Stimulated ACTH Release Research Articles

Arginine Vasopressin and Corticotropin Releasing Factor: Binding to Ovine Anterior Pituitary Membranes*

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.

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.

Effects of neuropeptide-Y and substance-P on the secretory activity of dispersed zona-glomerulosa cells of rat adrenal gland

Neuropeptide-Y (NPY) and substance-P (SP), two peptides contained in the chromaffin granules of adrenal medullary cells, were found to partially inhibit both basal and ACTH-stimulated release of aldosterone and 18-hydroxy-corticosterone by isolated rat zone-glomerulosa cells, without affecting the overall post-pregnenolone yield or basal progesterone output. Conversely, the exposure to both peptides increased 11-deoxycorticosterone and corticosterone secretion. These data indicate that NPY and SP are able to exert a direct suppression of 18-hydroxylase activity in rat zona-glomerulosa cells, without conceivably altering the earlier steps of aldosterone synthesis. The possible physiological implications of these findings are discussed in light of previous studies suggesting a net adrenoglomerulotrophic effect of NPY and SP in vivo.

Blockage of K+ channels reverses the inhibitory action of atriopeptin on secretagogue-stimulated ACTH release by perifused isolated rat anterior pituitary cells

The aim of the present study was to investigate how atriopeptin inhibits secretagogue-stimulated ACTH secretion in vitro. Perifused isolated rat anterior pituitary cells were used throughout; the ACTH content of the perifusate was measured by radioimmunoassay. In the presence of a constant (0.05 nmol/l) concentration of 41-residue corticotrophin-releasing factor (CRF), arginine vasopressin (AVP; 0.05-50 nmol/l) stimulated ACTH secretion in a concentration-dependent manner, the combination of 0.05 nmol CRF/l and 0.5 nmol AVP/l (CRF/AVP) stimulated ACTH release to six- to eightfold above baseline. The effect of CRF/AVP was not modified by tetrodotoxin, but was abolished by CoCl2 and reduced to about 70% of the control stimulus by nifedipine. Application of 103-126 residue atriopeptin for 10 min before and 2.5 min during the CRF/AVP stimulus strongly suppressed the evoked release of ACTH, the maximal inhibition was 75-90% at 10 nmol atriopeptin/l. The calcium ionophore ionomycin (200 nmol/l) reversed the effect of atriopeptin while it had no secretagogue activity of its own, and did not enhance the response to CRF/AVP. A variety of blockers of K+ channels, 4-amino pyridine, tetraethylammonium, apamine, quinine, but not tolbutamide, effectively antagonized the inhibitory action of atriopeptin (10 nmol/l). None of these drugs altered ACTH release evoked by CRF/AVP. In concentration-response experiments, the half effective concentration of 4-aminopyridine and tetraethylammonium were around 1 mmol/l and 10 nmol/l for apamine. Finally, tetraethylammonium and apamine also antagonized the inhibition of CRF/AVP-evoked ACTH release by 8-Br-cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophysial portal blood of conscious, unrestrained rams.

Insulin-induced hypoglycemia (IIH) is a strong stimulator of pituitary ACTH secretion. The mechanisms by which IIH activates the corticotrophs are still controversial. Indeed, in rats the variations of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) secretion in hypophysial portal blood (HPB) during IIH have been diversely appreciated. This may be due to the stressful conditions required for portal blood collection in rats. We studied the effects of IIH on the secretion of CRF and AVP in HPB and on the release of ACTH and cortisol in peripheral plasma in conscious, unrestrained, castrated rams. After the injection of a low (0.2 IU/kg) or high dose (2 IU/kg) of insulin, ACTH and cortisol levels in peripheral plasma increased in a dose-related manner. After injection of the low dose of insulin, CRF and AVP secretion in HPB were equally stimulated. After injection of the high dose of insulin, CRF secretion was further stimulated, while AVP release was dramatically increased. These results suggest that when the hypoglycemia is moderate, CRF is the main factor triggering ACTH release, and that the increased AVP secretion potentiates the stimulatory effect of CRF. When hypoglycemia is deeper, AVP secretion becomes predominant and may by itself stimulate ACTH release.

Secretion of ACTH by perifused isolated rat anterior pituitary cells: pulses of secretagogue enhance the secretory response and modify the effect of atriopeptin

The aim of the present study was to characterize the inhibitory action of atriopeptin on secretagogue-evoked ACTH release in vitro. Perifused isolated rat anterior pituitary cells were exposed to repeated pulses of 41-residue corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP). The net ACTH secretory response to both neurohormones increased progressively with the number of pulses applied, until a maximum hormonal response was reached which was stable for the subsequent period of observation (2-3 h). The maximal secretagogue-evoked hormone release eventually achieved was 4 and 1.7 times greater than the initial response to AVP and CRF-41 respectively. The size of the ACTH response elicited by 50 pmol CRF-41/l and 500 pmol AVP/l (CRF/AVP) given together also underwent progressive enhancement. The number of secretagogue pulses required to reach the maximal response to a particular stimulus depended upon the concentration of the secretagogue peptides, higher concentrations favoured a more rapid development of the stable secretory response. The potency of 103-126 residue atriopeptin to inhibit CRF/AVP-induced ACTH release varied by about 1000-fold depending upon the prior treatment of the cells. In general, cells not previously exposed to secretagogues appeared largely resistant, those under a moderate secretagogue drive were strongly inhibited, and those under intense stimulation were again refractory to inhibition by atriopeptin. In contrast, cortico-sterone suppressed stimulated ACTH release regardless of the state of the cells. The data demonstrate that the conditions of cell maintenance are pivotal determinants of the inhibitory effect of atriopeptin on secretagogue-stimulated ACTH release in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Basal and Corticotropin-Releasing Hormone-Stimulated Adenylate Cyclase Activity and Cytosolic Ca2+Levels by Somatostatin in Human Corticotropin-Secreting Pituitary Adenomas*

The effects of CRH and somatostatin (SRIH) on adenylate cyclase (AC) activity, intracellular free calcium concentrations [( Ca2+]i) and in vitro ACTH release were investigated in six human ACTH-secreting pituitary adenomas. In all tumors, CRH induced a marked stimulation (from 69-210% at 10 nM), whereas SRIH caused a definite inhibition (from 29-50% at 100 nM) of membrane AC. When added together, CRH and SRIH caused a purely additive effect on AC. In adenomatous corticotrophs CRH (10 nM) caused [Ca2+]i to rise from 160 +/- 30 nM (mean +/- SD) to 410 +/- 95 nM. CRH-induced transients were biphasic, with an initial peak predominantly due to redistribution from intracellular Ca2+ stores and a secondary phase due to Ca2+ influx. The effects of CRH on [Ca2+]i were totally independent of the stimulation of AC. In fact, cAMP-elevating agents other than CRH did not modify [Ca2+]i. SRIH (100 nM) decreased resting [Ca2+]i (approximately 20-40%) as well as [Ca2+]i rises induced by CRH, arginine vasopressin, or high K+. The effect of SRIH on [Ca2+]i was maintained in presence of high cAMP levels, while was totally abolished after pertussis toxin pretreatment. CRH (10 nM) stimulated ACTH release (from 22.5 +/- 3.5 to 45.0 +/- 8.5 pmol/L) by an extent similar to that elicited by calcium ionophore and forskolin. By contrast, SRIH (0.1 microM) inhibited both basal and CRH-stimulated ACTH release. In conclusion, in human adenomatous corticotrophs SRIH exerts an inhibitory action by reducing both AC activity and, independently, [Ca2+]i. In this way, SRIH can efficiently counteract the stimulatory action of CRH that in these cells involves activation of both cAMP and Ca2+ pathways.

Response to Corticotropin-Releasing Hormone during Pregnancy in the Baboon*

CRH is secreted by the placenta into human maternal and fetal plasma during gestation. In the present study plasma CRH was measured in the plasma of five pregnant baboons and their fetuses to ascertain whether the baboon is a suitable model for study of placental CRH. Studies were performed in chronically catheterized animals that exhibited no behavioral or endocrinological signs of stress; maternal animals moved freely about the cage. Mean maternal plasma CRH was 620 +/- 110 pmol/L (2970 pg/mL) at 146 +/- 11 days gestation, and mean fetal plasma CRH was 133 +/- 29 pmol/L (640 pg/mL) at delivery in four animals. Plasma CRH was undetectable (less than 8.5 pmol/L; less than 41 pg/mL) in nonpregnant animals and in animals 8 h after delivery. Maternal and fetal plasma CRH levels in the chronically catheterized baboon were very similar to human maternal and umbilical cord CRH levels at comparable gestational ages. In addition, the majority of maternal plasma CRH eluted in the same position as synthetic human CRH by gel filtration. CRH stimulation tests were performed in the chronically catheterized maternal baboon to investigate whether pituitary-adrenal function during pregnancy is similar to that observed after chronic CRH infusion; blunted ACTH and cortisol responses to acute injections of CRH are observed after chronic CRH infusion. The administration of 0.5 micrograms/kg ovine CRH (oCRH) failed to result in an ACTH or cortisol rise in four pregnant baboons. Baseline ACTH levels were 5.2 +/- 0.4 pmol/L (23.5 pg/mL), and baseline cortisol levels were 800 +/- 55 nmol/L (29.1 micrograms/dL); neither rose after CRH administration. In contrast, 0.5 micrograms/kg oCRH did result in significant ACTH and cortisol elevations in five nonpregnant baboons [ACTH: baseline, 5.9 +/- 1.4; peak, 16 +/- 4.8 pmol/L (P less than 0.05); cortisol: baseline, 430 +/- 55 nmol/L; peak, 960 +/- 200 nmol/L (P less than 0.05)]. In contrast, the administration of a larger dose of oCRH (5.0 micrograms/kg) led to stimulation of ACTH release in five pregnant baboons (baseline, 6.6 +/- 1.3 pmol/L; peak, 34.1 +/- 6.4; P less than 0.001). After this dose cortisol levels also rose in the pregnant animals (baseline = 1040 +/- 30 nmol/L; peak, 1620 +/- 130); however, this response was blunted compared to that in the nonpregnant animals (P less than 0.05). CRH (5.0 micrograms/kg) significantly stimulated both ACTH and cortisol in the nonpregnant animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of Serotonin Receptor Agonist-Induced Activation of the Hypothalamic-Pituitary-Adrenal Axis in the Rat

A substantial body of experimental evidence indicates that serotonin (5-HT) and several synthetic 5-HT receptor agonists activate the hypothalamic-pituitary-adrenal (HPA) axis. To explore the mechanism(s) by which 5-HT or 5-HT agonists enhance the activity of the HPA axis in vitro, we examined the stimulatory effects of the 5-HT1a agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT1c/5-HT1b agonist m-chlorophenylpiperazine (m-CPP), and the 5-HT2/5-HT1c agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI) on plasma ACTH and corticosterone secretion in the rat. To test whether 8-OH-DPAT, m-CPP, or DOI increase plasma ACTH levels by stimulating the release of endogenous CRH, catheterized conscious male Sprague-Dawley rats were pretreated with hyperimmune CRH rabbit serum (TS-6) or normal rabbit serum and subsequently challenged with a maximally stimulatory dose of the above 5-HT agonists. Pretreatment with TS-6 completely suppressed the ACTH response to m-CPP and significantly blunted the responses to 8-OH-DPAT or DOI. To examine whether the remaining ACTH response to 8-OH-DPAT or DOI was also mediated by a pituitary site of action, we administered each of these agents to pituitary stalk-transected or sham-operated rats. The ACTH responses to 8-OH-DPAT and DOI in stalk-transected rats were preserved, although significantly blunted, compared to those in sham-operated rats. This suggested that both of these 5-HT agonists may also act at the pituitary level to stimulate ACTH release in vivo. Although the ACTH responses to 8-OH-DPAT, m-CPP, and DOI were blunted after both TS-6 pretreatment and pituitary stalk transection, corticosterone responses were only slightly affected, suggesting that some of these compounds may cause corticosterone release in the rat through another mechanism. To evaluate this hypothesis, ACTH and corticosterone responses to 8-OH-DPAT, m-CPP, and DOI were examined in rats whose HPA axis had been suppressed by a single high dose injection of dexamethasone. The corticosterone responses to 8-OH-DPAT and DOI were blunted compared to those of saline-pretreated rats, but were inappropriately high compared to the ACTH responses observed in these rats. On the other hand, both ACTH and corticosterone responses to m-CPP were completely abolished by dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)

Sigma receptors modulate the hypothalamic-pituitary-adrenal (HPA) axis centrally: evidence for a functional interaction with NMDA receptors, [formula omitted

The present report investigates the modulation of the hypothalamic-pituitary-adrenal (HPA) axis in the rat by sigma receptors, using a selective ligand, (+) pentazocine, and comparing the effects with (+) SKF 10,047. Both compounds stimulate ACTH release potently after central and peripheral administration. These effects are centrally mediated, since they did not release ACTH from anterior pituitary primary cultures. The effects are not blocked by naloxone, but are blocked by the NMDA antagonist, CPP, indicating a centrally mediated functional interaction between NMDA and sigma receptors, in vivo .

Interleukin-1 Stimulates Corticotropin-Releasing Factor Gene Expression in Rat Hypothalamus*

To examine the effect of interleukin-1 (IL-1) on CRF and POMC gene expression, recombinant human IL-1 alpha and -beta were ip injected in rats. The plasma ACTH level showed a dose-related increase at 2 h after the injection of 0.5 and 2 micrograms IL-1 alpha and -beta, and also showed a sustained increase from 1 h until 5 h after the injection of 2 micrograms of IL-1 beta. CRF contents in the medial basal hypothalamus and ACTH contents in the anterior pituitary (AP) decreased at 2 h after the injection of 2 micrograms of IL-1 alpha and -beta, and such decreased levels were maintained until 5 h after the injection of 2 micrograms of IL-1 beta. The levels of CRF mRNA in the hypothalamus and POMC mRNA in AP significantly increased 3 h after the injection of 2 micrograms IL-1 alpha and -beta, and these levels were still higher at 5 h after the injection of 2 micrograms of IL-1 beta compared with those of the control. There was no significant change in the ACTH content and POMC mRNA levels in the intermediate-posterior pituitary or the hypothalamus or in the CRF contents and CRF mRNA levels in the cerebral cortex. These results indicate that acute administration of IL-1 alpha and -beta stimulates gene expression of hypothalamic CRF and CRF release, which causes the stimulation of ACTH release and POMC gene expression in AP.

Modulation of Basal and Corticotropin-Releasing Factor- Stimulated Proopiomelanocortin Gene Expression by Vasopressin in Rat Anterior Pituitary*

In several anterior pituitary hormone systems, factors that stimulate hormone release also stimulate hormone biosynthesis. In the corticotropes of the anterior pituitary, CRF stimulates ACTH release as well as cAMP accumulation and transcription of the POMC gene. Arginine vasopressin (AVP) is a well documented coregulator of ACTH release and potentiates CRF-stimulated cAMP accumulation. The present studies were undertaken to determine if AVP also potentiates the early effects of CRF on POMC gene expression in rat anterior pituitary primary cultures. We have measured the levels of the POMC primary transcript, processing intermediate, and mature mRNA in nuclear RNA samples and POMC mRNA in cytoplasmic RNA samples as well as ACTH release after treatment with CRF and/or AVP. After a 30-min treatment with 0.5 nM CRF, POMC primary transcript levels were increased by 200-400%. Thirty-minute or 1-h treatments with AVP alone (10 or 100 nM) did not affect primary transcript levels, but increased the amount of the processing intermediate, while a 2-h incubation with 100 nM AVP significantly decreased POMC primary transcript levels. The effects of CRF on the POMC primary transcript and nuclear processing intermediate were not potentiated by cotreatment with AVP, although a potentiation of CRF-stimulated ACTH release was observed. POMC nuclear and cytoplasmic mRNA levels were not affected by these 2-h or less treatments, while an 18-h incubation with 0.5 nM CRF alone or in combination with 100 nM AVP increased POMC cytoplasmic mRNA levels to about 140% of vehicle-treated control values. When a 1-h AVP treatment (100 nM) preceded presentation of CRF, we observed an attenuation of the stimulation of POMC primary transcript and processing intermediate levels by the 1-h CRF treatment. The effects of CRF on POMC primary transcript and processing intermediate levels in these experiments are consistent with a CRF-induced increase in POMC gene transcription. Our findings that AVP decreased primary transcript levels and attenuated the effects of CRF on this end point suggest that: 1) AVP clearly does not act in a synergistic manner with CRF to stimulate POMC gene expression; and 2) AVP possibly affects RNA stability and/or processing.

Thyrotropin-Releasing Factor-Induced Adrenocorticotropin Secretion Is Mediated by Corticotropin-Releasing Factor*

TSH-releasing factor (TRF), administered into the lateral cerebroventricle of adult male rats, elevated plasma concentrations of ACTH, epinephrine, and norepinephrine. TRF given iv was devoid of these activities. The CRF receptor antagonist, alpha-helical CRF9-41 (CRF9-41) given iv suppressed the TRF-induced increase in ACTH, but did not alter TRF-induced changes in plasma catecholamines. Intravenous administration of CRF antiserum totally blocked TRF-induced elevation of plasma ACTH concentrations. CRF receptor antagonists administered icv attenuated CRF-induced, but not TRF-induced elevation of plasma concentrations of ACTH, epinephrine, and norepinephrine. It is concluded from these results that TRF acts within the central nervous system to stimulate ACTH release through a CRF-dependent mechanism.

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The 'R'-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 microM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 microM respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.

Feedback control of vasopressin and corticotrophin secretion in conscious dogs: effect of hypertonic saline

Glucocorticoids are known to inhibit the ACTH response to a variety of stimuli. It has been suggested that vasopressin secretion is also inhibited by glucocorticoid negative feedback. The purpose of this study was to (1) determine the ACTH response to hypertonic saline and its sensitivity to glucocorticoid negative feedback and (2) to determine whether physiological elevations of plasma cortisol inhibit subsequent vasopressin responses to hypertonic saline. Five mongrel dogs (15-18 kg) were prepared with chronic arterial and venous catheters and studied while conscious. Ten experiments were performed on each dog in a randomized design separated by at least 5 days. Each experiment consisted of a pretreatment period (from -60 to -30 min except for dexamethasone administration) during which a glucocorticoid feedback signal was applied and a stimulus period (from 0 to 30 min) during which hypertonic saline was infused. The pretreatment and stimulus periods were separated by 30 min. Pretreatments were as follows: isotonic saline (control), half-maximal and maximal cortisol infusion (5.5 or 11 nmol/kg per min), ACTH(1-24) infusion (6.8 pmol/kg per min) which produces increases in endogenous cortisol, and dexamethasone (1.5 mg i.m.) given at 17.00 h the day before experimentation. Stimuli were as follows: hypertonic saline was infused at 0.2 or 0.4 mmol/kg per min which increased plasma sodium by about 6 or 12 mmol/l respectively. NaCl infusion at 0.2 mmol/kg per min had no effect on plasma ACTH or cortisol except when subsequent to ACTH(1-24) pretreatment when plasma ACTH actually increased to 41.4 +/- 2.9 pmol/l in response to hypertonic saline.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1β and Other Cytokines Stimulate Adrenocorticotropin Release from Cultured Pituitary Cells of Patients with Cushing's Disease*

Interleukin-1 (IL-1) is a cytokine that is secreted by macrophages and monocytes which stimulates rodent hypothalamic CRH release and possibly pituitary ACTH secretion. We studied the effect of IL-1 beta and other cytokines (gamma-interferon, thymosin fraction-5, and granulocyte colony-stimulating factor) on ACTH release from corticotroph adenoma tissue obtained from two patients with Cushing's disease. IL-1 beta (0.001-10 mumol/L) increased ACTH release 3-fold. Thymosin fraction-5 (10 mumol/L), gamma-interferon (1 mumol/L), and granulocyte colony-stimulating factor (1 mumol/L) also stimulated ACTH release. These cultured cells secreted little or no GH, PRL, TSH, LH, and FSH, and their release was not stimulated by any cytokine. These results suggest that ACTH release in patients with Cushing's disease may be responsive to stimulation by various cytokines.

Rapid as well as Delayed Inhibitory Effects of Glucocorticoid Hormones on Pituitary Adrenocorticotropic Hormone Release Are Mediated by Type II Glucocorticoid Receptors and Require Newly Synthesized Messenger Ribonucleic Acid as well as Protein*

Glucocorticoid hormones suppress the release of ACTH by the anterior pituitary gland: rapid feedback inhibits hormone secretion within 30 min of steroid application, delayed feedback is most effective at 1-2 h, and slow feedback becomes manifest in several hours. The aim of the present study was to determine the type of glucocorticoid receptor that mediates the rapid and delayed feedback actions of glucocorticoids and whether genomic activation occurs during the rapid and delayed time domains. Rat anterior pituitary cell columns were perfused with Dulbecco's minimum essential medium, 41-residue CRF (10(-9) M) was used as the secretagogue, which stimulated ACTH secretion to a peak of about 8- to 10-fold of basal release. The amount of ACTH released upon repeated 5 or 10 min stimulation with CRF was constant. Treatment with 10(-7) M corticosterone for 20 min immediately before and for 10 min during stimulation with CRF reduced ACTH release by about 50% (rapid feedback), while at 1 h and 2 h after the initial exposure to corticosterone the secretory response was 33% and 15% of control, respectively. The effect of corticosterone was prevented by the type II glucocorticoid/progesterone antagonist RU 38486 (10(-6) M). The selective type II receptor agonist RU 28362 (10(-7) M) was even more potent than corticosterone in inhibiting ACTH release; the time course of action was similar. When actinomycin D (10(-4) M) was applied in conjunction with RU28362 or corticosterone, no inhibitory effects appeared up to 2 h after the exposure to steroid. Puromycin (10(-4) M), given during and for 1 h after the administration of the steroid prevented the rapid as well as the delayed (1 h) inhibitory action of RU28362. When puromycin was removed from the system, a 75% inhibition of stimulated ACTH release developed at 2 h after the application of the steroid, indicating that translatable messenger RNA (mRNA) was still present in the cells. Cycloheximide (10(-4) M) was only partially effective at inhibiting rapid or delayed feedback, and increasing its concentration impaired the ACTH response to CRF-41. In summary, at the pituitary level the rapid as well as the delayed feedback inhibition of ACTH secretion by adrenal corticoids is exerted via type II glucocorticoid receptors. Furthermore, both rapid and delayed feedback require the synthesis of new mRNA and protein.

Effect of acute ether or restraint stress on plasma corticotropin-releasing hormone, vasopressin and oxytocin levels in the rat.

Ether and restraint stress-induced peripheral plasma corticotropin releasing hormone (CRH), arginine vasopressin (AVP), oxytocin (OXY) and adrenocorticotropin (ACTH) levels were measured by radioimmunoassays. Plasma CRH, AVP, OXY and ACTH rose to approximately twice the level of control rats 2 min after the onset of a 1-min exposure to ether. Plasma CRH rose further 5 min after the onset of ether stress, while plasma AVP and OXY returned to the baseline levels at 5 min. Plasma CRH, OXY and ACTH showed significant elevation 2 min after the onset of restraint stress, while plasma AVP did not show a significant change. Plasma OXY and ACTH rose further 5 min after the onset of restraint stress, whereas plasma CRH returned to baseline levels. CRH and OXY concentrations in the hypothalamic median eminence decreased 5 min after the onset of ether exposure and restraint, while the AVP concentration did not differ from control levels. The results, including the discrepancy between plasma CRH and ACTH 5 min after stress, suggest that CRH in the peripheral plasma is derived from both hypothalamic and extrahypothalamic tissues. The levels of stress-induced CRH in the peripheral plasma were sufficient to stimulate ACTH release. These results suggest that ether and restraint stress elevate plasma CRH shortly after the onset of the stress, and that this elevation in the plasma CRH level is at least partly responsible for stress-induced ACTH secretion.

Morphine Directly Modulates the Release of Stimulated Corticotrophin-Releasing Factor-41 from Rat Hypothalamusin Vitro

The actions of opioids and opiates on the hypothalamo-pituitary-adrenal axis are currently controversial. In the rat, morphine is reported to both stimulate and inhibit ACTH and corticosterone secretion, but the precise sites and mechanisms of these effects have remained unclear. To analyze further the hypothalamic actions of morphine, we have investigated its effect on hypothalamic fragments in vitro and measured the major CRF, CRF-41, by a specific RIA. The acute effects of morphine on both basal and stimulated ACTH release from dispersed pituitary cells were also investigated. Morphine (10(-8)-10(-6) M) did not significantly alter the basal secretion of CRF-41. However, similar concentrations of morphine inhibited CRF-41 release stimulated by norepinephrine in a dose-dependent manner. Similarly, morphine (10(-6) M) inhibited acetylcholine (10(-9) M)- and serotonin (10(-7) M)-stimulated CRF-41 release. The stimulatory effect on CRF-41 release induced by veratridine (10(-6) M) was inhibited by approximately 50% in the presence of morphine. KCl (28 nM)-mediated CRF-41 release was also significantly inhibited by morphine. Naloxone (10(-7)-10(-5) M) had no significant effect on either basal or norepinephrine-induced CRF-41 release, but reversed the inhibitory effect of morphine on norepinephrine-induced CRF-41 secretion in a dose-dependent manner. Morphine (10(-6)-10(-5) M) had no effect on either basal or CRF-41-stimulated ACTH release from dispersed pituitary cells. These data suggest that the predominant effect of morphine on hypothalamic CRF-41 release in vitro is suppression of the release induced by a variety of putative neurotransmitters and depolarizing agents. This inhibitory effect is reversed by naloxone, suggesting that it is mediated by opiate receptors, presumably situated directly on CRF-41 neurons.

Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)