31 Landa, L. R., Jr. Harbeck, M. Kaihara, K. Chepurny, O. Kitiphongspattana, K. Graf, O. Nikolaev, V. O. Lohse, M. J. Holz, G. G. Roe, M. W. 2005 J Biol Chem 280 35 31294-302 Sep 2 0021-9258 (Print) 15987680 Hypoglycemic Agents/metabolism Guanine Nucleotide Exchange Factors/genetics/metabolism Glucose/metabolism Fluorescent Dyes/metabolism Fluorescence Resonance Energy Transfer Cyclic AMP/analogs & derivatives/*metabolism Cell Line Calcium/*metabolism Animals Tolbutamide/metabolism Tetraethylammonium/metabolism Second Messenger Systems/physiology Recombinant Fusion Proteins/genetics/metabolism Potassium Chloride/metabolism Mice *Islets of Langerhans/cytology/metabolism Insulin/*metabolism 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. DK45817/DK/NIDDK NIH HHS/United StatesDK63493/DK/NIDDK NIH HHS/United StatesDK64162/DK/NIDDK NIH HHS/United StatesDK68822/DK/NIDDK NIH HHS/United StatesJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.United States http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15987680