Single cell Ca 2 + / cAMP cross - talk monitored by simultaneous Ca 2 + / cAMP fluorescence ratio imaging ( FICRhR / fura - 2 AM /

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2 , were simultaneously monitored in living cells by digital fluorescence ratio imaging using FICRhR, a single-excitation dualemission cAMP indicator, and fura-2, a dual-excitation singleemission Ca2+ probe. In single C6-2B glioma cells, isoproterenolor forskolin-evoked cAMP accumulation (measured in vivo as an increased FICRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level. cAMP concentration ([cAMP]i) to be continuously monitored in space and time in living cells microinjected with the probe (12-19). Given the physiological significance of the Ca2 /cAMP interplay, the potential ability to (i) simultaneously and continuously follow the single cell changes ofcAMP and Ca2' and (ii) identify any putative subcellular compartmentalization of these important regulatory molecules would certainly help gain insights into the biochemical mechanisms underlying their dynamic interactions. As a first step toward this novel in vivo approach, we successfully monitored Ca2+ and cAMP kinetics in the same cells by dual-excitation dual-emission fluorescence ratio imaging using fura-2 and FICRhR. For this study, C6-2B cells and REF-52 fibroblasts were chosen as model systems by virtue of the differential Ca2+ regulation of the cAMP pathway, the latter cell line displaying Ca2+-insensitive cAMP accumulation. Ca2l and cAMP are important second messengers that regulate a myriad of cell functions. In addition, there are numerous examples where regulatory interactions occur between them. Particularly interesting for their physiological relevance are the recent findings that (i) Ca2+ inhibits catecholamine-stimulated cAMP production and adenylyl cyclase activity in cardiac myocytes (1, 2) and (ii) a Ca2+-inhibited adenylyl cyclase (type V) is the predominant adenylyl cyclase isoform expressed in cardiac tissue (3, 4). These observations, together with the long-standing evidence that cAMP levels oscillate during the myocardial contraction cycle (5), have prompted the suggestion that, in heart, Ca2+/cAMP reciprocal effect (cAMP modulating Ca2+ homeostasis and Ca2+-inhibiting cAMP synthesis) could be the key mechanism for regulating cardiac rhythmicity and contractility (6). We have been specifically interested in the interplay between Ca2+ and cAMP in rat C6-2B glioma cells that, similar to heart cells, are highly responsive to catecholamines (7) and almost exclusively express type VI adenylyl cyclase (8) that is inhibited by Ca2+ in the low micromolar range (9). Thege biochemical features make C6-2B cells an ideal model for studying the Ca2+ regulation of hormone-stimulated cAMP accumulation (8-11) because virtually all cAMP produced, being synthesized by a Ca2+-inhibitable adenylyl cyclase, is under the potential control of Ca2+-mobilizing signals. While a number of imaging probes for Ca2+ exist, the recent introduction of FlCRhR, the first cAMP fluorescent probe (12), made it feasible to consider simultaneous imaging of both second messengers in the same living cell. FICRhR is a single-excitation dual-emission dye whose emission spectrum changes upon cAMP binding, allowing the intracellular free MATERIALS AND METHODS cAMP and Ca2+ Digital Fluorescence Ratio Imaging. Cells were grown on 25-mm round glass coverslips in Ham's F-10 nutrient mixture (C6-2B glioma cells) or Eagle's minimum essential medium (REF-52 fibroblasts; provided by R. Y. Tsien, University of California at San Diego, La Jolla) plus 10% calf serum, at 37°C in the presence of 95% air/5% CO2. FICRhR (recombinant fluoresceinand rhodamine-labeled cAMP-dependent protein kinase A; ref. 12) was directly microinjected (20 ,uM average stock solutions) into cells using an Eppendorf 5171 micromanipulator and 5246 transinjector. The final intracellular concentrations of FICRhR were between 0.2 and 2 ,uM, considering an injection volume of 1-10% of the cell volume (14). Cells were then loaded with 5 ,uM fura-2AM at room temperature for 30 min in Ham's F-10 medium supplemented with 20 mM Na/Hepes (pH 7.4), washed, and imaged in the same medium at 22°C. Drugs were diluted from stock solutions and added to the cells in serumfree Ham's F-10 medium containing 20 mM Na/Hepes (pH 7.4) and, in some experiments not shown, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (100 ,uM) and Ro20-1724 (100 ,uM) to prevent cAMP breakdown. Simultaneous imaging of Ca2+ and cAMP was accomplished by a combination of dual-excitation (fura-2) and dual-emission (FlCRhR) ratio imaging using the Zeiss Attofluor RatioVision workstation (Atto Instruments, Rockville, MD). This system includes a Zeiss Axiovert 135 microscope equipped for epifluorescence with a 510-nm dichroic mirror, a Fluar X40 1.3 na-oil immersion objective, and dual-emission ICCD cameras. Excitation filters (10 nm bandpass filters of 334, 380, and 488 Abbreviations: [Ca2+]i, cytosolic free Ca2+ concentration; [cAMP]i, intracellular free cAMP concentration; ISO, isoproterenol, FO, forskolin; TG, thapsigargin; IONO, ionomycin. *To whom reprint requests should be addressed. 4577 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4578 Cell Biology: DeBernardi and Brooker nm) located in the filter changer were automatically and alternately placed into the excitation light path while proper emission wavelengths were monitored with emission filters (510 to 530 nm bandpass and 570 nm long-pass) inserted in the respective ICCD cameras. Dual-emission ICCD cameras were aligned within 1 pixel of one another before each days experiments by using a fluorescent mixture of fluorescein and rhodamine to mimic the two emission wavelengths generated by FICRhR. Ca2+ and cAMP were simultaneously imaged by (i) exciting fura-2 at 334 and 380 nm with its emission monitored at 510-530 nm, and (ii) exciting FICRhR at 488 nm with the emission signals monitored at 510-530 nm and >570 nm. The 334/380 nm excitation ratio for fura-2 increases as a function of the cytosolic free Ca2+ concentration ([Ca2+],) whereas the FICRhR 510-530/>570 nm emission ratio (referred to as 520/580 nm emission ratio) increases upon intracellular free cAMP concentration ([cAMP]j) elevation. When microinjected into the cytoplasm of cells, FICRhR, due to its high molecular weight (172 kDa), does not enter the nucleus. Therefore, regions of interest that would be followed during the experiment were chosen within the cytoplasmic compartment of cells microinjected with the cAMP probe. However, similar to the native protein kinase A, FICRhR dissociates upon cAMP binding and the fluorescein-labeled catalytic subunit slowly migrates to the nucleus while the rhodamine-labeled regulatory subunit remains in the cytoplasm (12). In each cell being imaged, the regions of interest for fura-2 either were the same as for FICRhR or covered a larger area of the cell including the nucleus. Changes in [cAMP]i and [Ca2+]1 within the same cells were simultaneously monitored,with FICRhR being excited generally once every 30-60 sec and fura-2 every 5-10 sec. In vitro calibration for fura-2 was performed as described (20). Materials. FICRhR (cAMP Fluorosensor) was from Atto Instruments; fura-2AM, fura-2 pentapotassium salt, fluorescein, and rhodamine were from Molecular Probes; thapsigargin was from Research Biochemicals (Natick, MA); Ro2O1724 was from Biomol (Plymouth Meeting, PA). Cell culture products and all other drugs were from Sigma. RESULTS AND DISCUSSION Simultaneous cAMP/Ca2+ Imaging Feasibility Studies. The feasibility of cAMP/Ca2+ simultaneous fluorescence imaging in single cells was tested in situ by comparing the response of FICRhR and fura-2 to agents known to increase [cAMP]j and [Ca2+]i, respectively, in C6-2B cells labeled with either FICRhR or fura-2 versus cells colabeled with both dyes. As shown in Fig. 1 A and B, exposure of the cells to the synthetic catecholamine, isoproterenol (ISO), resulted in an increase in the FICRhR 520/580 nm emission ratio (reflecting increased cAMP synthesis) whose extent was virtually identical in cells labeled with FICRhR only (Fig. 1A) and in cells colabeled with FICRhR and fura-2 (Fig. 1B). Likewise, application of the Ca2+ ionophore, ionomycin (IONO), evoked an increase in fura-2 334/380 nm excitation ratio, reflecting a rise in [Ca2+]j, which was comparable between cells labeled with fura-2 only (Fig. 1C) and cells colabeled with both fura-2 and FICRhR (Fig. 1D). Therefore, this first set of experiments demonstrates that the detection of the fluorescent signal generated by either FICRhR or fura-2 is not appreciably affected by the simultaneous presence of both dyes in the same cell. We then investigated whether an increase in fura-2/FlCRhR excitation/emission ratio upon activation of the Ca2+/cAMP pathway would affect the response of either dye to a concomitant or subsequent challenge. Because Ca2+ is known to