Corticotropin-releasing factor (CRF) induced anorexia is not influenced by a melanocortin 4 receptor blockage.

CRF and melanocortin (MSH/ACTH) peptides share a number of central effects including anorexia and grooming. The effects of CRF may be secondary, due to CRF's effects on melanocortin peptide release. We investigated if the newly discovered selective melanocortin 4 receptor antagonist HS014 could influence CRF induced anorexia and grooming. The data show that ICV administration of CRF (3 mg/rat), significantly reduced food intake, feeding time and feeding episodes whereas it increased grooming time and grooming episodes. HS014 (5 mg/rat), that previously has been shown to antagonize the anorectic effect and the excessive grooming induced by alpha-MSH, did however not influence any of the behavioral effects induced by CRF when the peptides were administered together. The data indicate that the anorectic and grooming effects of CRF are independent of pathways involving the MC4 receptors. These data suggest that the anorectic and grooming effect of CRF are not due to a secondary effect caused by increase in release of melanocortins acting on the central MC receptors.     

Pressor, tachycardic and behavioral excitatory responses in conscious rats following ICV administration of ACTH and CRF are blocked by naloxone pretreatment

Experiments were conducted to compare the blood pressure and heart rate responses of conscious rats given intracerebroventricular (ICV) injections of adrenocorticotropin (ACTH 1-24) and corticotropin releasing factor (CRF). Under sodium pentobarbital anaesthesia, rats were implanted with a stainless-steel cannula into the lateral cerebral ventricle and had their right femoral artery and vein cannulated. Upon recovery (24-48 hr later) conscious, unrestrained rats were given ICV injections (total volume 5 microliter by gravity flow) of sterile saline, ACTH (1-24) (0.85 and 1.7 nmoles) or CRF (0.55 and 1.1 nmoles) and blood pressure and heart rate were monitored over the next 2 hr (from the abdominal aorta via the femoral arterial catheter). Both ACTH and CRF caused mean arterial pressure (MAP) to increase, which was paralleled with increases in mean heart rate (MHR). Moreover, these elevations in MAP and MHR were temporally associated with excessive grooming (for ACTH) and locomotor activity (for CRF), which occurred before and lasted as long as MAP and MHR were enhanced. Intravenous (IV) pretreatment whereby naloxone was given 10 min before ICV administration of ACTH (1.7 nmoles) or CRF (1.1 nmoles), showed that naloxone blocked the behavioral, pressor and tachycardic effects of both ACTH and CRF. The results demonstrate that the pressor, tachycardic and locomotor effects evoked in conscious rats by ICV administration of ACTH or CRF are antagonized by naloxone and that their hemodynamic changes may, in part, be mediated by prior behavioral activation.     

Site of calcium requirement for stimulation of ACTH release in rat anterior pituitary cells in culture by synthetic ovine corticotropin-releasing factor

Synthetic ovine corticotropin-releasing factor (CRF) causes a 6- to 8-fold stimulation of ACTH release and cAMP accumulation in rat anterior pituitary cells in culture at ED50 values of 1 and 4 nM, respectively. Removal of Ca2+ from the incubation medium reduces CRF-induced ACTH release by 70% but have no effect on cyclic AMP accumulation. ACTH release induced by 8-Br-cAMP is inhibited by 65% in the absence of Ca2+. The Ca2+ ionophore A23187 does not alter spontaneous ACTH release. Verapamil, a pharmacological agent that blocks Ca2+ entry into cells, has no influence on spontaneous or CRF-induced ACTH release. The present data clearly demonstrate a role of Ca2+ in CRF action at a step subsequent to cAMP formation and suggest that Ca2+ is mobilized from intracellular stores during CRF stimulation.     

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.     

Effects of adrenergic blockers on corticotropin-releasing factor-induced behavioral changes in rats

The effects of adrenoreceptor blocking agents on corticotropin-releasing factor (CRF)-induced behavioral changes in rats were examined. The i.c.v. injection of 1 micrograms ovine CRF significantly increased the grooming frequency, number of occurrences of rearing and total distance moved. I.c.v. administered phentolamine at a dose of 10 nmol completely suppressed the increase in rearing and total distance moved induced by CRF without affecting the grooming frequency, whereas 100 nmol phentolamine significantly decreased the grooming frequency as well as the rearing and total distance moved. In contrast, propranolol reduced the increase in rearing induced by CRF only at a dose which induced ataxia in rats. The increases in rearing and total distance moved induced by CRF were reduced by 10 nmol of yohimbine and 100 nmol of prazosin. S.c. injection of caffeine (10 mg/kg) produced a significant increase in grooming frequency, rearing, and total movement. Administration of 10 nmol phentolamine and yohimbine did not affect these behavioral changes induced by caffeine, while 100 nmol prazosin suppressed them. Therefore, prazosin depressed the behavior of rats non-specifically. These results suggest that CRF-induced behavioral hyperactivity is mediated at least in part by alpha-noradrenergic, mainly alpha 2-noradrenergic, systems in the brain.     

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.     

Effects of lithium on the hypothalamo-pituitary-adrenal axis

The effect of lithium on the hypothalamo-pituitary-adrenal axis was studied in vivo and in vitro. The levels of plasma vasopressin, ACTH and corticosterone increased after the administration of lithium (LiCl 4 mmol/kg BW, 11 days) in rats, while the tissue vasopressin concentration in the median eminence, the rest of the hypothalamus and the posterior pituitary was decreased. The CRF concentration in the posterior pituitary increased markedly, but it did not change significantly in the median eminence or the rest of the hypothalamus. The elevated plasma ACTH level might be at least partly due to the increased vasopression secretion. Lithium stimulated ACTH secretion per se and also enhanced vasopressin-induced ACTH secretion in cultured pituitary cells and in half pituitary incubations, while it did not affect CRF-induced ACTH secretion. Lithium inhibited CRF-induced cAMP accumulation in half pituitary incubations, while lithium and vasopressin did not affect cAMP accumulation per se or even when administered together. The results suggest that lithium-induced ACTH release is via a cAMP-independent mechanism. Thus, it is possible that lithium stimulates ACTH release by acting directly on the corticotroph, stimulating vasopressin release and potentiating vasopressin-induced ACTH release.     

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.     

Central corticotropin-releasing factor (CRF) may attenuate somatic pain sensitivity through involvement of glucocorticoids

Corticotropin-releasing factor (CRF) is an important regulator of physiological functions and behavior in stress. Analgesia is one of the characteristics of stress reaction and CRF is involved in providing stress-induced analgesia, however, the underlying mechanisms remain to be determined. Exogenous CRF mimics stress effects on pain sensitivity and causes analgesic effect. The present study was performed to investigate the participation of endogenous glucocorticoids in analgesic effects induced by central administration of CRF in anesthetized rats. The participation of glucocorticoids was studied by pharmacological suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis as well as an occupation of glucocorticoid receptors by its antagonist RU 38486. Since CRF administration causes the release of β-endorphin from the pituitary, the opioid antagonist naltrexone was used to determine the contribution of opioid-dependent mechanism to CRF-induced analgesia. An electrical current threshold test was applied for measurement of somatic pain sensitivity in anesthetized rats. Intracerebroventricular administration of CRF (2 μg/rat) caused analgesic effects (an increase of pain thresholds) and an increase in plasma corticosterone levels. Pretreatment with naltrexone did not change analgesic effects of central CRF as well as corticosterone levels in blood plasma. However, pharmacological suppression of the HPA axis leading to an inability of corticosterone release in response to CRF resulted in an elimination of CRF-induced analgesic effects. Pretreatment with RU 38486 also resulted in an elimination of CRF-induced effects. The data suggest that CRF-induced analgesic effects may be mediated by nonopioid mechanism associated with endogenous glucocorticoids released in response to central CRF administration.     

The effect of corticotropin-releasing factor (CRF) on autonomic and behavioral responses during shock-prod burying test in rats

When administered intracerebroventricularly (ICV) in rats, corticotropin-releasing factor (CRF) possesses arousing and anxiogenic properties, which may be found reflected in autonomic and behavioral activation. As these responses are dependent on dose and situation, ICV-injected CRF may affect behavioral responses to a defined stimulus in a different fashion than autonomic concomitants. Two experiments were conducted in order to test this hypothesis. In both experiments, rats were treated ICV with CRF or an artificial cerebrospinal fluid (aCSF) 5 min prior to a 15-min exposure to an electrified prod (shock-prod burying test, SPB test) in their home cages. In the first experiment, 0.3 ng CRF injected ICV in unhandled rats significantly reduced the prod-burying response to electric shock, in favor of immobility, whereas following 300 ng CRF ICV, the predominant behavioral response was grooming behavior. In contrast, habituated rats, implanted with telemetric devices to measure heart rate, core temperature, and gross activity in the second experiment, showed a significant increase of burying behavior after 0.3 ng CRF ICV, in comparison to vehicle-treated controls. However, simultaneous cardiac acceleration was of the same magnitude and duration in both groups. In addition, whereas similar rises in CT were observed in both groups during the SPB test, CRF-treated rats showed more marked rise in core temperature during the first 15 min of the posttest period. At the 24-h retention test, rats belonging to the CRF group showed burying behavior and HR responses, in onset, magnitude, and duration similar to day 1, whereas extinction of the burying response and tachycardia was found in controls. Changes in CT, although less marked, showed the same pattern as on day 1 in both groups. These results show a differential effect of central CRF on behavioral and autonomic activation induced by a well-defined stressful stimulus. The response to CRF seems to be not only situation related, but also dependent on the pretest experience of the animal.     

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.     

Regulation and role of p21-activated kinase 3 by corticotropin-releasing factor in mouse pituitary

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF, produced in the hypothalamic paraventricular nucleus, releases adrenocorticotropic hormone (ACTH) from the anterior pituitary (AP). ACTH in turn stimulates the release of glucocorticoid from the adrenal glands. Glucocorticoid then inhibits hypothalamic production of CRF and pituitary production of ACTH. Mice lacking a functional gene for CRF (CRF KO) showed severe impairment of the HPA axis, indicating that CRF is required for its regulation. We applied oligonucleotide microarray analysis to the AP of CRF KO to identify gene expression induced by CRF. Twenty-four genes showed less than 60% expression in CRF KO compared with normal mice. Real-time PCR analysis revealed that p21-activated kinase 3 (Pak3), prohormone convertase type 1 (PC1), and CRF-binding protein (BP) mRNA expression levels were increased by CRF in AP cells. Both Pak3 and PC1 were also increased by dexamethasone in AP cells, while CRF-BP mRNA levels were reduced. Therefore, both Pak3 and PC1 mRNA levels would be regulated by both CRF and glucocorticoids. Pak3 knockdown inhibited CRF-induced cell viability in AtT-20 cells, suggesting the important role of Pak3 in the proliferation of corticotrophs.     

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.     

Some functional aspects of canine corticotrophs

To examine the regulation and functional significance of canine pituitary pars intermedia corticotrophs, ACTH and cortisol responses to CRF were studied in healthy dogs before and after treatment with dexamethasone. In addition the effects of the dopamine agonist bromocriptine and the dopamine antagonist pimozide were investigated. In the latter two instances prolactin concentrations were also measured. Finally the pituitaries were studied immunocytochemically for ACTH and alpha-MSH. No response of ACTH or cortisol to bromocriptine was observed. Pimozide caused a slight rise in ACTH levels in some dogs. However, prolactin levels significantly decreased with bromocriptine and increased with pimozide. Injection of synthetic ovine CRF to dogs was followed by sharp increases in ACTH and cortisol values. These responses were obliterated by prior treatment with dexamethasone. In 1 of 4 dogs given dexamethasone before euthanasia, there were few pars distalis cells with ACTH(1-24) immunopositivity, although persistence of ACTH(1-24) reaction was noted within cells of the pars intermedia. The results indicate that none of the CRF-induced ACTH secretion in dogs is derived from pars intermedia corticotrophs. Dosages of bromocriptine and pimozide that clearly alter prolactin secretion do not consistently affect ACTH levels.     

Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs

Gastrointestinal motor activity following intracerebroventricular (ICV) and intravenous (IV) administration of corticotropin releasing factor (CRF), corticotropin (ACTH) and cortisol was investigated in fasted dogs with strain-gauge transducers chronically implanted on the antrum and proximal jejunum. ICV but not IV administration of CRF (20 to 100 ng/kg) suppressed the gastric cyclic migrating motor complex (MMC) for 3 to 6 hours without affecting the jejunum. Similar disruptive effects on the gastric MMC were observed after ICV administration of ACTH (0.5 U/kg) or cortisol (0.1 micrograms/kg) but not after IV administration of 10 times higher doses. These results suggest that in dog CRF may be involved in the central control of the interdigestive gastric motility, these effects were not probably due to the release of ACTH and cortisol the other hormones of the pituitary adrenocortical system change the gastric motility when centrally administered through a possible feed-back mechanism affecting brain CRF level.     

Intracerebroventricular ACTH activates the pituitary-adrenal system:dissociation from a behavioral response.

In the rat, intracerebroventricular injection of synthetic ACTH (ACTH_1–24, ACTH_1–16) elevated plasma corticosterone levels and induced the display of excessive grooming behavior. The grooming response could be elicited in hypophysectomized rats without concommittant elevation of plasma corticosterone. In intact rats subcutaneous injection of ACTH_1–24 and not of ACTH_1–16-NH₂ stimulated the release of adrenal corticosteroids, whereas no excessive grooming was observed. In contrast to the reduced effectiveness of a second icv injection of ACTH in inducing the behavioral response, no single-dose tolerance was observed for the effect of icv ACTH on the pituitary-adrenal system. Therefore it was concluded that two different central mechanisms underly the observed responses to the icv applied ACTH.     

A corticotropin-releasing factor antagonist reverses the stress-induced changes of exploratory behavior in mice

Corticotropin-releasing factor (CRF) administered intracerebroventricularly (ICV) to rats and mice has been shown to elicit a variety of behaviors resembling those that occur in stress. In a novel multicompartment chamber, ICV CRF altered the behaviors in a manner closely resembling that observed following a period of restraint. In particular, 75 ng CRF ICV or 30-40 min restraint markedly reduced the time mice spent in contact with novel stimuli. ICV injections of a peptide antagonist of CRF, alpha-helical CRF9-41 (ahCRF), reversed the effects of restraint on this measure. This effect of ahCRF was dose dependent, with a minimal effective dose of 10 micrograms. Other behavioral measures appeared normal, and ahCRF did not significantly alter the stimulus-contact time in unrestrained mice. These results provide strong evidence to support the hypothesis that endogenous CRF may be a factor affecting stress-induced changes in exploratory behavior in mice.     

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.     

The role of calcium in the mechanism of corticotropin releasing factor mediated ACTH release

To investigate the role of calcium (Ca+2) in CRF stimulated ACTH release, we studied the effect of the following conditions on CRF (10 nM) mediated ACTH release in primary pituitary monolayer culture: different concentrations of Ca+2; EGTA; lanthanum (La+3) and nifedipine, blockers of calcium cell influx and penfluridol, trifluoperazine, and pimozide, inhibitors of calmodulin activation. Higher concentrations of Ca+2 in the culture medium led to greater amounts of CRF induced ACTH release. EGTA at 3 mM decreased the amount of CRF stimulated ACTH release by 60% but did not alter the spontaneous release of ACTH. At 0.5 mM and 1.0 mM La+3, ACTH release induced by CRF was inhibited by 23% and 35% respectively (p less than 0.01). Nifedipine (both 10(-5) and 10(-4) M) inhibited CRF stimulated ACTH release but only to a maximum of 30%. This inhibition was completely overcome by the addition of 12 mM calcium. Penfluridol, pimozide, and trifluoperazine blocked the release of ACTH induced by CRF by 63%, 26%, and 0% respectively. In conclusion, extracellular Ca+2, Ca+2 influx, and calmodulin play a role in the mechanism of CRF stimulated ACTH in vitro.     

Role of cyclic AMP in corticotropin releasing factor mediated ACTH release

To elucidate the role of cAMP in the secretion of ACTH, the effect of (1) three phosphodiesterase inhibitors, (2) forskolin, and (3) 8Bromo-cAMP, on CRF mediated ACTH release was studied in rat pituitary cell culture. The action of glucocorticoids on CRF induced cAMP accumulation and ACTH release was investigated. Isobutyl-methylxanthine (IBMX), caffeine, and forskolin augmented the release of ACTH induced from CRF 1.0 nM by 17%, 39%, and 20%, respectively. Also IBMX and caffeine potentiated CRF 10 nM stimulated ACTH release by 32% and 20%. Doses of forskolin and 8Bromo-cAMP, which alone stimulate large amounts of ACTH release, did not increase the amount of ACTH released from CRF 100 nM stimulated cells. Cortisol (500 nM) and corticosterone (500 nM) inhibited CRF induced intracellular cAMP by 39% and 26% while inhibiting pituitary ACTH release by 40% and 52%. In conclusion, cAMP plays an important role in the mechanism of ACTH secretion and it appears the final intracellular mechanism of CRF stimulated ACTH is via cAMP. Also, glucocorticoids exert their inhibitory influence prior to cAMP generation.