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.     

Effects of posttrial hormone injections on memory processes

This experiment examined the effect on memory of posttrial injections of epinephrine, norepinephrine, ACTH, growth hormone, vasopressin and corticosterone. Rats were trained with a weak footshock (0.7 mA, 0.35 sec) in a one-trial inhibitory (passive) avoidance task. The animals received subcutaneous injections of one of the above hormones or saline immediately after training. On a retention test 24 hr after training, animals which received ACTH (0.03 or 0.3 IU/rat), epinephrine (0.1 mg/kg) or norepinephrine (0.1, 0.3 or 1.0 mg/kg) had retention performance which was significantly better than that of saline control animals. A higher posttrial ACTH dose (3.0 I.U./animal) impaired later retention performance. ACTH (0.3 I.U./animal) and norepinephrine (0.3 mg/kg) injections administered 2 hr after training had no significant effect on retention. Immediate posttrial injections of vasopressin (dose range 0.001–1.0 I.U./animal), growth hormone (0.5–1.0 mg/kg), or corticosterone (0.01–4 mg/kg) did not significantly enhance retention. These findings indicate that epinephrine, norepinephrine, and ACTH injections can enhance memory processes if the hormones are injected shortly after training. Such results are consistent with the view that hormonal consequences of an experience, particularly epinephrine, norepinephrine and ACTH release, may normally have a modulatory influence on memory processes in untreated animals. In addition, it is therefore possible that other posttrial treatments which enhance or impair later retention performance may act through hormonal mechanisms.     

The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). Evidence that protein kinase C mediates the action of AVP

This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.     

Angiotensin II increases ACTH release in the absence of endogenous arginine-vasopressin

Recent studies from our laboratory indicate a primary central site of action of Angiotensin II (AII) to release ACTH. The present studies were designed to test whether AII is able to release ACTH in vivo in a similar fashion in intact, cannulated, freely moving Long-Evans (LE) or in vasopressin (AVP)-deficient, Brattleboro (DI) female rats. The in vivo response to AII was compared with that elicited by synthetic CRF. AII injected i.v. (0.4 or 2 micrograms/100 g BW) induced a significant, dose-related increase in plasma ACTH values 5 and 15 min after injection, in both LE and DI rats. CRF given to LE and DI rats at 0.4 micrograms/100 g BW elicited a larger increase in ACTH plasma values than a similar dose of AII, 5 or 15 min after the injection. Moreover, ACTH levels after CRF in DI rats were significantly greater than those obtained in LE controls. In vitro studies using dispersed anterior pituitary cells indicate that the response of cells from either LE or DI rats to AII or AVP (both at 10(-9) and 10(-8)M) was similar. Cells from DI donors were hyperresponsive to CRF (2 X 10(-11) and 10(-10)M) in terms of ACTH release when compared with the response of cells from LE rats. The present results suggest that the presence of AVP is not essential to mediate the central response to AII and that AII may act centrally to stimulate CRF release from the hypothalamus in vivo, which would then enhance ACTH output. The results in the DI rat indicate that the increased response to CRF may be an important compensatory mechanism involved in the regulation of adrenocortical function in the DI rat.     

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.     

Corticotropin releasing factor activity of CRF 41 in normal man is potentiated by angiotensin II and vasopressin but not by desmopressin

Multiple hypothalamic factors seem to influence ACTH release. In vitro and/or in vivo animal models have shown that angiotensin II, vasopressin and some of its analogs are ACTH secretagogues capable of potentiating the corticotropin releasing activity of CRF41. Since these effects are controversial in man, we investigated in 3 groups of volunteers the corticotropin releasing activity of a 2h-infusion of angiotensin II (7 ng/kg/min), vasopressin (1 ng/kg/min) and desmopressin (1 ng/kg/min) given alone or in combination with a bolus injection of 100 micrograms CRF41 by measuring plasma concentrations of ACTH, cortisol, dehydroepiandrosterone and delta 4-androstenedione. Given alone angiotensin II and desmopressin had no significant effect in contrast to vasopressin which increased significantly the ACTH and steroid levels. Angiotensin II and vasopressin were both able to potentiate the corticotropin releasing activity of CRF41, whereas desmopressin was unable to produce such a potentiation. These results suggest that in man vasopressin and angiotensin II may well regulate the responsiveness of the pituitary-adrenal axis in various physiological or pathophysiological situations.     

Cortisol responsiveness to insulin-induced hypoglycemia in Cushing's syndrome with huge nodular adrenocortical hyperplasia

A 51-yr-old male patient with a 3 yr history of Cushing's syndrome is described. The baseline plasma cortisol level was elevated, while the plasma ACTH levels remained at an undetectable level. Dynamic testing of pituitary-adrenal function revealed no suppression after 8 mg of dexamethasone, and there was no response to metyrapone or CRF, while plasma cortisol showed a hyperresponse to synthetic ACTH. Plasma cortisol responded to insulin-induced hypoglycemia without an obvious ACTH response. These and the computerized tomography data suggested a "huge" bilateral nodular adrenocortical hyperplasia which was later confirmed by surgery. The left and right adrenal glands weighed 55 and 76 g, respectively. In vitro experiments, using the adrenal tissue, showed that there was an adrenal cortisol response to 1-39 ACTH but not to regular insulin, arginine vasopressin, angiotensin II, norepinephrine or epinephrine. These results indicate that plasma cortisol responded to a slight hypoglycemia-induced plasma ACTH change which was not detected in the ACTH radioimmunoassay or to factors other than ACTH which might be induced by hypoglycemia.     

In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin

The ability of arginine vasopressin (AVP) to potentiate the actions of synthetic ovine corticotropin-releasing factor (CRF) was examined using anterior pituitary fragments. Marked potentiation of ACTH release was observed upon incubating the fragments with a combination of 2 nM AVP and 1 nM CRF. Potentiation of CRF-induced ACTH release was also observed when the fragments were incubated with a combination of 1 nM AVP and 0.5 nM CRF. These results suggest that AVP may play a role in the release of ACTH from the adenohypophysis.     

Lack of effect of serotonin and norepinephrine on CRF release from hypothalami in vitro

A number of investigations utilizing hypothalami from adrenalectomized animals have provided conflicting results regarding the role of serotonin (5HT) and norepinephrine (NE) in CRF regulation. In order to further investigate these neurotransmitters, we performed three sets of experiments with hypothalami obtained from intact rats. In experiment I, freshly obtained rat hypothalami were randomly grouped and incubated in control medium and medium in the presence of 10(-11) M, 10(-10) M, and 10(-9) M serotonin. Aliquots of 200 microliters of these incubates were bioassayed for CRF activity using a dispersed anterior pituitary cell system, and ACTH secretion from the cells was determined by radioimmunoassay. A preliminary experiment had determined that a 200 microliters aliquot from hypothalami incubated in control medium resulted in a significantly (p less than 0.0001) greater ACTH release than obtained from cells alone. No significant effect of serotonin on hypothalamic CRF release was obtained. In experiment III, individual hypothalami were bisected longitudinally, and one half served as control. The contralateral half was incubated in medium containing 10(-11) M, 10(-10) M, and 10(-9) M serotonin. CRF release in this experiment again revealed no significant effect of serotonin. In experiment II, hypothalami were again randomly grouped and incubated with control medium and in the presence of 10(-8) M and 10(-6) M norepinephrine. This experiment resulted in no significant effect of norepinephrine on CRF release. These results suggest that serotonin and norepinephrine at the concentrations studied have no effect on CRF release from hypothalami obtained from intact rats.     

Mechanism involved in synergistic adrenocorticotropin response to activating protein kinase-A and -C in rat anterior pituitary cells

Activation of protein kinase C (PKC) stimulates adrenocorticotropin (ACTH) release synergistically in the presence of corticotropin releasing factor (CRF). We examined the effect of a cyclic nucleotide-specific phosphodiesterase inhibitor, 1-isoamyl-3-isobutylxanthine (IIX), on arginine vasopressin (AVP)-induced ACTH release and intracellular cAMP accumulation in normal rat anterior pituitary cells. IIX alone elevated intracellular cAMP accumulation. IIX potentiated AVP-induced ACTH release synergistically without further increase in cAMP accumulation, suggesting that synergistic ACTH release has an alternative mechanism other than the synergistic elevation of intracellular cAMP accumulation which has been reported. Phorbol 12-myristate-13-acetate (PMA) also induced synergistic ACTH release when incubated with IIX. IIX had no additional effect on ACTH response when incubated with maximal dose of CRF, forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Moreover, the combination of PMA and 8-Br-cAMP produced synergistic ACTH response. In conclusion, the synergistic ACTH release from rat pituitary corticotrophs occurs at least in the presence of directly activating events of PKC and PKA as well as PKC-induced inhibition of phosphodiesterase activity.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Oxytocin potentiates the ACTH-releasing activity of CRF(41) but not vasopressin

The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.     

Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system

Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G-100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10-min exposure to these secretagogues. More than 80% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF.     

Effect of angiotensin II on ACTH release in vivo: role of corticotropin-releasing factor

The ability of angiotensin II (ANG II) to release ACTH was studied in vivo. ANG II elicited dose-related increases in plasma ACTH levels in freely-moving non-anesthetized rats. The observation that this effect was totally abolished by immunoneutralization of endogenous CRF, as well as the lack of stimulatory activity of ANG II in pharmacologically blocked animals, suggest that the induction of CRF secretion by ANG II represents an essential modulator of the ACTH-releasing potency of this peptide.     

The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Effect of dexamethasone on the release of pituitary hormones in monolayer cultures of rat pituitary cells

The effect of dexamethasone on the release of ACTH, GH, PRL, LH and TSH was studied in monolayer cultures of rat pituitary cells in 4-hour incubation. With or without the addition of rat hypothalamic extract, the release of GH was significantly inhibited by dexamethasone at concentrations higher than 10(-9) M, although less remarkably than that of ACTH. Intracellular ACTH and GH were unchanged. PRL, LH and TSH were not affected. These results indicate that dexamethasone, when exerted for 4 hours, suppressed the release of GH as well as ACTH, at least in part, at the pituitary level.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Site of action of 5-hydroxytryptophan and 5-hydroxytryptamine on the hypothalamo-pituitary-adrenal system in vitro.

5-hydroxytryptamine at a concentration of 0.04 microgram/ml was able to block the ACTH release caused by hypothalamic tissue (CRF) while it was ineffective when a hypothalamic extract containing CRF was used. 5-hydroxytryptophan added to rat adrenal tissue caused a dose-dependent increase in corticosterone production. In a dose of 0.04 microgram/ml, 0.4 microgram/ml and 4.0 microgram/ml, 5-hydroxytryptophan was able to inhibit the ACTH release caused by hypothalamic tissue in vitro. However 0.04 microgram/ml was ineffective on the increase in ACTH secretion elicited by hypothalamic extract CRF. The data suggest that the inhibitory action of 5-hydroxytryptophan and 5-hydroxytryptamine is exerted at the hypothalamic level by inhibiting the release and/or synthesis of the corticotrophic releasing factor.