Extracellular Levels Of Cyclic AMP Research Articles

The positive allosteric modulator GS39783 enhances GABAB receptor‐mediated inhibition of cyclic AMP formation in rat striatum in vivo

We studied the effects of the positive allosteric modulator GS39783 on GABA(B) receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABA(B) receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABA(B) agonist R(-)-baclofen inhibited cyclic AMP formation stimulated by a water-soluble forskolin analogue in a concentration-dependent manner (EC50 7.3 microM, maximal inhibition 40%). The selective GABA(B) antagonist CGP56999 reversed R(-)-baclofen-induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(-)-baclofen (1 microM), significantly decreased cyclic AMP formation in a dose-dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABA(B) receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(-)-baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABA(B) receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein-coupled receptor-positive allosteric modulator.

Cyclic AMP-receptor proteins in human salivary glands.

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.

Involvement of P 1 receptors in the effect of Forskolin on Cyclic AMP accumulation and export in PC12 cells

In PC12 cells, forskolin as well as the adenosine receptor agonist 5′- N-ethylcarboxamido-adenosine (NECA) increased intracellular adenosine-3′,5′-cyclic monophosphate (cyclic AMP) levels, which peaked at 45–60 minutes and declined thereafter. Maximum levels were 3000 and 1700 pmol/10 6 cells during treatment with 10 μM forskolin or 0.1 μM NECA, respectively. Extracellular cyclic AMP rose with time, at mean rates of 24.7 (forskolin) and 11.3 (NECA) pmol/min/10 6 cells. With either drug, a linear correlation was obtained between the calculated time integral of intracellular cyclic AMP and the measured extracellular cyclic AMP levels, indicating that the outflow of cyclic AMP was sustained by a nonsaturated transport system. The ability of forskolin to increase intracellular and extracellular cyclic AMP levels was hindered in a concentration-dependent manner by 8-( p-sulfophenyl)theophylline (8-SPT). A similar inhibition was exerted by other two adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine. The concentration-response curve to adenosine was shifted to the right by 25 μM 8-SPT, whereas that of forskolin was shifted downwards. Adenosine deaminase (ADA, EC 3.5.44, 1 U/mL) reduced the intracellular cyclic AMP response to forskolin by 68%, whereas the adenosine transport inhibitor, dipyridamole (10 μM), significantly increased 1 and 10 μM forskolin-dependent cyclic AMP accumulation. Erythro-9-(2-hydroxy-3-nonyl)adenine (10 μM), an inhibitor of ADA, and α,β-methyleneadenosine 5′-diphosphate (100 μM), an inhibitor of ecto-5′-nucleotidase, did not alter forskolin activity. These results demonstrate that a cyclic AMP extrusion system operates in PC12 cells during adenylyl cyclase stimulation by forskolin and that this stimulation involves a synergistic interaction with endogenous adenosine. However, extruded cyclic AMP does not appear to significantly contribute to the formation of the endogenous adenosine pool.

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E 2 biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE 2 production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE 2 synthesis, and was without effect on IL-1β-stimulated PGE 2 levels. PGE 2 synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE 2 levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE 2 production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE 2 synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.

Regulation of cyclic AMP metabolism in bovine adrenal medullary cells

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 μM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca 2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specinc) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 μM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 μM forskolin. Rolipram had an EC 50 of ⩽ 1 μM and was equally effective at 100 μM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1mM than at 100 μM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 μM) enhanced the Ca 2+-dependent cyclic AMP response to K + depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 μM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 μM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2–3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.

Stress-induced increase of extracellular levels of cyclic AMP in rat cortex

Previous studies have suggested that stress may increase levels of cyclic AMP (cAMP) in the brain but these findings have been controversial due to the use of stressful procedures to inactivate brain enzymes. The present experiment therefore used a non-stressful technique, microdialysis, to assay extracellular levels of cAMP in the rat cortex after stress. Experiments were conducted 2 days after implantation of probes in the frontal cortex. Significant increases were found after the mild stressors of restraint or intraperitoneal injection of saline suggesting that increased tissue levels of cAMP had occurred. These responses were potentiated by local infusion of the phosphodiesterase (PDE) inhibitor, rolipram. It is concluded that one or more adenylate cyclase-coupled receptors in the cortex is activated by mild stress and that this activation can be detected in vivo by microdialysis.

Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells.

1. The regulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells. 2. Acetylcholine (100 microM), nicotine (10 microM) and dimethylphenylpiperazinium (20 microM) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl-beta-methylcholine (100 microM) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 microM) but were abolished by mecamylamine (5 microM). 3. Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1-20 microM), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5-10 microM nicotine and the EC50 was about 2 microM. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min. 4. The cellular cyclic AMP response to nicotine (10 microM) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.

Characterization of the stimulatory effect of galanin on growth hormone release from the rat anterior pituitary

The present study was undertaken to investigate the direct actions of rat galanin (R-GAL) on growth hormone (GH) release from the rat anterior pituitary in vitro . R-GAL modestly but significantly stimulated GH release without an increase in intra- and extracellular cyclic AMP levels in monolayer cultures of rat anterior pituitary cells. This stimulatory effect of R-GAL was dose-dependent but not additive with that of GH-releasing factor (GRF). R-GAL-stimulated GH release was less senstive to the inhibitory effect of somatostatin than was GRF-stimulated GH release. In perifusions of rat anterior pituitary fragments, R-GAL induced a gradual and sustained increase of GH release. Incremental GH release derived in part from preformed stored GH. These data confirm that R-GAL acts at the pituitary level to stimulate GH release by a mechanism distinct from that of GRF.

Intracellular and extracellular levels of cyclic AMP during the cell cycle of Saccharomyces cerevisiae

Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Saccharomyces cerevisiae there are stage-specific fluctuations in the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP). Results shown here indicate that the intracellular concentration of cAMP is at its highest during the division cycle, and at its lowest immediately prior to and just after cell separation. Results also show the extrusion of extracellular cAMP into the medium by Saccharomyces cerevisiae, extracellular cAMP levels being ten to one hundred times higher than intracellular levels. During the cell cycle of Saccharomyces cerevisiae the extracellular level of cAMP does not fluctuate.

Purification and properties of a cyclic-AMP phosphodiesterase that is active in only one cell type during the multicellular development of Dictyostelium discoideum.

Cyclic-AMP phosphodiesterase (PDE) accumulates during the aggregation stage of Dictyostelium where it functions in maintaining extracellular levels of cyclic AMP (cAMP). The activity decreases during the subsequent multicellular slug stage and then accumulates again during sorocarp construction, but the enzyme is active only in the developing stalk. Because of the possible significance of this localized activity in only one of the two cell types, we have purified the enzyme from the multicellular stage in order to understand its mode of regulation in vivo. We find that the enzyme which is localized in the prestalk cells is similar in many respects to the extracellular PDE which is active at the aggregation stage. The enzyme from both stages is inhibited by a low molecular weight protein. The mechanism of this inhibition is through a shift in the apparent Km for cAMP from micromolar to millimolar levels. The inhibited form of the enzyme can be activated by preincubation with MgSO4 and dithiothreitol (DTT). This activation treatment releases the inhibitor from the enzyme, thus restoring the low Km form, changes the molecular weight of the culmination stage enzyme from 95 000-100 000 to 68 000 by releasing the Mr 35 000-40 000 inhibitor protein, and causes irreversible loss of inhibitor activity. Although the inhibitor could be obtained in high yield from the aggregation stage by simply heating the extracellular fluid, it could not be detected from culmination stage extracts when prepared by this method. However, inclusion of calcium in the extraction buffer resulted in release of inhibitor from both heated and nonheated samples. The results indicate that the stalk cell specific PDE is regulated similarly to the aggregation stage PDE and opens the possibility of differential regulation of PDE in the two cell types.

Cyclic AMP levels in relation to membrane phospholipid variations in Neurospora crassa.

The correlation between membrane phospholipid composition and total cyclic AMP levels was investigated by using Neurospora lipid auxotrophs under various supplementation conditions. The lipid composition of the supplemented cultures was determined, and the intracellular and extracellular cyclic AMP levels were measured at various stages of the culture growth. Kinetic parameters and the thermostability of adenylate cyclase and of cyclic AMP-dependent phosphodiesterase were measured under all supplementation conditions. In inositol deficient inl cultures the levels of intracellular cyclic AMP decreased exponentially towards the end of the log phase and thereafter. In chol-l; chol-2 cultures, grown in N-monomethylethanolamine and low choline supplementation, the level of intracellular cyclic AMP decreased as function of decreasing exogenous choline supplement. Rates of cyclic AMP extrusion in all cultures were comparable on dry weight basis, and thus not affected by the mycelial lipid composition. Adenylate cyclase activity and thermostability decreased under those supplementation conditions resulting in reduction of cyclic AMP. Cyclic AMP-dependent phosphodiesterase was insensitive to phospholipid changes. Accordingly, it is suggested that specific perturbations in cellular phospholipid composition affect the membrane-bound adenylate cyclase and hence the cyclic AMP synthesis in vivo.

Folate deaminase and cyclic AMP phosphodiesterase in Dictyostelium discoideum: Their regulation by extracellular cyclic AMP and folic acid

Cyclic AMP and folic acid act as chemotactic factors in Dictyostelium discoideum. Both agents, when applied extracellularly, also control cell development from the growth stage to the acquisition of aggregation competence. Cyclic AMP phosphodiesterase and folate deaminase are extracellular enzymes whose activity is regulated during early differentiation of D. discoideum cells. The two enzymes help control the extracellular levels of cyclic AMP and folic acid. The substrates cyclic AMP and folic acid each increase the extracellular activity of folate deaminase as well as phosphodiesterase. The specificity of extracellular phosphodiesterase regulation by cyclic AMP indicates that the effect is mediated by specific cyclic AMP receptors rather than the catalytic site of cell surface phosphodiesterase. To some extent cyclic AMP and folic acid are interchangeable with respect to regulating differentiation and enhancing enzymatic inactivation of intercellular signals. Thus the two extracellular signals may share a common cellular pathway of signal transduction. The regulation of folate deaminase and phosphodiesterase by folic acid does not always parallel the folic acid effects on development. Pulses of folic acid stimulate development of aggregation competence, whereas a continuous flux inhibits. In contrast, either continuous flux or pulses of folic acid increase the deaminase and phosphodiesterase activities.

DISSOCIATION OF THE REGULATION OF FATTY ACID SYNTHESIS AND CYCLIC AMP LEVELS IN CULTURED GLIAL AND NEURONAL CELLS1

Abstract— Cultured C‐6 glia and neuroblastoma were utilized to investigate the relation of rates of fatty acid synthesis (from 3H2O) to levels of cyclic AMP under conditions of short‐term and long‐term regulation. The data demonstrate a consistent dissociation of alterations in rates of fatty acid synthesis and levels of cyclic AMP. Thus, marked alterations in the rate of fatty acid synthesis occurred when serum or albumin‐bound palmitic acid was present in the culture medium, but there were no accompanying alterations in levels of cyclic AMP. Similarly, when high intracellular and/or extracellular levels of cyclic AMP were induced by exposure of the cells to dibutyryl cyclic AMP or isoproterenol, no change in the rate of fatty acid synthesis occurred. Although the data raise serious doubt about an important role for cyclic AMP in the regulation of fatty acid synthesis, they do not rule out such a role. The findings do indicate that any such role must involve alterations in compartmentalization, metabolism or binding of the mononucleotide within the cell.

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.

Effect of hypoglycemia on extracellular levels of cyclic AMP in man

This study was designed to assess the response of cyclic AMP to “endogenous” hormonal stimulation resulting from insulin-induced hypoglycemia. Insulin was administered to four normal subjects and four adrenalectomized patients. Hypoglycemia resulted in four-fold increases in plasma cyclic AMP. This response is thought to be secondary to β-adrenergic stimulation for the following reasons: (1) the response was absent in adrenalectomized, cortisol-treated subjects; (2) it was abolished by propranolol; and (3) urinary excretion of cyclic AMP did not reflect the rise in plasma cyclic AMP.

Cell division and prophage induction in Escherichia coli: studies of nucleotide levels.

Cell division and prophage repression in the Escherichia coli mutant, T-44, are very sensitive to the levels of certain purine and pyrimidine derivatives in the media. The hypothesis that a change in the level of an adenine derivative in the small molecule pool of this strain was responsible for prophage induction and filament formation was tested. The nucleoside triphosphate pools in T-44 and C-600 nonlysogenic and lysogenic strains were labeled in experiments with (32)P and (33)P. Cultures were mixed, and the nucleotides were isolated. When adenine was present, the level of adenosine triphosphate (ATP) in T-44 compared to C-600 (as indicated by the isotope ratio) was increased up to twofold. Most of the other nucleotides increased but not to the same degree. In the lysogenic strain guanosine triphosphate and deoxycytidine triphosphate showed increases comparable to ATP, whereas increases noted in the deoxynucleotides in T-44 +/- lambda with adenine present were less. In experiments where T-44 and C-600 were incubated with (3)H- and (14)C-adenine, the levels of several compounds, including ATP, were slightly elevated in T-44. The combined data suggest that cultures of T-44 +/- lambda, grown in the presence of adenine, show a preferential increase in the level of ATP when compared to C-600 +/- lambda, but the increase in relation to the other nucleotides is less than twofold. In the experiment with (3)H- and (14)C-adenine, the level of inosine was found to be increased in T-44 relative to C-600. Cyclic AMP, when added to cultures of T-44 under various conditions, had no effect on prophage induction. Intracellular and extracellular levels of cyclic AMP in T-44 compared to C-600, incubated with had-acidin, guanosine, and cytidine (HGC) or with HGC plus adenine, were not significantly different. No compelling evidence for altered nucleotide metabolism in T-44 +/- lambda as a cause of prophage induction or filament formation was obtained.