GnRH action in rat anterior pituitary gland: regulation of protein, glycoprotein and LH synthesis.

The effect of synthetic GnRH on the synthesis of proteins and glycoproteins in the anterior pituitary and in vitro release of LH into the medium was studied. A maximal dose (25 ng/ml) of of synthetic GnRH caused optimum release of radioimmunoassayable LH into the medium after 2 h of incubation. A concomitant increase in cyclic AMP accumulation in the tissue and LH in the incubation medium was also observed under the influence of GnRH during different periods of incubation time. Incubation of the rat anterior pituitary with GnRH stimulated the incorporation of [3H] proline into acid precipitable proteins in a time- and dose-dependent manner, similar to radioimmunoassayable LH released into the medium. Similar results were obtained when pituitary was incubated with dibutyryl cyclic AMP. LH, in addition, enhanced the incorporation of [3H] glucosamine and [3H] amino acids mixture into acid-precipitable proteins suggesting that proteins including glycoproteins are synthesized by the rat anterior pituitary under the influence of GnRH. Approximately 10% of the radioactivity associated with proteins comigrated with radioimmunoassayable LH on the gels. GnRH also enhanced the incorporation of [3H] glucosamine and [3H] amino acid mixture into immunoprecipitable LH. The GnRH-induced incorporation of [3H] proline into anterior pituitary proteins was abolished by specific translation inhibitors.     

Basal and dibutyryl cyclic AMP-stimulated release of newly synthesized and stored growth hormone from perifused rat pituitaries.

The basal rate of pre-labeled stored 14C-labeled rat growth hormone ([14C]rGH) release from perifused rat pituitary explants is a constant fraction of pituitary GH content, suggesting random release from the storage pool. Basal release of newly synthesized [3H]rGH occurs in two phases: 1) immediate and associated with [3H]rGH synthesis, and 2) late (delayed by 60 min) and independent of concurrent [3H]rGH synthesis. Dibutyryl cyclic AMP (10(-2)M)-stimulated release of stored [14C]rGH is characterized by an initial acute rise followed by a second phase of continuous rapid release. Immediate and late release of new [3H]rGH is increased by dibutyryl cyclic AMP, and the late phase of [3H]rGH is less delayed. Simultaneous exposure of pituitary explants to [3H]alanine and [14C]leucine resulted in the release of immunoprecipitable rGH whose ratio of incorporated 3H and 14C varied with time. The observed changes suggest that after it is synthesized, a GH molecule may either be released directly or be processed into the somatotroph's storage compartment. In addition, stored GH is composed of two pools, one of which is immediately releasable. The differential incorporation of [3H]alanine and [14C]leucine into "big" and "small" rGH, together with the ability to differentially displace 3H-labeled "big" and 14C-labeled "small" rGH from the GH antibody suggest that "big" rGH is a heterogenous molecule including "small" rGH and another peptide rather than simply a dimer of "small" rGH.     

Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.     

Relationship between stimulated prolactin release from GH cells and cyclic AMP degradation and formation

We have studied the relationship between the prolaction (PRL) release induced by thyroliberin (TRH) and theophylline and the formation and inactivation of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) in cultured rat-pituitary cells (GH3 cells). TRH, which stimulated prolactin release, increased cyclic AMP formation and stimulated transiently both the low- and high-Km cyclic phosphodiesterases. The maximal effect on the phosphodiesterase was observed at 30 mM TRH. The stimulatory effect of TRH on the activity of the cyclic AMP phosphodiesterases was duplicated by incubation of the cells with cyclic AMP (2-10 mM). In washed particulate GH3 cell fractions, TRH increased the adenylyl cyclase activity up to 180%. Treatment of GH3 cells with theophylline stimulated the release of PRL and inhibited cyclic AMP degradation probably leading to the measured increase in cellular concentrations of the nucleotide. The effects of TRH and theophylline on cellular cyclic AMP concentrations and on PRL release were additive. There was a positive correlation between PRL release and cellular cyclic AMP concentration (r = 0.97). The elevations observed in cellular cyclic AMP concentration after TRH treatment are due to increased formation which in turn leads to phosphodiesterase activation. Therefore, cyclic AMP formation appears to be an intermediary step in the stimulus-secretion coupling caused by the tripeptide.     

Thyroxine secretion by isolated hog thyroid cells: a cyclic AMP independent pathway.

The release of 131I-labeled thyroxine (T4) from isolated hog thyroid cells was increased 1.5--2-fold by thyrotropin (TSH). Dibutyryl cyclic AMP failed to reproduce this TSH action. In this in vitro system another cell activity, T4 synthesis, was stimulated in an essentially identical fashion by TSH and dibutyryl cyclic AMP (time course of action, dose-response relationship). 3-Isobutyl-1-methylxanthine (IBMX), 0.5 mM, did not alter the basal [131I]T4 release whereas it enhanced the [131I]T4 synthesis. TSH, 60 MU/ml, increased the intracellular cyclic AMP concentration 3-4-fold. Chlorpromazine (5 X 10(-4)M) abolished the TSH stimulation of cyclic AMP accumulation but did not alter the TSH-induced increase in [131I]T4 secretion. It is concluded that the TSH action on [131I]T4 secretion by isolated thyroid cells is not mediated by the adenylate cyclase-cylic AMP system.     

Role of cyclic AMP in proliferation of lung tissue in organ culture

The role of cyclic AMP in cell proliferation and division has been the subject of study by a number of investigators in the past 30 years, but the argument of whether cyclic AMP is a negative or a positive regulator has not been settled. We studied the effect of cyclic AMP on proliferation of normal and postpneumonectomized lung tissues in young adult rats by measuring the incorporation of tritiated thymidine into lung DNA in organ culture. In normal lung tissues the incorporation of [3H]thymidine was increased by exogenous dibutyryl cyclic AMP, or by isoproterenol or forskolin to stimulate adenylate cyclase, or by caffeine, which inhibits cAMP phosphodiesterase. The effect of isoproterenol, but not forskolin, was abolished by the beta-adrenergic blocking agent propranolol. The effect of caffeine on [3H]thymidine incorporation was further enhanced in normal lung tissues in the presence of dibutyryl cyclic AMP and in postpneumonectomized lung tissues. Imidazole, a cAMP phosphodiesterase stimulator, also increased [3H]thymidine incorporation in culture, but the effect was not magnified in the presence of exogenous dibutyryl cyclic AMP, nor in postpneumonectomized lung tissues. The data suggest that cyclic AMP acts as a positive regulator in proliferation of lung tissues.     

Contribution of stored rat growth hormone to restoration of depleted rat pituitary immediate release pools

Stored rat pituitary growth hormone (GH) is functionally divided into immediately releasable and more stable compartments. These observations are consistent with either intracellular hormone compartmentalization within cells of a functionally homogeneous somatotroph population or summed responses from a heterogeneous population of functionally specialized cell subgroups. We investigated the pituitary's ability to recruit stored rGH to replenish depleted immediate release pools. We used perifused pituitary fragments whose stored rGH was labeled during pre-incubation in the presence of [3H]leucine. Initial immediate release pool depletion was accomplished by continuous exposure to combined 21 mM potassium ion (K+) and 1 mM dibutyryl cyclic AMP (dbcAMP). During a subsequent perifusion period in the presence of either no secretagogue, continuing 21 mM K+, or continuing 1 mM dbcAMP, we examined release of stored [3H]rGH in response to repetitive 15 min pulses of 21 mM K+ or 1 mM dbcAMP. Analysis was by specific immunoprecipitation. We had demonstrated that K+ and dbcAMP pulses can stimulate repetitive, albeit diminishing, stored [3H]rGH release responses. However, following pre-treatment with combined 21 mM K+ and 1 mM dbcAMP: (i) pulses of dbcAMP stimulated almost no stored [3H]rGH release in the presence of 21 mM K+ or in the absence of a background secretagogue; and (ii) stored [3H]rGH release in response to pulses of K+ was attenuated in the absence of background secretagogue but fully restored in the presence of dbcAMP. Conclusions: (i) at least some individual somatotrophs can compartmentalize stored hormone; and (ii) an active transport system facilitates restoration of somatotroph immediate release pools using stored rGH.     

Effect of thyrotropin-releasing hormone and dopamine on the in vitro secretion of prolactin by the bullfrog pituitary gland

Pituitary glands of bullfrogs (Rana catesbeiana) were incubated in medium containing thyrotropin-releasing hormone (TRH) and/or dopamine in order to see their effects on the release and synthesis of prolactin, which was measured by a homologous radioimmunoassay. Prolactin synthesis was measured by monitoring the incorporation of [3H]leucine into prolactin. TRH (0.1-10 ng/ml) stimulated the release of immunoassayable prolactin and newly synthesized [3H]prolactin into the medium in a dose-dependent manner; however, it was ineffective in increasing total prolactin (medium plus pituitary) and the incorporation of [3H]leucine into the total prolactin during the experimental period (20 hr). The TRH-induced elevation of prolactin release was suppressed by the addition of dopamine (5 X 10(-7) M) to the medium.     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

Release of Pituitary Growth Hormone by Prostaglandins and Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate in the Absence of Protein Synthesis

Effects of prostaglandins on the incorporation of [4,5-(3)H]leucine into growth hormone and its subsequent release into the incubation medium were studied. Incubation of rat anterior pituitary glands with 10(-6) M prostaglandin PGE(1) in tissue culture medium 199 for 7 hr caused a 40-300% increase in the release of labeled growth hormone into the incubation medium. PGE(1) at 10(-8) M increased growth hormone synthesis but not release. At 10(-6) M, PGE(2) had effects similar to PGE(1); PGA(1) increased growth hormone synthesis but not release. PGF(2alpha) was without effect on either synthesis or release of growth hormone.Prolactin synthesis and release were not affected by prostaglandins. All of the prostaglandins, at 10(-4) M, increased adenyl cyclase activity in the pituitary gland but phosphodiesterase activity was unaltered. Dibutyryl cyclic AMP, with or without caffeine, caused an up to 300% increase in labeled growth hormone release. No consistent effect of prolactin was observed. If potassium concentration was increased 10-fold, a 215% increase in growth hormone release was observed. A combination of hypertonic potassium and 10(-6) M PGE(1) increased growth hormone release 325%, suggesting that potassium and prostaglandins act by independent mechanisms. Addition of theophylline to pituitary gland, incubated in vitro, increased both the synthesis and release of growth hormone. Although fluoride greatly stimulated growth hormone release, it completely inhibited the incorporation of leucine into the hormone. Similarly, puromycin inhibited synthesis of growth hormone but did not block release induced by prostaglandin, dibutyryl cyclic AMP, theophylline, or fluoride. Prostaglandins increase pituitary adenyl cyclase activity and, presumably via cyclic AMP, increase growth hormone release, independently of protein synthesis.     

Studies on the mechanisms of somatostatin action on insulin release. IV. effect of somatostatin on cyclic AMP levels and phosphodiesterase activity in isolated rat pancreatic islets.

The effects of somatostatin on insulin release and cyclic AMP metabolism were studied in collagenase-isolated islets of Langerhans from the rat. Ceoncentrations from 500 to 2000 ng/ml significantly inhibited glucose stimulated insulin release, while 100 and 200 ng/ml were ineffective. Somatostatin (2000 ng/ml) inhibited insulin release and [3H]-cyclic AMP accumulation induced by 16.7 mM glucose after 10 and 30 min of incubation. In dose-response studies, the inhibition by somatostatin of the effect of glucose on [3H]cyclic AMP and insulin release could be overcome by a high concentration of the hexose (44.9 mM), suggesting competitive inhibition. In the absence of glucose, somatostatin inhibited [3H]cyclic AMP accumulation induced by the phosphodiesterase inhibitor, IBMX, while no inhibition was seen, again in the absence of hexose, when the [3H]cyclic AMP levels had been raised by the adenyl cyclase stimulator, cholera toxin. Somatostatin did not affect phosphodiesterase activity when added to islet homogenates, but preincubation of the islets with the peptide before homogenization decreased the activity by about 30%. It is suggested that somatostatin-induced inhibition of insulin release is, at least partially, mediated by cyclic AMP, probably through an action on islet adenyl cyclase.     

Failure of dopamine and bromocriptine to affect prolactin release and cell growth in the dopamine receptor-deficient 235-1 clone

The 235-1 clone was recently derived from the 7315a transplantable pituitary tumor and continues to secrete rat prolactin. The cells have a prominent Golgi apparatus which can be stained immunocytochemically for prolactin, but there were no 600–900 nm granules which are characteristic of normal mammotrophs. In a perfused cell-column apparatus, prolactin release from the clone was unchanged by dopaminergic agonists, thyrotropin-releasing hormone and estradiol but stimulated by dibutyryl cyclic AMP. Cellular cyclic AMP content was also not changed by dopamine but was dramatically enhanced by prostaglandin E₁, indicating that at least one hormone-adenylate cyclase coupling mechanism was functional. In radioligand binding studies using the dopamine antagonist [³H]spiperone, no evidence of a dopamine receptor was obtained. The [³H]spiperone binding present was not stereoselective, and exceedingly high concentrations of other ligands were required to displace the binding. In addition, the induction of a prolactin-secreting hard tumor in rats by subcutaneous innoculation of the 235-1 cells failed to induce measurable dopamine receptors associated with the tumor cells.In order to address the possibility that there were functional dopamine receptors on these cells, but that they could not be resolved with either the cell column and cyclic AMP studies or the radioreceptor assay, the clone cells were incubated with 0.1–100 nM bromocriptine for up to 8 days. Bromocriptine had no effect on the growth rate or prolactin secretion of the 235-1 clone but inhibited prolactin release from anterior pituitary cells by over 73% in control studies.We conclude that the 235-1 clone does not express dopamine receptors and that the presence of dopamine receptors is obligatory for the typical inhibitory effects of bromocriptine on prolactin release and pituitary cell growth.     

Studies of TRH-induced prolactin secretion and its inhibition by dopamine, using ovine pituitary cells

Prolactin secretion from ovine pituitary cell cultures was stimulated by thyrotropin-releasing hormone (TRH) (10(-10)-10(-7) M) with a half-maximal effect at approximately 2.5 X 10(-9) M. A maximally effective concentration of TRH produced a peak secretory response, 5-10-fold stimulation over basal release, within 15 min. Dopamine (10(-10)-10(-7) M) but not somatostatin caused a dose-related inhibition of TRH (10(-8) M) stimulated prolactin release. Both dopamine (10(-7) M) and somatostatin (10(-7) M) inhibited basal secretion from the cells. TRH did not significantly increase pituitary cell cyclic AMP levels under any of the conditions tested. Stimulation of prolactin secretion by TRH was not prevented when Ca2+ was omitted from the incubation medium. Dopamine inhibited secretion induced by TRH under low Ca2+ conditions. Our results are consistent with a hypothesis that TRH may stimulate prolactin secretion via release of intracellular Ca2+ rather than increased cellular Ca2+ uptake, and imply that dopamine inhibition involves a lowering of intracellular Ca2+ levels.     

Stimulation of colonic glycoprotein synthesis by dibutyryl cyclic AMP and theophylline.

The effects of dibutyryl cyclic AMP (B2cAMP) and theophylline on glyoprotein synthesis in rabbit colon were studied in mucosal organ cultures using [3H]glucosamine as a precursor. Addition of B2cAMP (1 mm) to culture medium caused a significant increase in glycoprotein synthesis after 12 and 24 hr compared with biopsies cultured in control medium. The increase in glycoprotein synthesis was observed only if the cyclic nucleotide was present continuously in the incubation medium for at least 12 hr. The stimulatory effect of B2cAMP on [3H]glucosamine incorporation was blocked by cycloheximide. B2cAMP also stimulated mucosal uptake of glucosamine into the intracellular pool and markedly enhanced specific activity of mucosa galactosyltransferase, an enzyme involved in glycoprotein synthesis. Addition of 5mM theophylline caused a greater than 2-fold increase in cAMP levels, which was also accompanied by an increase in glucosamine uptake and incorporation into mucosal glycoproteins. This study demonstrates that elevation of intracellular cAMP concentration in colon epithelium in vitro is associated with an increase in glycoprotein synthesis. These effects may be mediated in part by (1) increased uptake of glycoprotein precursors such as glucosamine, and (2) increased activity of glycoprotein synthetic enzymes.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

Actions of pertussis toxin on the inhibitory effects of dopamine and somatostatin on prolactin and growth hormone release from ovine anterior pituitary cells

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pretreatment of cells with pertussis toxin, in a dose-related fashion (1-100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.     

Effect of multiplication-stimulating activity (MSA) on the cyclic AMP level and proteoglycan synthesis in cultured chondrocytes

Multiplication-stimulating activity (MSA), a somatomedin purified from conditioned medium of Buffalo rat liver cells, had little effect on the intracellular level of cyclic AMP when it markedly enhanced the synthesis of sulphated glycosaminoglycans in rabbit chondrocytes in culture. In addition, MSA did not inhibit prostaglandin E1- or parathyroid hormone-induced accumulation of cyclic AMP in the chondrocytes. On the contrary, MSA slightly decreased stimulation of cyclic AMP accumulation by prostaglandin in human fibroblasts. Dibutyryl cyclic AMP and MSA increased the incorporation of [35S]sulphate and [3H]serine into proteoglycans synthesized by rabbit chondrocytes, and their effects were additive. These findings suggest that somatomedin and dibutyryl cyclic AMP enhance sulphate proteoglycan synthesis through different mechanisms. The lack of inhibitory effect of MSA on cyclic AMP accumulation may be favourable for producing additive effects with cyclic AMP on proteoglycan synthesis and DNA synthesis in chondrocytes.     

Effect of cyclic AMP on lipid accumulation and metabolism in human atherosclerotic aortic cells

The effects of dibutyryl cyclic AMP (db cAMP), cholera toxin, and methylisobutylxanthine on the content and metabolism of lipids in smooth muscle cells cultured from normal and atherosclerotic intima of human aorta have been studied. Db cAMP (0.1 mM) decreased the levels of triglycerides and esterified sterols 1.5-3-fold in cells cultured from fatty streaks and atherosclerotic plaques. Cholera toxin (100 micrograms/ml) and methylisobutylxanthine (0.1 mM) stimulated by 2-fold the cyclic AMP level in aortic smooth muscle cells and decreased the content of triglycerides and esterified sterols by 2-3-fold. Prolonged treatment with db cAMP and methylisobutylxanthine resulted in a fall in the intracellular phospholipids and free sterols. Using the labelled precursors, [3H]acetate and [14C]oleate, it was established that the agents increasing the intracellular cyclic AMP level inhibit the formation of sterols and fatty acids, impede the synthesis of phospholipids, triglycerides and esterified sterols, and stimulate their hydrolysis. The data demonstrate that cyclic AMP facilitates the regression of cellular lipoidosis by altering the intracellular lipid metabolism.     

Effect of dibutyryl cyclic AMP and oestradiol on incorporation of [3H]leucine into the proteins of luminal fluid of the rat uterus.

An injection of 12-5 nmol dibutyryl cyclic AMP (dbcAMP) into each uterine horn of ovariectomized rats maintained on progesterone alone, enhanced incorporation of [3H]leucine into uterine luminal proteins. A value of 11-73 X 10(3) c.p.m. [3H]leucine incorporated/rat was found after injection of dbcAMP compared with 3-69 X 10(3) c.p.m./rat after injection of saline. An injection of 1-0 microgram oestradiol, in contrast, gave 58-05 X 10(3) c.p.m./rat. Electrophoretic analysis showed that the radioactively labelled proteins found after an injection of dbcAMP or oestradiol were qualitatively similar to those detected on day 5 of pregnancy, the day of blastocyst implantation. The nucleotide thus apparently elicits a uterine response similar to that observed after an injection of oestradiol.