Adrenals Of Hypophysectomized Rats Research Articles

Induction of tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-mediated mechanism in immobilization stress.

Stress stimulates the sympathoadrenal system, causing activation of the catecholamine biosynthetic enzymes. Here we examine the changes of gene expression of tyrosine hydroxylase (TH; EC 1.14.16.2), the initial enzyme of catecholamine biosynthesis, with stress. A single immobilization of rats led to a large transient elevation in TH mRNA and a small elevation in TH immunoreactive protein and activity. Repeated daily immobilizations triggered more sustained changes in TH mRNA levels. After two immobilizations, the levels remained elevated even 3 days later. The rise in TH mRNA was followed by increased immunoreactive protein but only a small elevation in activity. With seven repeated immobilizations, the animals did not appear to adapt and still manifested a further rise in TH mRNA. TH activity was markedly elevated and returned to control levels 7 days after the immobilization. The rise in TH mRNA with a single immobilization occurred even in adrenals of hypophysectomized rats that underwent splanchnic nerve section. Immobilization for 30 min was sufficient to increase TH mRNA. The effect was abolished by the transcriptional inhibitor actinomycin D. Mobility gel-shift assays revealed increased binding of c-Fos and c-Jun to the AP-1 transcription factor site after a single immobilization, and the binding was not further elevated with repeated stress. This study shows that a single immobilization can activate TH gene expression by a nonneuronal nonpituitary-mediated pathway associated with increased binding of AP-1 transcription factors.

ACTH-induced mitochondrial DBI receptor (MDR) and diazepam binding inhibitor (DBI) expression in adrenals of hypophysectomized rats is not cause-effect related to its immediate steroidogenic action

Diazepan binding inhibitor (DBI) is a 10-kDa polypeptide that is enriched in steroidogenic cells such as adrenocortical, Leydig, and glial cells. In these cells, DBI and some of its processing products bind to the mitochondrial DBI receptor (MDR), located on the outer mitochondrial membrane, and stimulate pregnenolone formation by facilitating cholesterol access to the inner mitochondrial membrane where the cytochrome P-450 side chain cleavage enzyme is located. To determine whether the ACTH-induced increase in adrenal steroidogenesis occurs via changes in DBI and MDR expression the adrenal content of DBI-like immunoreactivity (DBI-LI), the MDR density, and the expression of mRNAs encoding for DBI and MDR were studied in hypophysectomized rats treated with vehicle or ACTH. After 9 days from the hypophysectomy, the levels of DBI-like immunoreactivity (DBI-LI) and DBI-mRNA declined to approximately 20% of their normal value; in contrast MDR-density and MDR-mRNA levels were reduced by 50–60% and were associated to a similar decrease in the activity of type A monoamine oxidase, a marker for mitochondrial proteins. Prolonged administration of ACTH-R (ACTH in saline containing 16% gelatin, 15 U/kg/day, from day 7 after surgery) to hypophysectomized rats, completely restored DBI and MDR adrenal expression to values similar to those of sham-operated rats. Our results indicate that ACTH, probably acting at the transcriptional level, is required for the normal expression of DBI and MDR in adrenal cortex. Changes in DBI and MDR expression after ACTH administration were not temporally related to the immediate steroidogenesis induced by ACTH, and may reflect its long-term trophic action on adrenocortical cells.

Diazepam-binding inhibitor (DBI)-processing products, acting at the mitochondrial DBI receptor, mediate adrenocorticotropic hormone-induced steroidogenesis in rat adrenal gland.

Diazepam-binding inhibitor (DBI) is a 9-kDa polypeptide that colocalizes in glial, adrenocortical, and Leydig cells with the mitochondrial DBI receptor (MDR). By binding with high affinity to the MDR, DBI and one of its processing products--DBI-(17-50)--regulate pregnenolone synthesis and have been suggested to participate in the immediate activation of adrenal steroidogenesis by adrenocorticotropic hormone (ACTH). In adrenals of hypophysectomized rats (1 day after surgery), ACTH failed to acutely affect the amount of adrenal DBI and the density of MDR but increased the rate of DBI processing, as determined by the HPLC profile of DBI-(17-50)-like immunoreactivity. The similar latency times for this effect and for ACTH stimulation of adrenal steroidogenesis suggest that the two processes are related. The ACTH-induced increase in both adrenal steroidogenesis and rate of DBI processing were completely inhibited by cycloheximide; this result suggests the requirement for the de novo synthesis of a protein with a short half-life, probably an endopeptidase. This enzyme, under the influence of ACTH, may activate formation of a DBI-processing product that stimulates steroidogenesis via the MDR. In support of this hypothesis is the demonstration that in hypophysectomized rats the MDR antagonist PK 11195 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide completely inhibited the adrenal steroidogenesis stimulated by ACTH and by the high-affinity MDR ligand 4'-chlorodiazepam.

Gamma 3-melanotropin promotes mitochondrial cholesterol accumulation in the rat adrenal cortex

Gamma 3-melanotropin (γ 3-MSH) facilitates a rapid, dose-dependent, and cycloheximide-insensitive increase in the concentration of mitochondrial free cholesterol in the adrenals of hypophysectomized rats. Physiological concentrations of various synthetic and native preparations of γ 3-MSH are potent, while γ-MSH is not. This cholesterol accumulation coincides with the activation of cholesteryl ester hydrolysis by γ 3-MSH, while the rates of cholesterol esterification and of mitochondrial cholesterol side-chain cleavage are unaffected. Conversely, ACTH inhibits cholesterol esterification. Therefore, γ 3-MSH and ACTH together may coordinate a substantial shift in the set-point of cholesteryl ester ag cholesterol cycling toward the right. Because ACTH also activates cholesterol side-chain cleavage, this coordinate effect on the flux of cholesterol substrate is manifest as a potentiation of Corticosteroidogenesis by γ 3-MSH. These data extend our previous studies demonstrating that pro-y-MSH polypeptides have an endocrine influence on the rat adrenal cortex.

Reversal of prostaglandin E2 effect on adrenal catecholamine release after hypophysectomy.

Prostaglandin E2 induced increased catecholamine release in vitro from adrenals of hypophysectomized rats, while in adrenals of intact rats catecholamine release was suppressed by prostaglandin E2.

Isolated adrenal cortex cells: hypersensitivity to adrenocorticotropic hormone after hypophysectomy.

Cells from the adrenals of hypophysectomized rats (up to 28 days after operation) require less adrenocorticotropic hormone to induce one-half maximal rate of production of 3',5'-adenosine monophosphate than do cells from the adrenals of intact rats. A corresponding increase in sensitivity is reflected in the steroidogenic response to adrenocorticotropic hormone up to 2 days after hypophysectomy.

Effect of ACTH and dibutyryl cyclic AMP on catecholamine synthesizing enzymes in the adrenals of hypophysectomized rats.

MAINTENANCE of normal levels of adrenal medullary enzymes concerned with the synthesis of catecholamines requires an intact pituitary–adrenocortical system. Wurtman and Axelrod1,2 showed that after hypophysectomy there is a striking decrease in adrenal levels of phenylethanolamine-N-methyl transferase (PNMT), the enzyme which converts noradrenaline to adrenaline. This decrease is prevented or reversed by treatment with ACTH or glucocorticoids. Dopamine-β-hydroxylase, which is responsible for the formation of noradrenaline from dopamine, was shown by Kvetňanský et al.3 to be decreased in the adrenal glands of hypophysectomized rats; levels of this enzyme are also restored by treatment with ACTH4 or dexamethasone (Gewirtz et al., unpublished observations). Tyrosine hydroxylase, the enzyme which converts tyrosine to 3,4-dihydroxyphenylalanine (DOPA), is also diminished in the adrenals of hypophysectomized rats and restored by ACTH treatment3,5. Unlike those of dopamine-β-hydroxylase and PNMT, however, tyrosine hydroxylase levels are not increased by treatment with dexamethasone5. Thus ACTH seems to have an effect on adrenal tyrosine hydroxylase which is not mediated by corticosteroids.

Acute effects of adrenocorticotropin on the levels of various lipids in the adrenals of hypophysectomized rats.

Male rats that were hypophysec-tomized 24 hr previously were injected iv with 100 mU or 10 U of ACTH. At brief intervals following the injection of the hormone, adrenal venous blood was collected to determine the rate of corticosterone secretion. Then the adrenals were excised, frozen, and subsequently extracted to obtain total lipids. The extracts were then analyzed to determine the concentrations of free and esterified cholesterol, triglycerides, free fatty acids and phospholipids in the adrenals. At 5, 10, 60 and 180 min after the injection of 100 mU of ACTH, no change occurred in the levels of any of the adrenal lipids examined, although the hormone produced the expected early rise in secretion of corticosterone. Again, with the larger dose of ACTH (10 U), no change occurred in the levels of adrenal lipids 60 min after the injection of the hormone. In contrast, 180 min after the administration of 10 U of ACTH, the concentrations of both free and esterified cholesterol decreased significantly (10 and 4...

Effect of adrenocorticotropin on the concentration of triglyceride in the adrenal gland of the hypophysectomized rat.

The concentration of triglyceride in the adrenal gland, measured by the method of Van Handel and Zilversmit, was found to be 2.2-3.2 mg/100 mg fresh tissue in the normal rat; this value increased to 3.5-4.8 mg/100 mg tissue 24-48 hr after hypophysectomy. Injection of 0.1-10 U of adrenocorticotropin in the rat 24-48 hr after hypophysectomy caused a 20-45% reduction in adrenal triglyceride concentration. This effect was apparent 3 hr after injection and persisted for at least 6 hr. {Endocrinology 78:1087, 1966) H observations first revealed that the adrenal cortex contains an abundant quantity of lipid, which is depleted by a single injection of adrenocorticotropin (ACTH) or by a variety of acute noxious stimuli (1). The chemical nature of the cortical lipid thus depleted has been the object of numerous studies, which agree that adrenal cholesterol esters are reduced by an adrenocorticotropic stimulus and that adrenal phospholipid concentration remains unchanged (1-4). Adrenal triglyceride content, however, has been reported increased (4), unchanged (3), or decreased (2) in animals subjected to acute stressful stimuli; these discordant data were obtained with indirect methods for calculating triglyceride content, since a specific analytic method for this lipid was not available until recently. The introduction by Van Handel and Zilversmit (5) of a direct method for determining triglyceride, and recognition of the influence of ACTH on the triglyceride metabolism of the fat cell, have led us to re-examine the possible effect of ACTH on the triglyceride content of the rat's adrenal gland. Materials and Methods Intact and hypophysectomized male Wistar rats weighing 250-260 g were obtained from Charles River Laboratories and were fed Purina Laboratory Checkers ad lib. The experiments were begun 2448 hr after hypophysectomy. Intact and hypophysectomized rats were injected subcutaneously with 0.5 ml normal saline or with 0.1-10 U clinical porcine ACTH (Wilson Laboratories) in a volume of 0.5 ml. At stated periods thereafter, the animals were killed by cervical fracture, the adrenal glands Received December 10,1965. Supported by USPHS Grants AM-06056, HE05741 and HE-00052, and by Grant U-1562 from the Health Research Council of New York City. 1 Career Scientist of the Health Research Council of New York City under Contract 1-118. were dissected free of extraneous tissue under a dissecting microscope, weighed and homogenized in chloroform/methanol 2 :1 (20 ml of solvent/pair of adrenals). The chloroform /methanol extract was washed according to Folch et al. (6) and then was analyzed for triglyceride by the method of Van Handel and Zilversmit (6), and for total cholesterol by the method of Abell et al. (7). Results The triglyceride concentration in the adrenal glands of normal rats ranged between 2.2 and 3.2 mg/100 mg fresh tissue, while the concentration in the adrenals of hypophysectomized rats (not treated with ACTH) ranged between 3.5 and 4.8 mg/100 mg tissue; the difference between these 2 groups was statistically significant (p <.025) in each of 3 experiments (Table 1). Injection of 10 U ACTH in the hypophysectomized rat caused a statistically significant (p <.01) reduction in adrenal triglyceride concentration 6 hr later; this decrease ranged between 33 and 44% of the preinjection concentration (Expt. 1, 2, 3, Table 1). The magnitude of the reduction in triglyceride concentration was proportional to the dose of ACTH in the range 0.1-10 U of hormone (Expt. 3, Table 1); following 0.1 U, however, the triglyceride decrease was of doubtful statistical significance. The depletion of adrenal triglyceride produced by 0.1-10 U of ACTH was associated with a parallel decline in adrenal cholesterol content (Expt. 3, Table 1), as originally described by Sayers and colleagues (1). The reduction in adrenal triglyceride following injection of 10 U ACTH was apparent at 3 hr and persisted for at least 6 hr (Table 2). Discussion These data demonstrate that ACTH reduces the concentration not only of cholesterol, but also of triglyceride, in the adrenal gland of the hypophysectomized rat.

Advantage of the triplet crossover design in the intravenous assay of corticotrophin (Sayers test).

The most sensitive and specific assay method for corticotrophin is still that which uses the fall of ascorbic acid concentration in the adrenals of hypophysectomized rats as the response to intravenously injected corticotrophin,* as described and introduced by Sayers et al. (1948). This method, however, has a relatively low precision due to a high experimental variance of the responses between animals. Data have been published from several laboratories (Sayers et al., 1948, Loraine, 1957, Rerup, 1958) concerning the index of precision of the method, λ, which ranges from 0.18 at Sayers laboratory to 0.5 found by Greenspan (1950). The reason for these widely different degrees of precision may be ascribed to the use of different strains of animals being more or less homogeneous genetically thus causing different response variances between animals, or being more or less sensitive to a given increase in successive doses resulting in differences in the slope

THE BIOASSAY OF CORTICOTROPHIN A

The adrenocorticotrophic hormone of the anterior pituitary gland may be assayed by many methods, of which the following have obtained wider acceptance: The adrenal repair method (Simpson, Evans, and Li, 1943; Sayers, White, and Long, 1943), in which the potency of a hormone sample is measured in terms of its ability to restore to normal the size and histology of the atrophied adrenals in hypophysectomized rats. The adrenal maintenance method (Simpson, Evans, and Li, 1943; Sayers, White, and Long, 1943), in which the potency of a sample is measured in terms of its ability to prevent adrenal atrophy after hypophysectomy in rats. The adrenal ascorbic acid depletion method (Sayers test) (Sayers et al., 1948), in which the potency is determined from the decrease in the amount of ascorbic acid present in the adrenals of hypophysectomized rats after intraveneous injection of the hormone. The Munson modification (Munson et al., 1948) of

The subcutaneous assay of corticotrophin A.

Since the introduction of corticotrophin (ACTH) into therapy in 1949 the hormone has usually been assayed by the ascorbic acid depletion method of Sayers et al. (1948), in which the fall in the concentration of ascorbic acid in the adrenals of hypophysectomized rats is a linear function of the logarithm of the dose ACTH, given intravenously. In therapy, however, corticotrophin is, as a rule, given subcutaneously or intramuscularly and only occasionally intravenously as a continuous infusion. In recent years clinical and experimental observations were made which demonstrated that the expected potency of ACTH-preparations did not always equal their clinical effect, when injected extravascularly, i. e. subcutaneously or intramuscularly. The therapeutic value of corticotrophin preparations obtained by different chemical procedures varied from 25 % to about 300 % of that expected from the determination of potency by the ascorbic acid depletion method. However, if used for continuous intravenous infusion, no variation in the clinical effect from the labelled potency greater than that within the error of the assay itself could be observed. These findings are extensively discussed by Wolfson (1953) and Fisher et al. (1953). Wolfson distinguishes three main types of corticotrophin: Crude corticotrophin (the first international corticotrophin standard La-l-A; the U. S. P. corticotrophin reference standard), which chemically is a glacial acetic acid extract of anterior pituitary of mammals. Corticotrophin A (ACTX, purified corticotrophin, high-potency corticotrophin, Type I-purified ACTH of Thompson and Fisher), which chemically is crude corticotrophin purified by adsorption on oxycellulose and subjected to little or no acid or peptic hydrolysis. Corticotrophin B (Actide corticotrophin), which is prepared by the use of acid or peptic hydrolysis or both.

“IN VIVO” AND “IN VITRO” EFFECT OF ASCORBIC ACID AND ACTH ON THE RAT ADRENAL CORTEX

Histochemical examination of adrenal glands of hypophysectomized rats given both ascorbic acid and ACTH showed an enlargement of the cortex and a decrease of sudanophilic substances, as compared to adrenals of hypophysectomized rats receiving ACTH alone. “In vitro” experiments on incubated slices of adrenal glands have shown that ascorbic acid and ACTH have a synergistic effect on the secretory activity of the cells of the adrenal cortex.

EFFECT OF SOME ANDROGENIC STEROIDS ON THE ADRENAL CORTEX OF HYPOPHYSECTOMIZED RATS1

MAINTENANCE and stimulation of the adrenal cortex of hypophysectomized animals by hormones from sources other than the pituitary gland have been reported. Testosterone propionate decreased the rate of adrenal cortical atrophy which normally follows hypophysectomy in the young male rat (Cutuly, Cutuly, and McCullagh, 1938), (Leonard, 1942), (Leatham).2 Thyroxin, insulin and non-hormonal toxic agents stimulated the adrenal cortex of hypophysectomized pigeons (Miller and Riddle, 1942) and the gonadotrophic principle from pregnant mare serum stimulated the adrenals of hypophysectomized ground squirrels (Zalesky, Wells, and Overholser, 1942). In view of these findings, the effects of androgens on the adrenals of hypophysectomized rats were investigated. This report is concerned with (a) the effects of different dosages of testosterone propionate and other androgens on the maintenance of adrenal weight in hypophysectomized rats, and (b) the nature of this stimulation of the adrenal cortical cells. Long-Evans ra...

PREPARATION OF ADRENOTROPIC EXTRACTS AND THEIR ASSAY ON TWO-DAY CHICKS11

IT is WELL KNOWN that the adrenal cortex shows enlargement and increased func-tion following various experimental procedures and treatments. It is also certain that marked cortical atrophy follows hypophysectomy. Many investigators be-lieve that a specific anterior pituitary (adrenotropic) hormone normally supports cortical tissue and its functions, though Grollman (i), Fazekas (a) and others have questioned the existence of a specific adrenotropic hormone. The first qualitative and quantitative studies in this field were largely based on histological changes in the adrenal cortex. An historical review of the earlier studies was made by Collip (3).Reiss (4) described a specific Sudanophobic zone which appears in adrenals of hypophysectomized rats and which disappears upon treatment with hypophyseal adrenotropic extracts. In 1937 Moon (5) described a methodof assay of adrenotropic hormone which utilizes increase of adrenal weight in ai-day rats.

Quantitative study on the adrenals of hypophysectomized rats

Quantitative study on the adrenals of hypophysectomized rats