Journal articles on the topic 'CAMP assay'
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1
Golla, Rajasree, and Ramakrishna Seethala. "A Homogeneous Enzyme Fragment Complementation Cyclic AMP Screen for GPCR Agonists." Journal of Biomolecular Screening 7, no. 6 (December 2002): 515–25. http://dx.doi.org/10.1177/1087057102238625.
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In the new high-throughput screening (HTS) campaign, receptor functional assays, 3’,5’-cyclic adenosine mono-phosphate (cAMP), intracellular [Ca2+]i, phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter™ enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Gαs-coupled receptor. In the EFC-cAMP assay, the β-galactosidase (β-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the β-gal enzyme acceptor (EA) fragment to form an active β-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Gαs to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC50 of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC50 ~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 μM in suspension cells. The assay is very robust, with a Z value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
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Elster, Lisbeth, Christian Elling, and Anders Heding. "Bioluminescence Resonance Energy Transfer as a Screening Assay: Focus on Partial and Inverse Agonism." Journal of Biomolecular Screening 12, no. 1 (November 12, 2006): 41–49. http://dx.doi.org/10.1177/1087057106295895.
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The reported data for compound screening with the bioluminescence resonance energy transfer (BRET2) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven β2 adrenergic receptor (β2-AR) agonists were tested for full or partial agonism in an improved version of the receptor/β-arrestin2 BRET2 assay and in 2 cyclic adenosine monophosphate (cAMP) assays (column cAMP assay and ALPHAscreen™ cAMP assay). Tested in the highly sensitive ALPHAscreen™ cAMP assay, all selected agonists behaved as full agonists, using isoproterenol as a reference compound. In the less sensitive column cAMP assay, ephedrine and dopamine had a clear partial response. For the BRET2 assay, a highly graded picture was obtained. Moreover, β2-AR antagonists were tested for inverse agonism. Pronounced inverse agonism was detected in the ALPHAscreen™ cAMP assay. Only marginal inverse agonistic responses were seen for alprenolol and pindolol in the column cAMP assay, and no inverse agonism was seen in the BRET2 assay. For the β2-AR, the BRET2 assay may be superior for secondary screening of agonists where a separation of full and partial agonists is needed and the ALPHAscreen™ cAMP assay may be preferred for primary screening of agonists where all receptor activating compounds are desired.
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Chiulli, Anthony C., Karen Trompeter, and Michelle Palmer. "A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels." Journal of Biomolecular Screening 5, no. 4 (June 2000): 239–47. http://dx.doi.org/10.1177/108705710000500406.
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The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.
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Kool, Jeroen, André Van Marle, Saskia Hulscher, Maurice Selman, Dick J. Van Iperen, Klaas Van Altena, Michel Gillard, et al. "A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production." Journal of Biomolecular Screening 12, no. 8 (December 2007): 1074–83. http://dx.doi.org/10.1177/1087057107308881.
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A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z′ factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H3 G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC50 values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements. ( Journal of Biomolecular Screening 2007:1074-1083)
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Sugiyama, Atsushi, Phi Wiegn, Scott McKnite, and Keith G. Lurie. "Enzymatic fluorometric assay for tissue cAMP." Journal of Clinical Laboratory Analysis 8, no. 6 (1994): 437–42. http://dx.doi.org/10.1002/jcla.1860080616.
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Martikkala, Eija, Anita Rozwandowicz-Jansen, Pekka Hänninen, Ulla Petäjä-Repo, and Harri Härmä. "A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay." Journal of Biomolecular Screening 16, no. 3 (February 22, 2011): 356–62. http://dx.doi.org/10.1177/1087057110397356.
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G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.
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Prystay, Linda, Annie Gagne, Patricia Kasila, Li-An Yeh, and Peter Banks. "Homogeneous Cell-Based Fluorescence Polarization Assay for the Direct Detection of cAMP." Journal of Biomolecular Screening 6, no. 2 (April 2001): 75–82. http://dx.doi.org/10.1177/108705710100600203.
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A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2cr (CRF2a) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 μM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.
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Tian, Ling, Rongsheng E. Wang, Ying Fei, and Yie-Hwa Chang. "A Homogeneous Fluorescent Assay for cAMP-Phosphodiesterase Enzyme Activity." Journal of Biomolecular Screening 17, no. 3 (November 7, 2011): 409–14. http://dx.doi.org/10.1177/1087057111426901.
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Cyclic adenosine monophosphate–phosphodiesterases (cAMP-PDEs) regulate the cellular level of cAMP by selectively catalyzing the hydrolysis of the phosphodiester bond in the cAMP molecule. They play important roles in modulating cellular and physiological functions. There is a growing interest in the study of cAMP-PDEs as therapeutic targets. We describe a novel method for measuring the enzyme activity of cAMP-PDEs that is based on a homogeneous fluorescence assay employing a cAMP-dependent DNA-binding protein (CAP). We demonstrate that the assay is quick and robust compared to traditional methods and is expected to be cost-effective for high-throughput screening of cAMP-PDE inhibitors. The usefulness of the assay is demonstrated by measuring IC50 values of three nonselective PDE inhibitors and by kinetic measurements of cAMP-PDEs from various rat tissues.
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Diana, Tanja, Paul D. Olivo, Yie-Hwa Chang, Christian Wüster, Michael Kanitz, and George J. Kahaly. "Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies." European Thyroid Journal 9, no. 2 (November 27, 2019): 67–72. http://dx.doi.org/10.1159/000504509.
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Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves’ disease (GD). We tested a novel homogeneous fluorescent 3′,5′ cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. Results: Both cell lines were stimulated with forskolin concentrations (0.006–200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8–25 and 3.1–50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006–50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8–12.5 and 0.2–3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. Conclusion: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.
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Danish, Azeem, Robin Gedschold, Sonja Hinz, Anke C. Schiedel, Dominik Thimm, Peter Bedner, Christian Steinhäuser, and Christa E. Müller. "A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators." International Journal of Molecular Sciences 22, no. 3 (January 31, 2021): 1417. http://dx.doi.org/10.3390/ijms22031417.
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Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.
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Nickolls, Sarah A., Alison Waterfield, Rachael E. Williams, and Ross A. Kinloch. "Understanding the Effect of Different Assay Formats on Agonist Parameters: A Study Using the µ-Opioid Receptor." Journal of Biomolecular Screening 16, no. 7 (May 5, 2011): 706–16. http://dx.doi.org/10.1177/1087057111406548.
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The correct interpretation of data is fundamental to the study of G-protein–coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at µ-opioid receptor agonists in three different assays: (1) [35S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) β-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist β-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z′ value was highest in the β-arrestin assay, followed by the [35S]GTPγS and cAMP assays. The cAMP data fitted well to the operational model, as did the [35S]GTPγS data, but the [35S]GTPγS assay led to an apparent overestimation of KA values. However, in the β-arrestin assay, data did not fit the operational model, as treatment with β-funaltrexamine reduced the Emax proportionally to receptor number, with no change in EC50. In addition, the EC50 values generated correlated well with affinity values. In conclusion, the β-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC50 value generated is a good approximation of affinity.
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Gilissen, Julie, Pierre Geubelle, Nadine Dupuis, Céline Laschet, Bernard Pirotte, and Julien Hanson. "Forskolin-free cAMP assay for Gi-coupled receptors." Biochemical Pharmacology 98, no. 3 (December 2015): 381–91. http://dx.doi.org/10.1016/j.bcp.2015.09.010.
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Allen, Michael, David Hall, Barbara Collins, and Keith Moore. "A Homogeneous High Throughput Nonradioactive Method for Measurement of Functional Activity of Gs-Coupled Receptors in Membranes." Journal of Biomolecular Screening 7, no. 1 (February 2002): 35–44. http://dx.doi.org/10.1177/108705710200700106.
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A method is described for measuring the activity of Gs-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of Gs-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high (~10%). Compared to complex cell-based assays, there is limited potential for non-specific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 μg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 μl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z’ for the assay is ~0.5.
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Hu, Shiau-Ting, Hsuan-Chen Wang, Guang-Sheng Lei, and Shao-Hung Wang. "Negative Regulation of IS2 Transposition by the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex." Journal of Bacteriology 180, no. 10 (May 15, 1998): 2682–88. http://dx.doi.org/10.1128/jb.180.10.2682-2688.1998.
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ABSTRACT Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS2 promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS2 transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS2 promoter. A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above. With an IS2 promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS2 promoter. IS2 was found to transpose at a frequency approximately 200-fold higher in an Escherichia coli host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS2 transposition.
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van der Lee, Miranda M. C., Marion Blomenröhr, Antoon A. van der Doelen, Jesse W. Y. Wat, Niels Smits, Bonnie J. Hanson, Chris J. van Koppen, and Guido J. R. Zaman. "Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1." Journal of Biomolecular Screening 14, no. 7 (June 11, 2009): 811–23. http://dx.doi.org/10.1177/1087057109337937.
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Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. ( Journal of Biomolecular Screening 2009:811-823)
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George, Samantha E., Peter J. Bungay, and Louise H. Naylor. "Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation." Journal of Biomolecular Screening 2, no. 4 (June 1997): 235–40. http://dx.doi.org/10.1177/108705719700200408.
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A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.
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Tewson, Paul, Scott Martinka, Nathan Shaner, Catherine Berlot, Anne Marie Quinn, and Thomas Hughes. "Assay for Detecting Gαi-Mediated Decreases in cAMP in Living Cells." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 9 (July 10, 2018): 898–906. http://dx.doi.org/10.1177/2472555218786238.
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Cell-based assays to detect Gαi signaling are often indirect, frequently involve complex pharmacological interventions, and are usually blind to the kinetics of the signaling. Our goal was to develop a simple, direct measure of Gαi signaling in living cells. We previously reported our fluorescent cADDis assay and showed that it reliably detects Gαs-mediated increases in cAMP levels. Agonists that stimulate a Gs-coupled receptor produce changes in the intensity of bright green or red fluorescent protein sensors that can be followed over time using automated fluorescence plate readers or fluorescence imaging systems. Since the cADDis sensors can monitor Gαs-mediated increases in adenylyl cyclase activity, in theory they should also be capable of detecting Gαi-mediated decreases. Here we apply our green fluorescent cADDis sensor to the detection of Gαi-mediated inhibition of adenylyl cyclase activity. We validated and optimized the assay in living HEK 293T cells using several known Gαi-coupled receptors and agonists, and we report robust Z′ statistics and consistent EC50 responses.
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Titus, Steven A., Xiao Li, Noel Southall, Jianming Lu, James Inglese, Michael Brasch, Christopher P. Austin, and Wei Zheng. "A Cell-Based PDE4 Assay in 1536-Well Plate Format for High-Throughput Screening." Journal of Biomolecular Screening 13, no. 7 (July 1, 2008): 609–18. http://dx.doi.org/10.1177/1087057108319977.
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The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,′5′-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5′nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein—coupled receptor as a driving force for cAMP production and a cyclic nucleotide—gated cation channel as a biosensor in 1536-well plates. ( Journal of Biomolecular Screening 2008:609-618)
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Berkowitz, L. A., K. T. Riabowol, and M. Z. Gilman. "Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter." Molecular and Cellular Biology 9, no. 10 (October 1989): 4272–81. http://dx.doi.org/10.1128/mcb.9.10.4272-4281.1989.
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Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.
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Berkowitz, L. A., K. T. Riabowol, and M. Z. Gilman. "Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter." Molecular and Cellular Biology 9, no. 10 (October 1989): 4272–81. http://dx.doi.org/10.1128/mcb.9.10.4272.
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Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.
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Huang, Qianwu, Jun Lv, Ting Dong, Haijun Liu, Lei Xu, and Mingcai Wu. "Cryptochrome 1 Alleviates the Antiproliferative Effect of Isoproterenol on Human Gastric Cancer Cells." Dose-Response 18, no. 3 (July 1, 2020): 155932582093902. http://dx.doi.org/10.1177/1559325820939022.
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Background: Cryptochrome 1 (CRY1) is a key protein that regulates the feedback loop of circadian clock. The abnormal expression of CRY1 was reported in numerous cancers, and contributed to tumorigenesis and progression. But the underlying mechanism remains undefined. Methods: CRY1 overexpression was constructed by lentivirus vector. Gene and protein expression was detected by reverse transcription quantitative polymerase chain reaction and Western blot. Cell proliferation was analyzed by CCK-8 assay. Cell migration ability was analyzed by scratch assay and transwell migration assay. The cAMP concentration was measured by intracellular cAMP assay. Results: Overexpression of CRY1 showed slightly effect on the proliferation and migration of HGC-27 cells. Upon exposure to isoproterenol (ISO), a β-adrenergic receptor agonist, cell proliferation, and migration were inhibited while the cAMP/PKA pathway was activated and ERK1/2 phosphorylation was suppressed. CRY1 overexpression reduced cAMP accumulation, retained ERK1/2 phosphorylation level and alleviated the antiproliferative effect upon exposure to ISO. However, CRY1 overexpression was inoperative on the antiproliferative effect of forskolin (FSK), a direct activator of adenyl cyclase (AC), or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor. Conclusions: Our results suggest CRY1 overexpression may protect cells from the antiproliferative effects via activation of the cAMP/PKA pathway through interrupting signal transduction from G protein-coupled receptors to AC.
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Matsumura, Y., S. Uchida, T. Rai, S. Sasaki, and F. Marumo. "Transcriptional regulation of aquaporin-2 water channel gene by cAMP." Journal of the American Society of Nephrology 8, no. 6 (June 1997): 861–67. http://dx.doi.org/10.1681/asn.v86861.
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Aquaporin-2 (AQP-2) water channel is a key molecule for urinary concentration whose expression is augmented by dehydration in vivo. To elucidate the regulatory mechanism of this phenomenon in vitro, mouse collecting duct cell lines were established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T antigen gene and then screened for the AQP-2 expression, using ribonuclease protection assay. In one cell line designated C4, the endogenous AQP-2 mRNA level measured by ribonuclease protection assay increased fourfold after treatment with chlorophenylthio-cAMP (cpt-cAMP) (400 microM). In contrast, phorbol 12-myristate 13-acetate did not affect the AQP-2 mRNA level. To identify the molecular mechanism(s) of cAMP-induced upregulation of AQP-2 mRNA in C4 cells, luciferase assay was performed using various 5'-flanking regions of the human AQP-2 gene. Luciferase activity in C4 cells transfected with constructs containing approximately 2.8-kbp or 224-bp 5'-flanking region showed a 3.5-fold increase by cpt-cAMP treatment, indicating that the 224-bp 5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms. This region contains cAMP-responsive element (CRE), and the deletion of the core sequence of CRE (GACGTCA) or introduction of mutation into CRE (GTGGTCA) completely abolished the responsiveness to cpt-cAMP, confirming the key role of CRE in the cAMP-induced transcriptional activation of the AQP-2 gene. Electrophoretic mobility shift assay revealed the existence of proteins binding to CRE in C4 cells and in rat kidney. The binding of CRE proteins to CRE was increased in the nuclear extract from cpt-cAMP-treated C4 cells and dehydrated rat kidney compared with those from controls. These results demonstrated that the CRE in the AQP-2 gene promoter is a key cis-element for cAMP-mediated transcriptional regulation of this gene and may be important for in vivo regulation of AQP-2 expression in a dehydrated state.
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Huang, Wei, Yan Zhang, and J. Richard Sportsman. "A Fluorescence Polarization Assay for Cyclic Nucleotide Phosphodiesterases." Journal of Biomolecular Screening 7, no. 3 (June 2002): 215–22. http://dx.doi.org/10.1177/108705710200700305.
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Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
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Vedel, Line, Hans Bräuner-Osborne, and Jesper Mosolff Mathiesen. "A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors." Journal of Biomolecular Screening 20, no. 7 (April 7, 2015): 849–57. http://dx.doi.org/10.1177/1087057115580019.
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Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein–coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)–based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the β2-adrenergic receptor (β2AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β2AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS.
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Tani, T., and Y. Naitoh. "Chemotactic responses of Dictyostelium discoideum amoebae to a cyclic AMP concentration gradient: evidence to support a spatial mechanism for sensing cyclic AMP." Journal of Experimental Biology 202, no. 1 (January 1, 1999): 1–12. http://dx.doi.org/10.1242/jeb.202.1.1.
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The motile responses of Dictyostelium discoideum amoebae to a cyclic AMP (cAMP) concentration gradient were examined using a novel assay system. In this system, a cAMP concentration gradient was generated, while the overall cAMP concentration could be either increased or decreased in a chamber containing amoebae. The chemotactic responses of amoebae were examined immediately after they had been subjected to the cAMP concentration gradient. Amoebae moving in random directions in a reference solution ascended a cAMP concentration gradient after they had been exposed to the gradient irrespective of whether there was an increase or a decrease in the overall cAMP concentration. This strongly supports the idea that D. discoideum amoebae can sense a spatial cAMP gradient around them and that this causes their chemoaccumulation behavior. Ascending locomotion became less conspicuous when the amoebae were treated with a homogeneous cAMP solution for approximately 8 min before exposure to a cAMP gradient. This cAMP pretreatment reduced the sensitivity of the amoeba to a cAMP concentration gradient. The cAMP concentration gradient could be reversed in less than 30 s in this assay system, allowing the generation of a cAMP wave by accumulating amoebae to be mimicked. The ascending amoebae continued to move in the same direction for 1–2 min after the gradient had been reversed. This is consistent with the well-known observation that reversal of a cAMP concentration gradient experienced by the amoebae passing through a cAMP wave does not negate their chemotactic movement towards the accumulation center.
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Manandhar, Preeti, Bridin Patricia Murnion, Natasha L. Grimsey, Mark Connor, and Marina Santiago. "Do gabapentin or pregabalin directly modulate the µ receptor?" PeerJ 9 (April 12, 2021): e11175. http://dx.doi.org/10.7717/peerj.11175.
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Background Pregabalin and gabapentin improve neuropathic pain symptoms but there are emerging concerns regarding their misuse. This is more pronounced among patients with substance use disorder, particularly involving opioids. Co-ingestion of gabapentinoids with opioids is increasingly identified in opioid related deaths, however, the molecular mechanism behind this is still unclear. We have sought to determine whether pregabalin or gabapentin directly modulates acute μ receptor signaling, or μ receptor activation by morphine. Methods The effects of pregabalin and gabapentin were assessed in HEK 293 cells stably transfected with the human μ receptor. Their effect on morphine induced hyperpolarization, cAMP production and ERK phosphorylation were studied using fluorescent-based membrane potential assay, bioluminescence based CAMYEL assay and ELISA assay, respectively. Pregabalin/gabapentin effects on morphine-induced hyperpolarization were also investigated in AtT20 cells. Results Pregabalin or gabapentin (1 µM, 100 µM each) did not activate the µ receptor or affect K channel activation or ERK phosphorylation produced by morphine. Neither drug affected the desensitization of K channel activation produced by prolonged (30 min) application of morphine. Gabapentin (1 µM, 100 µM) and pregabalin (1 µM) did not affect inhibition of forskolin-stimulated cAMP production by morphine. However, pregabalin (100 µM) potentiated forskolin mediated cAMP production, although morphine still inhibited cAMP levels with a similar potency to control. Discussion Pregabalin or gabapentin did not activate or modulate µ receptor signaling in three different assays. Our data do not support the hypothesis that gabapentin or pregabalin augment opioid effects through direct or allosteric modulation of the µ receptor. Pregabalin at a high concentration increases cAMP production independent of morphine. The mechanism of enhanced opioid-related harms from co-ingestion of pregabalin or gabapentin with opioids needs further investigation.
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Chen, Yongchang, Ying Wang, Hao Yu, Fengwei Wang, and Wenrong Xu. "The Cross Talk Between Protein Kinase A– and RhoA-Mediated Signaling in Cancer Cells." Experimental Biology and Medicine 230, no. 10 (November 2005): 731–41. http://dx.doi.org/10.1177/153537020523001006.
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The cross talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and RhoA-mediated signal transductions and the effect of this cross talk on biologic features of human prostate and gastric cancer cells were investigated. In the human gastric cancer cell line, SGC-7901, lysophosphatidic acid (LPA) increased RhoA activity in a dosedependent manner. The cellular permeable cAMP analog, 8-chlorophenylthio-cAMP (CPT-cAMP), inhibited the LPA-induced RhoA activation and caused phosphorylation of RhoA at serine188. Immunofluorescence microscopy, Western blotting, and green fluorescent protein (GFP)-tagged RhoA location assay in live cells revealed that RhoA was distributed in both the cytoplasm and nucleus of SGC-7901 cells. Treatment with LPA and/or CPT-cAMP did not induce obvious translocation of RhoA in the cells. The LPA treatment caused formation of F-actin in SGC-7901 cells, and CPT-cAMP inhibited the formation. In a modified Boyden chamber assay, LPA stimulated the migration of SGC-7901 cells, and CPT-cAMP dose-dependently inhibited the stimulating effect of LPA. In soft agar assay, LPA stimulated early proliferation of SGC-7901 cells, and CPT-cAMP significantly inhibited the growth of LPA-stimulated cells. In the prostate cancer cell line, PC-3, LPA caused morphologic changes from polygonal to round, and transfection with plasmid DNA encoding constitutively active RhoA(63L) caused a similar change. Treatment with CPT-cAMP inhibited the changes in both cases. However, in PC-3 cells transfected with a plasmid encoding mutant RhoA188A, LPA induced rounding, but CPT-cAMP could not prevent the change. Results of this experiment indicated that cAMP/PKA inhibited RhoA activation, and serine188 phosphorylation on RhoA was necessary for PKA to exert its inhibitory effect on RhoA activation. The cross talk between cAMP/PKA and RhoA-mediated signal transductions had significant affect on biologic features of gastric and prostate cancer cells, such as morphologic and cytoskeletal change, migration, and anchorage-independent growth. The results may be helpful in implementing novel therapeutic strategies for invasive and metastatic prostate and gastric cancers.
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Ofenloch-Hähnle, Beatus, and Karl Eisele. "Inhibition of cAMP-Phosphodiesterase by Molybdate." Zeitschrift für Naturforschung C 42, no. 1-2 (February 1, 1987): 162–64. http://dx.doi.org/10.1515/znc-1987-1-228.
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Abstract Inhibition of 3′:5′-cyclic-AMP-5′-nucleotidohydrolase (EC 3.1.4.17) type II (cAMP-phosphodiesterase) by sodium molybdate was studied: While determination of inorganic phosphate and 5′-ribonucleotide phosphohydrolase (5′-nucleotidase) (EC 3.1.3.5) activity was not disturbed by sodium molybdate in concentrations up to 10 mM , cAMP-phosphodiesterase was inhibited by millimolar concentrations of molybdate. The half maximal effect was observed at about 2 mM sodium molybdate (0.75 mM cAMP in the assay).
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Kirkman, Laura A., Louis M. Weiss, and Kami Kim. "Cyclic Nucleotide Signaling in Toxoplasma gondii Bradyzoite Differentiation." Infection and Immunity 69, no. 1 (January 1, 2001): 148–53. http://dx.doi.org/10.1128/iai.69.1.148-153.2001.
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ABSTRACT The ability of Toxoplasma gondii tachyzoites to differentiate into latent bradyzoite forms is essential for pathogenesis of clinical disease. We examined the effects of cyclic nucleotides on T. gondii bradyzoite differentiation in vitro. Differentiation of tachyzoites to bradyzoites was measured in an immunofluorescence assay using ME49 or its clonal derivative PLK, two well-characterized T. gondii strains. Treatment of human fibroblast cultures infected with T. gondii with 8-(4-chlorophenylthio)-cyclic GMP (CPT-cGMP), a membrane-permeable, nonhydrolyzable analogue of cGMP, resulted in an increased percentage of bradyzoite-positive vacuoles. Cyclic AMP (cAMP) also induced in vitro conversion of PLK, but the method of cAMP elevation was critical. Forskolin raises cAMP levels transiently and induced bradyzoites, whereas agents predicted to cause sustained elevation of cAMP were inhibitory to parasite conversion. Levels of cAMP were measured in host cells and extracellular tachyzoites. Forskolin, CPT-cGMP, and agents known to induce bradyzoite formation elevated cAMP in host cells and PLK parasites. These data suggest cyclic nucleotide signaling pathways are important in the stress-induced conversion of T. gondiitachyzoites to bradyzoites. Furthermore, because cAMP elevation was seen in PLK but not RH, a T. gondii strain that did not differentiate well in our assay, cAMP signaling within the parasite is likely to be critical.
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Terstappen, G. C., A. Giacometti, E. Ballini, and L. Aldegheri. "Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators." Journal of Biomolecular Screening 5, no. 4 (June 2000): 255–61. http://dx.doi.org/10.1177/108705710000500408.
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For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC50 of 0-9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.
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MAGIERA, Maria M., Mona GUPTA, Catherine J. RUNDELL, Nilima SATISH, Isabelle ERNENS, and Stephen J. YARWOOD. "Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A." Biochemical Journal 382, no. 3 (September 7, 2004): 803–10. http://dx.doi.org/10.1042/bj20040122.
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Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (ΔcAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with ΔcAMP-EPAC1 and ΔDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.
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Chen, Hen-Li, Laurie K. McCauley, and Nisha J. D’Silva. "cAMP Binding Protein Assay for Widespread Use in Cell Signaling Studies." BioTechniques 33, no. 1 (July 2002): 66–72. http://dx.doi.org/10.2144/02331st03.
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de Rave, S., H. M. J. Goldschmidt, Y. T. J. Somers-Pijnenburg, B. Bravenboer, and J. H. M. Lockefeer. "A comparison of porcine and human thyroid tissue in the assay of thyroid stimulating immunoglobulins." Acta Endocrinologica 113, no. 3 (November 1986): 335–39. http://dx.doi.org/10.1530/acta.0.1130335.
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Abstract. The central role of Thyroid Stimulating Immunoglobulins (TSI) in the pathogenesis of the hyperthyroidism of Graves' disease has become generally accepted and a wide variety of assays for the detection of these antibodies has been developed. The dependence on the availability of human thyroid tissue makes most of these assays unsuitable for routine clinical use, a problem circumvented by the use of nonhuman thyroid tissue in some TSI assays. We therefore compared porcine and human thyroid tissue in a TSI assay based on in vitro cAMP generation. No major differences in within and between run variation were found and, with some notable exceptions, a reasonable correlation could be demonstrated between the results in both assays (R = 0.89, P < 0.001). However, the sensitivity of the porcine TSI assay is only 60% of the estimated sensitivity of the human TSI assay. In spite of the practical advantages this porcine TSI assay, and possibly also other TSI assays using non-human thyroid tissue, cannot totally replace human TSI assays. The value of these assays in predicting the outcome of medical treatment of Graves' disease remains to be established.
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Mcguinness, Debra, Asra Malikzay, Richard Visconti, Karen Lin, Marvin Bayne, Frederick Monsma, and Charles A. Lunn. "Characterizing Cannabinoid CB2 Receptor Ligands Using DiscoveRx PathHunter™ β-Arrestin Assay." Journal of Biomolecular Screening 14, no. 1 (November 21, 2008): 49–58. http://dx.doi.org/10.1177/1087057108327329.
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The authors have characterized a set of cannabinoid CB2 receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/β-arrestin interaction assay (DiscoveRx PathHunter™). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE™). The authors show good correlation between the 3 assay systems tested, with the β-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB2 agonists. Further assay validation shows that DiscoveRx PathHunter™ HEK293 CB2 β-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB2 receptor and β-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB2 receptor. ( Journal of Biomolecular Screening 2009:49-58)
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Karasawa, Yusuke, Kanako Miyano, Hideaki Fujii, Takaaki Mizuguchi, Yui Kuroda, Miki Nonaka, Akane Komatsu, et al. "In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways." Molecules 26, no. 19 (October 8, 2021): 6079. http://dx.doi.org/10.3390/molecules26196079.
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Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.
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Bernecic, Naomi C., Bart M. Gadella, Simon P. de Graaf, and Tamara Leahy. "Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm." Reproduction 160, no. 2 (August 2020): 269–80. http://dx.doi.org/10.1530/rep-19-0430.
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Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20–23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.
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Kariv, Ilona, Michelle E. Stevens, Davette L. Behrens, and Kevin R. Oldenburg. "High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors." Journal of Biomolecular Screening 4, no. 1 (February 1999): 27–32. http://dx.doi.org/10.1177/108705719900400105.
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Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.
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Chen, Y., M. Allars, C. Abou-Seif, R. Smith, and R. C. Nicholson. "317. DIFFERENTIAL EFFECTS OF cAMP AND CRH IN CELL VIABILITY AND CELL FUSION OF CYTOTROPHOBLASTIC CELLS." Reproduction, Fertility and Development 22, no. 9 (2010): 117. http://dx.doi.org/10.1071/srb10abs317.
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Cell fusion of cytotrophoblasts is critical for a successful pregnancy, and is marked biochemically by proteins such as human chorionic gonadotrophin (hCG) and syncytin, and morphologically as large multi-nucleated cells. Placental corticotrophin releasing hormone (CRH) plays diverse roles during pregnancy and has been linked to the length of gestation. It is well accepted that CRH expression could be upregulated by some hormones, such as hCG via cellular cAMP, and that trophoblastic cell fusion can be induced by Forskolin (increases cAMP). We have shown, by reporter gene assay in human primary placental cells, that 8-Br-cAMP increased CRH gene expression. Thus, it is of interest to determine the roles of 8-Br-cAMP and CRH on trophoblast cell viability and cell fusion. Treatment with 8-Br-cAMP for 72 h significantly inhibited BeWo cell viability (MTT assay) by 29.7% (50 μM) and 54.8% (100 μM), and increased both apoptotic and necrotic cells (FACS analysis). 8-Br-cAMP (100 μM, 48 h) resulted in the appearance of nuclear fragments (DAPI stain) and Fas ligand gene expression by 6.8-fold (real-time RT-PCR). Syncytin 1 mRNA increased by 3.2-fold (real-time RT-PCR) and hCG secretion increased by 4-fold (ELISA assay). The formation of syncytium (CellMask and Hoechst co-stain) could clearly be seen by 72 h. CRH mRNA increased with 8-Br-cAMP treatment of BeWo cells (15-fold at 48 h). Treatment with CRH (100 nM) had a mild inhibitory effect on cell growth (~18%) but no effect on cell viability, up-regulated the expression of syncytin 1 mRNA (2.3-fold at 48 h) and induced cell fusion (72 h), but had no effect on hCG secretion. In summary, we show that 8-Br-cAMP and CRH are both potential inducers of cytotrophoblast cell fusion, and there is a dissociation between morphological and biochemical differentiation. Our data also indicates that the process of cell fusion can be associated with both apoptotic and non-apoptotic events.
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Schwede, Frank, Oleg G. Chepurny, Melanie Kaufholz, Daniela Bertinetti, Colin A. Leech, Over Cabrera, Yingmin Zhu, et al. "Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion." Molecular Endocrinology 29, no. 7 (July 1, 2015): 988–1005. http://dx.doi.org/10.1210/me.2014-1330.
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Abstract cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.
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McCoy, Eric S., Wendy A. Lea, Bryan T. Mott, David J. Maloney, Ajit Jadhav, Anton Simeonov, and Mark J. Zylka. "High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase." Journal of Biomolecular Screening 18, no. 4 (November 27, 2012): 481–89. http://dx.doi.org/10.1177/1087057112468613.
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The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well–based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.
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RAHIM, Z., S. I. KHAN, and A. K. CHOPRA. "Biological characterization of Aeromonas spp. isolated from the environment." Epidemiology and Infection 132, no. 4 (July 9, 2004): 627–36. http://dx.doi.org/10.1017/s0950268804002298.
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Cytotoxic enterotoxin (Act) is a key virulence factor in the pathogenesis of infections caused by Aeromonas spp. The cytotoxic enterotoxin gene (act) was detected in 32 out of 69 environmental isolates of Aeromonas spp. by hybridization with the act gene probe. To evaluate the pathogenic potential of the act gene probe-positive isolates, 32 act gene probe-positive and 31 randomly selected act gene probe-negative isolates were tested for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a Vero cell line. The act gene probe-positive isolates significantly differed from the toxin gene probe-negative ones with respect to enterotoxicity in the SMA (P=0·009) and haemolytic activity (P=0·005). The CAMP–haemolysin phenotype was significantly associated with the rabbit ileal loop assay (P=0·08), Vero cell assay (P=0·064), and haemolysin production under the microaerophilic conditions (P=0·056) of the act gene probe-positive isolates of Aeromonas spp. These data indicated the role of Act in the pathogenesis of Aeromonas infections and that the enterotoxic potential of Aeromonas spp. could be assessed by simply performing a CAMP–haemolysin assay.
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Tereba, Allan. "High Density Protein Kinase Assay With Subattomole Sensitivity." Journal of Biomolecular Screening 3, no. 1 (February 1998): 29–35. http://dx.doi.org/10.1177/108705719800300104.
Full textAbstract:
A rapid assay system based on incorporation of [γ-32P]ATP into biotinylated peptide substrates and their subsequent capture onto a high capacity streptavidin-coated membrane, SAM2™, has been developed for the detection of protein kinases. The system uses prenumbered and partially cut membrane squares for analyzing a limited number of samples or can be formatted to analyze up to 1,536 samples per microtiter plate footprint of 7.0 cm X 10.6 cm. The high biotin-binding capacity and low background of the membrane allows the use of nearly saturating amounts of most substrates, giving this system very high signal-to-noise ratios at low enzyme concentrations. Using cAMP-dependent Protein Kinase A (PKA) as a model system, as little as 0.3 amol of purified enzyme in 0.2 μl can be detected with a linear response range of over 3 orders of magnitude. cAMP-dependent kinase activity can be measured directly in tissue extracts by using a specific substrate and harsh washing procedures to reduce nonspecific backgrounds from proteins phosphorylated by other kinases. For increased assay flexibility, results can be analyzed either by PhosphorImager™ quantitation or by scintillation counting.
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Beck, Elizabeth M., Euan Parnell, Angela Cowley, Alison Porter, Jonathan Gillespie, John Robinson, Lindsay Robinson, et al. "Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1." Cells 8, no. 11 (November 12, 2019): 1425. http://dx.doi.org/10.3390/cells8111425.
Full textAbstract:
Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.
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Molden, Brent M., Kimberly A. Cooney, Kirk West, Lex H. T. Van Der Ploeg, and Giulia Baldini. "Temporal cAMP Signaling Selectivity by Natural and Synthetic MC4R Agonists." Molecular Endocrinology 29, no. 11 (November 1, 2015): 1619–33. http://dx.doi.org/10.1210/me.2015-1071.
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Abstract The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain, where it controls energy balance through pathways including α-melanocyte-stimulating hormone (α-MSH)-dependent signaling. We have reported that the MC4R can exist in an active conformation that signals constitutively by increasing cAMP levels in the absence of receptor desensitization. We asked whether synthetic MC4R agonists differ in their ability to increase intracellular cAMP over time in Neuro2A cells expressing endogenous MC4R and exogenous, epitope-tagged hemagglutinin-MC4R-green fluorescent protein. By analyzing intracellular cAMP in a temporally resolved Förster resonance energy transfer assay, we show that withdrawal of α-MSH leads to a quick reversal of cAMP induction. By contrast, the synthetic agonist melanotan II (MTII) induces a cAMP signal that persists for at least 1 hour after removal of MTII from the medium and cannot be antagonized by agouti related protein. Similarly, in mHypoE-42 immortalized hypothalamic neurons, MTII, but not α-MSH, induced persistent AMP kinase signal, which occurs downstream of increased cAMP. By using a fluorescence recovery after photobleaching assay, it appears that the receptor exposed to MTII continues to signal after being internalized. Similar to MTII, the synthetic MC4R agonists, THIQ and BIM-22511, but not LY2112688, induced prolonged cAMP signaling after agonist withdrawal. However, agonist-exposed MC4R desensitized to the same extent, regardless of the ligand used and regardless of differences in receptor intracellular retention kinetics. In conclusion, α-MSH and LY2112688, when compared with MTII, THIQ, and BIM-22511, vary in the duration of the acute cAMP response, showing distinct temporal signaling selectivity, possibly linked to specific cell compartments from which cAMP signals may originate.
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Shearer, W. T., C. L. Patke, E. B. Gilliam, H. M. Rosenblatt, K. S. Barron, and F. M. Orson. "Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism." Journal of Immunology 141, no. 5 (September 1, 1988): 1678–86. http://dx.doi.org/10.4049/jimmunol.141.5.1678.
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Abstract A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.
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Pakshir, Keyvan, Mahboubeh Bordbar, Kamiar Zomorodian, Hasti Nouraei, and Hossein Khodadadi. "Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination ofCandida africanafrom VaginalCandida albicansSpecies." Journal of Pathogens 2017 (2017): 1–5. http://dx.doi.org/10.1155/2017/7126258.
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Candida africana asa species recovered from female genital specimens is highly close toC. albicans. The present study was conducted to discriminateC. africanafrom presumptive vaginalC. albicansstrains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP) with vaginal bacterial flora. A total of 110 stock vaginalC. albicansisolates were examined byHWP1gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively.Staphylococcus aureus,Staphylococcus epidermidis, andStreptococcus agalactiaewere used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp), all isolates were identified asC. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7%) were biofilm-positive, 54 out of 110Candidaisolates (49%) demonstrated cohemolytic effects withS. agalactiae, and 48 out of 110 showed this effect withS. aureus(43.6%). All isolates were CAMP-negative withS. epidermidis. We detected all isolates asCandida albicansand almost half of the isolates were CAMP-positive withS. aureusandS. agalactiae, suggesting that these bacteria increase the pathogenicity ofCandidain vaginal candidiasis.
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KAMTHONG, Pisate J., Fu-mei WU, and Ming-chi WU. "cAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression." Biochemical Journal 350, no. 1 (August 9, 2000): 115–22. http://dx.doi.org/10.1042/bj3500115.
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Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1α (IL-1α), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1α or LPS, but not by PMA. Electromobility shift assays showed that IL-1α predominantly activated nuclear factor κB (NF-κB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-κB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1α than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-κB signalling pathway.
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Cheng, Jingfei, Michael A. Thompson, Henry J. Walker, Catherine E. Gray, Gina M. Warner, Wei Zhou, and Joseph P. Grande. "Lixazinone Stimulates Mitogenesis of Madin-Darby Canine Kidney Cells." Experimental Biology and Medicine 231, no. 3 (March 2006): 288–95. http://dx.doi.org/10.1177/153537020623100308.
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Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.
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Libersan, Danielle, Guy Rousseau, and Yahye Merhi. "Differential regulation of P-selectin expression by protein kinase A and protein kinase G in thrombin-stimulated human platelets." Thrombosis and Haemostasis 89, no. 02 (2003): 310–17. http://dx.doi.org/10.1055/s-0037-1613448.
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SummaryP-selectin is rapidly translocated from platelet α-granules following activation. Intracellular cyclic AMP (cAMP) is a potent inhibitory pathway that results in global downregulation of platelet activation. While cAMP-dependent protein kinase (PKA) has long been considered as the main mediator of cAMP-dependent effects, no study has yet evaluated its effect on P-selectin expression in human platelets. Pretreatment of thrombin-stimulated platelets with forskolin resulted in a concentration-dependent inhibition of P-selectin expression that correlated with adenylyl cyclase activity. Inhibition of PKA with H-89 reversed cAMP-induced inhibition of P-selectin while cGMP-dependent protein kinase (PKG) inhibition with KT5823 significantly potentiated cAMP-dependent inhibition of P-selectin. Similar results were also observed in a platelet/neutrophil binding assay. In conclusion, cAMP-induced inhibition of P-selectin expression is, in large part, mediated through activation of PKA. PKG appears to be sollicited for P-selectin expression when cAMP levels are elevated which suggest a cAMP/PKG-dependent pathway of platelet activation.
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Fujita, Hidetoshi, Ryouji Fujii, Satoko Aratani, Tetsuya Amano, Akiyoshi Fukamizu, and Toshihiro Nakajima. "Antithetic Effects of MBD2a on Gene Regulation." Molecular and Cellular Biology 23, no. 8 (April 15, 2003): 2645–57. http://dx.doi.org/10.1128/mcb.23.8.2645-2657.2003.
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ABSTRACT DNA methylation is essential for epigenetic gene regulation during development. The cyclic AMP (cAMP)-responsive element (CRE) is found in the promoter of many cAMP-regulated genes and plays important roles in their gene expression. Methylation occurs on the CRE site and results in transcriptional repression via a direct mechanism, that is, prevention by the methyl group of binding of the cAMP-responsive factor CREB to this site. A recent study indicated that the nucleosome is also important in repressing transcription. In this study, we investigated the regulation of transcriptional repression on methylated CRE. We focused on methyl-CpG binding domain protein 2 (MBD2). MBD2 consists of two forms, MBD2a and MBD2b, the latter lacking the N-terminal extension of MBD2a. Unexpectedly, we found that MBD2a, but not MBD2b, promoted activation of the unmethylated cAMP-responsive genes. An in vivo binding assay revealed that MBD2a selectively interacted with RNA helicase A (RHA), a component of CREB transcriptional coactivator complexes. MBD2a and RHA cooperatively enhanced CREB-dependent gene expression. Interestingly, coimmunoprecipitation assays demonstrated that MBD2a binding to RHA was not associated with histone deacetylase 1. Our results indicate a novel role for MBD2a in gene regulation.