Cyclic AMP is a ubiquitous intracellular second messenger that transmits information to several proteins including cyclic nucleotide-gated ion channels and protein kinase A (PKA). In turn, these effectors regulate such diverse cellular functions as Ca2+ influx, excitability, and gene expression, as well as cell-specific processes such as glycogenolysis and lipolysis. The enzymes known to regulate cAMP levels, adenylyl cyclase and phosphodiesterase, have been studied in detail. Unfortunately, an understanding of how information is encoded within cAMP signals has been elusive, because, until recently, methods for measuring cAMP lacked both spatial and temporal resolution. In this paper, we describe two recently developed methods for detecting cAMP levels in living cells. The first method measures fluorescence energy transfer between labeled subunits of PKA. This method is particularly useful for monitoring cellular localization of PKA activity following increases in cAMP levels. However, the slow activation and deactivation rates, the necessarily high concentrations of labeled subunits, and the redistribution of labeled subunits throughout the cell, all intrinsic to this method, limit its utility as a cAMP sensor. The second method uses genetically modified cyclic nucleotide-gated channels to measure plasma membrane-localized cAMP levels in either cell populations or single cells. The rapid gating kinetics of these channels allow real-time measurement of cAMP concentrations. These methods have given us the first glimpses of cAMP signals within living cells. © 2002 Biomedical Engineering Society.
PAC2002: 8716Uv, 8780-y, 8716Sr
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Adams, S., B. Bacskai, A. T. Harootunian, M. Mahaut-Smith, P. J. Sammak, S. S. Taylor, and R. Y. Tsien. Imaging of cAMP signals and A-kinase translocation in single living cells. Adv. Second Messenger Phosphoprotein Res. 28:167-170, 1993.
Adams, S. R., A. T. Harootunian, Y. J. Buechler, S. S. Taylor, and R. Y. Tsien. Fluorescence ratio imaging of cyclic AMP in single cells. Nature (London) 349:694-697, 1991.
Allen, D. G., D. A. Eisner, and C. H. Orchard. Characterization of oscillations of intracellular calcium concentration in ferret ventricular muscle. J. Physiol. 352:113-128, 1984.
Bacskai, B. J., B. Hochner, M. Mahaut-Smith, S. R. Adams, B. K. Kaang, E. R. Kandel, and R. Y. Tsien. Spatially resolved dynamics of cAMP and protein kinase A subunits in aplysia sensory neurons. Science 260:222-226, 1993.
Beavo, J. A. Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiol. Rev. 75:725-748, 1995.
Beavo, J. A., P. J. Bechtel, and E. G. Krebs. Activation of protein kinase by physiological concentrations of cyclic AMP. Proc. Natl. Acad. Sci. U.S.A. 71:3580-3583, 1974.
Berridge, M. J., P. H. Cobbold, and K. S. Cuthbertson. Spatial and temporal aspects of cell signalling. Philos. Trans. R. Soc. London, Ser. B 320:325-343, 1988.
Brooker, G. Oscillation of cyclic adenosine monophosphate concentration during the myocardial contraction cycle. Science 182:933-934, 1973.
Brostrom, C. O., J. D. Corbin, C. A. King, and E. G. Krebs. Interaction of the subunits of adenosine 38:58-cyclic monophosphate-dependent protein kinase of muscle. Proc. Natl. Acad. Sci. U.S.A. 68:2444-2447, 1971.
Brown, R. L., R. Gramling, R. J. Bert, and J. W. Karpen. Cyclic GMP contact points within the 63 kDa subunit and a 240 kDa associated protein of retinal rod cGMP-activated channels. Biochemistry 34:8365-8370, 1995.
Brown, R. L., S. D. Snow, and T. L. Haley. Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels. Biophys. J. 75:825-833, 1998.
Brunton, L. L., J. S. Hayes, and S. E. Mayer. Functional compartmentation of cAMP and protein kinase in heart. Adv. Cyclic Nucleotide Res. 14:391-397, 1981.
Chen-Izu, Y., R. P. Xiao, L. T. Izu, H. Cheng, M. Kuschel, H. Spurgeon, and E. G. Lakatta. G i-dependent localization of ? 2-adrenergic receptor signaling to L-type Ca2+ channels. Biophys. J. 79:2547-2556, 2000.
Cooper, D. M. F., N. Mons, and J. W. Karpen. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature (London) 374:421-424, 1995.
Cuthbertson, K. S., D. G. Whittingham, and P. H. Cobbold. Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature (London) 294:754-757, 1981.
Davare, M. A., V. Avdonin, D. D. Hall, E. M. Peden, A. Burette, R. J. Weinberg, M. C. Horne, T. Hoshi, and J. W. Hell. A ? 2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science 293:98-101, 2001.
De Koninck, P., and H. Schulman. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279:227-230, 1998.
de Rooij, J., F. J. Zwartkruis, M. H. Verheijen, R. H. Cool, S. M. Nijman, A. Wittinghofer, and J. L. Bos. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature (London) 396:474-477, 1998.
DeBernardi, M. A., and G. Brooker. Single cell Ca2+/cAMP cross talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging. Proc. Natl. Acad. Sci. U.S.A. 93:4577-4582, 1996.
Detlev, S., and D. Restrepo. Transduction mechanisms in vertebrate olfactory receptor cells. Physiol. Rev. 78:429-466, 1998.
Dhallan, R. S., K.-W. Yau, K. A. Schrader, and R. R. Reed. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature (London) 347:184-187, 1990.
DiFrancesco, D. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55:455-472, 1993.
Dolmetsch, R. E., K. Xu, and R. S. Lewis. Calcium oscillations increase the efficiency and specificity of gene expression. Nature (London) 392:933-936, 1998.
Doskeland, S. O., and D. Ogreid. Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I. J. Biol. Chem. 259:2291-2301, 1984.
Fagan, K. A., T. C. Rich, S. Tolman, J. Schaack, J. W. Karpen, and D. M. F. Cooper. Adenovirus-mediated expression of an olfactory cyclic nucleotide-gated channel regulates the endogenous Ca2+-inhibitable adenylyl cyclase in C6-2B glioma cells. J. Biol. Chem. 274:12445-12453, 1999.
Fagan, K. A., J. Schaack, A. Zweifach, and D. M. F. Cooper. Adenovirus encoded cyclic nucleotide-gated channels: A new methodology for monitoring cAMP in living cells. FEBS Lett. 500:85-90, 2001.
Fesenko, E. E., S. S. Kolesnikov, and A. L. Lyubarsky. Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature (London) 313:310-313, 1985.
Fewtrell, C. Ca2+ oscillations in nonexcitable cells. Annu. Rev. Physiol. 55:427-454, 1993.
Finn, J. T., M. E. Grunwald, and K.-W. Yau. Cyclic nucleotide-gated ion channels: An extended family with diverse functions. Annu. Rev. Physiol. 58:395-426, 1996.
Francis, S., and J. D. Corbin. Cyclic nucleotide-dependent protein kinases: Intracellular receptors for cAMP and cGMP action. Crit. Rev. Clin. Lab Sci. 36:275-328, 1999.
Goaillard, J. M., P. V. Vincent, and R. Fischmeister. Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J. Physiol. 530:79-91, 2001.
Gorbunova, Y. V., and N. C. Spitzer. Dynamic interactions of cyclic AMP transients and spontaneous Ca2+ spikes. Nature (London) 418:93-96, 2002.
Gordon, S. E., M. D. Varnum, and W. N. Zagotta. Direct interaction between amino-and carboxyl-terminal domains of cyclic nucleotide-gated channels. Neuron 19:431-441, 1997.
Gray, P. C., J. D. Scott, and W. A. Catterall. Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins. Curr. Opin. Neurobiol. 8:330-334, 1998.
Hagen, V., C. Dzeja, S. Frings, J. Bendig, E. Krause, and U. B. Kaupp. Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: Synthesis and application to cyclic nucleotide-gated channels. Biochemistry 35:7762-7771, 1996.
Hall, D. D., and J. W. Hell. The fourth dimension in cellular signaling-Response. Science 293:2205, 2001.
Hanson, P. I., T. Meyer, L. Stryer, and H. Schulman. Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron 12:943-956, 1994.
Harootunian, A. T., S. R. Adams, W. Wen, J. L. Meinkoth, S. S. Taylor, and R. Y. Tsien. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. Mol. Biol. Cell 4:993-1002, 1993.
Hempel, C. M., P. Vincent, S. R. Adams, R. Y. Tsien, and A. I. Selverston. Spatiotemporal dynamics of cyclic AMP signals in an intact neural circuit. Nature (London) 384:166-169, 1996.
Hofmann, F., P. J. Bechtel, and E. G. Krebs. Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues. J. Biol. Chem. 252:1441-1447, 1977.
Houge, G., R. A. Steinberg, D. Ogreid, and S. O. Doskeland. The rate of recombination of the subunits (RI and C) of cAMP-dependent protein kinase depends on whether one or two cAMP molecules are bound per RI monomer. J. Biol. Chem. 265:19507-19516, 1990.
Jurevicius, J., and R. Fischmeister. cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by b-adrenergic agonists. Proc. Natl. Acad. Sci. U.S.A. 93:295-299, 1996.
Karpen, J. W., and T. C. Rich. The fourth dimension in cellular signaling. Science 293:2204-2205, 2001.
Karpen, J. W., A. L. Zimmerman, L. Stryer, and D. A. Baylor. Gating kinetics of the cyclic-GMP-activated channel of retinal rods: Flash photolysis and voltage-jump studies. Proc. Natl. Acad. Sci. U.S.A. 85:1287-1291, 1988.
Kurahashi, T., and A. Kaneko. Gating properties of the cAMP-gated channel in toad olfactory receptor cells. J. Physiol. 466:287-302, 1993.
Li, J., and H. A. Lester. Single-channel kinetics of the rat olfactory cyclic nucleotide-gated channel expressed in Xenopus oocytes. Mol. Pharmacol. 55:883-893, 1999.
Liu, M., T. Y. Chen, B. Ahamed, J. Li, and K.-W. Yau. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science 266:1348-1354, 1994.
Lowe, G., and G. H. Gold. Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells. Nature (London) 366:283-286, 1993.
Mehats, C., C. B. Andersen, M. Filopanti, S. L. Jin, and M. Conti. Cyclic nucleotide phosphodiesterases and their role in endocrine cell signaling. TRENDS Endocrinol. Metab. 13:29-35, 2002.
Molday, R. S. Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases: The Friedenwald Lecture. Invest. Ophthalmol. Visual Sci. 39:2493-2513, 1998.
Mons, N., A. Harry, P. Dubourg, R. T. Premont, R. Iyengar, and D. M. F. Cooper. Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses. Proc. Natl. Acad. Sci. U.S.A. 92:8473-8477, 1995.
Nakamura, T., and G. H. Gold. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature (London) 325:442-444, 1987.
O'Dea, R. F., M. K. Haddox, and N. D. Goldberg. Interaction with phosphodiesterase of free and kinase-complexed cyclic adenosine 38,58-monophosphate. J. Biol. Chem. 246:6183-6190, 1971.
Ogreid, D., and S. O. Doskeland. Cyclic nucleotides modulate the release of [3H] adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I by the catalytic subunit of the kinase. Biochemistry 22:1686-1696, 1983.
Ogura, A., T. Iijima, T. Amano, and Y. Kudo. Optical monitoring of excitatory synaptic activity between cultured hippocampal neurons by a multisite Ca2+ fluorometry. Neurosci. Lett. 78:69-74, 1987.
Orchard, C. H., D. A. Eisner, and D. G. Allen. Oscillations of intracellular Ca2+ in mammalian cardiac muscle. Nature (London) 304:735-738, 1983.
Pugh, Jr., E. N. Transfected cyclic nucleotide-gated channels as biosensors. J. Gen. Physiol. 116:143-145, 2000.
Rapp, P. E., and M. J. Berridge. Oscillations in calciumcyclic AMP control loops form the basis of pacemaker activity and other high frequency biological rhythms. J. Theor. Biol. 66:497-525, 1977.
Reisert, J., and H. R. Matthews. Responses to prolonged odour stimulation in frog olfactory receptor cells. J. Physiol. 534:179-191, 2001.
Reisert, J., and H. R. Matthews. Simultaneous recording of receptor current and intraciliary Ca2+ concentration in salamander olfactory receptor cells. J. Physiol. 535:637-645, 2001.
Rich, T. C., K. A. Fagan, H. Nakata, J. Schaack, D. M. F. Cooper, and J. W. Karpen. Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J. Gen. Physiol. 116:147-161, 2000.
Rich, T. C., K. A. Fagan, T. E. Tse, J. Schaack, D. M. F. Cooper, and J. W. Karpen. A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc. Natl. Acad. Sci. U.S.A. 98:13049-13054, 2001.
Rich, T. C., T. E. Tse, J. G. Rohan, J. Schaack, and J. W. Karpen. In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J. Gen. Physiol. 118:63-77, 2001.
Roos, W., C. Scheidegger, and G. Gerisch. Adenylate cyclase activity oscillations as signals for cell aggregation in Dictyostelium discoideum. Nature (London) 266:259-261, 1977.
Schwartz, J. H. The many dimensions of cAMP signaling. Proc. Natl. Acad. Sci. U.S.A. 98:13482-13484, 2001.
Smith, S. B., H. D. White, J. B. Siegel, and E. G. Krebs. Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits. Proc. Natl. Acad. Sci. U.S.A. 78:1591-1595, 1981.
Steinberg, S. F., and L. L. Brunton. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. 41:751-773, 2001.
Stojilkovic, S. S., and K. J. Catt. Calcium oscillations in anterior pituitary cells. Endocr. Rev. 13:256-280, 1992.
Sunahara, R. K., C. W. Dessauer, and A. G. Gilman. Complexity and diversity of mammalian adenylyl cyclases. Annu. Rev. Pharmacol. Toxicol. 36:461-480, 1996.
Sutherland, E. W. Studies on the mechanism of hormone action. Science 177:401-408, 1972.
Trivedi, B., and R. H. Kramer. Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists. Neuron 21:895-906, 1998.
Tsien, R. Y., T. Pozzan, and T. J. Rink. T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature (London) 295:68-71, 1982.
Varnum, M. D., K. D. Black, and W. N. Zagotta. Molecular mechanism for ligand discrimination of cyclic nucleotidegated channels. Neuron 15:619-625, 1995.
Walsh, D. A., and S. M. Van Patten. Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 8:1227-1236, 1994.
Yau, K.-W. Phototransduction mechanism in retinal rods and cones: The Friedenwald lecture. Invest. Ophthalmol. Visual Sci. 35:9-32, 1994.
Zaccolo, M., F. De Giorgi, C. Y. Cho, L. Feng, T. Knapp, P. A. Negulescu, S. S. Taylor, R. Y. Tsien, and T. Pozzan. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2:25-29, 2000.
Zaccolo, M., and T. Pozzan. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295:1711-1715, 2002.
Zhang, J., Y. Ma, S. S. Taylor, and R. Y. Tsien. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl. Acad. Sci. U.S.A. 98:14997-15002, 2001.
Zong, X., H. Zucker, F. Hofmann, and M. Biel. Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels. EMBO J. 17:353-362, 1998.
About this article
Cite this article
Rich, T.C., Karpen, J.W. Review Article: Cyclic AMP Sensors in Living Cells: What Signals Can They Actually Measure?. Annals of Biomedical Engineering 30, 1088–1099 (2002). https://doi.org/10.1114/1.1511242
- G-protein signaling
- Cyclic nucleotide-gated channel
- Protein kinase A
- Adenylyl cyclase
- Second messengers
- cAMP signals