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FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells

Abstract

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3–6 d.

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Figure 1: Schematic structure of the cAMP sensor Epac1-camps and equipment setup for FRET imaging.
Figure 2: Measurements of basal cAMP concentrations in various cell types.
Figure 3: Examples of experiments showing measurements of ligand-induced changes in intracellular cAMP concentrations, as well as in kinetics and permeabilities for cAMP analogs.

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References

  1. Beavo, J.A. & Brunton, L.L. Cyclic nucleotide research—still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3, 710–718 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik. 437, 55–75 (1948).

    Article  Google Scholar 

  3. Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846 (1978).

    Article  CAS  PubMed  Google Scholar 

  4. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Lohse, M.J. et al. Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol. Sci. 29, 159–165 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Nikolaev, V.O. & Lohse, M.J. Monitoring of cAMP synthesis and degradation in living cells. Physiology (Bethesda) 21, 86–92 (2006).

    CAS  Google Scholar 

  8. Berrera, M. et al. A toolkit for real-time detection of cAMP: insights into compartmentalized signaling. Handb. Exp. Pharmacol. 186, 285–298 (2008).

    Article  CAS  Google Scholar 

  9. Willoughby, D. & Cooper, D.M. Live-cell imaging of cAMP dynamics. Nat. Methods 5, 29–36 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Zaccolo, M. & Pozzan, T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711–1715 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. DiPilato, L.M., Cheng, X. & Zhang, J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. USA 101, 16513–16518 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ponsioen, B. et al. Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep. 5, 1176–1180 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nikolaev, V.O., Bünemann, M., Hein, L., Hannawacker, A. & Lohse, M.J. Novel single chain cAMP sensors for receptor-induced signal propagation. J. Biol. Chem. 279, 37215–37218 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Nikolaev, V.O., Bünemann, M., Schmitteckert, E., Lohse, M.J. & Engelhardt, S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ. Res. 99, 1084–1091 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Brooker, G., Harper, J.F., Terasaki, W.L. & Moylan, R.D. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic. Nucleotide Res. 10, 1–33 (1979).

    CAS  PubMed  Google Scholar 

  16. Williams, C. cAMP detection methods in HTS: selecting the best from the rest. Nat. Rev. Drug Discov. 3, 125–135 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Lehnart, S.E. et al. Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. Cell 123, 25–35 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lefkimmiatis, K., Moyer, M.P., Curci, S. & Hofer, A.M. 'cAMP sponge': a buffer for cyclic adenosine 3′, 5′-monophosphate. PLoS ONE 4, e7649 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Norris, R.P. et al. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136, 1869–1878 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mironov, S.L. et al. Imaging cytoplasmic cAMP in mouse brainstem neurons. BMC Neurosci. 10, 29 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Leroy, J. et al. Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ. Res. 102, 1091–1100 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Iancu, R.V. et al. Cytoplasmic cAMP concentrations in intact cardiac myocytes. Am. J. Physiol. Cell Physiol. 295, C414–C422 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nikolaev, V.O. et al. Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science 327, 1653–1657 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. von Hayn, K. et al. Gq-mediated Ca2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells. Am. J. Physiol. Cell Physiol. 298, C324–C332 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Jacobs, S., Calebiro, D., Nikolaev, V.O., Lohse, M.J. & Schulz, S. Real-time monitoring of somatostatin receptor-cAMP signaling in live pituitary. Endocrinology 151, 4560–4565 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Aoki, K. & Matsuda, M. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat. Protoc. 4, 1623–1631 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Palmer, A.E. & Tsien, R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1, 1057–1065 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Werthmann, R.C., von Hayn, K., Nikolaev, V.O., Lohse, M.J. & Bünemann, M. Real-time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6. J. Physiol. 587, 4091–4104 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Nikolaev, V.O., Gambaryan, S., Engelhardt, S., Walter, U. & Lohse, M.J. Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis. J. Biol. Chem. 280, 1716–1719 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Lissandron, V. et al. Transgenic fruit-flies expressing a FRET-based sensor for in vivo imaging of cAMP dynamics. Cell Signal. 19, 2296–2303 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Shafer, O.T. et al. Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron 58, 223–237 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Di Benedetto, G. et al. Protein kinase A type I and type II define distinct intracellular signaling compartments. Circ. Res. 103, 836–844 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Willemse, M., Janssen, E., de Lange, F., Wieringa, B. & Fransen, J. ATP and FRET—a cautionary note. Nat. Biotechnol. 25, 170–172 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Wachten, S. et al. Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells. J. Cell Sci. 123, 95–106 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Kawasaki, H. et al. A family of cAMP-binding proteins that directly activate Rap1. Science 282, 2275–2279 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Mei, F.C. et al. Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation. J. Biol. Chem. 277, 11497–11504 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Bartsch, M. et al. Bioactivatable, membrane-permeant analogs of cyclic nucleotides as biological tools for growth control of C6 glioma cells. Biol. Chem. 384, 1321–1326 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Schultz, C. et al. Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP. J. Biol. Chem. 268, 6316–6322 (1993).

    CAS  PubMed  Google Scholar 

  40. Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The work in authors' laboratories is supported by the Deutsche Forschungsgemeinschaft (grants SFB487 and SFB688 to M.J.L.; grant NI 1301/1-1 to V.O.N.), European Research Council (to M.J.L.) and University of Göttingen Medical Center ('pro futura' grant to V.O.N.). We thank H.-G. Genieser for critical reading of the manuscript.

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Authors and Affiliations

Authors

Contributions

S.B., F.S., F.B. and V.O.N. performed experiments and analyzed the data. A.S. and D.C. prepared primary cardiomyocytes and thyroid cells. F.S., M.J.L. and V.O.N. developed the protocol. S.B. and V.O.N. wrote the paper.

Corresponding author

Correspondence to Viacheslav O Nikolaev.

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Competing interests

F.S. is an employee and a shareholder of the Biolog Life Science Institute, a company that sells chemicals including cAMP analogs.

Supplementary information

Supplementary Figure 1

Experiments aimed to determine the concentration of MDL-12,330A which is capable of fully inhibiting adenylyl cyclase activity in cells and which can be used to reliably measure Rmin values. (PDF 56 kb)

(a) Concentration-response dependence of MDL-12,330A effect on the basal FRET ratio in cardiomyocytes shows that 100 µM of this compound can fully inhibit the sensor, and application of higher inhibitor concentrations does not lead to lower Rmin values (means ± SEM, n=7), which was also true for thyroid cells (not shown). (b) In cells with low basal adenylyl cyclase activity, inhibitory effect of MDL-12,330A can be tested after stimulation of cAMP production, as shown here for 293A cells treated with the _-adrenergic receptor agonist isoproterenol. 100 µM of MDL-12,330A were also sufficient to fully block the stimulated adenylyl cyclase activity in these cells. A grey trace shows a control experiment where the buffer A was added instead of MDL-12,330A, which did not result in a rapid decrease of signal. Representative experiments, n=5.

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Börner, S., Schwede, F., Schlipp, A. et al. FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6, 427–438 (2011). https://doi.org/10.1038/nprot.2010.198

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