scholarly journals Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

2008 ◽  
Vol 190 (11) ◽  
pp. 3824-3834 ◽  
Author(s):  
Bob Kennedy M. Dass ◽  
Ritu Sharma ◽  
Avinash R. Shenoy ◽  
Rohini Mattoo ◽  
Sandhya S. Visweswariah
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Mycobacterium Smegmatis ◽  
Functional Characterization ◽  
Acid Stress ◽  
Logarithmic Phase ◽  
Intracellular Camp ◽  
Genes Encoding ◽  
Camp Levels

ABSTRACT Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3′,5′-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ΔMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the ΔMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ΔMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.

scholarly journals Cyclic AMP Promotes Neuronal Survival by Phosphorylation of Glycogen Synthase Kinase 3β

2000 ◽  
Vol 20 (24) ◽  
pp. 9356-9363 ◽  
Author(s):  
Mingtao Li ◽  
Xiaomin Wang ◽  
Mary Kay Meintzer ◽  
Tracey Laessig ◽  
Morris J. Birnbaum ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Glycogen Synthase ◽  
Neuronal Survival ◽  
Glycogen Synthase Kinase ◽  
Intracellular Camp ◽  
Glycogen Synthase Kinase 3Β ◽  
Gsk 3Β ◽  
Camp Levels

ABSTRACT Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3β (GSK-3β). The increased phosphorylation of GSK-3β by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3β in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3β mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3β and inhibits its apoptotic activity in neurons.

Effect of Cyclic AMP and Cyclic GMP on Thromboplastin (Factor III) Synthesis in Human Monocytes In Vitro

1983 ◽  
Vol 50 (04) ◽  
pp. 804-809 ◽  
Author(s):  
Torstein Lyberg
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Immune Complexes ◽  
Phosphodiesterase Inhibitors ◽  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Human Monocytes ◽  
Purified Protein Derivative ◽  
A 23187

SummaryHuman monocytes in vitro respond to various agents (immune complexes, lectins, endotoxin, the divalent ionophore A 23187, 12-0-tetradecanoyl-phorbol 13-acetate [TPA], purified protein derivative [PPD] of Bacille Calmette-Guerin) with an increased synthesis of the protein component of thromboplastin. The effect of cyclic AMP and cyclic GMP on this response has been studied. Dibutyryl-cyclic AMP, prostaglandin E1 and the phosphodiesterase inhibitors 3-butyl-1-methyl-xanthine (MIX) and rac -4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 201724), separately and in combination have a pronounced inhibitory effect on the response to immune complexes and PPD, and a moderate effect on the response to endotoxin and lectins. The effect on TPA response and on the response to A 23187 was slight. Dibutyryl-cyclic GMP (1 mM) gave a slight inhibition of the TPA arid IC response, but had essentially no effect on the response to other inducers. The intracellular cAMP level increased when monocytes were incubated with IC, TPA or A 23187 followed by a decrease to basal levels within 1-2 hr, whereas lectin (PHA) and PPD did not induce such changes. The cAMP response to endotoxin varied. Stimulation with IC induced an increase in monocyte cGMP levels, whereas the other stimulants did not cause such changes.

Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes

1982 ◽  
Vol 243 (2) ◽  
pp. G117-G126
Author(s):  
R. Fogel ◽  
G. W. Sharp ◽  
M. Donowitz
Keyword(s):  
Cholera Toxin ◽  
Calcium Content ◽  
Intracellular Camp ◽  
Rabbit Ileum ◽  
Camp Content ◽  
Serosal Surface ◽  
Ileal Absorption ◽  
Camp Levels

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

scholarly journals Alteration of Platelet Cyclic AMP (cAMP) by Ethanol

1977 ◽  
Author(s):  
D.H. Cowan ◽  
M. Kikta ◽  
D. Baunach
Keyword(s):  
Cyclic Amp ◽  
Platelet Function ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Platelet Dysfunction ◽  
Camp Metabolism ◽  
Subnormal Level ◽  
Camp Levels ◽  
Stimulation Of

Studies of cAMP in human platelets exposed to ethanol were done to assess one possible mechanism for ethanol-related platelet dysfunction. Ingestion of ethanol by 3 subjects produced blood ethanol levels from 65-76 mM. Thrombocytopenia occurred in 1 subject and impaired platelet function occurred in all. Platelet cAMP decreased 36,51, and 59% below control levels. Infusion of ethanol to 2 normals produced blood ethanol levels of 43 mM and decreased platelet cAMP by 15% and 22%. Incubation of normal platelets with 86 mM ethanol in vitro decreased cAMP from 13.8 ± 2.9 (1 SD) to 9.4 ± 3.5 (p<0.02). By contrast, ethanol did not impair the increase in cAMP that occurred with 1.3 μM PGE1. Further, ethanol enhanced the increase in cAMP produced by 2.0 mM papaverine (Pap) by 160-220% and that produced by Pap + PGE1 by 58%. Dopamine, 0.1 mM, caused a 23% decrease in the basal level of cAMP, a 31% decrease below the subnormal level of cAMP seen with ethanol alone, and a 41% reduction in the increased level of cAMP produced by Pap + ethanol. The effect of ethanol on platelet cAMP metabolism is complex. Ethanol reduces basal levels of cAMP, does not decrease elevated levels that result from PGE1 stimulation of adenylate cyclase, and augments the inhibitory effect of Pap on platelet phosphodiesterase (PDE). Despite causing a decrease in basal cAMP levels, ethanol may impair platelet function by potentiating the effect of agents or other conditions which increase cAMP. The effect of ethanol on Pap-stimulated PDE activity may be blocked by dopamine, a neuropharmacologic agent that is actively accumulated by platelets.

scholarly journals Cyclic AMP enhances the sexual agglutinability of Chlamydomonas flagella.

1989 ◽  
Vol 109 (1) ◽  
pp. 247-252 ◽  
Author(s):  
U W Goodenough
Keyword(s):  
Cell Wall ◽  
Cyclic Amp ◽  
Mating Type ◽  
Feedback System ◽  
Intracellular Camp ◽  
Camp Generation ◽  
Sexual Agglutinability ◽  
Single Mating ◽  
Wall Loss ◽  
Camp Levels

Sexual adhesion between Chlamydomonas reinhardtii gametes elicits a rise in intracellular cAMP levels, and exogenous elevation of intracellular cAMP levels in gametes of a single mating type induces such mating responses as cell wall loss, flagellar tip activation, and mating structure activation (Pasquale, S. M., and U. W. Goodenough. 1987. J. Cell Biol. 105:2279-2292). Here evidence is presented that sexual adhesion mobilizes agglutinin to the flagellar surface, and that this mobilization can be induced by exogenous presentation of cAMP to gametes of a single mating type. It is proposed that Chlamydomonas adhesion entails a positive feedback system--initial contacts stimulate the presentation of additional agglutinin--and that this feedback is mediated by adhesion-induced cAMP generation.

A Novel Loss of Function Melanocortin-4-Receptor Mutation (MC4R-F313Sfs*29) in Morbid Obesity

2020 ◽  
Author(s):  
Elisabetta Trevellin ◽  
Marnie Granzotto ◽  
Cristina Host ◽  
Francesca Grisan ◽  
Diego De Stefani ◽  
...  
Keyword(s):  
Early Onset ◽  
Functional Characterization ◽  
Gene Mutations ◽  
Melanocortin Receptor ◽  
Hek293 Cells ◽  
Intracellular Camp ◽  
Loss Of Function ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor

Abstract Context Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity, and the identification of potential pathological variants is crucial for the clinical management of patients with obesity. Objective To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (body mass index [BMI] 38.8 kg/m2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development. Design Setting and Patients The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically encoded biosensors for cAMP and Ca2+. Results The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca2+ levels after stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0 = -90% ± 8%; P &lt; 0.001). In silico modeling showed that the F313Sfs*29 mutation causes a major reorganization in the cytosolic domain of MC4R, thus reducing the affinity of the putative GalphaS binding site. Conclusions The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca2+ signaling, blunting intracellular G protein-mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.

scholarly journals Adenosine 3':5'-monophosphate content and actions in the division cycle of synchronized HeLa cells.

1976 ◽  
Vol 71 (2) ◽  
pp. 515-534 ◽  
Author(s):  
C E Zeilig ◽  
R A Johnson ◽  
E W Sutherland ◽  
D L Friedman
Keyword(s):  
Cell Cycle ◽  
Cyclic Amp ◽  
Hela Cells ◽  
S Phase ◽  
Intracellular Camp ◽  
Specific Effects ◽  
Low Concentrations ◽  
High Concentrations ◽  
Camp Levels ◽  
Stimulation Of

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.

scholarly journals A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum.

1990 ◽  
Vol 10 (7) ◽  
pp. 3297-3306 ◽  
Author(s):  
P C Ma ◽  
C H Siu
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Dictyostelium Discoideum ◽  
Surface Glycoprotein ◽  
Intracellular Camp ◽  
Cell Surface Glycoprotein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Camp Receptor

The EDTA-resistant cell-cell adhesion expressed at the aggregation stage of Dictyostelium discoideum is mediated by a cell surface glycoprotein of Mr 80,000 (gp80). The expression of gp80 is developmentally regulated by cyclic AMP (cAMP). In vitro nuclear run-on experiments show that transcription of the gp80 gene is initiated soon after the onset of development. The basal level of gp80 transcription is significantly augmented by exogenous cAMP pulses. Interestingly, in analog studies, 2'-deoxy-cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP are all capable of inducing a rapid accumulation of gp80 mRNA, suggesting the presence of a unique cAMP receptor that responds equally well to these analogs. To determine whether intracellular cAMP plays a role in the regulation of gp80 expression, caffeine was used to block cAMP-induced receptor-mediated adenylate cyclase activation. Expression of gp80 mRNA was blocked in caffeine-treated cells but could be substantially restored by treatment with exogenous cAMP pulses, suggesting that adenylate cyclase activation is not required. gp80 expression was also examined in the signal transduction mutants synag 7 and frigid A. In both mutants, gp80 was expressed at the basal level. Pulses of cAMP as well as 2'-deoxy-cAMP and N6-monobutyryl-cAMP were capable of restoring the normal level of gp80 expression in synag 7 cells. These results, taken together, indicate bimodal regulation of gp80 expression during development and the involvement of a novel cAMP receptor in the transmembrane signalling pathway that regulates gp80 gene expression.

Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes

1999 ◽  
Vol 276 (2) ◽  
pp. C487-C496 ◽  
Author(s):  
Azeddine Ibrahimi ◽  
Nada Abumrad ◽  
Hengameh Maghareie ◽  
Michael Golia ◽  
Ilana Shoshani ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Morphological Changes ◽  
Intracellular Camp ◽  
Specific Marker ◽  
Marker Enzyme ◽  
Adenylyl Cyclases ◽  
Consequent Reduction ◽  
Elevated Expression ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Camp Levels

Differentiation of Ob1771 preadipocytes to adipocytes was characterized by morphological changes and elevated expression of the specific marker enzyme, glycerol-3-phosphate dehydrogenase. A differentiation response substantially more complete and rapid than that obtained with insulin and 3,5,3′-triiodothyronine was observed with established inhibitors of adenylyl cyclases: 2′,5′-dideoxyadenosine (2′,5′-dd-Ado), 9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by N 6-(2-phenylisopropyl)adenosine or 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively, for adenosine A1 receptors. Also ineffective were IBMX or forskolin, agents that can raise intracellular cAMP levels. Potency of the differentiation response followed the order 2′,5′-dd-Ado (1–20 μM) > 9-CP-Ade (10–100 μM) = 9-Ara-Ade (10–100 μM) >> inosine, consistent with their potencies to inhibit adenylyl cyclases. The data suggest that inhibition of adenylyl cyclase via the P-site and the consequent reduction in cell cAMP levels facilitate the induction of differentiation in Ob1771 cells. The findings raise the question whether the known endogenous P-site ligands participate in the differentiation response induced by hormones.

Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum

Development ◽  
1990 ◽  
Vol 109 (3) ◽  
pp. 715-722
Author(s):  
B.B. Riley ◽  
S.L. Barclay
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Species ◽  
Spore Formation ◽  
Intracellular Camp ◽  
Simultaneous Exposure ◽  
High Ph ◽  
Camp Hydrolysis ◽  
Extracellular Camp ◽  
Camp Levels

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.

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