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.     

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.     

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.     

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.     

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.     

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)     

Stimulatory effects of Gila monster venom on rat pancreatic acini

The effects of Gila monster venom on dispersed rat pancreatic acini were compared with those of secretin and VIP. The efficacy of the venom in terms of amylase release was much higher (a 24-fold increase over basal secretion) than that of secretin (a 4-fold increase) and VIP (+ 40% only). On the other hand, cyclic AMP levels increased 12-fold with the venom, as compared to 18-fold with secretin and 16-fold with VIP. The venom, VIP and secretin all displaced 125I-VIP and the competition curve with the venom was steeper, suggesting that all VIP-recognizing receptors bound the venom with the same affinity. VIP receptors were, however, not responsible for the release of amylase provoked by the venom since VIP (and secretin) did not inhibit the secretory action of the venom. The venom exerted no effect on 45Ca efflux and its secretory effect did not depend on the presence of external calcium. Besides, the effect of CCK-8 on amylase release was additive with the effect of the venom. A first exposure to the venom induced a refractoriness to itself with respect to amylase release but not in terms of cyclic AMP increase. In conclusion, Gila monster venom may contain one component binding to VIP/secretin receptors with resulting cyclic AMP elevation. A second venom component may be responsible for the high secretory efficacy, without involving cyclic AMP or calcium efflux.     

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.     

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.     

Cellular Mechanisms Regulating Adrenocorticotropin Release

Abstract

Using a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16), the various factors regulating ACTH release and the intracellular mechanisms mediating this response were investigated. CRF, catechola-mines and VIP stimulate ACTH release whereas glucocorticoids and SRIF block secretion. Glucocorticoids block both ACTH synthesis and release. SRIF acts through multiple mechanisms to prevent stimulated ACTH release. Cyclic AMP and Ca⁺⁺ are important second messengers in the receptor mediated release of ACTH but other mediators may also be involved. The interaction of these various CRF-like substances and inhibitors of ACTH release may result in a fine-tune regulation of corticotroph activity Such regulation may be important in the organism response to stress.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

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.     

Inhibition of protein synthesis on homologous and heterologous desensitization in the rat adipocyte

Desensitization of lipolysis was induced in isolated rat adipocytes by incubation with isoproterenol 10^?5M or ACTH 250 mU/ml for two and three hours, respectively. Those cells desensitized with isoproterenol were restimulated with either isoproterenol 10^?7M or ACTH 6 mU/ml and those cells desensitized with ACTH were restimulated with isoproterenol 10^?7M. Lipolysis was quantitated by the release of cyclic AMP and glycerol. No effect on either homologous or heterologous desensitization was observed when either cycloheximide 2 μg/ml or puromycin 10^?4M was included in the incubation media during the induction of desensitization. These findings support the conclusion that protein synthesis plays no role in the desensitization of lipolysis in the isolated rat adipocyte.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Control of endogenous triglyceride breakdown in the mouse diaphragm

The control of endogenous triglyceride breakdown was studied in vitro, in the incubated intact mouse diaphragm. Isoproterenol (2 microgram/ml) produced parallel increases in glycerol and free fatty acid release, and in tissue cyclic AMP levels, suggesting that cyclic AMP mediates the action of the catecholamine on triglyceride mobilization. In addition to cyclic AMP, calcium seems to be involved in the action of isoproterenol because preincubation of hemidiaphragms in the presence of the calcium ionophore A23187 decreased the lipolytic effect of the drug. Insulin (12.5 mU/ml) antagonized the action of isoproterenol on triglyceride breakdown (it decreased glycerol and free fatty acid release) without altering its stimulatory effect on cyclic AMP levels. On the other hand, no detectable effect on lipolysis was observed with carbachol in control and denervated hemidiaphragms, although the latter possess acetylcholine receptors over the entire surface area of the muscle. It was concluded that catecholamines control triglyceride breakdown in muscle while the cholinergic system does not seem to be involved. Cyclic AMP, calcium, and insulin all affect lipolysis in muscle and the interrelationships remain to be elucidated.     

High concentrations of catecholamines selectively diminish the sensitivity of CRF-stimulated ACTH release by cultured rat pituitary cells to the suppressive effects of dexamethasone

ACTH-release by primary cultures of rat anterior pituitary cells in response to CRF, vasopressin, epinephrine, norepinephrine and VIP is readily suppressible by dexamethasone. Rat hypothalamic extract-induced ACTH release is less sensitive to the inhibitory effect of dexamethasone than that elicited by CRF and the other secretagogues mentioned above. In studying the additive and potentiating effect on ACTH release of CRF in combination with vasopressin, VIP and the catecholamines it became evident that only the combination of micromolar concentrations of epinephrine or norepinephrine together with nanomolar concentrations of CRF will make ACTH release significantly less sensitive to the suppressive effect of dexamethasone. Other combinations of CRF and vasopressin or CRF and VIP will render ACTH release as suppressible to dexamethasone, as that elicited by each of these compounds by itself. This observation in the rat might explain at least in part the observation that a diminished suppressibility of the pituitary-adrenal axis to dexamethasone can be found in patients with psychiatric disease, especially depression.     

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.