The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

A cyclic AMP binding-protein from barley seedlings

A protein fraction extracted from barley seedlings was shown to bind 3′:5′-cyclic AMP. The binding effect is real and not due to interference with the standard binding-protein assay used. Evidence is presented that this is a specific binding-protein; even at high concentrations other protein fractions from the same source showed no affinity for cyclic AMP. None of a range of cyclic and non-cyclic nucleotides that were examined exhibited a degree of binding with the protein comparable to that with cyclic AMP. The cyclic AMP/binding protein complex has a K_d of 8 nM. This complex eluted at an identical position in the elution sequence from a Sephadex G-150 column as the uncomplexed binding-protein. The barley binding-protein is in a fraction which also exhibits the enzymic activities of glucose 6-phosphatase, ATPase, 5′-nucleotidase, and fructose 1,6-diphosphatase.     

Dibutyryl cyclic AMP effects on calcium metabolism by mouse mastocytoma cells

The ability of mouse mastocytoma cells to take up 45Ca2+ was measured in normal growth medium. As previously observed in physiological buffers with succinate and Pi, cells grown for 18h with N6,O2'-dibutyryladenosine 3',5' cyclic monophosphate (DB cyclic AMP) to inhibit growth took up more 45Ca2+ than untreated cells. However 45Ca2+ uptake by cells in growth medium was less sensitive to respiratory inhibitors or uncouplers than 45Ca2+ uptake in physiological buffer. Increased 45Ca2+ uptake by 18h cyclic nucleotide-treated cells was not a result of tighter mitochondrial coupling since mitochondria prepared from cyclic nucleotide-treated cells were less coupled than those from untreated cells. Nevertheless studies with uncouplers suggested that the bulk of the intracellular Ca2+ was associated with mitochondria. DB cyclic AMP-treated cells contained less total Ca2+ than untreated cells indicating that net Ca2+ efflux occurred during the 18h period of drug treatment. These observations suggest that Ca2+ fluxes increase in DB cyclic AMP-treated PY815 cells and that a net efflux of Ca2+ occurs during growth inhibition by the cyclic nucleotide.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Effect of hydrocortisone on the desialylation of intestinal brush-border enzymes of the rat during postnatal development

High affinity cyclic AMP phosphodiesterase activity in preparations of acini isolated from mammary tissue of lactating rats is shown to be stimulated by the addition of physiological concentrations of insulin to incubations of acini in vitro. This effect is expressed specifically on membrane-associated phosphodiesterase and occurs in the absence of concurrent protein synthesis. The possible functional role of this aspect of insulin's action on mammary tissue is discussed and compared with the well-known reversal by this hormone of the effects of lipolytic agents in adipose tissue and liver.     

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE‐Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos‐2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH‐induced activation of the CRE. Activators of non‐cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE‐luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H‐89 (10 µM) almost completely blocked PTH‐induced activation of the CRE‐reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time‐dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH‐induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN‐93 had no significant effect on the response to PTH. We conclude that non‐cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH. J. Cell. Biochem. 106: 887–895, 2009. © 2009 Wiley‐Liss, Inc.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

A possible role of cyclic 3′,5′-adenosine monophosphate in the germination of Cicer arietinum seeds

Changes in the activities of adenyl cyclase, cyclic AMP phosphodiesterase, protein phosphokinase, RNase, protease, DNA, RNA and protein synthesis during the initial imbibition phase of the germination cycle of Cicer arietinum (chick pea, Bengal gram) are reported. Activation of adenyl cyclase and phosphorylation of cellular proteins appears to precede RNA and protein synthesis in the imbibed seeds.     

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function, such as adhesion, aggregation, and secretion. Elevation of intracellular cAMP, which induces the activation of PKA, results in the inhibition of platelet function. Thus, tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy. In this review, we summarize the PKA substrates and their contributions to platelet function, especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology. In addition, we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis.Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function,such as adhesion,aggregation,and secretion.Elevation of intracellular cAMP,which induces the activation of PKA,results in the inhibition of platelet function.Thus,tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy.In this review,we summarize the PKA substrates and their contributions to platelet function,especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology.In addition,we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

Urinary cyclic AMP excretion is increased in Macaca fascicularis and Macaca mulatta compared to humans

Changes in parathyroid hormone and its second messenger cyclic AMP have been implicated in the pathogenesis of osteoporosis. Nonhuman primate models have been useful in the study of osteoporosis, but the physiology of mineral metabolism in certain species is different than in humans. We investigated parameters of mineral metabolism in 15 normal adult female cynomolgus and 14 normal adult female rhesus monkeys. In both species, urinary cyclic AMP was increased compared with humans, and the nephrogenous cyclic AMP in the cynomolgus monkeys was also elevated. Despite this, there was no evidence for hyperparathyroidism in either species as evaluated by serum or plasma phosphorus and midregion-specific and/or aminoterminal-specific immunoreactive parathyroid hormone. Given the increasing use of nonhuman primates in the study of osteoporosis, understanding basic changes in mineral metabolism is important before pathologic effects of bone loss can be understood.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H₂ utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H₂ uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H₂ uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H₂ metabolism.     

A comparison of the membrane-bound and extracellular cyclic AMP phosphodiesterases of Dictyostelium discoideum

The Dictyostelium discoideum membrane-bound and extracellular cyclic nucleotide phosphodiesterases (EC 3.1.4.17) shear several properties including the ability to react with a specific glycoprotein inhibitor and small inhibitory molecules. We have partialy purified the membrane-bound enzyme and compared its properties to those of the extracellular form. The kinetic properties of the two forms were similar except that, while associated with membrane particles, the membrane-bound form exhibited non-linear kinetics when assayed ove a broad substrate range. The isoelectric point of the membrane-bound phosphodiesterase was identical to that of the extracellular enzyme when isoelectrofocusing was done in the presence of 6 M urea. The molecular weights of membrane-bound and extracellular enzyme, determined by gel filtration, were the same following isoelectrofocusing in the presence of 6 M urea. When precipitated with an antiserum prepared against purified extracellular phosphodiesterase, the partially purified membrane-bound enzyme preparation was shown to contain a M_r 50 000 polypeptide comigrating with the extracellular enzyme during SDS polyacrylamide gel electrophoresis. When the iodinated extracellular enzyme and the iodinated M_r 50 000 polypeptide from membrane-bound enzyme were subjected to partial proteolytic digestion, similar profiles were obtained indicating extensive regions of homology.