Uppsala university: Faculty of Medicine: Department of medical cell biology: Research: Erik Gylfe: Spatio-temporal dynamics of cAMP signals
Spatio-temporal dynamics of cAMP signals
Cyclic AMP is a prototype second messenger that transduces signals from a variety of cell surface receptors to multiple intracellular targets. In pancreatic beta cells, cAMP strongly enhances insulin secretion by potentiating Ca2+-dependent exocytosis. cAMP formation is catalyzed by adenylyl cyclases and the degradation mediated by phosphodiesterases. Protein kinase A (PKA) and cAMP-dependent guanine nucleotide exchange factors are the major cAMP effectors in beta cells. Little is known about the kinetics of cAMP signals. The lack of information stems from the difficulty to measure cAMP in individual living cells. We have recently developed a method that allows recording of cAMP concentration changes in the sub-plasma membrane space of individual cells. The technique is based on fluorescent protein-tagged PKA subunits, modified so that the catalytic subunit undergoes translocation to or from the plasma membrane upon changes in cAMP concentration. Fluorescence is selectively detected from a small volume adjacent to the membrane using evanescent wave microscopy. This approach allowed us to demonstrate that stimulation of beta cells with glucagon and glucagon-like peptide-1 (GLP-1) often triggered cAMP oscillations. We have also shown that different temporal patterns of cAMP signals could contribute to selective regulation of downstream events. Brief elevations of cAMP were sufficient to trigger Ca2+ spikes, but only prolonged cAMP elevation induced PKA translocation into the nucleus. The aim of ongoing work is to understand how the concentration of cAMP is regulated in beta cells by nutrients, hormones and neurotransmitters, and how the spatio-temporal pattern of the messenger is involved to control beta cell function.
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This experiments demonstrates sequential increases of cAMP in individual insulin-secreting beta cells after inhibition of phosphodiesterases with IBMX and activation of adenylyl cyclases with forskolin. cAMP was monitored by measuring fluorescence in the submembrane space by evanescent-wave-microscopy. The blue fluorescence comes from cyan fluorescent protein fused to the regulatory subunit of PKA. Yellow fluorescent protein was fused with the catalytic subunit of PKA. Rise of cAMP triggers dissociation of the regulatory and catalytic subunits. Since the catalytic subunit was anchored to the plasma membrane the blue fluorescence remains membrane-associated whereas there is a loss of yellow florescence as the catalytic subunit diffuses into the cytoplasm. The black trace shows the plasma membrane-associated blue/yellow fluorescence ratio as a measure of cAMP.
Publications
Dyachok D, Idevall-Hagren O, Sågetorp J, Tian G, Wuttke A, Arrieumerlou C, Akusjärvi G, Gylfe E, Tengholm A. 2008. Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion. Cell Metab 8:26-37
Tengholm A, Gylfe E. 2009. Oscillatory control of insulin secretion. Mol Cell Endocrinol 15;297(1-2):58-72..
Tengholm A. 2007. Cyclic AMP: Swing that message! Cell Mol Life Sci 64:382-385.
Dyachok O, Isakov Y, Sågetorp J, Tengholm A. 2006. Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells. Nature 439:349-52.
Dyachok O, Sågetorp J, Isakov Y, Tengholm A. 2006. cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells. Biochem Soc Trans 34:498-501.
For further information contact:
Anders.Tengholm@mcb.uu.se
©2003 Department of Medical Cell Biology | Last updated: 2006-11-08
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