Pharmacological Adrenalectomy Research Articles

Pharmacological suppression of endogenous glucocorticoid synthesis attenuated blood pressure and heart rate response to acute restraint in Wistar rats.

Glucocorticoids (GCS) are known to modulate cardiovascular response during stress conditions. The present study was aimed to test the hypothesis that permissive and/or stimulating effect of GCs is essential for the maintenance of peripheral vascular resistance and for the adequate response of cardiovascular system to stressor exposure. The effects of acute pharmacological adrenalectomy (PhADX) on humoral and cardiovascular parameters were studied in adult Wistar rats under the basal conditions and during the acute restraint stress. Acute PhADX was performed by the administration of metyrapone and aminoglutethimide (100 mg/kg s.c. of each drug) resulting in a suppression of endogenous glucocorticoid synthesis. Blood pressure (BP), heart rate (HR) and core body temperature were measured using radiotelemetry. BP responses to administration of vasoactive agents were determined in pentobarbital-anesthetized animals. PhADX considerably attenuated stress-induced increase of BP, HR and core body temperature. PhADX did not abolish BP and HR lowering effects of ganglionic blocker pentolinium indicating preserved sympathetic function in PhADX rats. BP response to exogenous norepinephrine administration was attenuated in PhADX rats, suggesting reduced sensitivity of cardiovascular system. Suppression of corticosterone synthesis by PhADX increased basal plasma levels of ACTH, aldosterone and plasma renin activity in unstressed animals but there was no further increase of these hormones following stressor exposure. In conclusion, PhADX attenuated stress-induced rise of blood pressure, heart rate and core body temperature indicating an important permissive and/or stimulating role of glucocorticoids in the maintenance of the adequate response of cardiovascular system and thermoregulation to several stimuli including acute exposure to stressor.

Glucocorticoid withdrawal affects stress-induced changes of urocortin 2 gene expression in rat adrenal medulla and brain.

Corticotropin-releasing factor is well known activator of the hypothalamic-pituitary-adrenocortical axis, that represents crucial system participating on stress response of the organism. Urocortins are members of the corticotropin-releasing factor family of peptides with proposed effects on neuroendocrine and behavioral stress response mechanisms. Urocortin 2, one of three known urocortins, is present in central and peripheral stress response system and its expression can be augmented by glucocorticoids. In the present study we have examined how glucocorticoid withdrawal affects urocortin 2 gene expression after acute immobilization in the adrenal medulla and selected brain areas in rats. We used pharmacological adrenalectomy to block synthesis of corticosterone. Our results show that the immobilization-induced rise in urocortin 2 mRNA levels in rat adrenal medulla was not inhibited by glucocorticoid withdrawal. On the other hand, observed changes in the brain indicate that the effect of stress and pharmacological adrenalectomy on urocortin 2 gene expression is site-specific. While in the paraventricular nucleus and locus coeruleus the immobilization induced rise of urocortin 2 was not inhibited by pharmacological adrenalectomy in the arcuate nucleus and central amygdala it was. Moreover, we have seen a significant depletion of urocortin 2 plasma levels after immobilization. The immobilization induced rise of urocortin 2 gene expression in rat adrenal medulla and brain areas regulating stress response pathways and preservation of its induction after adrenalectomy suggests a role of urocortin 2 in the neuroendocrine stress response of an organism. This article is protected by copyright. All rights reserved.

Etomidate, pharmacological adrenalectomy and the critically ill: a matter of vital importance

Etomidate is a potent suppressant of adrenal steroidogenesis, effectively inducing reversible pharmacological adrenalectomy. Recent evidence suggests that for every five patients with septic shock given etomidate without corticosteroid supplementation, one patient will die as a consequence. Other critically ill patients are also at possible risk, and this risk requires further exploration. Etomidate will also confound investigations into the effects of disease states on adrenal function, and should therefore be avoided. A moratorium on the use of etomidate in critically ill patients outside clinical trials may be prudent until its safety is established.

Ketoconazole increases cocaine and opioid use in methadone maintained patients

Stress plays an important role in substance abuse problems. For example, in studies with rodents stress induces reinstatement of opioid and cocaine self-administration. In addition, attenuation of the stress response by pharmacological adrenalectomy using ketoconazole, a cortisol synthesis inhibitor, reduces cocaine self-administration in rodents. In contrast, studies in primates and humans have produced conflicting results using cortisol synthesis inhibitors for attenuating cocaine-related behaviors and subjective effects. To explore the treatment implications of these findings, ketoconazole's (600–900 mg daily) ability to reduce heroin and cocaine use was compared with placebo in 39 methadone maintained patients with a history of cocaine abuse or dependence during a 12-week double blind trial. Contrary to the predicted effects, both heroin and cocaine use increased after patients were stabilized on methadone and ketoconazole. Depressive and withdrawal symptoms improved no more with ketoconazole than with placebo treatment, and side effects were greater on ketoconazole than placebo. As reported before with methadone treatment, morning cortisol levels were significantly lower than normal values throughout the clinical trial, but were not lower with ketoconazole than placebo treatment. Thus, in agreement with the negative results from acute dosing studies in primates and humans, chronic ketoconazole treatment does not appear to reduce cocaine or opioid use in humans maintained on methadone.

Pharmacological Adrenalectomy with Mitotane

The potential of mitotane (ortho, para′-DDD, commonly used to treat adrenal carcinomas in humans and dogs) was investigated as an alternative to surgical adrenalectomy in birds, salamanders, and lizards. House sparrows (Passer domesticus) were injected twice daily with vehicle or one of two doses of mitotane (225 or 450 mg/kg), and basal and stress-induced levels of corticosterone (CORT) were measured 3 and 5 days after injections. Mitotane reduced basal CORT levels to nondetectable and abolished stress-induced CORT increases by the 3rd day of treatment. In another study, a single injection of mitotane was effective in lowering endogenous CORT levels 36 h later, but levels had apparently recovered by 10 days after the injection. Mitotane did not effect testicular weights and had no detectable effect on testosterone levels. In contrast to its effects on house sparrows, mitotane did not lower endogenous CORT levels in either tiger salamanders (Ambystoma tigrinum) or tree lizards (Urosaurus ornatus), even at doses much higher than those used in house sparrows.

Attenuation of Hypothalamic‐Pituitary‐Adrenal Axis Stress Responses in Late Pregnancy: Changes in Feedforward and Feedback Mechanisms

The hypothalamic-pituitary-adrenal axis is hyporesponsive to stress in late pregnancy, exemplified as reduced adrenocorticotropic hormone (ACTH) and corticosterone responses to restraint, but the mechanisms are unknown. We investigated forward drive and negative feedback upon the hypothalamic-pituitary-adrenal axis in pregnant rats. Corticotropin-releasing hormone (CRH) and vasopressin mRNA expression in the parvocellular paraventricular nucleus and mineralocorticoid and glucocorticoid receptor expression in the paraventricular nucleus and hippocampus were quantified with in situ hybridization. Because it can enhance the corticosterone negative feedback signal, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) bioactivity in these brain regions and anterior pituitary was measured in vitro, and ACTH and corticosterone stress responses were measured after intracerebroventricular glycyrrhetinic acid, an 11beta-HSD inhibitor. Changes in corticosterone feedback on ACTH secretion were examined after pharmacological adrenalectomy by metyrapone and aminoglutethimide. Parvocellular paraventricular nucleus CRH mRNA content was reduced on day 21 and the CRH mRNA : vasopressin mRNA ratio was unaltered, indicating decreased production of both CRH and vasopressin. An increase in glucocorticoid receptor mRNA expression in the dentate gyrus (mineralocorticoid receptor mRNA expression was unaltered) and increased 11beta-HSD1 activity in the paraventricular nucleus and anterior pituitary suggest an increase in slow negative feedback mechanisms in pregnancy, but glycyrrhetinic acid did not modify the stress response. After metyrapone/aminoglutethimide treatment, corticosterone decreased ACTH secretion more slowly in pregnancy, indicating a decrease in rapid feedback sensitivity. Thus, reduced forward drive rather than increased effectiveness of glucocorticoid negative feedback may underlie stress hyporesponsiveness of the hypothalamic-pituitary-adrenal axis in pregnancy.

Impaired basal and restraint-induced epinephrine secretion in corticotropin-releasing hormone-deficient mice.

CRH is thought to play a role in responses of the adrenocortical and adrenomedullary systems during stress. To investigate the role of CRH in stress-induced secretions of corticosterone and epinephrine, we subjected wild-type (WT) and CRH-deficient (knockout, KO) mice to restraint, and analyzed plasma corticosterone, plasma catecholamines, and adrenal phenylethanolamine N-methyltransferase (PNMT) gene expression and activity before and during 3 h of restraint. Plasma corticosterone increased over 40-fold in WT mice, but minimally in CRH KO mice. Adrenal corticosterone content tended to increase in CRH KO mice, although to levels 5-fold lower than that in WT mice. CRH KO mice had significantly lower plasma epinephrine and higher norepinephrine than WT mice at baseline, and delayed epinephrine secretion during restraint. Adrenal PNMT messenger RNA content in CRH KO mice tended to be lower than that in WT mice, though the degree of induction was similar in both genotypes. PNMT enzyme activity was significantly lower in CRH KO mice. Pharmacological adrenalectomy abolished restraint-induced corticosterone secretion and PNMT gene expression in WT mice, consistent with an absolute requirement of glucocorticoids for PNMT gene expression. We conclude that glucocorticoid insufficiency in CRH KO mice leads to decreased basal and restraint-induced plasma epinephrine and adrenal PNMT gene expression and enzyme activity.

Glucocorticoid negative feedback on the HPA axis in five inbred rat strains.

The aim of the present work was to study the influence of altering glucocorticoid negative feedback on both basal activity of the hypothalamic-pituitary-adrenal (HPA) axis and its response to acute stress (tail shock) in five inbred rat strains known to differ in some depression-like behaviors: Brown Norway (BN), Fischer 344 (F344), Lewis (Lew), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) rats. Two complementary approaches were used: 1) enhancement of negative feedback by administration of 0.05 and 0.2 mg/kg dexamethasone (Dex) and 2) attenuation of negative feedback by pharmacological adrenalectomy (PhADX). The results indicate that 1) Lew rats consistently show adrenocorticotropic hormone (ACTH) and corticosterone hyporesponsiveness to stress, 2) interstrain differences in the effect of Dex on the HPA axis were very weak and not related apparently to differences in the metabolism of the steroid, 3) the suppressive effect of the highest dose of Dex on basal corticosterone levels was lower in BN rats than in the other strains, and 4) after PhADX, an increase in ACTH levels was observed in response to acute stress in BN, F344, and WKY but not in Lew and SHR rats, suggesting possible interstrain differences in pituitary sensitivity to neural stimuli induced by stress. In summary, our results indicate that there are differences among the strains with regard to both 1) the suppressive effect of Dex on the HPA axis, BN rats showing a certain degree of resistance, and 2) the capability of PhADX rats to respond to acute stress, which suggests a defective release of ACTH in Lew and SHR rats. The biological meaning of these alterations of corticosteroid negative feedback among the five inbred strains studied remains to be established.

Impact of adrenalectomy on 5-HT6 and 5-HT7 receptor gene expression in the rat hippocampus.

Both glucocorticoid excess and decreased serotonergic (5-HT) transmission may cause depression. The recently cloned 5-HT6 and 5-HT7 receptors have high affinity for antidepressants. Here, we show that pharmacological adrenalectomy increases 5-HT6 and 5-HT7 receptor mRNA expression in specific hippocampal subfields, effects partly reversed by corticosterone replacement. Increased 5-HT6 and 5-HT7 receptor expression may provide a basis, in part, for the therapeutic actions of adrenal steroid synthesis inhibitors in resistant depression.

Effects of surgical and pharmacological adrenalectomy on the initiation and maintenance of intravenous cocaine self-administration in rats

Previous research has suggested the potential involvement of the hypothalamic-pituitary-adrenal (HPA) axis in psychostimulant reinforcement. In particular, we have found significant correlations between electric footshock-induced increases in plasma corticosterone and the acquisition, or lack thereof, of intravenous cocaine self-administration in rats. The experiments presented here were designed to further determine the role for corticosterone in cocaine reinforcement in rats by decreasing plasma levels of the hormone with surgical and pharmacological adrenalectomies. Bilateral adrenalectomy completely abolished the acquisition of intravenous cocaine self-administration over a wide range of doses (0.03125 to 1.0 mg/kg/infusion) without affecting food maintained responding. This suppression of self-administration was partially reversed by adding corticosterone (100 μg/ml) to the rats' drinking water. In another group of rats, pretreatment with metyrapone, which blocks the synthesis of corticosterone, resulted in dose-related decreases in ongoing cocaine self-administration. These data suggest that corticosterone is not only important, but may also be necessary for both the acquisition and maintenance of cocaine reinforcement in rats.

Effects of metyrapone infusion on corticotropin-releasing factor and arginine vasopressin secretion into the hypophysial portal blood of conscious, unrestrained rams.

The effects of rapid changes of circulating cortisol levels on ACTH secretion and on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) concentrations into hypophysial portal blood were studied in six adult rams. Pharmacological adrenalectomy was obtained by 3 h metyrapone infusion (100 mg.kg-1.h-1). Blockade of cortisol synthesis induced a tenfold increase of plasma ACTH levels accompanied by a moderate increase of CRF secretion (150% vs preinjection levels) and a large increment of AVP secretion (535% vs preinjection levels). ACTH levels remained high during the 3 h following the end of metyrapone infusion. During the same period, CRF secretion was still elevated (231% vs preinjection levels), while AVP secretion was further stimulated (2,151% vs preinjection levels). Subsequent hydrocortisone infusion (66 micrograms.kg-1.h-1) for 2 h induced a rapid decrease of both ACTH and AVP secretion, while CRF levels in hypophysial portal blood still remained elevated. These data suggest that changes in ACTH secretion induced by acute modifications of the negative glucocorticoid feedback are, in addition to the well documented direct effect of cortisol on the corticotropes, mainly mediated by variations of hypothalamic AVP secretion.

CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy

A regimen of twice daily metyrapone injections (100 mg/kg), resulted in pharmacological adrenalectomy of pregnant rats and fetuses in utero, i.e. depression of plasma corticosterone and elevation of plasma adrenocorticotropic hormone (ACTH). Toxicity was minimal on days 14–17 of pregnancy, and increased with higher maternal weight and pregnancy progression. Corticotropin releasing hormone (CRH) messenger RNA abundance in the pregnant adults increased significantly within 48 h of metyrapone initiation. No change in CRH gene expression in the paraventricular nucleus of fetuses (days 17–18) was seen, even after 72 h of the regimen. This is compatible with the independence of CRH gene expression of glucocorticoid feedback in the fetal rat.

Effect of pharmacological adrenalectomy on corticotropin-releasing factor-like and arginine vasotocin immunoreactivities in the brain and pituitary of the eel: Immunocytochemical study

Metopirone, a blocker of the 11β-hydroxylase, was used to suppress cortisol synthesis in the eel, producing a pharmacological adrenalectomy. After 1 and 2 days, corticotropin-releasing factor (CRF) immunoreactivity was reduced in the preoptic nucleus (PON) and was increased in the rostral neurohypophysis (NH) where CRF fibers ended in close proximity to ACTH cells. After 4 days, immunoreactive (ir) CRF increased in the PON or was similar to that of controls, and was reduced in the rostral NH. It was not affected in the caudal NH among intermediate lobe (IL) ramifications. Arginine vasotocin (AVT) immunostaining was often slightly increased in the PON, but changes were not apparent in the pituitary and AVT-ir fibers remained very scarce in the rostral NH. Metopirone significantly increased the cross-sectional area of CRF-and AVT-ir perikarya in the parvocellular region and the number of these perikarya as well as the ratio of CRF to AVT cell bodies in the totality of the PON. Colocalization of CRF and AVT, barely detected in control eels, occurred in 9.6% of CRF perikarya after 1–2 days, but in only 2.4% after 4 days. ACTH cells were rapidly and markedly stimulated. These immunocytochemical studies suggest that CRF synthesis, axonal transport, and release are increased in metopirone-treated eels, acting on the pituitary to stimulate ACTH release and interrenal cell activity. The participation of AVT in ACTH cell stimulation is less clear. These data are compared to those reported in mammals.

Fetal and maternal levels of corticosterone and ACTH after pharmacological adrenalectomy

A paradigm of pharmacological adrenalectomy of pregnant rats and fetuses in utero is described. A regimen of twice daily metyrapone injections (10mg/100gm body weight), results in marked depression of serum corticosterone in pregnant and in fetal rats without surgical trauma and stress. The technique should be useful in a wide variety of studies involving the developing brain-adrenal axis.

The Adrenocortical System Responds Slowly to Removal of Corticosterone in the Absence of Concurrent Stress*

After removal of corticosteroid feedback by surgical or pharmacological adrenalectomy, plasma ACTH increases more rapidly than can be explained by changes in receptor-mediated gene expression. In aminoglutethimide-treated rats, plasma ACTH increased only at doses much higher than those inhibiting plasma corticosterone, suggesting that adrenal enzyme blockers may themselves be stressful. To determine the adrenocortical system response to stressless corticosterone removal, adrenalectomized rats maintained for 5 days on corticosterone in the drinking water were switched to steroid-free fluid (-B) or again given steroid (+B); additional rats were adrenalectomized (ADX). Plasma ACTH did not differ between -B and +B rats until 18-24 h after steroid removal, regardless of whether steroid was withdrawn at the circadian maximum or minimum. Plasma ACTH was similar between -B and ADX rats 0.5-14 days after corticosterone removal, although morning plasma ACTH was more stable in -B rats at 4-7 days. Evening plasma ACTH increased significantly after day 3 in ADX and -B rats. Unlike ADX rats, -B rats did not exhibit pituitary ACTH depletion at 12 and 24 h, but both -B and ADX groups had significantly elevated pituitary ACTH by 6.5 days. We conclude that 1) rapid increases in ACTH secretion after surgical or pharmacological adrenalectomy result from interaction between stress and loss of corticosteroid feedback; 2) no immediate interaction occurs between loss of feedback and circadian stimuli; and 3) the effects of steroid withdrawal may require at least 3 days to be stably expressed.

Enhanced uptake of HRP by hypophysiotropic neurons following stress and adrenalectomy

Following systemic injections of horseradish peroxidase (HRP), the uptake of HRP by parvocellular hypophysiotropic neurons of the nucleus preopticus (NPO) was enhanced in response to stress or pharmacological adrenalectomy. Following lesions of the NPO, pharmacological adrenalectomy resulted in an enhanced uptake of HRP by parvocellular hypophysiotropic neurons of the nucleus lateral tuberis (NLT). The activation of hypophysiotropic neurons of the NPO and NLT in response to stimulation of the hypothalamo-corticotroph axis suggests that these neurons are involved in the stimulation of adrenocorticotropic hormone secretion.

Cytological evidence for activation of neuroendocrine cells in the parvocellular preoptic nucleus of the goldfish hypothalamus following pharmacological adrenalectomy

The nucleus preopticus (NPO) of the goldfish hypothalamus is composed of parvocellular (NPOpc) and magnocellular (NPOmc) neurosecretory neutrons. The cytology of NPOpc and NPOmc neurons was examined with light and electron microscopy following pharmacological adrenalectomy with the adrenocortical inhibitor, metopirone. After five days of metopirone administration, light microscopy revealed a significant increase in nuclear area of NPOpc, but not of NPOmc, neurons. Ultrastructural examination of NPOpc neurons revealed two cell types, PC1 and PC2 neurons, which could be distinguished by the relative abundance and the size of the neurosecretory granules in the cytoplasm. The ultrastructural appearance of the NPOmc neurons revealed a single cell type containing abundant neurosecretory granules. Following five days of metopirone administration, the ultrastructural appearance of the PC1 neurons indicated a state of enhanced secretory activity. Metopirone had no observable effect on the appearance of the PC2 or NPOmc neurons. These observations demonstrate that PC1 neurons are activated under the conditions of pharmacological adrenalectomy and suggest that the secretory activity of these neurons is inhibited by adrenocorticosteroids.