Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca2+ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH3) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca2+ concentrations and inhibited by higher (microM range) concentrations of the cation. A 2-3-fold stimulation of the activity in response to Ca2+ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca2+ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca2+ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca2+ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca2+ X Mg2+ concentrations in the incubation also influenced the Ca2+ concentration dependence of adenylate cyclase; at high Mg2+ more Ca2+ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH3 cells only in the presence of Ca2+; as Ca2+ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca2+ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH3 cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca2+. Increasing free Ca2+ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells

Although relaxin acts at several abdominal sites and mammary tissue associated with pregnancy and parturition, the scope of target tissues and the signals conveying the relaxin message into the cell are poorly defined. We found that human relaxin rapidly elevates the cyclic AMP content of cultured rat anterior pituitary cells. This is a graded response (EC50 0.3 nM relaxin) that can be blocked by anti-relaxin antibodies or the hormones somatostatin and dopamine. Furthermore, other hormones with some sequence homology to relaxin, such as insulin and insulin-like growth factor-I, have no such action. We conclude that the anterior pituitary may be a target tissue for relaxin and that cyclic AMP may act as an intracellular messenger for relaxin in these cells.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Thyrotropin-releasing hormone and cyclic AMP activate distinctive pathways of protein phosphorylation in GH pituitary cells

The studies reported here were undertaken to clarify the cellular mechanism of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), in clonal, hormone-responsive GH pituitary cells and to assess the possibility of a role for cyclic AMP as a mediator of TRH action. We investigated patterns of protein phosphorylation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of high speed supernatant and pellet fractions from untreated and treated GH cells. Brief treatment of cells with agents which elevate or mimic cellular cyclic AMP (8-bromo cyclic AMP, dibutyryl cyclic AMP, vasoactive intestinal polypeptide or cholera toxin) stimulated the phosphorylation of five supernatant peptides (41, 45, 47, 72, and 82 kilodaltons) and one pellet peptide (135 kilodaltons) and decreased the phosphorylation of one supernatant peptide (55 kilodaltons). In contrast, TRH promoted the phosphorylation of four different supernatant peptides (two 59, 65, and 80 kilodaltons). In addition, TRH also stimulated the phosphorylation of cyclic AMP-responsive 41-, 45-, and 82-kilodalton supernatant peptides and 135-kilodalton pellet protein and decreased the phosphorylation of 55-kilodalton supernatant peptide. Altered labeling of 47- and 72-kilodalton supernatant peptides, however, was not observed with TRH. Time course studies, as well as the overlapping biological action of TRH and vasoactive intestinal polypeptide, lead us to conclude that these peptide hormones utilize distinct, parallel pathways which converge at some late step. Furthermore, the results indicate that effects of TRH are mediated by a cyclic AMP-independent pathway.     

Inhibitory effect of clonidine upon adenylate cyclase activity,cyclic AMP production,and insulin release in rat pancreatic islets

Conflicting opinions were recently expressed concerning the possible effect of ₂-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca²⁺), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.     

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.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2–3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low K_m (0.17 μM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study. Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

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.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Desensitization of adenylate cyclase and cyclic AMP flux during the early stages of liver regeneration

Liver regeneration is controlled by a complex network of interactions between hormones, growth factors, and a variety of hepatotrophic factors. Transient increases in cAMP in the early stages of liver regeneration that are necessary for DNA synthesis and subsequent mitosis have been reported; however, studies on the mechanisms that control cellular cAMP levels during liver regeneration, namely adenylate cyclase activity, cAMP-dependent phosphodiesterase activity, and cAMP efflux from the cell, have been generally incomplete. In this study we have shown that although there are three peaks in intracellular cAMP levels in the first 24 hours after partial hepatectomy, the adenylate cyclase activity stimulated by glucagon, prostaglandin E2, adrenaline, and fluoride in vitro decreases with time. KD and BMAX of hepatocyte glucagon and beta receptors were similar to the sham controls. Our results are consistent with a mixed homologous/heterologous desensitization of the adenylate cyclase system. There was also a loss of cAMP-dependent phosphodiesterase activity after partial hepatectomy. We speculate that even though the hormone-stimulated adenylate cyclase system has been desensitized, the system retains the ability to respond to the transient pulses of the variety of hormones secreted after partial hepatectomy and thus raise the intracellular concentration of cAMP. The decrease in cAMP-dependent phosphodiesterase may be necessary to prevent rapid breakdown of cAMP.