal deviation-type hepatomas (Morris hepatomas 7316A and 7794A). The absence of Peak C in these tumors coincided with the inability of the glucocorticoid to induce tryptophan oxygenase in these tumors and in the liver of rats during early postnatal development. Peak B was consistently observed in various hepatomas and immature rat liver with capability to induce tyrosine aminotransferase. Thus Peak C appeared to be a highly differentiated type of glucocorticoid receptor mediating specific hormone actions and to be present in mature liver cells, but not in immature liver or tumor cells, even of the minimal deviation type.

Physicochemical characterization of a new glucocorticoid receptor.

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 micrograms/100 g body wt.), while the 'classic' glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specifically to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25 degrees C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000-100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.34 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the 'classic' glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.

A new glucocorticoid receptor species: relation to induction of tryptophan dioxygenase by glucocorticoids.

When rats were treated with a high dose (greater than or equal to 20 micrograms/100 g BW) of dexamethasone, their liver cytosol showed a predominant peak of specific binding protein of glucocorticoid eluting with 0.13-0.14 M NaCl on diethylaminoethyl cellulose chromatography. On the other hand, when rats were treated with a low dose (approximately 2 micrograms/100 g BW) of dexamethasone, the cytosol showed only the same peak as that of untreated rats, which is widely thought to be that of the typical glucocorticoid receptor. The appearance of the new binding peak depended both on the dose of hormone and the time (from 30 min to 20 h) after dexamethasone treatment; it disappeared 40 h after treatment. A peak similar to that in the cytosol also appeared in the nuclear fraction after treatment with a high dose of hormone. The glucocorticoid-inducible enzymes, tryptophan dioxygenase [(TO) EC 1.13.11.11] and tyrosine aminotransferase (EC 2.6.1.5) were assayed as physiological markers of receptor function. TO induction correlated well with the appearance of the new binding peak in terms of dose- and time dependence on glucocorticoid, whereas tyrosine aminotransferase induction did not. The new peak of glucocorticoid-binding protein detected in the cytosol and nuclear fractions may thus represent the glucocorticoid receptor species involved in TO induction, physiological changes under stress, and pharmacological changes after therapy with high doses of glucocorticoid hormones.

Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor.

When young rats (less than 14 days old) were treated once a day for 2 days with 100 micrograms/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 micrograms/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.

Appearance of new hepatic glucocorticoid binding proteins under various stressful conditions: relation to endogenous glucocorticoid secretion.

When rats were subjected to the stress of burns, tumors, or partial hepatectomy, a notable new peak of glucocorticoid binding protein appeared on DEAE-cellulose chromatography. This peak accompanied the original peak, which was the only dominant peak in intact rats. The appearance of the new binding protein was concomitant with a high rise in serum corticosterone levels. The new peak was eluted with 0.12-0.14 M NaCl and another, small new peak with 0.02-0.03 M NaCl, while the original peak of intact rats was eluted with 0.05-0.08 M NaCl. In rats adrenalectomized prior to the stress, the new peaks did not appear. To mimic these stressful conditions which provoked a burst of endogenous glucocorticoid, rats were administered with an exogenous high dose of dexamethasone (100 micrograms/100 g B.W.) in vivo. The new peak eluted with 0.12-0.14 M NaCl was again observed and was more dominant in the hormone-treated rats than the stressed rats. These three peaks eluted with 0.02-0.03 M, 0.05-0.08 M, and 0.12-0.14 M NaCl are called here Peak A, B, and C, respectively. This is the first demonstration of the effect of physiological changes in serum levels of glucocorticoid hormone on the nature of glucocorticoid binding protein by DEAE-cellulose chromatography.

Studies on antianaphylactic agents. 7. Synthesis of antiallergic 5-oxo-5H-[1]benzopyrano[2,3-b]pyridines.

5-Oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carboxylic acids 23 and their tetrazole analogues 24 were synthesized from 4-oxo-4H-1-benzopyran-3-carbonitriles 3 or 2-amino-4-oxo-4H-1-benzopyran-3-carboxaldehydes 4. When administered intravenously, they exhibited antiallergic activity in a reaginic PCA test in rats. In the carboxylic acid series, the activity was influenced by the substituents at the 2-position and increased substantially in the following order: Me, OMe less than NH2 less than OH, H less than NHOMe. On the other hand, in the tetrazole series, 2-unsubstituted derivatives showed the highest activity. Regardless of the kinds of substituents at positions 2 and 3, compounds bearing an alkyl group, especially an isopropyl group at the 7-position, were superior in activity to the corresponding unsubstituted compounds. Among these alkyl derivatives, 3-carboxylic acid derivatives, i.e., 23c (7-ethyl), 23g (2-amino-7-isopropyl), 23r [2-(methoxyamino)-7-isopropyl], and a 3-tetrazole derivative 24c (7-isopropyl), were 41-184 times as potent as disodium cromoglycate. They also exhibited remarkable activity when administered orally; clinical studies on 23g (AA-673) are in progress.

Studies on antianaphylactic agents. 6. Synthesis of some metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone and their analogues.

The metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone (AA-344) (1), an orally effective antiallergic agent, and their analogues were synthesized to confirm the proposed structures and to determine their activity in the rat passive cutaneous anaphylaxis (PCA) test. A glucuronic acid metabolite (6) was assigned the structure 24b, 1-deoxy-1-[5-(6-ethylchromon-3-yl)tetrazol-1-yl]-beta-D-glucopyranuronate, by the comparison of 13C NMR, mass spectra, and TLC of isomeric compounds. In 13C NMR spectra, the shift difference of the tetrazole ring carbons between a pair of isomers was more remarkable than that of the glycosidic carbons. Therefore, the former is a useful criterion for distinguishing between such isomers. Some of the metabolities and analogues were active when administered intravenously, and two metabolites (2 and 3) were also effective upon oral administration.

Hybrid isozymes of rat pyruvate kinase. Their subunit structure and developmental changes in the liver.

Thin-layer polyacrylamide gel electrophoresis of various rat tissues revealed three major isozymes (types L, M1 and M2) and various intermediate forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). In vitro dissociation and reassociation of purified enzymes showed that the three major isozymes had homotetrameric structures. L.M2 hybrids and M1.M2 hybrids closely resembled some naturally occurring intermediates; the subunit structure of intermediates isolated from the small intestine (form 3 or form 4) were estimated to be (L)2(M2)2 and (L)(M2)3, respectively. Pyruvate kinase activity after electrophoresis could be estimated quantitatively from densitometric measurements of the electrophoretic pattern. Type L activity in fetal liver was separated from type R activity derived from intrahepatic erythropoietic cells. It changes in three distinct steps during development: it increased during the late fetal period, remained steady during the neonatal period and increased again after weaning. Some of the intermediates found in extracts of early fetal iver were shown to cross-react with both anti-L and anti-M1 serum, suggesting that they might be L.M2 or R.M2 hybrids. These hybrid enzymes were shown to appear only during early fetal and neonatal periods.

Studies on antianaphylactic agents. 5. Synthesis of 3-(1H-tetrazol-5-yl)chromones, a new series of antiallergic substances.

A number of 3-(1H-tetrazol-5-yl)chromones were synthesized and found to have antiallergic activity in the rat passive cutaneous anaphylaxis (PCA) test. These compounds are active when administered orally in rats and of possible value for the treatment of asthma.