Opposite effects of dexamethasone and ACTHon the adrenal cortex response to ethane dimethanesulphonate (EDS)

Recently we have reported that ethane dimethane-sulphonate (EDS), the Leydig cell cytotoxin, caused marked atrophy of the adrenal cortex of adult male rats. The aim of this work was to examine whether a 9-day treatment with dexamethasone (0.25 mg/kg/d) or ACTH (40 IU/kg/d), which started 4 days prior to administration of a single dose of EDS (75 mg/kg), influenced the response of the inner adrenocortical zones to the toxin. On day 15 after administration of EDS, adrenal weight was significantly decreased in saline treated rats, but glandular and serum corticosterone levels were not altered. In dexamethasone-suppressed rats, the effect of EDS was augmented; an additional decrease in adrenal weight was accompanied by reduced adrenal and serum corticosterone levels. In ACTH-treated animals EDS was ineffective. These results demonstrate that the deleterious effects of EDS on rat adrenal cortex can be prevented by ACTH and potentiated by dexamethasone.     

Nucleolar segregation as an early marker for DNA damage; an experimental study in rats treated with 4-hydroxyaminoquinoline 1-oxide

Male 6-week-old Sprague Dawley rats were given a single intravenous injection of 4-hydroxyamino-quinoline 1-oxide (4HAQO) at a dose of 20 mg/kg in order to produce ultrastructural changes as possible morphological biomarkers for toxicity. Immunohistochemically demonstrated formation of 4HAQO-DNA adduct was correlated with the changes found. Nucleolar alteration, demonstrable by electron microscopy as segregation of nucleolar components into granular and fibrillar compartments, was evident in cells of the target organs, exocrine pancreas and adrenocortex, but not of the non-target liver parenchyma. Sequential observation clarified that such alteration was highest in frequency 6 h and 4 h after 4HAQO administration in pancreatic acinar cells and adrenocortical cells respectively. Electron microscopically, apoptotic changes of acinar cells were evident 2 h after injection of 4HAQO. DNA adduct formation was consistently demonstrated in the same target organs showing nucleolar segregation, the highest frequency being noted 4 h after 4HAQO treatment in both pancreatic acinar cells and adrenocortical cells. Our results thus indicate an identity of the target cells for nucleolar segregation and 4HAQO-DNA adduct formation which correlates with 4HAQO-toxicity. We suggest that nucleolar segregation occurs subsequent to the generation of DNA damage.     

Body weight gain, food intake and adrenal development in chronic noise stressed rats

Wistar chronic treated rats (30 days) were used to investigate the effect of hypothalamic-pituitary-adrenal activity on growth, food intake and adrenal development (weight and DNA content). The animals were submitted to noise stress, ACTH administration and dexamethasone suppression test. Noise stress decreased body weight gain and food intake. No adrenal hypertrophy was observed but an increase in relative DNA content by stress has been found. ACTH and dexamethasone treated rats showed a body weight and food intake decrease vs. controls. The effect on body weight was higher in dexamethasone treated rats. Adrenal hypertrophy and hyperplasia were found in ACTH treated rats, whereas dexamethasone provoked adrenal atrophy with a decrease in DNA content.     

Modifications of adrenocortical responses following frontal cortex simulation in rats with hypothalamic deafferentations and medial forebrain bundle lesions

With the purpose of delineating the neural pathways in the rat which mediate adrenocortical responses following frontal cortex stimulation, the effects of partial hypothalamic deafferentations and medial forebrain bundle lesion were studied. In intact and sham-operated animals, cortical stimulation through permanently implanted electrodes caused a significant increase in plasma corticosterone levels. In rats with anterior hypothalamic deafferentation and bilateral medial forebrain bundle lesions the adrenal response to cortical stimulation was blocked completely, while in animals with posterior hypothalamic deafferentation there occurred a normal rise in plasma corticosterone. These studies demonstrate that the frontal cortex effects on adrenocortical secretion are neurally mediated and involve an anterior hypothalamic input, more specifically the medial forebrain bundle.     

Morphological correlates of hormone secretion in the rat adrenal cortex and the role of filopodia

Rat adrenals in different states of stimulation were examined by transmission electron microscopy following perfusion fixation using an in situ isolated-circulation technique. In unstimulated glands, intracortical capillaries were constricted and the cells of the cortex were pressed closely together with little development of filopodia or intercellular spaces. Glands fixed during the period of operative stress, or following a 1 hr perfusion with Adrenocorticotropic hormone (ACTH) showed that the radially orientated capillaries of the cortex were massively expanded, and the cells of both the glomerulosa and fasciculata exhibited an extensive development of filopodia on their surfaces. These filopodia extended into large intercellular spaces, where they often entered into complex relationships with filopodia from neighboring cells. The development of filopodia by cells of the adrenal cortex was also observed using scanning electron microscope techniques. In cells either incubated with ACTH in vitro or isolated from adrenals of rats treated with ACTH in vivo, the filopodia were numerous, often branched, and could reach as much as 1 micro m in length. In contrast, adrenal cells obtained from animals pretreated with cortisol were smooth surfaced. Other cell characteristics, including mitochondria, smooth endoplasmic reticulum, dense granules, and coated vesicles did not show such dramatic correlations with the state of stimulation. It is considered that the development of filopodia and intercellular space is related to secretory mechanisms in the rat adrenal cortex.     

Effects of early experience on the metabolism and production of corticosterone in rats.

Rats were handled for 3 min daily for 3 weeks before or after weaning or were left totally undisturbed. At 80 days of age, animals from each group were selected for corticosterone half-life determination. Adrenal tissue from thr remaining animals was incubated in vitro in the presence or absence of ACTH. Handled and unmanipulated animals did nto differ in half-life or in adrenocortical output. These data do not support the hypothesis that the reduced adrenocortical reactivity that results from manipulating animals is due to either the metabolsim of corticosterone or the response of the adrenal cortex to ACTH.     

Electron microscopic studies of the effect of ACTH and flavin-adenine dinucleotide on adrenocortical atrophy of rats treated with dexamethasone phosphate.

In a previous paper the authors described the morphologic observations that the concomitant administration of ACTH and flavin-adenine dinucleotide (FAD) to hypophysectomized rats exerted a more potent preventive effect on atrophy of the adrenal cortex of the animals than the single administration of ACTH. The present study was made to electron-microscopically observe the effect of concomitant administration of ACTH and FAD on atrophy of the adrenal cortex induced with the administration of dexamethasone (Dx). The zona fasciculata of the adrenal gland of rats treated with Dx+ACTH+FAD was morphologically closer in cell organelles such as smooth-surfaced endoplasmic reticulum, mitochondria and chylomicrons to that of control animals than the counterpart of animals treated with Dx+ACTH only. The zona fasciculata of the adrenal cortex of animals treated with Dx+FAD was morphologically similar to that of animals treated with Dx only. These findings suggested that FAD would potentiate the adrenocorticotropic action of ACTH through its physiologic action.     

ELECTRON MICROSCOPIC STUDIES OF THE EFFECT OF ACTH AND FLAVIN-ADENINE DINUCLEOTIDE ON ADRENOCORTICAL ATROPHY OF RATS TREATED WITH DEXAMETHASONE PHOSPHATE

In a previous paper the authors described the morphologic observations that the concomitant administration of ACTH and flavin-adenine dinucleotide (FAD) to hypophysectomized rats exerted a more potent preventive effect on atrophy of the adrenal cortex of the animals than the single administration of ACTH. The present study was made to electron-microscopically observe the effect of concomitant administration of ACTH and FAD on atrophy of the adrenal cortex induced with the administration of dexamethasone (Dx). The zona fasciculata of the adrenal gland of rats treated with Dx+ACTH+FAD was morphologically closer in cell organelles such as smooth-surfaced endoplasmic reticulum, mitochondria and chylomicrons to that of control animals than the counterpart of animals treated with Dx+ACTH only. The zona fasciculata of the adrenal cortex of animals treated with Dx+FAD was morphologically similar to that of animals treated with Dx only. These findings suggested that FAD would potentiate the adrenocorticotropic action of ACTH through its physiologic action.     

The effect of previous stimulation of the adrenal cortex by adrenocorticotrophin on the function of the pituitary—adrenocortical axis in response to stress

1. There appear to be at least two peripheral regulators affecting normal physiological adrenal function during repeated stimulation with adrenocorticotrophin (ACTH).2. There is a tachyphylaxis to ACTH if the stimulation of the adrenal gland is frequent. This may be due to alteration or saturation of the trapping mechanism for acceptance of ACTH by the tissue. There also may be inhibitors at the adrenal level. The results obtained could also be explained on the basis of end product inhibition by corticosterone on the process of synthesis or by the presence of a destructive enzyme for ACTH.3. If the amounts of ACTH released or injected are sufficiently great the gland is capable of synthesizing more steroid but adrenocortical dynamics do not allow an immediate release of the steroid into the peripheral circulation. Thereby high levels of steroid in the plasma are maintained for longer periods rather than increased.     

Rat adrenocortical dynamics.

1. The dynamics of the adrenocortical response to adrenocorticotrophic hormone (ACTH) was studied in anaesthetized, acutely hypophysectomized male rats. 2. ACTH test-signals were applied either in a jugular vein ('intact infused preparation') or in the aorta through the coeliac artery with the aorta ligated below this artery ('isolated in situ perfused preparation'). The adrenocortical responses were measured directly in samples serially taken from the left adrenal vein. 3. Tested ACTH signals were either impulses (injections of 0-05--1-2 mu. ACTH) or step functions (constant infusions of 0-025--1-6 mu. ACTH/min). 4. All responses showed a 3-6 min delay, larger delays corresponding to smaller input signals. The step responses reached steady-state level without overshoot. 5. The impulse responses of the isolated perfused and of the intact infused glands, as well as their step responses, were similar as to dynamic form. 6. It is concluded that there is an inherent delay in the responses of both the isolated perfused and of the intact infused rat adrenal gland. Further, unlike what has been reported for the canine adrenal gland, the intact rat adrenal gland does not appear to be appreciably 'faster' in its response than the isolated gland. Finally, the amplification factor from ACTH to corticosterone could be estimated for both preparations in the case of ACTH injections; a value of 1-3 X 10(6) (one ACTH molecule activates synthesis and release of 1-3 million corticosterone molecules) was found.     

Steroid production and membrane potential measurement in cells of the adrenal cortex

1. The relation between corticosteroid production and the cellular membrane potential of adrenocortical cells has been investigated using a flow system for the incubation in vitro of adrenal glands from neonatal rabbits. Corticosteroid production by the adrenal cells was determined by a fluorescence method; membrane potentials were measured with glass micro-electrodes.

2. Adrenocorticotrophic hormone (ACTH) increased corticosteroid production without altering the membrane potential of the adrenocortical cells. Conversely, a 5-fold increase in [K]_o did not increase corticosteroid production but decreased the cellular membrane potential. The increase in [K]_o did not impair the stimulation of corticosteroid production by ACTH.

3. The log dose—response relation between the concentration of ACTH in the incubation medium and the amount of corticosteroid produced by the adrenal tissue was linear.

4. No evidence was found of a direct relation between the production of corticosteroid by adrenocortical cells and the cellular membrane potential. Indeed, the output of steroid appeared to be relatively independent of the polarization of the cell membrane.

     

Adrenocortical Response Profiles to Corticotrophin-Releasing Hormone and Adrenocorticotrophin Challenge in the Chronically Catheterized Adult Guinea-Pig

The guinea-pig has been used extensively to investigate adrenal steroidogenesis. However, very little is known about adrenocortical responses to corticotrophin-releasing hormone (CRH) and adrenocorticotrophin (ACTH) in this species, in vivo. In the present study, we have developed a stress-free sampling system, in the chronically catheterized adult guinea-pig, that has allowed us to investigate basal and activated adrenocortical activity. Indwelling carotid artery and jugular vein catheters were surgically implanted into female guinea-pigs (n = 5). Each animal was treated with vehicle, human CRH (0.2 or 2 microg kg-1) and ACTH1-24 (0.2 or 2 microg kg-1), and serial plasma samples removed for analysis of ACTH and cortisol concentrations by radioimmunoassay. There was no effect of serial sampling on pituitary-adrenocortical activity, indicating that the animals remain in an unstressed state. Basal plasma ACTH and cortisol concentrations were 703.9 +/- 24.5 pg ml-1 and 117.9 +/- 5.2 ng ml-1, respectively. Both CRH and ACTH significantly increased adrenocortical activity in a dose-dependent manner. ACTH (2 microg kg-1) was the most potent activator leading to plasma cortisol concentrations of 647 +/- 116 ng ml-1. In conclusion, we have shown that basal plasma cortisol concentrations in the guinea-pig are low compared to those obtained in previous studies by cardiac puncture or following decapitation. However, plasma ACTH concentrations are high compared to other species. We have also shown that human CRH and ACTH1-24 act as potent activators of the guinea-pig pituitary-adrenocortical axis, leading to response profiles consistent with mild cortisol resistance.     

Different therapeutic efficacy of ketoconazole in patients with Cushing's syndrome

Summary The property of ketoconazole to inhibit adrenal biosynthesis of cortisol was used in a clinical study of 14 patients with Cushing's syndrome (pituitary-dependent Cushing's disease,n=10; adrenocortical adenoma,n=2; adrenocortical carcinoma,n=1; ectopic ACTH syndrome,n=1). Five patients were treated in a short-term manner (1000 mg over 24 h) and nine patients for a longer period (600 mg/die from 1 week up to 12 months). After short-term administration of ketoconazole, serum cortisol levels fell distinctly only in the patient with adrenocortical adenoma, but not at all or only slightly in the other patients, whereas serum levels of progesterone and 11-deoxy-compounds increased markedly in all patients, with the exception of the patient with adrenocortical carcinoma. Plasma ACTH levels increased in the patients with Cushing's disease but not in the patients with tumor. After long-term treatment of three patients with Cushing's disease over 3, 10, and 12 months, the clinical signs of hypercortisolism persisted or were only slightly ameliorated. In these three patients as well as in three other patients with Cushing's disease treated for a shorter period of 1 to 4 weeks, serum and urinary cortisol levels decreased, but were not normalized, whereas plasma ACTH levels increased variably. Only in one patient with Cushing's disease, in the second patient with adrenocortical adenoma, and in the patient with ectopic ACTH syndrome, serum and urinary cortisol levels returned to normal. We concluded from our data, that the antimycotic drug inhibits biosynthesis of cortisol by blocking adrenal 11- and 17-hydroxylase activity. This effect was compensated in part by a rebound increase of pituitary ACTH secretion in most patients with Cushing's disease. Therefore, ketoconazole treatment is above all effective in patients with Cushing's syndrome due to an adrenal tumor or in patients with ectopic ACTH syndrome, who cannot respond with an increased pituitary ACTH secretion.Abbreviations ACTH Adrenocorticotropic hormone - AA Adrenocortical adenoma - AC Adrenocortical carcinoma - B Corticosterone - CRH Corticotropin-releasing hormone - EAS Ectopic ACTH syndrome - PDCD Pituitary-dependent Cushing's disease - P Progesterone - 17OH-P 17-hydroxyprogesterone - RIA Radioimmunoassay - S 11-deoxycortisol - DOC 11-deoxycorticosterone     

Electron microscopic studies on the effect of ACTH and flavin adenine dinucleotide on adrenocortical atrophy of hypophysectomized rat.

Electron microscopic observation was made on the outer fasciculata cells in the adrenal cortex of hypophysectomized rats receiving 10 mg of FAD and/or 0.3 mg of ACTH intraperitoneally once a day for 5 consecutive days from 24 hours after hypophysectomy. The simultaneous administration of FAD and ACTH to the hypophysectomized rat was more effective for preventing adrenocortical atrophy induced than the administration of ACTH alone. This effect appeared as clear cells with low electron density. While the characteristics induced by hypophysectomy were the decrease in number of smooth-surfaced endoplasmic reticulum (SER) and mitochondria and also crista of mitochondria being tubular. The clear cells showed a less degree of their characteristic. From this fact, it is considered that the external FAD acts against fasciculata cells in the adrenal cortex of hypophysectomized rats as a coenzyme for flavin enzyme under ACTH and decreases oxidation-reaction in mitochondria and oxidative phosphorilation reaction in SER, being induced by hypophysectomy.     

微囊包膜肾上腺组织体外培养研究

为了解微囊包膜大鼠肾上腺组织体外培养存活状况,使用海藻酸钠、氯化钙及多聚-L-赖氨酸制成微囊包膜。取24只大鼠肾上腺组织,其中12只鼠的肾上腺组织用微囊包膜（包膜组）,12只鼠不用包膜（未包膜组）,两组在体外培养36h后,取出培养液;加入促肾上腺皮质激素（ACTH）继续培养24、36h,用放免法分别测ACTH刺激前后的醛固酮、皮质醇浓度。共培养72h取两组肾上腺组织进行光镜和电镜观察。结果提示包膜组醛固酮、皮质醇分泌量显著高于未包膜组（P<0.01）;包膜组经ACTH刺激36h的皮质醇分泌量明显高于刺激前的分泌量（P<0.01）;包膜组经ACTH刺激24、36h醛固酮和皮质醇的分泌量显著高于未包膜组（P<0.01）;未包膜组ACTH刺激前、后的醛固酮、皮质醇分泌量无显著差异（P>0.05）。光镜和电镜观察见肾上腺组织细胞结构完整,细胞存活良好。结论微囊包膜肾上腺组织在体外培养条件下细胞存活良好,微囊不影响组织的分泌功能,并对ACTH刺激有良好的反应。     

Differential effects of medial forebrain bundle lesions on adrenocortical responses following limbic stimulation

Adult male rats had electrolytic lesions placed bilaterally in the medial forebrain bundle and were subsequently implanted with stimulating electrodes in one of the following limbic regions: (1) dorsal hippocampus; (2) ventral hippocampus; (3) medial septal nucleus; (4) basolateral amygdala; (5) mesencephalic reticular formation. Following electrical stimulation, blood was drawn by acute venesection, under either, for plasma corticosterone determinations. In non-lesioned animals, electrical stimulation in all of the limbic regions led to elevated plasma corticosterone levels. In rats with lesions in the medial forebrain bundles, the adrenocortical response to stimulation in the dorsal hippocampus, the basolateral amygdala or the reticular formation was markedly attenuated. On the contrary, the same lesions were without effect upon the corticosterone secretory response to medial septal stimulation, and had only a slight inhibitory effect upon the response to electrical stimulation in the ventral hippocampus. The results demonstrate that the medial forebrain bundle plays a major role in the transmission of impulses to the mediobasal hypothalamus, originating in the dorsal hippocampus, basolateral amygdala or mesencephalic reticular formation, which activate adrenocortical secretion; its role in the transmission of cues arising in the ventral hippocampus or medial septum is, however, minor.     

Immunohistochemical localization of the ACTH (MC-2) receptor in the rat placenta and adrenal gland

The adrenocorticotropic hormone (ACTH) acts on adrenocortical cells and promotes steroidogenesis by specific binding to the ACTH (MC-2) receptor (ACTHR). To gain an insight into ACTH action on local steroidogenic organs, we examined the immunohistochemical expression of ACTHR in rat adrenal glands and placentas during the mid-late gestation period. Antibodies against synthetic ACTHR peptides were raised in rabbits, and Western blot analysis showed that the antibody reacted with specific proteins in the rat adrenal glands and placentas. The peroxidase-labeled antibody method revealed that ACTHR was distributed in the plasma membrane and cytoplasm of the parenchymal cells of the adrenocortical zona fasciculata. In the placenta, ACTHR was distributed in the junctional spongiotrophoblasts at day 13 of gestation--with a gradual decrease in the staining during the gestational period, whereas ACTHR appeared in the placental labyrinthine cells from days 15 to 19 of gestation. Immunoelectron microscopy revealed that ACTHR was also localized in the ribosomes of the fasciculata cells and the labyrinthine cells. Our findings suggest that ACTHR may play a physiological role in steroidogenesis in the adrenal cortical parenchymal cells as well as in the trophoblasts of rat placentas during mid-late gestation.     

Immunohistochemical localization of cytochrome P-450C21 in human adrenal cortex and its relation to endocrine function

Cytochrome P-450C21 was successfully demonstrated in the human adrenal glands by a peroxidase-antiperoxidase method. All three cortical layers were stained in the normal adrenal glands, particularly the zonae glomerulosa and reticularis. Well-stained and faintly stained cells were intermingled in the zona fasciculata, suggestive of intrazonal variations. The immunoreactivity was particularly intense at the site of ACTH action, i.e., cells in micronodules and cells around myelolipomatous lesions in adrenocortical hyperplasia of Cushing's disease and sites of regeneration in the normal adrenal glands. In adrenocortical adenomas with Cushing's syndrome and primary aldosteronism, cells with large nuclei were generally stained well. In the adrenocortical tissue adjacent to a functioning adenoma, the immunoreactivity was observed only in the zona glomerulosa, especially in cases of primary aldosteronism. This finding is consistent with morphologic observations.