Subsensitive pituitary cyclic AMP response to stress following adrenalectomy is not caused by loss of adrenal epinephrine

We have previously reported that various stressors acutely elevate levels of pituitary cyclic AMP in vivo and that this stress response is not seen in animals tested 7 or 30 days post-adrenalectomy. In this report we present data that demonstrate that the loss of the pituitary cyclic AMP stress response following adrenalectomy is not the result of the loss of stress-induced adrenal epinephrine release. These data show that (1) although administration of epinephrine to intact rats does not elevate levels of pituitary cyclic AMP, administration of epinephrine to adrenalectomized animals does not elevate pituitary cyclic AMP levels in vivo; (2) splanchnic denervation prevents stress-induced adrenal epinephrine release but does not abolish stress-induced increases in pituitary cyclic AMP; and (3) the time course of the developing subsensitive pituitary cyclic AMP response to stress following adrenalectomy is much slower (2 to 3 days) than the loss of circulating epinephrine.     

Site of inhibitory action of CRE 9-41 on ACTH release from isolated rat pituitary cells

The corticotropin-releasing factor (CRF) analog CRF 9-41 inhibits CRF, but not forskolin or dibutyryl cyclic AMP, stimulated release of ACTH from isolated pituitary cells. CRF 9-41 also blocks CRF-stimulated accumulation of cyclic AMP in a parallel dose dependent fashion. CRF 9-41 has no effect on basal ACTH release or cAMP levels. This substantiates that the analog acts as a direct CRF antagonist and that the site of this inhibition is most likely at the level of binding of CRF to its receptor on the corticotrope. Various substances, including most prominently glucocorticoids, inhibit release of ACTH from the pituitary. In an effort to develop another class of inhibitors, Rivier et al recently synthesized analogs of corticotropin releasing factor (CRF). One among these, alpha-helical ovine CRF 9-41 blunts adrenalectomy and stress induced ACTH release in non-anesthetized rats. At micromolar concentrations, CRF 9-41, shifts rightward the dose response of isolated pituitary cells to ovine CRF. Thus, the authors suggested that CRF 9-41 acts as a competitive antagonist to CRF-induced ACTH secretion. CRF appears to act through stimulation of adenylate cyclase. To determine the potential site of action of CRF 9-41 in the activation sequence for adenylate cyclase, we studied its effects on pituitary cyclic AMP formation and ACTH secretion from dispersed anterior pituitary cells derived from normal adult rats, as well as, its interaction with cyclic nucleotide agonists.     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.     

葆包牡丹碱对垂体前叶组织块释放促肾上腺皮质激素的刺激效应

本实验利用垂体组织块离体灌流技术,观察到γ－氨基丁酸Ａ受体拮抗剂荷包牡丹笃切除双侧肾上腺９６ｈ后的大鼠垂体前叶ＡＣＴＨ的分泌具有强烈的刺激作用。但同样浓度的荷包牡丹笃分离的垂体前叶细胞的ＡＣＴＨ分泌无影响,提示肾上腺切除后,γ－氨基丁酸在垂体前叶直接或通过间接途径抑制ＡＣＴＨ分泌。     

Dexamethasone suppresses ACTH release without attenuating pituitary cyclic AMP response to stress in vivo

Dexamethasone, a synthetic glucocorticoid, has been shown to decrease basal and stress-elevated levels of the pituitary hormone ACTH. Glucocorticoids are known to bind to multiple sites within the brain and pituitary and it is not known which site(s) is most important in mediating the observed inhibition of ACTH release. At the level of the corticotroph, there is contradictory data from in vitro studies regarding whether dexamethasone acts proximal or distal to the formation of the cyclic AMP second messenger that has been shown to be involved in CRF-stimulated ACTH release. In the present report, we have examined the effects of dexamethasone pretreatment on stress-induced elevations in pituitary cyclic AMP and the release of ACTH in vivo. Acute stress (15 min of intermittent footshock) elevated levels of pituitary cyclic AMP and plasma ACTH consistent with previous studies. Dexamethasone administration (0.4 mg/kg 24 hr prior to sacrifice plus 0.2 mg/kg 2 hr prior to sacrifice) inhibited stress-induced elevations in plasma ACTH but did not affect pituitary cyclic AMP response to acute stress. These findings suggest that dexamethasone inhibits the release of ACTH via an action distal to the generation of cyclic AMP.     

Monensin inhibition of corticotropin releasing factor mediated ACTH release

Monensin is a sodium selective carboxylic ionophore that has been helpful in studying the intracellular mechanisms of protein secretion by its ability to inhibit transport of secretory proteins, particularly through the Golgi apparatus, and by its capacity to block intracellular posttranslational processing events. We studied in rat anterior pituitary cell culture the effects of monensin on: CRF stimulated ACTH release; presynthesized (stored) ACTH release; and on forskolin- (activator of adenylate cyclase) and KCl- (a membrane depolarizer which does not stimulate ACTH synthesis) induced ACTH release. Monensin inhibited CRF stimulated ACTH release in a dose-dependent fashion. The ED50 was 2.7 x 10(-8) M and maximal inhibition was 52% at 1.5 x 10(-7) M. Inhibition at 40 minutes of CRF incubation was similar to the percent inhibition noted at 1 hr 40 min and 2 hr 40 min. Monensin (1.5 x 10(-6) M) decreased the amount of ACTH release from cells incubated with cycloheximide plus CRF by 32% (p less than 0.01). Monensin individually inhibited forskolin (2 x 10(-6) M) and dibutyryl cyclic AMP (3 x 10(-3) M) mediated ACTH release in a dose-dependent fashion. The inhibition of forskolin and dibutyryl cyclic AMP mediated ACTH release by 1.5 x 10(-6) M monensin was 48% and 46% respectively. Monensin (1.5 x 10(-6) M) also reduced KCl (50 mM) stimulated ACTH release by 48%. This study demonstrates that monensin inhibits CRF mediated ACTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of glucocorticoids on adipocyte corticotropin receptors and adipocyte responses

A specific and sensitive assay for determining the binding of adrenocorticotropin (ACTH) to isolated rat adipocytes has been developed and utilized to study the effect of glucocorticoids on ACTH receptor. Measurement of the binding of tritiated ACTH (spec. act. 90 Ci/mmol) to adipocytes isolated from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats indicated that there are no differences among these three populations in either the magnitude or the affinity of the binding reaction. The binding interaction was found to be of high affinity (K_d = 5.23 + 1.92 · 10^?9 M) and paralleled closely the stimulation of lipolysis (K_m = 2.09 ± 0.35 · 10^?9 M). About 16 300 receptors were calculated to be present per adipocyte. Hormone-induced cyclic 3′,5′-adenosine monophosphate production remained intact after adrenalectomy, thereby confirming that receptors are not lost during steroid deprivation. The lipolytic response did, however, become less sensitive to both ACTH and epinephrine following adrenalectomy. Pre-treatment of adrenalectomized rats with dexamethasone resulted in an increase in basal and hormone-stimulated levels of cyclic AMP and glycerol production to super-normal values. In adipocyte ghost preparations, ACTH and epinephrine sensitive adenylate cyclase activity was not decreased by adrenalectomy and dexamethasone administration did not result in a selective enhancement of ACTH sensitive adenylate cyclase activity. Our results indicate that glucocorticoids do not cause their permissive effects by specific regulation of the ACTH receptor on the adipocyte.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.     

Multireceptor-induced release of adrenocorticotropin from anterior pituitary tumor cells

Corticotropin releasing factor (CRF), (?) isoproterenol and vasoactive intestinal peptide (VIP) induced cyclic AMP synthesis and the release of immunoreactive adrenocorticotropin hormone (ACTH) from clonal mouse AtT-20 pituitary tumor cells. CRF and (?) isoproterenol together produced an additive increase in cyclic AMP formation but a less than additive effect on ACTH secretion. VIP with either CRF or (?) isoproterenol produced additive increases in both cyclic AMP and ACTH secretion. Forskolin, an activator of adenylate cyclase stimulated the release of ACTH suggesting that cyclic AMP mediates some of the effects of hormone-receptor activation on ACTH secretion. The action of all three receptor agonists and forskolin on ACTH release was blocked by dexamethasone treatment. The release process, but not the changes in cyclic AMP synthesis was calcium dependent with all these hormones. The calcium ionophore, A-23187, increased ACTH secretion without altering intracellular cyclic AMP content. Its effect on secretion was not additive with either CRF, (?) isoproterenol or VIP. These observations indicate that hormone-induced regulation of ACTH secretion converges at varying intracellular locations.     

荷包牡丹碱对垂体前叶组织块释放促肾上腺皮质激素的刺激效应

本实验利用垂体组织块离体灌流技术,观察到－氨基丁酸Ａ受体拮抗剂荷包牡丹碱对切除双侧肾上腺９６ｈ后的大鼠垂体前叶ＡＣＴＨ的分泌具有强烈的刺激作用。但同样浓度的荷包牡丹碱对分离的垂体前叶细胞的ＡＣＴＨ分泌无影响。提示肾上腺切除后,－氨基丁酸在垂体前叶直接或通过间接途径抑制ＡＣＴＨ分泌。     

Comparison of the effects of CRF and stress on levels of pituitary cyclic AMP and plasma ACTH in vivo

We have previously reported that acute stress increases levels of rat pituitary cyclic AMP in vivo. The present study was conducted to test the hypothesis that stress-induced increases in pituitary cyclic AMP in vivo were mediated via CRF. We compared the effects of various stressors with the effects of CRF or epinephrine administration on pituitary cyclic AMP and plasma ACTH responses in vivo. Stressors, epinephrine or CRF increased levels of pituitary cyclic AMP. Pituitary cyclic AMP response to either immobilization or CRF was much greater at light onset than at lights off in rats maintained on at 12 hr light: 12 hr dark lighting regimen. In rats with pituitary stalk transections, footshock did not increase levels of pituitary cyclic AMP, suggesting that some factor of central origin was required for this stress response. Exogenous CRF administration did increase levels of pituitary cyclic AMP in stalk-transected rats, while epinephrine increased levels in sham-operated but not in stalk-transected rats. Antisera to CRF markedly decreased pituitary cyclic AMP response to exogenous CRF administered 6 min following antisera and partially attenuated pituitary cyclic AMP response to forced running. Taken as a whole these data support a major role for CRF in the pituitary cyclic AMP response to stress.     

Formation of gamma-aminobutyric acid from glutamate and putrescine in rat hypothalamic and hippocampal synaptosomes and regulation of these processes by glucocorticoids]

It has been shown that single or multiple hydrocortisone and ACTH administrations to intact rats increased GABA content and its synthesis from glutamate and putrescine in synaptosomes of hypothalamus. The letter content was increased by single hormonal administration while multiple hormonal administration and adrenalectomy decreased it. Ornithine decarboxylase activity was increased by single hydrocortisone administration to intact animals, following adrenalectomy, and it was decreased by single hormonal administration to adrenalectomized rats. GABA synthesis in synaptosomes of hippocampus from putrescine was increased by single hydrocortisone and multiple hormonal administrations. GABA content was increased by multiple administration of both hormones and was decreased by adrenalectomy. Putrescine level was decreased by multiple hydrocortisone administration to intact and single administration to adrenalectomized rats; ornithine decarboxylase activity was decreased by multiple administration of both hormones.     

Effect of 41-CRF antiserum on the secretion of ACTH, B-endorphin and alpha-MSH in the rat

In order to elucidate the physiological role of the 41 amino-acid residue corticotropin-releasing factor (41-CRF) on the secretion of ACTH, B-Endorphin and alpha-MSH, plasma levels of these peptides were measured by radioimmunoassay in intact and adrenalectomized rats, two hours after the injection of either 41-CRF antiserum (CRF-AS) or normal rabbit serum for controls. The administration of CRF-AS strikingly lowered the plasma ACTH levels in both intact and adrenalectomized rats. A statistically significant reduction of plasma levels of B-Endorphin was also observed in the same rats. However, the effect of CRF-AS on B-Endorphin release was less pronounced than the effect on ACTH release. No changes in plasma alpha-MSH levels were observed after passive immunization with CRF-AS. We conclude that, in the rat, 41-CRF plays a physiological role in the regulation of ACTH and B-Endorphin secretion, but is not involved in the regulation of alpha-MSH release from the pituitary gland.     

Adrenocorticotropic (ACTH) cells of rats after head irradiation.

The heads of intact rats and of rats 15 days after adrenalectomy were irradiated with 1200 R. Some of the adrenalectomized animals with irradiated heads were treated with 5 mg of deoxycorticosterone acetate. In adrenocorticotropic cells of adrenalectomized rats 15 and 30 days after irradiation, the decreased amount of the granular endoplasmic reticulum and the Golgi regression imply a considerably decreased protein synthesis. The finding of an increased number of granules and of granules larger than normal suggests that they are the result of slowed down transport and secretion of the products of these cells' activities. After head irradiation an increased number of degenerative cells was evident in the pituitary of both intact and adrenalectomized rats. The degree of the degenerative changes and the number of degenerative cells was higher 30 than 15 days after irradiation and both were decreased if the adrenalectomized rats were treated with deoxycorticosterone acetate.     

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.     

The effect of somatostatin and of prolyl-leucyl-glycinamide (MIF) on ACTH release in dispersed pituitary cells.

The hypothalamic releasing and release-inhibiting peptides have multiple effects on more than one pituitary hormone. In this study the action of the two hypothalamic inhibiting factors, somatostatin (GH-IH) and MSH release-inhibiting factor, prolyl-leucyl-glycinamide (MIF), on ACTH release were studied. Increasing concentrations of GH-IH and MIF were added to 1 ml of a suspension of dispersed anterior pituitary cells from male rats. Both GH-IH and MIF (10^?5 to 10^?11 M) were without effect on basal ACTH secretion of normal and of adrenalectomized rats. However, both peptides, within certain concentration ranges, inhibited the ACTH release stimulated by rat hypothalamic extracts or by arginine vasopressin. The most effective concentrations were 35 nM MIF or 6 nM GH-IH. Beyond these concentrations no further suppression was observed. Our results indicate that somatostatin and MIF can inhibit ACTH release, but only in a state of steroid deprivation and within a limited dose range.     

Adrenalectomy abolishes the stress-induced increase in pituitary cyclic AMP

We have previously demonstrated that various stressors increase pituitary cyclic AMP in vivo in the rat. In the course of studying the mechanisms mediating this response, we examined the effect of bilateral adrenalectomy on footshock-induced increases in pituitary cyclic AMP. In unoperated rats, intermittent footshock markedly increased pituitary levels of cyclic AMP and plasma levels of corticosterone and prolactin. Adrenalectomy completely abolished the stress-induced increase in pituitary cyclic AMP. The marked increase in plasma prolactin following footshock was not affected by adrenalectomy. Our results indicate that adrenal factors are involved in the stress-induced increase in pituitary cyclic AMP.     

Inhibition of ACTH secretion in mouse pituitary tumor cells by activation of muscarinic cholinergic receptors

Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C-terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism.     

Pituitary ACTH responsiveness in the vasopressin deficient rat.

ACTH concentration and the responsiveness of dispersed anterior pituitary cells to hypothalamic extract and to vasopressin were studied in homozygous (DI), heterozygous (HTZ), DI-pitressin treated (DIP) Brattleboro rats, and in control Long-Evans rats.Absolute, but not relative, anterior pituitary weights of HTZ, DI, and DIP animals were significantly smaller than those of controls. The concentration of immunoreactive (I) and bioreactive (B) ACTH in dispersed anterior pituitary cells was greater in DI and DIP than in HTZ or in control animals, although the total amount of ACTH was greater in control than DI or HTZ animals. Media from incubates of pituitary cells derived from DI and DIP animals contained less I and B ACTH than those from HTZ or control animals. Pituitary cells derived from DI animals secreted markedly less ACTH following incubation with hypothalamic extract (NIH-HE-RP-1) than did cells from HTZ animals. The response in DIP animals was intermediate between that of DI and HTZ animals. In contrast, pituitary cells derived from DI and DIP animals were significantly more responsive to vasopressin than those from control or HTZ animals.