Exogenous Cyclic AMP Research Articles

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.

Phosphate inhibition of secondary metabolism in Streptomyces hygroscopicus and its reversal by cyclic AMP

Inorganic phosphate inhibited the biosynthesis of the macrolide antibiotic turimycin in different strains of Streptomyces hygroscopicus. In the wild type strain a depression was observed with increasing phosphate concentrations. A total inhibition was found at 0.1 M phosphate. In a high producing mutant a minimum of turimycin production occured when the phosphate concentration was between 5 mM and 10 mM. Above this concentration the antibiotic synthesis increased again but the production period shifted to a later period of cultivation. Addition of inorganic phosphate resulted in an initial increase of intracellular cyclic AMP content. But a second elevation characterizing the normal level of cyclic AMP throughout the growth phase was prevented by phosphate. Exogenous cyclic AMP as well as positive effectors of the adenylyl cyclase system were able to overcome the phosphate suppression. Cyclic AMP abolished the reduction of protein synthesis following phosphate addition and caused the reappearance of a protein band which may be responsible for the turimycin biosynthesis.

Effect of metabolic inhibitors on vasopressin‐stimulated transport systems in the toad bladder

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes

The addition of glucagon (10 −6 M) to an incubation mixture containing 32Pi and hepatocytes isolated from livers of rats fed ad libitum results in both a 3-fold increased incorporation of 32P into L-type pyruvate kinase and a decreased catalytic activity. The 32P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10 −3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10 −7 M) had essentially no effect on the incorporation of 32P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase invivo is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP

A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.

Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum

When amoebae of Dictyostelium discoideum, suspended in buffer, were treated with 100 n M pulses of cAMP, the extracellular cAMP phosphodiesterase (ePD) activity increased dramatically and the synthesis of the phosphodiesterase inhibitor (PDI) was repressed. In addition, the time of appearance on the cell surface of contact sites A, membrane-bound cAMP phosphodiesterase, and cAMP binding sites was accelerated by 3–4 hr and the concentration of intracellular cAMP increased ⋍20-fold. When the concentration of the cAMP pulse was reduced to 1 n M, the effect of the pulses on membrane differentiation and on the cAMP pool was virtually the same, while the effect on the ePD-PDI system was reduced. When cAMP was added to the suspension continuously, the nucleotide had no effect on membrane differentiation and failed to stimulate the intracellular cAMP pool, however, the ePD-PDI system was regulated normally. When the developmental mutant, HC112, was treated with cAMP pulses, membrane differentiation and the level of the cAMP pool were unaffected, while the ePD-PDI system responded to the exogenous cAMP. In another mutant, HC53, membrane differentiation was stimulated by cAMP pulses and this response was accompanied by a sharp increase in the concentration of the cAMP pool. These results suggest that the ePD-PDI system and membrane differentiation are regulated independently by exogenous cAMP and that regulation of the ePD-PDI system does not require activation of the adenylyl cyclase.

Neurotransmitter and cyclic nucleotide modulation of frog cardiac contractility

The experiments performed in this study were designed to test the hypothesis that cyclic nucleotides mediate the effects of adrenergic and cholinergic neurotransmitters on frog ventricle. A physiological approach was to measure the twitch tension and contracture tension of ventricular strips in the presence of exogenous neurotransmitter agonists. These observations were correlated to biochemical measurements of cyclic AMP and cyclic GMP levels in ventricular strips exposed to the same physiological conditions. Epinephrine augmented twitch tension, depressed contracture tension, and augmented cyclic AMP levels. Phosphodiesterase inhibitors potentiated the effects of epinephrine. Exogenous cyclic AMP usually mimicked the epinephrine effect on both twitch and contracture tension. Carbachol and acetylcholine depressed twitch tension without affecting contracture, and carbachol transiently augmented cyclic GMP while depressing cyclic AMP levels. These muscarinic agonists inhibited the effects of epinephrine in depressing contracture tension and increasing cyclic AMP levels. Cyclic GMP did not mimic the twitch depressant effect of carbachol but did tend to mimic the cholinergic antagonism of epinephrine. Our observations generally support the idea that cyclic AMP mediates the effects of epinephrine but do not clearly relate cyclic GMP to the action of cholinergic agonists.

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

Physiological and biochemical characterization of aggregation-deficient mutants of Dictyostelium discoideum: detection and response to exogenous cyclic AMP

Forty independently isolated aggregation-deficient (Agg−) mutants of Dictyostelium discoideum, which have been partially characterized genetically, were examined for the ability to respond chemotactically to exogenous cAMP and for certain cellular and extracellular activities thought to be involved in this process. Only five of the mutants failed to produce a statistically significant chemotactic response to cAMP. When shaken in buffer, most of the Agg− mutants produced reduced levels of the cAMP-binding sites and the cell-bound cAMP phosphodiesterase reported to be components of a cell-surface cAMP receptor involved in the chemotactic response. In addition, most of the mutants failed to form contact sites A and were unable to develop on water agar plates. When the mutants were pulsed with 100 nM cAMP, 36 of 40 mutants produced detectable contact sites A and three-quarters of these strains underwent further development when placed on a solid substratum. Treatment with cAMP pulses also stimulated certain mutants to form higher levels of cAMP receptors. These observations suggest that the impaired differentiation of the plasma membrane of many Agg− mutants is due, at least in part, to the abnormal synthesis and (or) secretion of cAMP by these strains. When grown in bacterial suspension, all 40 of the mutants produced extracellular phosphodiesterase (ePD) activity, and all but 8 of the mutants secreted detectable amounts of the ePD inhibitor. In general, Agg− strains carrying mutations at the same aggregation locus (aggF–J) exhibited very similar physiological and biochemical properties.

3′,5′-cyclic AMP binds to and promotes polymerisation on platelet tubulin

IN spite of considerable research, the regulatory mechanism of the reversible assembly–disassembly of microtubules remains obscure. Of the various processes and agents which have been examined as potential regulators probably none has received as much attention as cyclic nucleotides. Indeed, the number of reports which indicate that adenosine 3′,5′-cyclic monophosphate (cyclic AMP) exerts a stimulatory effect in the formation of microtubules, or maintains the latter in the presence of dissociative influences is steadily growing1–13. Most of these studies used intact cells, however, leaving unclear whether the cyclic nucleotide was directly or indirectly involved in this process. To try to resolve this problem, I have used tubulin extracted from platelets: addition of exogenous cyclic AMP has been reported to prevent cold-induced disassembly of microtubules in about 50% of the platelets14. I show here that cyclic AMP stimulates the assembly of microtubules and binds to platelet tubulin, primarily to the α-subunit.

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in [formula omitted

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of 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.

The control of enzyme secretion from fly salivary glands

1. Stimulation of fluid secretion from fly salivary glands by 5-hydroxytryptamine (5-HT) is known to involve calcium and cyclic AMP. Isolated salivary glands were used to investigate the role of these second messengers in the control of enzyme (sucrase) secretion.2. The protein component of secretion from isolated glands treated with 5-HT appears to be identical to that of saliva secreted by flies during feeding.3. Stimulation of fluid secretion by 5-HT follows a definite dose-response curve, but there is no consistent relationship between the rate of enzyme secretion and the stimulating concentration of 5-HT.4. Exogenous cyclic AMP causes secretion of enzymes as well as of fluid, thus mimicking the action of 5-HT. The phosphodiesterase inhibitor theophylline enhances the rate of 5-HT-stimulated enzyme secretion.5. Removal of calcium from the bathing medium enhances enzyme secretion in response to 5 or 10 nM-5-HT but has no effect on enzyme secretion stimulated by 100 nM-5-HT or by cyclic AMP.6. Addition of 0.1 mM-lanthanum to medium containing 2 mM-calcium mimics the effect of calcium-free solution on 5-HT-stimulated enzyme secretion.7. The ionophore A 23187 causes secretion of both fluid and enzyme. The secretory rate is initially high but soon declines and ceases after about 40 min.8. Enzyme secretion in response to 5-HT or to cyclic AMP is progressively inhibited as the concentration of potassium is increased from 10 to 80 mM. Secretion in response to A 23187 is initially inhibited by 80 mM-potassium but then partially recovers.9. The rate of enzyme secretion appears to be affected by the intracellular concentrations of both calcium and cyclic AMP. It is possible that the rate of enzyme secretion increases as the intracellular calcium concentration rises, until the optimal calcium concentration is reached when further increase in the level of calcium progressively inhibits secretion. The optimal calcium concentration for enzyme secretion is lower than that for fluid secretion, and 5-HT normally causes maximal fluid secretion and submaximal enzyme secretion.

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic AMP

The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.

Cyclic AMP Induced Incorporation of 33Pi into Human Spermatozoa Membrane Components

Three protein components of human spermatozoa membranes served as substrates for protein kinases. Protein III (“-‘50,000 M W) was phosphorylated only under stimulation by exogenous cyclic-AMP, while protein II (�80,000 M W) phosphorylation was independent of the presence of the cyclic nucleotide. Protein 1 (“105,000 M W) that accounts for an important percentage of the total spermatozoa membrane protein, showed the highest phosphorylation rate under cyclic-AMP stimulation. These observations suggest that the cyclic-AMP mediated alterations in the structural and biochemical properties of the human spermatozoa are produced through phosphorylauon of specific membrane proteins. It may also be suggested that modifications of the tertiary and quatennary structure of the plasma membrane proteins, through cyclic-AMP stimulated phosphorylation, may be requirements for the initial steps of capacitation.

Stimulation of active uptake of 2-aminoisobutyric acid in Streptomyces hydrogenans by exogenous dibutyryl cyclic AMP

Stimulation of active uptake of 2-aminoisobutyric acid in Streptomyces hydrogenans by exogenous dibutyryl cyclic AMP

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E 1 (PGE 1), PGE 1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B 2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE 1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.

In vivo and in vitro phosphorylation of the adenovirus type 5 single-strand-specific DNA-binding protein

The type 5 adenovirus single-strand-specific DNA-binding protein can be labeled with 32PO 4 added as inorganic phosphate to the medium of infected monkey or human cells. The DNA binding protein in the infected cell can be isolated as a phosphoprotein at early and late times after infection. Most or all of the [ 32P]phosphate can be removed from the DNA-binding protein by alkaline phosphatase or hydrolysis at alkaline pH. The dephosphorylated protein retains the ability to bind specifically to single-stranded DNA. H5 ts125 is a mutation in the structural gene of the 72,000 MW single-strand-specific DNA-binding protein. When cells infected with this viral mutant at the permissive temperature are shifted to the nonpermissive temperature, the phosphorylation of the 72,000 MW protein decreased rapidly (within 30 min) even though approximately normal (wild type) levels of this protein (labeled with 35S]methionine for 30 min) were detected in H5 ts125-infected cells shifted to the nonpermissive temperature. An in vitro protein kinase assay utilizing nuclei from infected or uninfected cells has been developed. This in vitro assay can detect the phosphorylation of at least three adenovirus-induced phosphoproteins (100,000 MW; 72,000 MW; 36,000 MW) that are also observed when adenovirus-infected cells are labeled with inorganic [ 32P] phosphate in vivo. The protein kinase activity detected in this in vitro assay is not dependent upon or stimulated by the addition of exogenous cyclic AMP or cyclic GMP. The 72,000 M W protein phosphorylated in vitro by this phosphokinase activity has been shown to be the adenovirus single-strand-specific DNA-binding protein by a specific immunoprecipitation test.

The regulation of cyclic AMP-phosphodiesterase and its specific inhibitor by cyclic AMP in dictyostelium

The levels of cellular and extracellular cyclic AMP-phosphodiesterase activity in preaggregation cells of three species of the cellular slime mold Dictyostelium were found to be elevated by the addition of exogenous cyclic AMP. This increase in enzyme activity appeared to be due largely to the ‘turn-off’ of the synthesis of its specific inhibitor. In addition to regulating the production of cyclic AMP-phosphodiesterase and its inhibitor, elevated concentrations of cyclic AMP also prolonged cell aggregation. These observations suggests that the extracellular phosphodiesterase-inhibitor system of Dictyostelium might be involved in regulating the level of extracellular cyclic AMP in cell populations prior to aggregation.

Cyclic AMP in action of antidiuretic hormone: effects of exogenous cyclic AMP and its new analogue

Exogenous cyclic 3',5'-adenosine monophosphate (cAMP) stimulates the effect of the antidiuretic hormone, vasopressin (VP), only in pharmacologic quantities and results have often been inconsistent. The present study examined the ability of a new analogue, 8-[p-Cl-phenylthio]cyclic 3',5'-adenosine monophosphate (C1-PheS-cAMP) to mimic the effect of VP, both biochemically (protein kinase activation) and functionally (hydrosomatic response of perfused collecting tubules) in mammalian kidney tissue. C1PheS-cAMP was found to be about 100 times as effective as cAMP both biochemically and functionally. The increased effectiveness of C1PheS-cAMP is probably is probably due to a greater permeability across the cell membrane and to the resistance of C1PheS-cAMP to enzymatic degradation, Cyclic AMP phosphodiesterase inhibition was observed with C1PheS-cAMP, but its contribution to overall effect was minor. C1PheS-cAMP was found to be more effective than exogenous vasopressin, an effect probably due primarily to its resistance to catabolism. The results provide further new evidence that cAMP and protein kinase are involved in the cellular action of vasopressin. C1PheS-cAMP proved to be a useful tool in the study of hormone action, especially in steps subsequent to cAMP generation.