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J. Biol. Chem. 280 (35): 31294-31302

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 {beta}-Cell Line*

Luis R. Landa, Jr.{ddagger}§, Mark Harbeck{ddagger}§, Kelly Kaihara{ddagger}, Oleg Chepurny¶, Kajorn Kitiphongspattana{ddagger}, Oliver Graf{ddagger}, Viacheslav O. Nikolaev||, Martin J. Lohse||, George G. Holz¶, , and Michael W. Roe{ddagger}**

{ddagger}Department of Medicine, The University of Chicago, Chicago, Illinois 60637, the Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York 10016, and the ||Institute of Pharmacology and Toxicology, University of Würzburg, D-97078 Würzburg, Germany

Abstract: Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

Received for publication May 24, 2005. Revision received June 24, 2005.

* This work was supported by research grants from the American Diabetes Association (to G. G. H. and M. W. R.) and by National Institutes of Health Grant DK45817 (to G. G. H.) and Grants DK63493, DK64162, and DK68822 (to M. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at contains supplemental Fig. S1 and Table 1.

§ Both authors contributed equally to this work.

** To whom correspondence should be addressed: Dept. of Medicine MC-1027, The University of Chicago, 5841 South Maryland Ave., Chicago, IL 60637. Tel.: 773-702-4965; Fax: 773-834-0486; E-mail: mroe{at}

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