Ca and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca -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 Ca 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 Ca and inhibited by nitrendipine, a Ca 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 Ca and cAMP oscillations that were abolished in the absence of extracellular Ca and blocked by 2 ,5 -dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca and cAMP signaling in electrically excitable endocrine cells and suggest that Ca -dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase. Ca and cAMP are ubiquitous second messengers that regulate many cellular functions, including gene expression, protein biosynthesis, and exocytosis. In eukaryote cells, Ca and cAMP signaling cascades are interconnected and often exhibit subcellular concentration gradients that are spatially and temporally heterogeneous (1–4). Intracellular cAMP concentration can be affected by activation of Ca -sensitive isoforms of adenylate cyclase (5–7) and phosphodiesterase (8, 9). Conversely, cAMP and protein kinase A modulation of Ca channels and plasma membrane Ca -ATPases influences Ca signal transduction (4, 10–14). The way in which these dynamic signals are integrated in single cells remains unresolved. Only two studies have reported measurements of both second messenger signaling pathways simultaneously in single cells (15, 16). Concurrent imaging of cAMP and Ca in C6-2B glioma cells and REF-52 fibroblasts loaded with FlCRhR, a recombinant fluorescence resonance energy transfer (FRET)-based cAMP indicator, and Fura-2 revealed that the increase in intracellular Ca following application of thapsigargin and ionomycin causes a decrease in isoproterenol-stimulated cAMP accumulation in C6-2B glioma cells, but not in REF-52 fibroblasts (15). In Rana esculenta ventricular cells, simultaneous detection of cAMP with FlCRhR and whole cell L-type Ca current by patch-clamp electrophysiology indicated that increases in cAMP production activate Ca channel conductance (16). Spontaneous Ca and cAMP increases have been observed in embryonic Xenopus neurons loaded with Fluo-4, a fluorescent Ca indicator, and FlCRhR (17). The cells generate three to four pulses of cAMP/h, each lasting 3–7 min. cAMP accumulation appears to affect Ca spiking; the frequency of spontaneous Ca transients (3–10/h) is increased by application of forskolin, an activator of adenylate cyclase (AC), and decreased by inhibition of protein kinase A with KT5720. Stimulation of neurons with pulses (12/min) of forskolin and 3-isobutyl-1-methylxanthine (IBMX) also increases Ca spike frequency. On the other hand, elimination of the spontaneous Ca transients inhibits cAMP production, whereas transient Ca spikes evoked by pulses of 100 mM KCl generate cAMP transients in embryonic Xenopus neurons. Although these elegant studies suggested a role for AC and protein kinase A in mediating the cAMP-induced effects on Ca signaling and confirmed that the formation of cAMP is Ca -dependent, the temporal interrelationships were * 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. □S The on-line version of this article (available at http://www.jbc.org) 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@ medicine.bsd.uchicago.edu. 1 The abbreviations used are: FRET, fluorescence resonance energy transfer; AC, adenylate cyclase; IBMX, 3-isobutyl-1-methylxanthine; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; [cAMP]c, cytoplasmic cAMP concentration; [Ca 2 ]c, cytoplasmic Ca concentration; DDA, 2 ,5 -dideoxyadenosine; PDE, phosphodiesterase; TEA, tetraethylammonium chloride. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 35, Issue of September 2, pp. 31294–31302, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.