Stimulation of guanosine 3',5'-cyclic monophosphate accumulation in rat anterior pituitary gland in vitro by synthetic somatostatin

Synthetic somatostatin stimulated cyclic GMP accumulation with dose dependency (10 ng/ml – 10 μg/ml in a dose examined) in rat anterior pituitary gland in vitro. The stimulation of cyclic GMP levels in the gland was observed after 2 min incubation with somatostatin. Cyclic AMP production induced by TRH or PGE₁ was supressed by this GH release inhibiting factor, while cyclic GMP concentration in the gland was elevated. The present results seem to suggest that inhibitory effect on GH release by somatostatin in anterior pituitary gland is mediated through change in concentration of cyclic AMP and cyclic GMP in the target cells.     

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.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland.

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Dexamethasone is a potent stimulator of growth hormone-releasing factor-induced cyclic AMP accumulation in the adenohypophysis

The stimulatory effect of maximal concentrations of synthetic human pancreatic growth hormone (GH)-releasing factor (GRF)(1-40)NH on cyclic AMP accumulation in rat anterior pituitary cells in culture is 4.5-fold increased following a 48-h preincubation with the potent glucocorticoid dexamethasone while the sensitivity of GRF action is increased by approximately 4-fold. Dexamethasone pretreatment, on the other hand, has no effect on basal cyclic AMP levels but approximately doubles both basal and GRF-induced GH release. The present data suggest that the potent stimulatory effect of glucocorticoids on GH secretion is exerted on the adenylate cyclase system at a step preceding cyclic AMP formation.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Effect of prostaglandins on hypothalamic-pituitary-testicular function in vitro.

The effect of prostaglandins (PG) A1, E1, E2 and F2 alpha in the concentration range of 10(-7)--10(-4) M were studied in vitro on a rat hypothalamic tissue, collagenase-digested isolated anterior pituitary cell and Leydig cell suspension system by measuring the testosterone production of incubated Leydig cells. PGs did not change the testosterone production and the hCG sensitivity of the Leydig cells, nor the LH secretion and the LHRH sensitivity of the anterior pituitary cells. PGE2 at concentrations of 10(-6), 10(-5) and 10(-4) M significantly increased the hypothalamic tissue-induced pituitary-testicular activation, and this stimulatory effect of PGE2 was dose dependent. PGA1, PGE1 and PGF2 alpha did not alter hypothalamic LHRH release measured in vitro. The results suggest that PGE2 has a direct stimulatory effect on hypothalamic LHRH release.     

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.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. d-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only d-glucose and d-mannose were able to stimulate insulin release and cyclic [³H]AMP accumulation in the absence of other substrate. d-fructose had a stimulatory effect in the presence of 3.3 mM d-glucose only at a high concentration (38.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. d-Galactose was effective only together with 8.3 mM d-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [³H]AMP accumulation, was glucose-mannose-fructose-galactose.l-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM d-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [³H]AMP response.d-mannoheptulose and d-glucosamine inhibited the insulin and cyclic [³H]-AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s.Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [³H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the β-cell.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration.

Five min following a single iv injection of PGE2 into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE2 injection. Plasma LH le-el increased at 2 min after PGE2 and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

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.     

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration

Five min following a single iv injection of PGE₂ into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE₂ injection. Plasma LH level increased at 2 min after PGE₂ and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Protein kinase C enhances growth hormone releasing factor (1-40)-stimulated cyclic AMP levels in anterior pituitary. Actions of somatostatin and pertussis toxin

The brain peptide human growth hormone releasing factor (1-40) (GRF), which stimulates adenylate cyclase activity in the anterior pituitary, is the predominant hormone signal for pituitary growth hormone (GH) release. Activators of protein kinase C such as teleocidin and 4 beta-phorbol 12-myristate 13-acetate (PMA) double the cyclic AMP accumulation induced by GRF, with no apparent effect on GRF potency; an inactive 4-alpha-PMA has no such action in cultured anterior pituitary cells. This PMA potentiation can be measured as early as 60 s, is maximal by 15 min, and wanes such that by 3-4 h there is no such amplifying effect of PMA. PMA, phorbol 12,13-dibutyrate, and teleocidin ED50 values for potentiating GRF activity are similar to those obtained for direct protein kinase C activation. The major inhibitory peptide somatostatin reduced both GRF- and GRF + PMA-stimulated cyclic AMP accumulation. Pertussis toxin totally blocked this somatostatin action without affecting the degree of maximal GRF potentiation achieved with PMA. Thus, the pertussis toxin target(s) are required for somatostatin inhibition of the cyclic AMP generating system, but may not be involved in the PMA potentiation of GRF-stimulated cyclic AMP accumulation.     

The effect of prostaglandins on ox pituitary content of adenosine 3′:5′-cyclic monophosphate and the release of growth hormone

1. An assay, based on competition between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and cyclic [(3)H]AMP for binding to a rabbit skeletal muscle protein, has been used to measure tissue contents of cyclic AMP. The assay has a sensitivity of 0.05pmol of cyclic AMP. Cyclic GMP and cyclic CMP have 0.5%, and cyclic IMP 6.5%, of the ability of cyclic AMP to displace cyclic [(3)H]AMP from binding protein; AMP, ADP and ATP have no effect. 2. By using this method, the cyclic AMP content of ox pituitary slices exposed to prostaglandin was determined; release of growth hormone was measured by radioimmunoassay. 3. Release of growth hormone was increased by 45min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 10nm-prostaglandin E(2), 0.1mum-prostaglandin A(1) or 1mum-prostaglandin B(1) in the presence of 0.5mm-theophylline. 4. Pituitary cyclic AMP content was increased by 10min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 0.1mum-prostaglandin E(2) in the presence of 0.5mm-theophylline. 5. The maximum increase in cyclic AMP content was observed 10min, and significant changes in growth hormone release 30min, after introduction of prostaglandin E(2). 6. The increase in pituitary cyclic AMP content, but not in the rate of release of growth hormone, was observed in the absence of external Ca(2+). 7. The stimulation of release of growth hormone by prostaglandin was decreased by preincubation of tissue for 2h in colchicine (100mum) or cytochalasin B (10mug/ml). 8. These results support the suggestion that increased release of growth hormone after treatment with prostaglandin is the result of increased tissue cyclic AMP content, and possibly involves a microfilamentous or microtubular protein.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Effects of thyrotropin and N6,O2′-dibutyryl cyclic 3′,5′-adenosine monophosphate on prostaglandin levels in thyroid

We have shown that TSH increases PG levels in isolated bovine thyroid cells. We now report that TSH also increases PG levels in rat and mouse thyroid, and that these effects may be mediated via cyclic AMP. PG and cyclic AMP levels in intact rat and mouse thyroid lobes were measured by radioimmunoassay. During 60-min incubations at 37°C, 25 mU/ml TSH effected a 75–83% increase in PGE₁ and PGF_2α ”equivalents“ in rat thyroid; parallel measurements of endogenous cyclic AMP in these intact thyroid lobes revealed that maximal TSH-induced increase in cyclic AMP also required 60-min incubations. In mouse thyroid, 5 mU/ml TSH increased PGE₁ and PGF_2α levels 38–82% above basal; this TSH effect was evident within 15 min of incubation, thus mimicking the time-course of TSH-induced increase in mouse thyroid cyclic AMP. Exogenous DBcAMP, 0.5 to 3 mM, effected a dose-related increase in mouse thyroid PG levels. The stimulatory effects of both TSH and DBcAMP on mouse thyroid PG levels were abolished by aspirin and indomethacin. These studies suggest that TSH-induced increase in endogenous PG levels in thyroid may be mediated by cyclic AMP.     

The regulation of growth hormone secretion from the isolated rat anterior pituitary in vitro. The role of adenosine 3′:5′-cyclic monophosphate

1. The release of growth hormone from isolated fragments of rat anterior pituitary tissue incubated in vitro was studied by employing a double-antibody radioimmunoassay. 2. In the absence of added stimuli, two phases of hormone release could be distinguished, an early phase of 2h duration and a subsequent late phase. In the early phase, hormone release was rapid but could be significantly decreased by calcium depletion and by 2,4-dinitrophenol whereas the rate of release in the late phase was uninfluenced by these incubation conditions. These results have been interpreted as indicating the existence of a secretory component in the early phase of release. 3. In subsequent experiments, the effects of various agents on the rate of hormone output during the late phase of incubation were investigated. Hormone release was increased by theophylline and by dibutyryl cyclic AMP (N(6)-2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate), the response to both of these agents being related to the concentration of the stimulant employed. 4. The stimulation of growth hormone output by theophylline was significantly decreased by calcium deprivation and by 2,4-dinitrophenol. The response to dibutyryl cyclic AMP was diminished by 2,4-dinitrophenol, iodoacetate and 2-deoxyglucose but not by malonate or colchicine. 5. Arginine, beta-hydroxybutyrate, albumin-bound palmitate and variation in the glucose concentration of the incubation medium over a wide range were without any statistically significant effect on the rate of hormone release from either control pituitary fragments or those subject to secretory stimulation by dibutyryl cyclic AMP. 6. It is suggested that the regulation of growth hormone secretion is mediated by cyclic AMP (adenosine 3':5'-cyclic monophosphate). The secretion observed in response to cyclic AMP requires the presence of ionized calcium and a source of metabolic energy but is independent of pituitary protein synthesis de novo. The integrity of the glycolytic pathway of glucose metabolism appears to be essential for cyclic AMP-stimulated growth hormone secretion to occur.