Proc. Natl. Acad. Sci. USAVol. 93, pp. 4577-4582, May 1996Cell Biology

Single cell Ca2+/cAMP cross-talk monitored by simultaneousCa2+/cAMP fluorescence ratio imaging

(FICRhR/fura-2AM/dual dye fluorescence imaging)

MARIA A. DEBERNARDI AND GARY BROOKER*Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, 3900 Reservoir Road N.W., Washington, DC 20007, and AttoInstruments, Inc., 1450 Research Boulevard, Rockville, MD 20850

Communicated by Lutz Birnbaumer, University of California, Los Angeles, School of Medicine, Los Angeles, CA, December 26, 1995 (received forreview November 21, 1995)

ABSTRACT The spatial and temporal dynamics of twointracellular second messengers, cAMP and Ca2 , were si-multaneously monitored in living cells by digital fluorescenceratio imaging using FICRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isopro-terenol- or forskolin-evoked cAMP accumulation (measuredin vivo as an increased FICRhR emission ratio) was reducedwhen cytosolic free Ca2+ concentration was elevated before,simultaneously with, or after cAMP activation. However, inREF-52 fibroblasts, Ca2+ neither prevented nor reducedforskolin-stimulated cAMP production. These results providenovel in vivo evidence for the Ca2+ modulation of the cAMPtransduction pathway in C6-2B cells. The simultaneous mi-croscopic measurement of cAMP and Ca2+ kinetics in singlecells makes it now possible to study the regulatory interactionsbetween these second messengers at the cellular and even thesubcellular level.

cAMP concentration ([cAMP]i) to be continuously monitoredin 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 con-tinuously follow the single cell changes ofcAMP and Ca2' and(ii) identify any putative subcellular compartmentalization ofthese important regulatory molecules would certainly helpgain insights into the biochemical mechanisms underlying theirdynamic interactions. As a first step toward this novel in vivoapproach, we successfully monitored Ca2+ and cAMP kineticsin the same cells by dual-excitation dual-emission fluorescenceratio imaging using fura-2 and FICRhR. For this study, C6-2Bcells and REF-52 fibroblasts were chosen as model systems byvirtue of the differential Ca2+ regulation of the cAMP path-way, the latter cell line displaying Ca2+-insensitive cAMPaccumulation.

Ca2l and cAMP are important second messengers that regu-late a myriad of cell functions. In addition, there are numerousexamples where regulatory interactions occur between them.Particularly interesting for their physiological relevance are therecent findings that (i) Ca2+ inhibits catecholamine-stimulatedcAMP production and adenylyl cyclase activity in cardiacmyocytes (1, 2) and (ii) a Ca2+-inhibited adenylyl cyclase (typeV) is the predominant adenylyl cyclase isoform expressed incardiac tissue (3, 4). These observations, together with thelong-standing evidence that cAMP levels oscillate during themyocardial contraction cycle (5), have prompted the sugges-tion that, in heart, Ca2+/cAMP reciprocal effect (cAMPmodulating Ca2+ homeostasis and Ca2+-inhibiting cAMP syn-thesis) could be the key mechanism for regulating cardiacrhythmicity and contractility (6). We have been specificallyinterested in the interplay between Ca2+ and cAMP in ratC6-2B glioma cells that, similar to heart cells, are highlyresponsive to catecholamines (7) and almost exclusively ex-press type VI adenylyl cyclase (8) that is inhibited by Ca2+ inthe low micromolar range (9). Thege biochemical featuresmake 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 aCa2+-inhibitable adenylyl cyclase, is under the potential con-trol 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 bothsecond messengers in the same living cell. FICRhR is asingle-excitation dual-emission dye whose emission spectrumchanges upon cAMP binding, allowing the intracellular free

MATERIALS AND METHODScAMP and Ca2+ Digital Fluorescence Ratio Imaging. Cells

were grown on 25-mm round glass coverslips in Ham's F-10nutrient mixture (C6-2B glioma cells) or Eagle's minimumessential medium (REF-52 fibroblasts; provided by R. Y.Tsien, University of California at San Diego, La Jolla) plus10% calf serum, at 37°C in the presence of 95% air/5% CO2.FICRhR (recombinant fluorescein- and rhodamine-labeledcAMP-dependent protein kinase A; ref. 12) was directlymicroinjected (20 ,uM average stock solutions) into cells usingan Eppendorf 5171 micromanipulator and 5246 transinjector.The final intracellular concentrations of FICRhR were be-tween 0.2 and 2 ,uM, considering an injection volume of 1-10%of the cell volume (14). Cells were then loaded with 5 ,uMfura-2AM at room temperature for 30 min in Ham's F-10medium supplemented with 20 mM Na/Hepes (pH 7.4),washed, and imaged in the same medium at 22°C. Drugs werediluted from stock solutions and added to the cells in serum-free Ham's F-10 medium containing 20 mM Na/Hepes (pH7.4) and, in some experiments not shown, the phosphodies-terase inhibitors 3-isobutyl-1-methylxanthine (100 ,uM) andRo20-1724 (100 ,uM) to prevent cAMP breakdown.

Simultaneous imaging of Ca2+ and cAMP was accomplishedby a combination of dual-excitation (fura-2) and dual-emission(FlCRhR) ratio imaging using the Zeiss Attofluor RatioVisionworkstation (Atto Instruments, Rockville, MD). This systemincludes a Zeiss Axiovert 135 microscope equipped for epi-fluorescence with a 510-nm dichroic mirror, a Fluar X40 1.3na-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, for-skolin; TG, thapsigargin; IONO, ionomycin.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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nm) located in the filter changer were automatically andalternately placed into the excitation light path while properemission wavelengths were monitored with emission filters(510 to 530 nm bandpass and 570 nm long-pass) inserted in therespective ICCD cameras. Dual-emission ICCD cameras werealigned within 1 pixel of one another before each days exper-iments by using a fluorescent mixture of fluorescein andrhodamine to mimic the two emission wavelengths generatedby FICRhR. Ca2+ and cAMP were simultaneously imaged by(i) exciting fura-2 at 334 and 380 nm with its emissionmonitored at 510-530 nm, and (ii) exciting FICRhR at 488 nmwith the emission signals monitored at 510-530 nm and >570nm. The 334/380 nm excitation ratio for fura-2 increases as afunction of the cytosolic free Ca2+ concentration ([Ca2+],)whereas the FICRhR 510-530/>570 nm emission ratio (re-ferred to as 520/580 nm emission ratio) increases uponintracellular free cAMP concentration ([cAMP]j) elevation.When microinjected into the cytoplasm of cells, FICRhR, dueto its high molecular weight (172 kDa), does not enter thenucleus. Therefore, regions of interest that would be followedduring the experiment were chosen within the cytoplasmiccompartment of cells microinjected with the cAMP probe.However, similar to the native protein kinase A, FICRhRdissociates upon cAMP binding and the fluorescein-labeledcatalytic subunit slowly migrates to the nucleus while therhodamine-labeled regulatory subunit remains in the cyto-plasm (12). In each cell being imaged, the regions of interestfor fura-2 either were the same as for FICRhR or covered alarger area of the cell including the nucleus. Changes in[cAMP]i and [Ca2+]1 within the same cells were simultaneouslymonitored,with FICRhR being excited generally once every30-60 sec and fura-2 every 5-10 sec. In vitro calibration forfura-2 was performed as described (20).

Materials. FICRhR (cAMP Fluorosensor) was from AttoInstruments; fura-2AM, fura-2 pentapotassium salt, fluores-cein, and rhodamine were from Molecular Probes; thapsigar-gin was from Research Biochemicals (Natick, MA); Ro2O-1724 was from Biomol (Plymouth Meeting, PA). Cell cultureproducts and all other drugs were from Sigma.

RESULTS AND DISCUSSIONSimultaneous cAMP/Ca2+ Imaging Feasibility Studies.

The feasibility of cAMP/Ca2+ simultaneous fluorescence im-aging in single cells was tested in situ by comparing theresponse of FICRhR and fura-2 to agents known to increase[cAMP]j and [Ca2+]i, respectively, in C6-2B cells labeledwith either FICRhR or fura-2 versus cells colabeled with bothdyes. As shown in Fig. 1 A and B, exposure of the cells to thesynthetic catecholamine, isoproterenol (ISO), resulted in anincrease in the FICRhR 520/580 nm emission ratio (reflectingincreased cAMP synthesis) whose extent was virtually identicalin cells labeled with FICRhR only (Fig. 1A) and in cellscolabeled with FICRhR and fura-2 (Fig. 1B). Likewise, appli-cation of the Ca2+ ionophore, ionomycin (IONO), evoked anincrease in fura-2 334/380 nm excitation ratio, reflecting a risein [Ca2+]j, which was comparable between cells labeled withfura-2 only (Fig. 1C) and cells colabeled with both fura-2 andFICRhR (Fig. 1D). Therefore, this first set of experimentsdemonstrates that the detection of the fluorescent signalgenerated by either FICRhR or fura-2 is not appreciablyaffected by the simultaneous presence of both dyes in the samecell.We then investigated whether an increase in fura-2/FlCRhR

excitation/emission ratio upon activation of the Ca2+/cAMPpathway would affect the response of either dye to a concom-itant or subsequent challenge. Because Ca2+ is known to

Time, sec Time, sec

FIG. 1. The detection of either FICRhR (cAMP) or fura-2 (Ca2+) fluorescent signal by digital imaging microscopy is unaffected by the presence ofboth dyes in the same cells. Single C6-2B cells were microinjected with FlCRhR (A) and imaged as described. After the baseline FICRhR fluorescenceemission ratio (indicating basal [cAMP]j) was recorded, the ,B-adrenergic receptor agonist ISO (10 ,uM) was applied to the cells to raise [cAMP]j. [cAMP]ielevation resulted in a 1.41-fold increase in FICRhR 520/580 nm emission ratio. When a parallel set of cells microinjected with the same batch of FlCRhRwere also labeled with fura-2 (B), ISO-induced cAMP accumulation resulted in a virtually identical (1.39-fold) increase in FICRhR 520/580 nm emissionratio. Likewise, the [Ca2+]i rise evoked by IONO (1 ,uM) elicited a 3.10- and 2.83-fold increase in fura-2 334/380 nm excitation ratio in cells labeled withfura-2 (C) and colabeled with both dyes (D), respectively. Although the extent of the increase in fura-2 excitation ratio in cells colabeled with FlCRhRand fura-2 was found to be slightly smaller than in cells labeled with fura-2 only, the kinetics of the Ca2+ responses were virtually identical in both cellpopulations. In this and following figures, the experimental results showing single cell [cAMP]i and [Ca2+]i are presented as population means (2-10 cellsbeing imaged per field) of FICRhR 520/580 nm emission ratio and fura-2 334/380 nm excitation ratio, respectively, plotted against time.

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inhibit cAMP synthesis in C6-2B cells (8-11), REF-52 fibro-blasts, whose agonist-stimulated cAMP production is notaffected by Ca2+ (unpublished data), were chosen to addressthis question. Addition of the adenylyl cyclase activator for-skolin (FO; 50 ,tM) to REF-52 cells colabeled with FICRhRand fura-2 stimulated new cAMP synthesis as indicated by anincreased 520/580 nm FICRhR emission ratio (Fig. 24). WhileFO-mediated increased FICRhR signal remained elevated, arise in [Ca2+]i evoked by the microsomal Ca2+-ATPase inhib-

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itor thapsigargin (TG; 1 ,uM) could be detected in the samecells as an expected fura-2 334/380 nm excitation ratio in-crease. Likewise, as shown in Fig. 2B, when [Ca2+]i was firstmaximally activated by IONO (5 ,M), a subsequent exposureof the cells to FO increased FICRhR emission ratio to anextent comparable to that detected when FO was added beforeany [Ca2+]i elevation. Finally, REF-52 fibroblasts were coin-cubated with TG (1 ,M) and FO (50 ,uM) and the kinetics ofboth [Ca2+]i and [cAMP]1 were monitored by following thechanges in the excitation and emission spectrum of fura-2 andFICRhR, respectively, at the same time and in the same cells(Fig. 2C). The transient increase in [Ca2+]i by TG was detectedas a 1.58-fold increase in fura-2 excitation ratio, whereasFO-stimulated new cAMP synthesis resulted in a long-lasting1.57-fold increase in FICRhR emission ratio. In control ex-periments (performed with the same batches of FICRhR andfura-2), FO and TG, added separately, had elicited a 1.47-foldincrease in FICRhR emission ratio and a 1.60-fold increase infura-2 excitation ratio, respectively. Therefore, a pharmaco-logically induced change in the emission/excitation spectrumof FlCRhR/fura-2 neither prevents nor impairs the detectionof a concomitant or subsequent change in the fluorescencesignal of either dye, as also shown in Fig. 3. Moreover, theseresults demonstrate that agonist-stimulated cAMP productionin single REF-52 fibroblasts is insensitive to elevated [Ca2+]ibeing neither reduced nor prevented by Ca2+-generating stim-uli, consistent with our recent in vitro finding that FO-stimulated REF-52 cell cAMP production (measured in cellextracts by radioimmunoassay) is not affected by TG-evoked[Ca2+1] increase (unpublished data).Ca2+/cAMP Cross-Talk in C6-2B Cells. Having established

that neither basal nor agonist-stimulated FICRhR and fura-2fluorescent signals appreciably affect each other's detection,

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FIG. 2. Time course of cAMP-induced FICRhR emission ratio andCa2+-evoked fura-2 excitation ratio in single REF-52 fibroblasts.Three separate sets of REF-52 fibroblasts were microinjected withFICRhR, loaded with fura-2, and imaged as described. After basalFICRhR 520/580 nm emission ratio (resting cAMP levels) and fura-2334/380 nm excitation ratio (resting Ca2+ levels) were recorded insingle fibroblasts, cells were treated (A) first with FO (50 ,uM) andsubsequently with TG (1 ,uM) or (B) first with IONO (5 ,uM) and thenwith FO (50 jiM). (C) Simultaneous addition of FO (50 ,uM) and TG(1 ,iM) to REF-52 fibroblasts evoked an immediate elevation in both[cAMP]i and [Ca2+]i, as reflected by a rapid increase in both FICRhRand fura-2 fluorescence ratio signals in the same cells.

FIG. 3. Simultaneous fluorescence digital imaging of cAMP/FICRhR and Ca2+/fura-2 responses in single REF-52 fibroblasts.Pseudocolor images of FICRhR 520/580 nm emission ratio afterexcitation at 488 nm (A-C) and fura-2 334/380 nm excitation ratio withemission monitored at 520 nm (D-F) in two REF-52 cells. Note that,while both fibroblasts had been loaded with fura-2 (as shown in D-F)only the one on the left was microinjected with FICRhR and this cellonly shows up in A-C. FICRhR (A) and fura-2 (D) resting ratios,indicating basal cAMP and Ca2+ levels before stimulation. (B and E)Ratio images taken one min after addition of 50 ,uM FO: FICRhRemission ratio (B) has increased as a consequence of increased[cAMP]j, while fura-2 excitation ratio (E) in both fibroblasts has notchanged because [Ca2+]i has not been affected by FO. Two minuteslater, cells were treated with 1 ,uM TG, which increased [Ca2+]i andfura-2 excitation ratio (F) in both fibroblasts without affecting[cAMP]i and FICRhR emission ratio (C). Note that the change infura-2 excitation ratio in the cell labeled with fura-2 only is similar tothat detected in the FICRhR and fura-2 colabeled cell (F). IncreasingFICRhR and fura-2 ratios, reflecting increasing [cAMP]i and [Ca2+],are coded in pseudocolor hues ranging from violet to green, as shownin the color scale on the right.

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.0U) ~~~~~~~~~~~~1.6 '

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FIG. 4. Inverse relationship between TG-elevated [Ca2+]s andFO-stimulated cAMP accumulation in single C6-2B cells. Cells werelabeled with both FICRhR and fura-2 and imaged. FO (50 j,M) andTG (1 ,uM) were added together to the cells to simultaneously increase[cAMP]i and .[Ca2+]j, respectively, and FICRhR and fura-2 responsesfrom seven single C6-2B cells were separately and automaticallycollected. An analysis of the data revealed that, among the cells beingimaged in this experiment, four of the cells (A) exhibited a smallerincrease in fura-2 334/380 nm excitation ratio, reflecting a smallerTG-evoked [Ca2+]i increase, and a greater FICRhR 520/580 nmemission ratio, indicating a greater cAMP response to FO. Conversely,in three cells where TG induced a greater increase in [Ca2+]i, theFO-evoked cAMP/FlCRhR response was smaller (B).

we then proceeded to characterize the dynamics of the regu-lation by Ca2+ of cAMP accumulation in single living C6-2Bcells. To this end, we exploited the heterogeneity in the Ca2+response that individual C6-2B cells display upon exposure toTG. Thus, C6-2B cells microinjected with FICRhR and loadedwith fura-2AM were treated with FO together with TG and thechanges in the fluorescence emission/excitation ratio ofFlCRhR/fura-2 were recorded (Fig. 4). Interestingly, amongthe cells followed throughout the experiment, those thatdisplayed a lower [Ca2+], increase in response to TG exhibiteda greater FO-elicited cAMP accumulation and increase inFICRhR emission ratio (Fig. 4A), while in cells most respon-sive to TG, and showing a greater increase in [Ca2+]j, theFO-induced cAMP formation and FICRhR response werereduced by 40-50% (Fig. 4B). This is consistent with ourprevious in vitro finding showing that when C6-2B cells arechallenged with FO together with TG, cAMP accumulationmeasured in cell extracts is reduced by about 60% comparedto cells exposed to FO alone (10). Similar results were alsoobtained when [Ca2+]i was elevated before exposure of thecells to either FO or ISO (data not shown). Moreover, whenC6-2B cells were coincubated with FO and IONO, a massive[Ca2+], increase was detected in virtually all the cells beingimaged while cAMP accumulation was only minimally stimu-lated in these same cells, as reflected by a very small increasein FICRhR 520/580 nm emission ratio (Fig. 5A). Ca2+ inhi-bition ofcAMP-mediated FICRhR response was also observedin the presence of phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and Ro20-1724, 100 ,uM; data not shown).Importantly, the inhibitory effect of Ca2+ on cAMP kinetics

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FIG. 5. IONO-evoked [Ca2+]i increase prevents FO-induced singleC6-2B cell cAMP accumulation. (A) FICRhR and fura-2 colabeledC6-2B cells were exposed simultaneously to FO (100 jiM) and IONO(5 AiM). While a marked [Ca2+]i increase was observed, a minimalcAMP/FlCRhR response was detected. However, upon exogenouselevation of [cAMP]i by the cell-permeable cAMP analog, N6,02'-dibutyryl cAMP (dbcAMP; 1 mM, which maximally mimics cAMPaction in the cells), the FICRhR emission ratio did increase, providingevidence that, under conditions where [Ca2+]j was elevated, the cAMPfluorosensor was still functional. (B) Control experiment performed inparallel where C6-2B cells challenged with FO in the absence ofIONO successfully responded to the increased cAMP production withan increased FICRhR emission ratio.

appears to be specific for C6-2B cells because in REF-52fibroblasts IONO-induced [Ca2+], increase did not prevent sub-

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FIG. 6. Elevated [Ca2+]i reduces the cAMP accumulation previ-ously induced by ISO in single C6-2B cells. Cells were microinjectedwith FICRhR, loaded with fura-2, and imaged. Upon addition of 10,iM ISO, [cAMP]i, measured as FICRhR emission ratio, rapidlyincreased (solid squares), remained at peak level for about 2 min, andprogressively decreased once [Ca2+]1 was elevated with TG (1 ,uM,open circles). Solid diamonds represent the time course of thecAMP/FlCRhR response induced by ISO in the absence of Ca2+-mobilizing agents from a parallel experiment, performed the same dayon the same batch of C6-2B cells microinjected with the same lot ofFlCRhR. The steady cAMP/F1CRhR signal (solid diamonds) can beappreciated and compared to the short-lasting response detectedinstead in the presence of TG (solid squares).

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sequent cAMP accumulation by FO (Fig. 2B) nor did TG impairthe FlCRhR response evoked by FO (Fig. 2C). Therefore,simultaneous fluorescence imaging of cAMP and Ca2+ allowedfor the first time a single cell analysis of the negative effect of Ca2+on C6-2B cell cAMP kinetics.The acute consequences of increased [Ca2+], on the ongoing,

agonist-induced cAMP production in C6-2B cells were thenexplored by first stimulating cAMP accumulation with ISOand subsequently elevating [Ca2+], with TG (Fig. 6). ISOevoked an immediate 1.4-fold increase in the FICRhR 520/580nm emission ratio which remained at peak level for at least 2min (solid squares). However, when [Ca2+], was increased withTG (open circles; basal [Ca2+],: -70 nM; peak after TG: -300nM), the FICRhR signal progressively decreased to about 50%of its peak value by about 9 min from TG addition (or 13 minafter ISO addition). In the absence of Ca2+-generating stimuli(solid diamonds), the ISO-induced 1.4-fold increase in Fl-CRhR 520/580 nm emission ratio was longer lasting such that,after about 15 min from ISO addition, the FICRhR signal wasstill 85% of its peak value. A similar Ca2+-mediated reductionof the cAMP-induced FICRhR response was observed inexperiments where C6-2B cells were first challenged with ISO(Fig. 7) or FO (Fig. 84) and subsequently with IONO, whichelevates [Ca2+], to putatively maximal levels (resting [Ca2+]was 50-100 nM; the [Ca2+]i peak after IONO was 500-1000nM). These results indicate that, in C6-2B cells, Ca2+ cannotonly inhibit agonist-stimulated cAMP accumulation on itsstart, but it is also able to modulate a previously activated

cAMP transduction signal by instantaneously inhibiting fur-ther cAMP synthesis.The hypotheses that, in C6-2B cells, Ca2+ decreases cAMP-

mediated FICRhR response by impairing the dye performanceor enhancing cAMP degradation (rather than by inhibitingagonist-promoted cAMP accumulation) were also considered.To test the first possibility, C6-2B cells colabeled with Fl-CRhR and fura-2AM were treated with the cell-permeablecAMP analog N6,02'-dibutyryl cAMP (dbcAMP), which ex-ogenously increases [cAMP]j and, therefore, induces a changein FICRhR emission ratio without newly stimulated cAMPsynthesis being involved. In the presence of highly elevated[Ca2+]i, which had either prevented (Fig. 5A) or antagonized(Fig. 8A) FO-mediated FICRhR response, dbcAMP was able toevoke (Fig. SA) or completely restore (Fig. 8A) the FICRhRresponse. Moreover, the dbcAMP-elicited increase in [cAMP]jand FICRhR emission ratio was not affected by a subsequent[Ca2+]i rise (Fig. 8B), thus showing that (i) changes in thefura-2 excitation spectrum per se do not impair FICRhRresponse in C6-2B cells, and (ii) FICRhR responsiveness toincreased [cAMP]i caused by providing cells with preformedcAMP is indeed insensitive to elevated [Ca2+],. With regard tothe second possibility, a Ca2+-dependent stimulation of phos-phodiesterase activity appears to be the primary mechanismfor the inhibition ofcAMP accumulation in certain cell systems(21). However, in C6-2B cells as well as other cell lines andtissues (1, 2, 8-11, 22-26) mostly expressing Ca2+-inhibitableadenylyl cyclases, a Ca2+-mediated enhancement of phosphod-

FIG. 7. Ca2+ modulation of single C6-2B cell cAMP response followed by simultaneous fura-2 and FICRhR fluorescence ratio imaging. C6-2Bcells colabeled with FICRhR and fura-2 were treated with ISO (10 ,tM) which, by promoting new cAMP synthesis, increased FICRhR emissionratio (yellow trace). When [Ca2+]i was elevated by IONO (1 ,uM, blue trace), the FICRhR response started to decline (reflecting reduced cAMPaccumulation) such that by 5 min from IONO addition [cAMP]i/FlCRhR emission ratio was 30% of its peak value. At the bottom are pseudocolorimages of FICRhR 520/580 nm emission ratio after excitation at 488 nm (Upper) and fura-2 334/380 nm excitation ratio with emission monitoredat 520 nm (Lower) from the experiment shown in the graph. Green arrows indicate the time at which the images in sets A, B, and C were taken.(Set A) Basal FICRhR emission ratio and fura-2 excitation ratio, indicating resting cAMP and Ca2+ levels, respectively. (Set B) Three minutes afteraddition of ISO, FICRhR emission ratio has increased because of the increased [cAMP]j while fura-2 excitation ratio has not changed, reflectingunmodified [Ca2+]i. (Set C) About 5 min after IONO treatment, fura-2 excitation ratio has increased as a function of the increased [Ca2+]i, whileF1CRhR emission ratio has now decreased, indicating reduced cAMP production. Increasing FICRhR and fura-2 ratios (reflecting increasing[cAMP]i and [Ca2+]j) are coded in pseudocolor hues ranging from violet to red, as shown in the color scale on the right.

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-E1.71.6. 3-

.25 deoxFO

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400 800 1200 1600 2000 ff3.2 1.3

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FIG. 8. Elevated [Ca2M]P inhibits the cAMP/FlCRhR response

evoked by FO but not by dbcAMP in single C6-2B cells. InA, glioma

cells, colabeled with FlCRhR and fura-2, were treated first with

1-deoxyforskolin (deoxFO; 50 ,uLM), an inactive FO analog that, as

expected, failed to affect cAMP/FlCRhR response, and then with FO

(50 ,uM) which increased the FlCRhR emission ratio, reflecting

increased cAMP content. cAMP/FlCRhR peak response lasted for

about 4 min but underwent an abrupt reduction to virtually baseline

value at the same time as [Ca2+]i was greatly elevated by IONO (1

1±sM). However, when [cAMP]j was increased by exogenous addition of

N6,02'-dibutyryl cAMP (dbcAMP; 250 ,uLM, which would be expected

to totally dissociate FlCRhR and yield a maximal response), the

change in FlCRhR emission ratio was completely restored, proving

that, under conditions where [Ca2+]i was above homeostatic values,

the dye was still functional. In B, C6-2B cells, colabeled with FlCRhRand fura-2, were treated with dbcAMP (250 ,uM) to elevate [cAMP]jwithout activating new cAMP synthesis. [cAMP]i in single C6-2B cells

slowly increased and remained at peak level even when [Ca2+]i was

elevated by TG (1 ,uM) and, later, IONO (1 ,uM), showing that

dbcAMP-evoked cAMP/FlCRhR response in not affected by Ca2+.

iesterase activity has been precluded as the main mechanism

responsible for Ca2+ inhibition of agonist-induced cAMP

formation. In these cases, a direct negative effect of Ca2+ on

adenylyl cyclase has been demonstrated to be the likely

mechanism (1, 2, 8-11, 22-26). Thus, in light of these consid-

erations, it is appropriate to interpret the inhibitory effect of

elevated [Ca2+]j on the FlCRhR response elicited in single

C6-2B cells by cAMP-generating signals as the result of a true

negative regulation by Ca2+ of the cAMP signaling pathway,

whose dynamics could now be followed in vivo. The cell

specificity of such a cross-talk is supported by the lack of effect

of elevated [Ca2+]i on the kinetics of FO-induced FlCRhRresponse in REF-52 fibroblasts.

To conclude, first time simultaneous imaging of cAMP and

Ca2+ by fluorescence digital microscopy of single living cells (i)

allows for an immediate and continuous analysis of the kinetics

of these two ubiquitously important second messengers, (ii)

provides in vivo evidence for a dynamic regulation by Ca2+ ofagonist-induced cAMP production in specific cell types, and(iii) demonstrates the utility of this dual-dye ratio imagingtechnique for the study of the spatial and temporal interactionsbetween Ca2+ and cAMP, and potentially other intracellularregulatory messengers, at the single cell and subcellular level.

We are grateful to J. Scott Mc Donald for his help with the softwareand graphics. This work was supported by Grant lR43GM50642 fromthe National Institute of General Medical Sciences to Atto Instru-ments, Inc.

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Proc. Natl. Acad. Sci. USA 93 (1996)

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