Characteristics of action of prostaglandins on cyclic AMP accumulation in rat anterior pituitary gland.

Prostaglandins (PGs) were found to lead to a marked stimulation of cyclic AMP accumulation in rat anterior pituitary gland in vitro in the following decreasing order of potency: PG E-1 E-2 GREATER THAN A-1 A-I GREATER THAN F-1ALPHA F-2ALPHA. The effect of PGs is potentiated by theophylline. The stimulatory effect of PGs on cyclic AMP accumulation is already detected 2min after the addition of 1-x 10-7 to 1-x 10-6 M PG E-2 and its maximal effect is reached after approximated 30 min of incubation, with a progressive decrease toward basal cyclic AMP levels at later time intervals. Increased intracellular cyclic AMP concentrations are accompanied by an increased release of the nucleotide into incubation medium. Complete removal of Ca-e+ from the incubation medium by addition of EGTA was found to increase the stimulatory effect of PG E-2 ON CYCLIC AMP accumulation. The action of PGs on hormonal release and cyclic AMP accumulation support the hypothesis of a role of PGs in the mechanism of anterior pituitary hormone (particularly growth hormone) release.     

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.     

Effects of colchicine on the morphology and prolactin secretion of rat anterior pituitary cells in monolayer culture

The effects of incubation of rat anterior pituitary cells in monolayer culture with 10(-6) M colchicine have been investigated during time-intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration at later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and at later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: at short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "bleb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment.     

Localization of microfilaments in prolactin cells of the rat anterior pituitary gland

Summary Prolactin cells from anterior pituitary glands of normal non-lactating female rats, and lactating animals, some of which were separated from their pups for 48 hours, were examined ultrastructurally for the presence of microfilaments. Microfilaments were found in specific intracellular locations in all cells examined. They were in association with the nuclear envelope, the Golgi complex, the endoplasmic reticulum, small vesicles of the endoplasmic reticulum, and secretory granules. The possible role of microfilaments in the movement of intracellular organelles is considered.This investigation was supported by the National Institutes of Health grants AM 12583 and TW 02023.The authors wish to express their gratitude to Mr. M. G. Williams and Miss Pauline Cisneros for their excellent technical assistance.     

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.     

Effects of VIP, TRH, GABA and dopamine on prolactin release from superfused rat anterior pituitary cells

Effects of VIP, TRH, dopamine and GABA on the secretion of prolactin (PRL) from rat pituitary cells were studied in vitro with a sensitive superfusion method. Dispersed anterior pituitary cells were placed on a Sephadex G-25 column and continuously eluted with KRBG buffer. Infusion of TRH (10(-11) - 10(-8)M) and VIP (10(-9) - 10(-6)M) resulted in a dose-related increase in PRL release. LHRH (10(-8) - 10(-5)M) had no effect on PRL release. On the other hand, infusion of dopamine (10(-9) - 10(-6)M) and GABA (10(-8) - 10(-4)M) suppressed not only the basal PRL release from dispersed pituitary cells but also the PRL response to TRH and VIP. The potency of TRH to stimulate PRL release is greater than that of VIP, and the potency of dopamine to inhibit PRL secretion is stronger than that of GABA on a molar basis. These results indicate that TRH and VIP have a stimulating role whereas dopamine and GABA have an inhibitory role in the regulation of PRL secretion at the pituitary level in the rat.     

Pertussis toxin blocks somatostatin's inhibition of stimulated cyclic AMP accumulation in anterior pituitary tumor cells

Somatostatin inhibits both forskolin and (-) isoproterenol-stimulated cyclic AMP accumulation in AtT-20 cells. Pretreatment of these cells with pertussis toxin prevents somatostatin's inhibitory effects on cyclic AMP production. This pretreatment also enhances the cyclic AMP response to forskolin and (-) isoproterenol without affecting basal cyclic AMP levels. The blockade of somatostatin's inhibitory effect was dependent both on the time of preincubation and concentration of pertussis toxin used. The rise in forskolin-stimulated cyclic AMP formation following pertussis toxin treatment preceded the blockade of somatostatin's inhibitory actions. The results suggest that somatostatin acts through an inhibitory guanine nucleotide regulatory protein to affect adenylate cyclase activity.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.     

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.     

Size heterogeneity of prolactin secreted from and stored in the rat anterior pituitary gland in vitro

The size heterogeneity of rat pituitary prolactin was investigated using anterior pituitary glands from female rats incubated in vitro and gel filtration on Sephadex G-100. Monomeric prolactin was preferentially secreted compared with dimeric and trimeric material. When glands were incubated with dopamine, prolactin secretion was inhibited and the relative proportion of dimer in the gland (but not the medium) was decreased. Morphine sulphate reversed the effect of dopamine on prolactin secretion and on the proportion of prolactin in the gland that was in the dimeric form. The results suggest that monomeric prolactin is more readily secreted than dimer, and that dopamine decreases the production or stability of the dimer.     

Inhibition by prolactin of gonadotrophin-induced cyclic AMP accumulation in the preovulatory ovary

We investigated whether prolactin acts at the ovarian level by interfering with the accumulation of gonadotrophin-induced ovarian cyclic AMP. Mouse ovaries were incubated with hCG and varying doses of prolactin. At the end of the incubation, the cyclic AMP which accumulated in the tissue + medium was measured. In ovaries devoid of corpora lutea, a significant inverse correlation (r = -0.93, P less than 0.05) was obtained between the doses of prolactin (0.1-25.6 micrograms ovine prolactin) and hCG-induced accumulation of ovarian cyclic AMP. In the presence of the phosphodiesterase inhibitor IBMX, however, the same doses of prolactin failed to exhibit any restricting influence on the accumulation of cyclic AMP. In luteinized ovaries, the same doses of prolactin in the absence of IBMX did not inhibit the hCG-induced cyclic AMP accumulation.     

Effects of adrenalectomy and hydrocortisone on DNA synthesis in the rat anterior pituitary gland

The effect of adrenalectomy and hydrocortisone on /³H/-thymidine uptake by rat anterior pituitary cells in the short-time organ culture was investigated. Adrenalectomy significantly increases the incorporation tritiated thymidine into anterior pituitary cells nuclei, whereas hydrocortisone has an opposit effect. The existence on negative feedback between the intracellular hormone content and DNA synthesis in ACTH-secreting anterior pituitary cells is suggested.     

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.     

Studies of growth hormone secretion VIII. Effects of alpha,beta-methylene-ATP on prostglandin induced GH release and cyclic AMP accumlation in rat anterior pituitary glands.

Alpha, beta-methylene-ATP, a competitive inhibitor of adenylate cyclase of liver and fat cell membrane preparations, caused a dose related inhibition of PGE1 and PGE2-induced cyclic AMP accumulation in rat anterior pituitary explants. At the same time, this ATP analog potentiated PGE1 and PGE2-promoted growth hormone secretion. The possible functional role of prostaglandins and cyclic nucleotides in the regulation of growth hormone secretion remains to be defined.