fluctuations in the concentration of extracellular atp synchronized with intracellular ca ...

FLUCTUATIONS IN THE CONCENTRATION OF EXTRACELLULAR

ATP SYNCHRONIZED WITH INTRACELLULAR Ca21 OSCILLATORY

RHYTHM IN CULTURED CARDIAC MYOCYTES

Koichi Kawahara and Yukako Nakayama

Laboratory of Cellular Cybernetics, Graduate School of Information Science andTechnology, Hokkaido University, Sapporo, Japan

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically.The intracellular concentration of free Ca2þ also changes rhythmically in associationwith the rhythmic contraction of myocytes (Ca2þ oscillation). Both the contractionand Ca2þ oscillatory rhythms are synchronized among myocytes, and intercellularcommunication via gap junctions has been considered primarily responsible for thesynchronization. However, a recent study has demonstrated that intercellular com-munication via extracellular ATP-purinoceptor signaling is also involved in the inter-cellular synchronization of intracellular Ca2þ oscillation. In this study, we aim toelucidate whether the concentration of extracellular ATP changes cyclically and con-tributes to the intercellular synchronization of Ca2þ oscillation among myocytes. Inalmost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellularCa2þ oscillations were synchronized with each other. The simultaneous measurementof the concentration of extracellular ATP and intracellular Ca2þ revealed the extra-cellular concentration of ATP actually oscillated concurrently with the intracellularCa2þ oscillation. In addition, power spectrum and cross-correlation analyses suggestedthat the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purino-ceptors, resulted in the asynchronization of Ca2þ oscillatory rhythms among cardiacmyocytes. Treatment with suramin also resulted in a significant decrease in the ampli-tudes of the cyclic changes in both intracellular Ca2þ and extracellular ATP. Takentogether, the present study demonstrated the possibility that the concentration ofextracellular ATP changes cyclically in association with intracellular Ca2þ, contributingto the intercellular synchronization of Ca2þ oscillation among cultured cardiac myo-cytes. (Author correspondence: [email protected])

Keywords Contraction rhythm, Ca2þ oscillation, Gap junction, Purinoceptor,Extracellular ATP

Submitted August 22, 2007, Returned for revision September 4, 2007, Accepted September 11,2007

Address correspondence to Koichi Kawahara, Laboratory of Cellular Cybernetics, GraduateSchool of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan.Fax: þ81-11-706-7591; E-mail: [email protected]

Chronobiology International, 24(6): 1035–1048, (2007)Copyright # Informa HealthcareISSN 0742-0528 print/1525-6073 onlineDOI: 10.1080/07420520701800843

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INTRODUCTION

Isolated and cultured neonatal cardiac myocytes contract spontaneouslyand cyclically (Harary & Farley, 1963). The contraction rhythms of twoisolated cardiac myocytes, each of which beats at different frequencies atfirst, become synchronized after the establishment of mutual contacts(Jongsma et al., 1987), suggesting mutual entrainment occurs due to elec-trical and/ormechanical interactions between twomyocytes. The intracellu-lar concentration of free Ca2þ also changes rhythmically in association withthe rhythmic contraction of myocytes (Ca2þ oscillation). Such a Ca2þ oscil-lation was also synchronized among cultured cardiac myocytes (Nakayamaet al., 2005). It is generally believed that gap junctional intercellularcommunication plays an important role in the intercellular synchronizationof intracellular Ca2þ oscillation (Kimura et al., 1995). However, a recentstudy revealed the extracellular ATP-purinoceptor signaling system is alsoinvolved in the intercellular synchronization of the intracellular Ca2þ oscil-lation among cultured cardiac myocytes (Kawahara & Nakayama, 2007;Nakayama et al., 2007).

Extracellular ATP acts as a potent agonist on a variety of different celltypes, including cardiomyocytes (Kunapuli & Daniel, 1998), inducing abroad range of physiological responses. The cellular effects mediated byATP are determined by the subtypes of P2 purinergic receptors expressedin the particular cell type. In cardiomyocytes, the expression of ionotropicP2X1–P2X7 receptors and metabotropic P2Y1, P2Y2, P2Y4, P2Y6, andP2Y11 receptors have been described (Vassort, 2001). The diversity ofATP receptors expressed in cardiomyocytes reflects the variety of ATPeffects described for single cells as well as the whole organ. In the singlecardiomyocyte, micromolar levels of extracellular ATP increase plasmamembrane permeabilities for cations (Puceat et al., 1991), intracellularcalcium transients (Vassort, 2001; Podrasky et al., 1997), and contractionamplitude (Mei & Liang, 2001; Podrasky et al., 1997). Moreover, ATPcan stimulate phospholipase C (PLC) (Podrasky et al., 1997). On theorgan level, ATP acts as a positive inotropic agent (Mei & Liang, 2001)and can induce various forms of arrhythmia (Vassort, 2001).

Extracellular ATP in the cardiovascular system may originate fromdifferent cellular sources. Under ischemic conditions, myocytes releaseATP (Dutta et al., 2004), and it has also been speculated that exercisingheart muscle cells are a source of extracellular ATP, similar to what hasbeen reported for the working skeletal muscle cell (Forrester, 1972).Mechanical strain leads to the release of ATP from a variety of cell types,including cardiac myocytes. Uozumi et al. (1998) reported that in culturedneonatal cardiomyocytes, ATP release can be stimulated by the applicationof mechanical force. During the cycle of systolic contraction and diastolicrelaxation, the sarcolemmal membrane is subjected to continuous and

K. Kawahara and Y. Nakayama1036

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pronounced mechanical deformations. Thus, there is a possibility thatspontaneously contracting cardiac myocytes cyclically release ATP.

This study investigated whether the concentration of extracellular ATPpossibly changes cyclically in association with intracellular Ca2þ oscillationand contributes to the intercellular synchronization of Ca2þ oscillationamong cardiac myocytes. Here it is shown that the extracellular concen-tration of ATP actually oscillated concurrently with the intracellular Ca2þ

oscillation, contributing to the intercellular synchronization of Ca2þ oscil-lation among cardiac myocytes.

METHODS

The animal experiments conformed to the principles of laboratoryanimal care (NIH publication No. 85-23, revised 1996), as well as theguide for the care and use of laboratory animals, Hokkaido UniversitySchool of Medicine. In addition, the experimental protocol conformedto international ethical standards in chronobiology (Touitou et al., 2006).

Culture of Cardiac Myocytes

Themethod of culture is described elsewhere in detail (Kawahara et al.,2002, 2006; Kohashi et al., 2003; Yamauchi et al., 2002; Yoneyama &Kawahara, 2004). In short, cardiacmyocytes were prepared fromventriclesof one- to three-day-old neonatal Wistar rats removed after decapitation.The ventricles were rinsed in a 25 mMHEPES-buffered minimum salt sol-ution (MSS) to remove contaminating blood cell components and thenminced with scissors into fragments for digestion with 0.1% collagenase(Wako Chemical, Tokyo, Japan) in MSS at 378C for 10 min. The digestedfragments were centrifuged at 1000 rpm for 2 min (LC-100, TOMY,Japan), and precipitated cell components were washed twice with MSS toterminate the effects of collagenase. The cell components were suspendedin MCDB 107 (Research Institute for Functional Peptides, Yamagata,Japan) containing 5% FCS (MBL, Nagoya, Japan), and then passedthrough a wire mesh screen (90 mm porosity) to remove large aggregatesof cells; the filtered suspension contains cardiac myocytes and fibroblasts.To separate cardiac myocytes from fibroblasts based on the selectiveadhesion technique, the cell suspension was poured into petri dishes (f60 mm, Falcon) and incubated for 60 min at 378C, in 5% CO2 and 95%air. By virtue of the procedure, most of the fibroblasts adhere to the dish.After incubation, the suspension, mostly containing cardiac myocytes, wascollected. The suspension was centrifuged at 700 rpm for 5 min to separatethe remaining blood cell components in the supernatant. The precipitatedcells were resuspended in MCDB 107 containing 5% FCS, transferrin(10 mg/mL, Sigma, St. Louis, Missouri, USA), and insulin (10 mg/mL,

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Yamanouchi, Tokyo, Japan). The cell suspensionwas passed through a finewire mesh screen (25 mm porosity) to remove remaining small aggregatesof myocytes, and finally the isolated myocytes remaining were cultured ata density of about 3.0 � 105 cells/ml in a petri dish (f 30 mm, Falcon)coated beforehand with fibronectin (10 mg/mL, Sigma).

For the simultaneous measurement of the concentrations of intracellu-lar Ca2þ and extracellular ATP, isolated cardiac myocytes were cultured ata density of about 3.0 � 105 cells/mL on glass coverslips (Matsunami,Tokyo, Japan) in a petri dish (f30 mm, Falcon). The glass coverslipswere coated beforehand with fibronectin (10 mg/mL, Sigma). Cardiacmyocytes cultured for four days (4 DIV) were used in this measurement.The measurement area of the simultaneous chemiluminescence (CL)/flu-orescence (FL) measurement system contained about 15–20 culturedcardiac myocytes. Prior to the simultaneous measurements, cardiac myo-cytes were loaded with 5 mM fluo 4/AM (Molecular Probes) in MCDBmedium at room temperature for 30 min. Following the addition of a luci-ferin-luciferase solution (Lucifer 250, Kikkoman Corp., Chiba, Japan), theglass coverslip was placed in the simultaneous CL/FL measuring system.Kikkoman’s ATP Determination Kit (Lucifer 250/HS) offers a convenientbioluminescence assay for the quantitative determination of ATP withrecombinant firefly luciferase and its substrate D-luciferin. Cardiac myo-cytes cultured for four days were used in this study.

Cellular Ca21 Measurements

Changes in the cytosolic concentration of free Ca2þ were measuredusing fluo 4. Cardiac myocytes in culture were loaded with the fluorescentcalcium indicator during a 30 min incubation with acetoxymethyl ester offluo 4 (fluo 4/AM, 5 mM; Molecular probes, Eugene, Oregon, USA) inMCDB medium at room temperature. Fluo 4 was excited at 490 nm,and emission intensity was measured at 525 nm. Fluorescent imageswere acquired at approximately 200 msec intervals with a cooled CCDcamera (C4880-80; Hamamatsu Photonics, Hamamatsu, Japan). An analy-sis of the acquired images was completed with an image processing andmeasuring system (AQUACOSMOS; Hamamatsu Photonics). Fluorescentintensity (F) was normalized with the initial value (F0), and changes in therelative fluorescent intensity (F/F02 1) were used to assess those in cellu-lar free Ca2þ.

Simultaneous Measurement of Intracellular Ca21

and Extracellular ATP

Dynamic changes in the concentrations of intracellular Ca2þ in cardiacmyocytes and extracellular ATP were simultaneously measured with


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the simultaneous chemiluminescence and fluorescence measurementsystem (Hamamatsu Photonics, Hamamatsu, Japan). The measurementsystem is described in detail elsewhere (Satozono et al., 2006). In brief, ahigh-intensity blue LED flashed in synchronization with a pulse generator.Band-pass filters with a very sharp width at 488 nm are used because of thebroad LED spectrum. Cultured cardiac myocytes emit chemiluminescencefrom luciferin/luciferase (560 nm) and fluorescence from Fluo-4 (530 nm).The emission is guided to a Photomultiplier (R1635P, Hamamatsu Photo-nics) through a band-rejection filter. The band-rejection filter removes theexcitation scattering at 488 nm. Signals from the PMT were amplified anddiscriminated by a Photon Counting Unit (C6465, Hamamatsu Photonics).The photon pulses from C6465 were input into a multi-channel universalcounter (PCI32-8M, CONTEC, Tokyo, Japan) via a custom-made gatecircuit. The circuit has two gates and divides the signal into two accordingto the timing pulse. The branched signals from the gate circuit are inputinto the universal counter. The universal counter is inserted into PCIslots of a PC and controlled by the measurement software. In the measure-ment, the timing pulse frequency is 1 kHz and is faster than the cellresponse. The measurement of fluorescence and chemiluminescenceis therefore almost simultaneous. The excitation width is 0.2 ms and isshorter than the timing pulse width because the fluorescence intensityis strong enough to measure within a short excitation time. Data acqui-sition was performed at 200 ms intervals. The detection threshold ofATP with this system was estimated at about 1 f M (1 � 102 15 M).

Statistics

The data are expressed as the mean+ SD. Comparisons were per-formed using the one-way analysis of variance (ANOVA) followed by apaired t-test. A p value of less than either 0.01 or 0.05 was considered stat-istically significant.

RESULTS

We first investigated whether the spontaneous cyclic changes in theconcentration of free Ca2þ (Ca2þ oscillation) in cultured cardiac myocyteswere synchronized among myocytes using a fluorescent Ca2þ indicator,fluo 4/AM (see Figure 1). The relative intensity of fluo 4 fluorescence inthree cells (see Figure 1a and 1b) fluctuated cyclically, and the Ca2þ oscil-lation was synchronized among myocytes (see Figure 1c). In almost all thecultures tested, the Ca2þ oscillation in cardiac myocytes was synchronizedamong cultured cells at 4 DIV.

This study aimed to clarify whether the concentration of extracellularATP actually oscillated in association with the cyclic changes in the


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intracellular concentration of free Ca2þ (Ca2þ oscillation) in cultures ofcardiac myocytes. To address this issue, a simultaneous measurement ofthe concentrations of intracellular Ca2þ and extracellular ATP was con-ducted in cultured cardiac myocytes using the simultaneous chemilumi-nescence (CL)/fluorescence (FL) measurement system (HamamatsuPhotonics, Hamamatsu, Japan).

The study first investigatedwhether the cyclic changes in the extracellu-lar concentration of ATP are detected by the simultaneousCL/FLmeasure-ment system. Cultures were pre-treated with a drug for the extinction ofextracellular ATP for 5 min. After that, the simultaneous measurementwas started. The treatment resulted in a significant reduction in chemilumi-nescence (CL) compared with that in cultures without treatment (seeFigure 2a and 2b), suggesting themeasuredCL reflected the concentrationof extracellular ATP.To confirm this further, the cultureswere treatedwithtriton-X 100 to disrupt the integrity of plasma membranes of cardiac myo-cytes and evaluated for the release of intracellular ATP. We found thattreatment with triton-X markedly increased CL (see Figure 2c).

To terminate the cyclic contractile activity of cardiac myocytes, cultureswere treated with 7.5 mM BDM for more than 5 min. This concentrationof BDM almost completely terminates the spontaneous contractile activityof cultured cardiac myocytes (Nakayama et al., 2005). The treatment did

FIGURE 1 Intercellular synchronization of intracellular Ca2þ oscillation in cardiac myocytes in cul-ture. The Ca2þ oscillation in cultured neonatal cardiac myocytes at four days in vitro (4 DIV) indicatedby arrows in the phase-contrast image of the culture (a) and in the fluorescence image (b) was synchro-nized among myocytes (c). Cyclic changes in the intracellular concentration of free Ca2þ weremeasured by loading myocytes with the fluorescent Ca2þ probe fluo 4/AM (5 mM). The scale barsindicate 100 mM.


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not significantly change the chemiluminescence signals (see Figure 2d and2e), suggesting that the measured changes in the chemiluminescencereflected the concentration of extracellular ATP, not artifacts caused bythe cyclic movements of the extracellular fluid.

The measurement area of the simultaneous CL/FL measurementsystem contains about 15–20 cultured cardiac myocytes. Under normalconditions, almost all of the myocytes in the culture exhibited synchro-nized Ca2þ oscillation, as shown in Figure 1. The study next investigatedwhether the macroscopic changes in the fluorescence signals reflecting

FIGURE 2 Validity for the estimation of changes in the concentration of extracellular ATP in culturedcardiac myocytes using the simultaneous chemiluminescence (CL)/fluorescence (FL) measurementsystem. Extracellular ATP was removed by treating the cultures with an ATP extinction drug. Thetreatment resulted in a significant reduction of the chemiluminescence signal (i.e., the signal indicatedby grey in parts a and b). Data are expressed as themeanþ SD (n ¼ 4 different coverslips). ��p , 0.01.Treatment of cultures with triton-X 100 (0.2%) resulted in amarked increase in chemiluminescence (c),reflecting a massive release of ATP from the intracellular to extracellular space. Treatment with BDM(7.5 mM) did not produce significant changes in the chemiluminescence (the signal indicated by grey ind), suggesting that fluctuation of the extracellular fluid in association with the rhythmic contraction ofcardiac myocytes did not affect the chemiluminescence. Data are expressed as the meanþ SD (n¼4different coverslips). Abbreviations: CL ¼ chemiluminescence, ns ¼ not significant.


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the intracellular concentration of free Ca2þ could be detected using thesimultaneous CL/FL measurement system. The fluo 4 fluorescencereflecting the intracellular concentration of Ca2þ changed cyclically (seeFigure 3a). Auto-correlation (see Figure 3b) and FFT (see Figure 3c) ana-lyses of the fluorescence signals indicated the dominant oscillation fre-quency was about 0.4 Hz. These results suggested that the macroscopic

FIGURE 3 Intracellular Ca2þ and extracellular ATP detected by using the simultaneous CL/FLmeasurement system. Intracellular Ca2þ oscillation was measured by using the CL/FL measurementsystem (a–c). The fluo 4 fluorescence reflecting the intracellular concentration of Ca2þ changed cycli-cally (a). Auto-correlation (b) and FFT (c) analyses of the fluorescence signals indicated the dominantoscillation frequency was about 0.4 Hz. Extracellular ATP oscillation was verified by the simultaneousCL/FL measurement system (d–f). The chemiluminescence (CL) reflecting the extracellular concen-tration of ATP changed cyclically (d). Auto-correlation (e) and FFT (f) analyses of the CL signals indi-cated the dominant oscillation frequency was about 0.4 Hz. Abbreviations: FL ¼ fluorescence,CL ¼ chemiluminescence, ACC ¼ auto-correlation coefficient, PSD ¼ power spectrum density.


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changes in the fluorescence signal reflecting the concentration of free Ca2þ

could be detected using the simultaneous CL/FL measurement system.The study investigated the possibility the concentration of extracellularATP changes cyclically as well. The CL reflecting the extracellular concen-tration of ATP changed cyclically (see Figure 3d). Auto-correlation (seeFigure 3e) and FFT (see Figure 3f) analyses of the CL signals indicatedthe dominant oscillation frequency was about 0.4 Hz.

It was recently demonstrated that treatment with suramin, a blocker ofP2 purinoceptors, results in the asynchronization of intracellular Ca2þ

oscillation among myocytes (Nakayama et al., 2007), suggesting that thefluo 4 fluorescence signal detected by using the simultaneous CL/FLmeasurement system would be markedly reduced by treatment withsuramin. Whether and how suramin treatment resulted in a reduction inamplitude of the oscillation of the concentrations of both extracellularATP and intracellular Ca2þ were investigated. Treatment of cultureswith suramin (100 mM) for about 5 min resulted in a significant reductionin the oscillation amplitude of the fluo-4 fluorescence reflecting the intra-cellular concentration of Ca2þ (see Figure 4a and 4b). The suramin treat-ment also resulted in a significant reduction in the chemiluminescencereflecting the extracellular concentration of ATP (see Figure 4c and 4d).

FIGURE 4 Changes in the amplitude of intracellular Ca2þ and extracellular ATP oscillations caused byblocking purinoceptors. Treatment of cultures with suramin (100 mM) for about 5 min resulted in sig-nificant reduction in the oscillation amplitudes of the fluo 4 fluorescence, reflecting the intracellularconcentration of Ca2þ (the signal indicated by grey in a), and the chemiluminescence, reflecting theextracellular concentration of ATP (the signal indicated by grey in c). Data are expressed as themeanþ

SD (n ¼ 4 different coverslips). �p , 0.05, ��p , 0.01. Abbreviations are the same as those in Figure 3.


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An FFT analysis demonstrated that treatment of cultures with suramin(100 mM) for about 5 min resulted in a marked reduction in the regularityof the Ca2þ oscillatory rhythm. The spectrum profile, with a single domi-nant peak (see Figure 5a) before the onset of suramin treatment, changed

FIGURE 5 Spectrum and cross-correlation analyses of intracellular Ca2þ and extracellular ATPdetected by the simultaneous CL/FL measurement system. Intercellular asynchronization of intra-cellular Ca2þ oscillation on the blocking of purinoceptors (a and b). An FFT analysis demonstratedthat treatment of cultures with suramin (100 mM) resulted in a marked reduction in the regularityof the Ca2þ oscillatory rhythm. The spectrum profile, with a single dominant peak (a) before theonset of suramin treatment, changed to one with multiple spectral peaks (b) following the treatment.Asynchronization occurred between intracellular Ca2þ and extracellular ATP oscillations on the block-ing of purinoceptors (c–f). Cross-correlation (c and d) and cross-FFT (e and f) analyses revealed thatthe treatment of cultures with suramin resulted in a marked reduction in the correlation between thecyclic changes in the concentration of extracellular ATP and intracellular Ca2þ in cardiac myocytes.The cross-spectrum profile, with a single dominant peak (e) before the onset of suramin treatment,changed to one without detectable spectral peaks (f) following the treatment. Abbreviation: CCC ¼

cross-correlation coefficient. Other abbreviations are the same as those in Figure 3.


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to one with multiple spectral peaks (see Figure 5b) following the treatment,possibly reflecting the suramin-induced asynchronization of Ca2þ oscil-lation among myocytes.

In a final step, the present study investigated whether the synchroniza-tion between oscillations of intracellular Ca2þ and extracellular ATP wasinterrupted by the blocking of purinoceptors. Cross-correlation (seeFigure 5c and 5d) and cross-FFT (Figure 5e and 5f) analyses revealedthat treatment of cultures with suramin (100 mM) for about 5 min resultedin a marked reduction in the correlation between the cyclic changes in theconcentrations of extracellular ATP and intracellular Ca2þ in cardiac myo-cytes. The cross-spectrum profile, with a single dominant peak (seeFigure 5e) before the onset of suramin treatment, changed to onewithout detectable spectral peaks (see Figure 5f) following the treatment.These results suggest the synchronization between oscillations of intra-cellular Ca2þ and extracellular ATP collapsed when purinoceptors wereblocked with suramin.

DISCUSSION

The present study demonstrated that extracellular concentration ofATP possibly changes cyclically in association with the oscillatory rhythmin the intracellular concentration of Ca2þ (Ca2þ oscillation) and that theATP-purinoceptor signaling pathway is critical to the intercellular syn-chronization of intracellular Ca2þ oscillation in cultured cardiac myocytes.Although the detailed mechanisms responsible for the release of ATPfrom the intracellular to extracellular space are not yet fully understood,previous studies have reported that under ischemic conditions, myocytesrelease ATP (Dutta et al., 2004) and that mechanical strain leads torelease of ATP from a variety of cell types, including cardiac myocytes(Uozumi et al., 1998). A recent study has demonstrated the possibilityin electrically stimulated isolated adult cardiac myocytes that deformationof the contracting cardiac myocytes is not a key stimulus for the release ofcellular ATP (Godecke et al., 2005). In this study, the complete termin-ation of cyclic contractile activity of cardiac myocytes by treatment withan un-coupler of E-C coupling did not affect the intercellular synchroniza-tion of intracellular Ca2þ oscillation (see Figure 2d & 2e), suggesting thepossibility that cyclic changes in free intracellular Ca2þ was a key factor,not a mechanical deformation itself, for the release of cellular ATP.

Exogenously applied ATP evoked an increase in the concentrationof intracellular Ca2þ in astrocytes (Bruner & Murphy, 1993) and incardiac myocytes (Vassort, 2001; Zhang et al., 1996). Studies on thepotency of ATP analogs suggest that an increase in the concentrationof intracellular Ca2þ in astrocytes is due to the activation of the P2Ysubtype (Kastritsis et al., 1992; Salter & Hicks, 1994). ATP-evoked


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Ca2þ responses could play important roles in physiological or patho-logical processes within the central nervous system. Increases in theconcentration of intracellular Ca2þ produced by ATP have been corre-lated with increases in inositol phospholipids turnover and with inositol1,4,5 trisphosphate (IP3) itself (Kastritsis et al., 1992; Pearse et al.,1989). This correlation has led to the speculation that IP3 may be amediator of Ca2þ release from the intracellular Ca2þ store (sarcoplasmicreticulum, SR) produced by ATP, although a causal relationship has notbeen established. Evidence indicates that P2Y receptors may be coupledto additional signal transduction pathways that might affect intracellularCa2þ secondarily (Bruner & Murphy, 1993). In addition, a previousstudy revealed that the activation of P2 purinergic receptors raisesthe concentration of intracellular Ca2þ via the phospholipase C/IP3pathway (Podrasky et al., 1997).

In conclusion, the present study demonstrated for the first time thepossibility that the concentration of extracellular ATP changes cyclicallyin association with the oscillatory rhythm in the concentration of intra-cellular Ca2þ (Ca2þ oscillation) in cultured cardiac myocytes. Inaddition, the study also demonstrated that the ATP-purinoceptor sig-naling pathway is critical to the intercellular synchronization of intra-cellular Ca2þ oscillation. The involvement of this extracellularsignaling system in the intercellular synchronization of Ca2þ oscillationin myocytes seems dependent on the period of the culture. Treatmentof cardiac myocytes cultured for more than five days (.5DIV) withsuramin did not produce an asynchronization of Ca2þ oscillation (datanot shown), probably because of the full development of intercellularcommunication via gap junction channels. However, there is a possi-bility that the extracellular ATP-purinoceptor system, which once disap-pears as myocytes mature, would become functional again and play animportant role in the coordination of oscillatory rhythm among cardiacmyocytes when the intercellular communication via gap junction chan-nels ceases under conditions such as hypoxia or ischemia. This emer-gent property of the intercellular communication system is now underinvestigation.

ACKNOWLEDGMENTS

The authors would like to express cordial thanks to Mr. Tanigawa andMr. Sugiyama, Hamamatsu Photonics Inc., for permitting us to use thesimultaneous CL/FL measurement system, which is not commerciallyavailable at present. This work was supported by a grant-in-aid for scien-tific research from the Ministry of Education, Science, and Culture ofJapan (16300145, 19300153) to the lead author.


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fluctuations in the concentration of extracellular atp synchronized with intracellular ca ...

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