Proliferative response of mouse submandibular gland to androgen but not to thyroid hormone

Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and 3H-thymidine labelling index, respectively. Injections of 5 alpha-dihydrotestosterone (4 micrograms/g body weight/day) or L-thyroxine (0.5 microgram/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5 alpha-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of L-thyroxine for any duration did not induce a significant increase in the labelling index. The present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Regulation of trypsin-like esteroprotease synthesis by 5 alpha-dihydrotestosterone and triiodothyronine in mouse submandibular gland.

The hormonal regulation of trypsin-like esteroprotease synthesis in mouse submandibular gland was studied at the isozyme level. Antiserum to a mixture of two purified esteroproteases precipitated all the esteroproteases in a crude extract of this gland. Measurement of incorporation of [3H]leucine showed that total esteroprotease synthesis was stimulated by both 5 alpha-dihydrotestosterone and triiodothyronine and that the two hormones had synergistic effects. The observed correlation between the increases of synthetic rate and specific activity of this enzyme suggests that the enzyme level is regulated mainly by the rate of enzyme synthesis. Newly synthesized esteroprotease-antibody complexes gave four peaks of radioactivity with esteroprotease activity and one peak without enzyme activity on isoelectric focusing in acrylamide gel containing 8 M urea. The radioactivities of these five peaks were increased similarly by the two hormones separately or in a combination. These results suggest that the actions of androgens and thyroid homrones in esteroprotease synthesis are indistinguishable at the isozyme level.     

Precocious differentiation of mouse parotid glands and pancreas induced by hormones

Changes of alpha-amylase activity (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland and pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induced by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone is involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland.

The effects of 5 alpha-dihydrotestosterone (DHT) and thyroxine (T4) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administration of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T4 increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T4 in adrenalectomized mice suggests that T4 has a direct effect on the submandibular gland.     

Precocious differentation of mouse parotid glands and pancreas induced by hormones

Changes of α-amylase activity (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) in mouse parotid gland pancreas were investigated during embryonic and postnatal development. Amylase activity in the parotid gland increased from around day 12 and reached the adult level on day 30. On the other hand, the activity in the pancreas increased during the last stage of gestation, decreased after birth, and then gradually increased from around day 15, reaching the adult level on day 35. Precocious differentiation of the parotid gland was induceed by injections of hydrocortisone or thyroxine after birth, but these hormones did not have additive effects on the parotid gland. Injection of insulin had little effect when given alone, but suppressed the effects of the other two hormones on the gland. Only hydrocortisone increased the amylase activity in mouse pancreas during postnatal development, the other two hormones causing slight decrease in pancreatic amylase. Adrenalectomy and injection of hydrocortisone affected the parotid gland but not the pancreas of adult mice. These results suggest that hydrocortisone involved in cytodifferentiations of the parotid gland and pancreas, and in maintenance of the parotid gland. Thyroxine may also be important in differentiation of the parotid gland.     

Precocious induction of pepsinogen in the stomach of suckling mice by hormones

Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5–10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15–20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23.These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.     

Dexamethasone and glucagon cause synergistic increases of urea cycle enzyme activities in livers of normal but not adrenalectomized rats.

Adrenalectomized and intact rats were given constant high-dose infusions of glucagon, 0.3 mg/kg per day for 7 days, with or without low-dose dexamethasone, 0.01 mg/kg daily, to test whether glucocorticoids potentiate glucagon induction of the 5 urea cycle enzymes as they do in cultured rat hepatocytes. Glucagon did not induce any of the urea cycle enzymes in adrenalectomized Sprague-Dawley rats and only induced argininosuccinate lyase (EC 4.3.2.1) in adrenalectomized inbred Wistar-Furth rats. Dexamethasone alone induced arginase in adrenalectomized and in intact Wistar-Furth rats and restored the other enzymes to normal levels in adrenalectomized rats. In intact Wistar-Furth rats, the combination of hormones gave synergistic increases of all 5 enzymes over the responses to each hormone alone, but in adrenalectomized rats the combination was only additive or less than additive compared with the sum of single hormone responses. The lack of synergism between the two hormones in adrenalectomized rats suggest that other factors play a role in glucagon induction of this cycle.     

Proliferative Response of Mouse Submandibular Gland to Androgen But Not to Thyroid Hormone

Abstract. Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and ³H-thymidine labelling index, respectively. Injections of 5α-dihydrotestosterone (4 μg/g body weight/day) or l -thyroxine (0.5 μg/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5α-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of l -thyroxine for any duration did not induce a significant increase in the labelling index. the present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

The action of 5 alpha-dihydrotestosterone and antiandrogens on the activities of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the pituitary gland of gonadectomized rats

In gonadectomized rats of either sex s.c. administration of 5 alpha-dihydrotestosterone (DHT) reversed, in a dose dependent manner, effects brought about by gonadectomy: it decreased pituitary wet weight and serum levels of LH and FSH and suppressed microsomal enzyme activities involved in testosterone and progesterone metabolism in the pituitary gland, NADPH-linked 5 alpha-reductase and NADH-linked 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH). Concomitantly administered nonsteroidal antiandrogen, flutamide (5 mg/day), antagonized some of the suppressive effects induced by a 14-day treatment of gonadectomized rats with high dose (1 mg/day) of DHT. It completely blocked DHT action on pituitary 5 alpha-reductase activity in the female rat and, in the male, inhibition was found to be 30-35%. In male, but not female rats, it completely blocked DHT suppression of serum FSH level whereas it slightly, but significantly inhibited DHT suppression of serum LH in rats of either sex. However, flutamide did not prevent DHT suppression of pituitary wet weight or NADH-linked 3 alpha-HSDH activity. Concomitantly administered progestational antiandrogen, cyproterone acetate (5 mg/day), inhibited DHT-induced weight increase of seminal vesicles by 50-55% and completely blocked the weight decrease of pituitary gland but did not antagonize DHT suppression of serum gonadotropins or pituitary enzyme activities. The results obtained with flutamide suggest that DHT-induced suppression of pituitary NADPH-linked 5 alpha-reductase, but not NADH-linked 3 alpha-HSDH activity, might involve an androgen receptor mechanism.     

Comparative study of the effect of hydrocortisone and thyroxine on suckling mouse small intestine in organ culture

The influence of hydrocortisone (10(-8)--10(-5) M) and thyroxine (10 (-9)--10(-6) M) on intestinal epithelial cell differentiation and proliferation have been studied using explants of suckling mouse jejunum maintained in serum-free organ culture. Hydrocortisone induced the appearance of sucrase activity and increased trehalase, glucoamylase, lactase and alkaline phosphatase activities. Thyroxine was completely ineffective at all the concentrations used. None of these hormones affected the mitotic activity or the 3H-thymidine incorporation into DNA. These results demonstrate that hydrocortisone but not thyroxine acts directly on intestinal brush border membrane differentiation and that both hormones do not influence the proliferation of the epithelial cells during postnatal development.     

Hormonal activation of adenylate cyclase and its role in cyclic AMP mediated regulation of RNA synthesis in the mouse mammary gland.

The activity of adenylate cyclase (EC 4.6.1.1) in the mouse mammary gland increases during late pregnancy and reaches its maximum value at one day pre partum. In the mouse mammary gland explant culture the adenylate cyclase activity is stimulated by a cooperative action of insulin, prolactin and hydrocortisone. The effect of these hormones can be demonstrated in intact cells, but not in a cell-free system. In the explants, RNA synthesis is stimulated by dibutyryl cyclic AMP, insulin and prolactin. The effects of both protein hormones and cyclic AMP are additive. The results obtained suggest that insulin and prolactin in cooperation with hydrocortisone are involved in the regulation of RNA synthesis in the mammary gland by activation of the adenylate cyclase system, independently of their effect on this process not mediated by cyclic AMP.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.     

Comparison of the properties of L-threonine dehydratase isolated from the livers of intact, adrenalectomized or cortisol-treated rats]

L-Threonine dehydratase preparations were isolated from liver of intact, treated with hydrocortisone and adrenalectomized rats. These preparations had different properties in stability, sensitivity to proteases and kinetic patterns. The preparations possessed also serine dehydratase activity, and the ratio threonine: serine activities was modified during the procedure of enzyme purification. It appears that the hormones affect not only the amount of enzyme proteins, but the qualitative properties of these proteins.     

Effect of ACTH and its fragment on brain catecholamines in intact and adrenalectomized rats.

Brain catecholamine metabolism was monitored by distribution of labelled noradrenaline (3H-NA) after intraventricular injection to intact and adrenalectomized rats. The adrenalectomy produced an increased disappearance rate of the labelled pool in the hypothalamus, hippocampus and neocortex. These changes could be prevented by hydrocortisone pretreatment. Painful stimuli resulted in an increased disappearance of the labelled pool in both intact and adrenalectomized rats. The implantation of hydrocortisone into the tuberoinfundibular region prevented the stress-induced changes of the catecholamine metabolism. Intraventricular administration of ACTH1-24 and ACTH4-10 produced a significant increase of the disappearance rate in different brain regions of adrenalectomized rats. The blocking of catecholamine synthesis by intraventricular injection of alpha-methyl-m-tyrosine resulted in a marked decrease of the labelled pool but did not prevent the ACTH-induced decrease of the tracer pool. On the other hand, the blocking of monoamine-oxydase activity by Pargyline led to a marked increase of the labelled pool but intraventricular administration of ACTH led to an increase of the disappearance rate. The mechanism of ACTH action on brain catecholamine metabolism is still obscure, however, an increased release of the NA to ACTH peptides is very likely in the light of the present observations.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.