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.     

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.     

Monensin inhibition of corticotropin releasing factor mediated ACTH release

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

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.     

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.     

Desensitization of corticotropin-releasing factor receptors

Pretreatment of rat anterior pituitary cells with corticotropin releasing factor (CRF) rapidly and markedly reduced the ability of CRF to restimulate cyclic AMP formation and adrenocorticotropic hormone (ACTH) release. The effect was dependent on the length of time of pretreatment as well as the concentration of CRF. Neither basal nor intracellular immunoreactive ACTH levels nor basal cyclic AMP content were affected. CRF's stimulatory action on cyclic AMP formation and ACTH release recovered within one hour following CRF pretreatment. Forskolin, a compound that directly activates adenylate cyclase also releases ACTH from these cells. Pretreatment with CRF did not alter forskolin-stimulated cyclic AMP accumulation or ACTH secretion. Furthermore, CRF pretreatment did not change epinephrine's ability to increase the release of ACTH. These results indicate that CRF can regulate the responsiveness of its own receptor.     

Effects of adrenalectomy on CRH regulation of ACTH release: adenylate cyclase activity, cyclic AMP-dependent protein kinase activity and ACTH release

Corticotropin releasing hormone (CRH) stimulation of ACTH release and cyclic AMP-mediated events involved in the control of ACTH release were compared in sham-operated and adrenalectomized rats. CRH-stimulated adenylate cyclase activity was decreased in pituitary homogenates from adrenalectomized animals. CRH-stimulated cyclic AMP accumulation was essentially abolished and CRH-stimulated cyclic AMP-dependent protein kinase (A-kinase) activity was decreased in freshly prepared anterior pituitary cells from adrenalectomized animals. Basal and CRH-stimulated ACTH release was elevated in these cells. Since ACTH release is increased in adrenalectomized rats despite the down regulation of CRH-linked pituitary mechanisms, we speculate that the site of action of disinhibition by corticosterone of ACTH release (or synthesis) following adrenalectomy is distal to the generation of cyclic AMP and/or that non-CRH mediated mechanisms assume a greater role in ACTH regulation following adrenalectomy.     

Indirect evidence that protein kinase C plays a critical role in signal transduction of both vasopressin and corticotropin-releasing factor on pituitary cells in culture

The possible role of protein kinase C (PKC) in the cyclic AMP-dependent mechanism of action of corticotropin-releasing factor (CRF) on proopiomelanocortin cells of anterior and intermediate pituitary glands was examined after pretreatment of cells in culture with the PKC inhibitor retinal or the phorbol ester PMA, which depletes cell stores of the kinase. We found that these drugs not only abolished ACTH response to PMA and vasopressin, which both activate PKC, but unexpectably also dampened by 80-90% the stimulatory effect of CRF. Cell treatment with retinal failed to prevent CRF-induced accumulation of cyclic AMP. Retinal and PMA pretreatments of intermediate pituitary cells likewise inhibited alpha-MSH secretion stimulated by CRF. These data provide evidence to suggest that the mechanism of action of CRF on pituitary cells involves both cyclic AMP and PKC messenger systems.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

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.     

Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/D16-16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu-cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.     

Second Messengers Associated with the Action of AII and Dopamine D2 Receptors in Anterior Pituitary. Relationship with Prolactin Secretion

Abstract

We have shown that DA receptors of the D2 subtype inhibit prolactin release by several mechanisms. DA receptors inhibit cyclic AMP production through a GTP binding protein sensitive to the Bordetella pertussis toxin. However, this mechanism cannot be involved in the blockade of the AII stimulated prolactin secretion by DA. This blockade is probably partly due to the inhibition of the AII-stimulated inositol phosphate production by DA. This inhibition is also sensitive to the Bordella pertussis toxin. The toxin is able to ADP-ribosylate three substrates in anterior pituitary cells (39, 40 and 41 kDa). In addition, we show here that AII receptors inhibit adenylate cyclase of anterior pituitary cell homogenates, but not in intact cells.     

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

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

Muscarinic cholinergic inhibition of cyclic AMP formation and adrenocorticotropin secretion in mouse pituitary tumor cells

Cholinergic muscarinic receptors were identified in AtT-20/D16-16 (AtT-20) cell membranes by receptor binding techniques and the effect of carbachol on basal and stimulated cyclic AMP formation and ACTH release was investigated. Carbachol markedly decreased the stimulatory effect of the adenylate cyclase activator, forskolin, on both cyclic AMP formation and ACTH secretion. Carbachol also reduced forskolin-stimulated adenylate cyclase activity. The stimulatory effects of (-) isoproterenol on cyclic nucleotide formation and ACTH secretion were also blocked by carbachol. The inhibitory effects of carbachol on (-) isoproterenol-stimulated cyclic AMP synthesis and ACTH secretion were reversed by the muscarinic antagonist, atropine, and not by the nicotinic antagonist, gallamine. These data suggest that in AtT-20 cells, inhibition of ACTH secretion may be regulated by activation of muscarinic receptors coupled negatively to adenylate cyclase.     

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.     

Protein kinase C potentiates corticotropin releasing factor stimulated cyclic AMP in pituitary

The hypophysiotrophic hormone corticotropin releasing factor (CRF) stimulates the anterior pituitary corticotroph to export stress hormones such as adrenocorticotrophic hormone (ACTH). In rat anterior pituitary cells, CRF-induced elevation of cyclic AMP was profoundly potentiated (by an order of magnitude) by stimulators of protein kinase C. This effect occurred within minutes, was concentration dependent, and exhibited the appropriate pharmacological specificity to attribute the effects to protein kinase C. Phorbol myristate acetate (PMA), phorbol dibutyrate (PDB) and teleocidin were active with appropriate EC50's, while 4-alpha-PMA was inactive. PMA and PDB were also ACTH secretagogues in their own right. We suggest that protein kinase C can modulate CRF receptor coupling to the adenylate cyclase holoenzyme in anterior pituitary cells.     

Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells

Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others.     

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 µM, 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 µ-M 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.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine