Biochem. J. (1991) 274, 55-62 (Printed in Great Britain)
55
Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes Michel PUCEAT, Odile CLEMENT, Frederique SCAMPS and Guy VASSORT Laboratoire de Physiologie Cellulaire Cardiaque, INSERM U-241, Universite Paris-Sud, Bat. 443, 91405 Orsay Cedex, France
The origin of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by extracellular ATP was investigated in single isolated cardiac myocytes loaded with indo- 1. The nucleotide added at a concentration of 10 uM triggers a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations, increasing [Ca2+]1 to around 200 nm from a basal value of 70 nm. Neither caffeine nor ryanodine affects the magnitude of the Ca2+ transient, but both shorten it by preventing the Ca2+ oscillations. This indicates that the latter must be related to the release of Ca2+ from the sarcoplasmic reticulum. Since ATP also induces cell depolarization (as shown by experiments using the potential sensitive dye bis-oxonol), the initial Ca2+ spikes were attributed to the opening of voltage-dependent Ca2+ channels. A small Ca2+ transient still remains under experimental conditions designed to prevent Ca2+ influx from external medium (low-Ca2+ high-Mg2+ medium containing La3+) and after depletion of the sarcoplasmic-reticulum Ca2+ load with caffeine. Under these conditions, when this Ca2+ transient was buffered by 1,2-bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid, ATP was unable to trigger the initial Ca2+ spikes. These results indicate that ATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum and that this Ca2+ release is responsible for the depolarization. The effects of ATP on [Ca2+]i share the same characteristics as the acidification simultaneously induced by the nucleotide (as shown by experiments using the pH-sensitive probe snarf-1). These ionic variations are highly specific to ATP and its hydrolysis-resistant analogues. They both require the presence of Mg2+ and Cl- ions in the extracellular medium, and they are prevented by pretreatment of the cells with 4,4'-di-isothiocyanostilbene or probenecid. These results suggest that: (1) the ATP-induced acidification leads to displacement of Ca2+ ions from or close to the internal face of sarcolemma; (2) the Ca2+ ions activate a nonspecific membrane conductance responsible for the depolarization of the cells; (3) the depolarization leads to a Ca2+ influx, owing to the opening of the voltage-dependent Ca2+ channels; (4) this increase in Ca2+ triggers the release of Ca2+ from the sarcoplasmic reticulum, which is facilitated by the increase in inositol trisphosphate following P2-purinergic stimulation.
INTRODUCTION ATP has been reported to be a neurotransmitter released by sympathetic nerves [1]. Its concentration can also increase significantly in the coronary vessels during cardiac hypoxia [2], and could be responsible for the early increase in intracellular Ca2+ during ischaemia [3]. The effects of purinergic agonists on cardiac contractility are well described. Adenosine and ATP decrease mechanical activity of the atria, probably by stimulating P,-purinergic receptors coupled via G-proteins to both adenylate cyclase and K+ conductance [4-6]. ATP also induces a positive
found only a slow rise in intracellular Ca2". More recently, ATP demonstrated to trigger a membrane depolarization associated with the rapid Ca2+ transient [27]. These effects of ATP on [Ca2+]i were reported in cells in suspension, and the mechanism by which ATP induces this Ca2+ transient remains unclear. We previously showed that ATP applied to a single cardiac cell induced a transient acidification, owing to the activation of the HCO3-/Cl- exchanger [28], which is followed by an alkalinization due to the activation of the Na+/H+ antiport [28,29]. The purpose
inotropic effect on the rat ventricle [7,8] by activation of P2purinergic receptors leading to the hydrolysis of phospho-
a
inositides [9]. -Several effects of ATP were reported in various tissues. ATP was shown to release Ca2+ from intracellular stores sensitive to InsP3 [10-12]. This rise in [Ca21]i is associated with biphasic changes in cytoplasmic pH in endothelial cells [13]. Furthermore, in the same study, it was shown that the nucleotide also increased membrane ionic permeabilities as well as it does in thymocytes [14] and in macrophages [15]. More particularly, ATP activates a cationic conductance in cultured muscle cells [16-18], in smooth-muscle cells [19,20] and in lacrimal acinar cells [21]. Some of these effects of ATP were also reported in isolated cardiac ventricular myocytes. Thus the nucleotide also induces a depolarization due to a non-specific inward current in cardiac cells [22,23]. Moreover, Sharma & Sheu [24] and De Young & Scarpa [25,26] showed that ATP triggered a large Ca2+ transient, followed by a weaker sustained increase, whereas others [7]
was
of this study was to characterize the effects of ATP on [Ca2+]i in single cardiac cell and to examine the relationship between
changes in
pH, and [Ca2+]1. We propose a mechanism by which the ATP-induced acidification triggers the release of Ca2+ from intracellular stores other than the sarcoplasmic reticulum. This Ca2+ release leads to the opening of a non-specific conductance responsible for cell depolarization and the subsequent large Ca2+ transient.
MATERIALS AND METHODS Preparation of cardiac cells Cardiac ventricular cells were isolated from 200 g male Wistar rats by the method previously described [30].
Measurement of [Ca2+1i and pHi in single cells The experiments on a single cardiac myocyte were performed with an epifluorescence Nikon Diaphot microscope. The cells were loaded in a Hepes buffer containing (in mM) NaCl 117,
Abbreviations used: [Ca2"]i, free cytosolic calcium concentration; pHp, intracellular pH; BAPTA, 1,2-bis-(O-aminophenoxy)ethane-NNNN''-tetraacetic acid;,CCCP, earbonyl cyanide m-chflorophenylhydrazone; DIDS, 4,4'-di-isothiocyanostilbene-2,2'-disulphonate; EIPA, ethylisopropylamiloride.
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KCl 5.7, MgCl2 1.7, NaH2PO4 1.2, NaHCO3 4.4, CaC12 1.8, Hepes 20, glucose 10 and 0.5 % BSA at pH 7.4, either with 4 uM-indo1/AM [31] for 15 min or with 5 juM-snarf-I/AM for 25 min at room temperature. The fluorescent probes were prepared in accordance with ref. [32], except that serum was omitted. After at least 1 h to allow for hydrolysis of the ester, the myocytes were placed in a small bath (500 ,l) on the stage of the microscope. The bath was then continuously washed at 1.5 ml/min with the different solutions. All experiments on single cells were performed at 32 °C in the same buffer as that used during the indicator loading procedure, except that BSA was omitted. A 100 W xenon lamp provided the illumination; the excitation intensity was, however, attenuated by two neutral-density filters to decrease photobleaching of the dyes. After passing through the excitation filter (360 nm for indo- 1 or 514 nm for snarf- 1), the light was reflected by a dichroic mirror (DM 400 for indo- 1 or DM 565 for snarf-l) and was focused on to the cell by a CF Fluor immersion x40 Nikon objective. The emitted light of longer wavelengths was transmitted by the dichroic mirror and reflected by a second one (DM 455 for indo- 1 or DM 605 for snarf- 1) to separate the two emission wavelengths with two filters (405 nm and 470-490 nm for indo- 1 or 580 nm and 640 nm for snarf-1) before they reached two photomultiplier tubes. The basal fluorescence of the medium and the autofluorescence of the cell were negligible; they were not detected by the photomultiplier tubes, owing to the low excitation intensity. The signals from the photomultiplier tubes were filtered at a frequency of 10 Hz, processed by a Compaq computer and simultaneously displayed on a video tape and a chart recorder. The computer program allowed us to calculate on line the ratio of the two wavelengths (405/480 nm for [Ca2ll1 measurement, or 640/580 nm for pH1 measurement) and to plot this ratio as a function of the time. Photographs of the indo- 1- and snarf- I-loaded cells revealed a quite homogeneous distribution of the probes into the cells. Calibration of pH; pHi was calibrated by the nigericin method [33]. The cell was superfused with Mes buffer at pH 5.5 or with Hepes buffer at pH 8.5 containing 140 mM-KCl, 1.7 mM-MgCl2, 1.2 mM-KH2PO4 and 10/lM-nigericin. The fluorescence ratio R (F640/F580) was calculated at each pH; pHi was then determined from the
equation:
pH, = PKa + log [(R - Rmin.)/(Rmax - R)] + log ,# Rmin and Rmax are respectively the fluorescence ratios measured at pH 5.5 and 8.5, and /3 is the ratio of fluorescence yield of the H-free/H-bound indicator at 640 nm. We assumed that the PKa of snarf is 7.6.
Calibration of ICa2+jj At the end of the experiment, the bath was washed with the Hepes solution without glucose, and 3 mM-amytal and 5, MCCCP were added to deplete the cellular ATP pool as described in [34]. When the cell became squared (rigor contracture), 5 /tMionomycin was added to the bath to obtain the maximum fluorescence ratio (Rmax ). The minimum fluorescence ratio (Rmin.) was obtained by bathing some cells for 1 h with 5 mM-EGTA and 5 /M-ionomycin. The Ca2+ concentration was calculated from the following
equation:
[Ca2]1i = Kd 3 (R-Rmin.)/(Rmax. - R) , is the ratio of fluorescence yield of the Ca2+-free/Ca2+-bound indicator at 480 nm, and K, is 250 nM. The absolute values of [Ca21], must be taken with caution because of the possible incomplete hydrolysis of the indo- 1 ester.
However, these values allowed us to compare the effects of ATP under different experimental conditions. Cell stimulation The cells were externally stimulated with a Teflon-coated 60 um tungsten electrode. A pulse of 2 V and 2 ms duration was applied to the cell at a frequency of 1 Hz. Measurement of membrane potential in cell suspension The membrane potential was recorded in a cell suspension with a Jobin Yvon JY3D fluorimeter by using the fluorescent dye bis-oxonol [35]. The cells were bathed in the same solution used during the loading protocol for indo-1 and snarf-1, except that BSA was omitted; 20 nm of the dye was added to the fluorimeter cuvette containing 4 x 105 myocytes. Experiments began after only 5 min of equilibration and redistribution of oxonol between intra- and extra-cellular medium. Monochromator wavelengths used were 540 nm for excitation and 580 nm for emission (both 4 nm slit width). Membrane potential was calibrated by adding KCI to the cell suspension from a 3 M stock solution to give different final concentrations. The potential was calculated as a function of extracellular KCI concentration by the Nernst equation, assuming an intracellular concentration of K+ of 140 mm [36]. Variations of fluorescence appeared to be a linear function of potential. These experiments were performed at 37 °C.
Materials DIDS, probenecid and bumetanide were obtained from Sigma. Indo-1/AM, BAPTA/AM, nigericin and ionomycin were purchased from Calbiochem, and snarf-1/AM and bis-oxonol were from Molecular Probes. Collagenase and hexokinase were obtained from Boehringer. All other chemicals were obtained from commercial sources. Statistical analysis Data are presented as means+S.E.M. Statistical significances were estimated by Student's t test. RESULTS General effects of ATP on ICa2l;i Under resting conditions, the [Ca2"], of single isolated cardiac cells was 69 + 5 nM (n = 17) (Table 1). Fig. 1 (a) shows that a fast application to the cell of a Hepes-buffered solution containing 10,uM-ATP first induced a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations. The damped oscillations formed a plateau at which [Ca2l], reached about 200 nm. The plateau was maintained even after removal of ATP. Then [Ca2+]i decreased within 1 min to 150 nm, a value significantly higher than the resting one, and recovered the basal value within 3-5 min. Similar results were obtained on electrically driven cells (1 Hz). After ATP application, the cell ceased to follow the stimulation and developed a few Ca2+ spikes at its own rhythm; then [Ca2+]i slowly decreased to 120 nm (Fig. lb). Similar effects of ATP on [Ca2+]i were observed when cells were superfused with a Krebs-Henseleit solution gassed with 02/CO2 (19: 1), with 20 mM-Hepes replaced by 20 mM-NaHCO3 (results not shown). Sources of Ca2+ mobilized by ATP The increase in [Ca2+]i could originate from various intracellular compartments and from Ca2+ entry. We first measured how much the sarcoplasmic reticulum could contribute, since ATP increases InsP3 [9], which in many tissues is reported to release Ca2+ from intracellular stores. When cells were incubated
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ATP-induced rise in HI, Ca2+ and depolarization
57
ICa2"Ii
with 10 M-ryanodine to prevent Ca2+ release from the sarcoplasmic reticulum, or when 10 mM-caffeine was applied to deplete the sarcoplasmic-reticulum Ca2+ load fully (as indicated by the ineffectiveness of a second application of the drug), ATP still triggered some Ca2` spikes, and the magnitude of the Ca2+ transient was unchanged (Table 1). However, under these conditions [Ca2+]1 rapidly returned to its basal value (Fig. 2a). This last result suggested that the cluster of Ca2+ oscillations which maintained a high [Ca2+]1 value during the plateau could be related to the release of Ca2+ from the sarcoplasmic reticulum [37]. To test this hypothesis further, in the experiment shown in Fig. 2(b) we applied 10 mM-caffeine to a cell after [Ca2+1] had reached the plateau after ATP application. Caffeine induced a large rise in [Ca2+]1, after which Ca2+ oscillations stopped and [Ca2+]1 rapidly fell back to its basal concentration. To reinforce the view that MgATP triggered a Ca2+ influx from the external medium, we used the 'Mn2+-quench' technique [38]. Fig. 3 shows that application of ATP to a single cell in a Ca2+-free solution containing 1 mM-MnCl2 induced a quenching of the indo-1 fluorescence at the two emission wavelengths. The superfusion of MnCl2 alone did not significantly alter the fluorescence, nor did it alter the caffeine-induced release of Ca2+ from intracellular stores. It might be expected that the initial large Ca2+ spikes triggered by ATP were related to the activation of voltage-dependent Ca2+
Table 1. Effects of MgATP on single-cardiac-cell
Single indo- l-loaded cells were rapidly superfused with 10 ztMgATP in control medium (first line), in the presence of 10 UMryanodine after preincubation for 30 min or of 10 mM-caffeine (second line), or after superfusion with a low-Ca2" (0.1 mM)/highMg2+ (3.4 mM) medium containing 1 ,sM-La3" after Ca2+ depletion ofthe sarcoplasmic reticulum with 10 mM-caffeine (third line). [Ca2+]i was determined as described in the Materials and methods section. Since after MgATP application [Ca2+], was not at a steady-state level, the magnitude of the Ca2+ transient was arbitrarily estimated at the top of the oscillations during the plateau or at the top of the spikes for ryanodine and caffeine experiments. Numbers of experiments performed in each experimental condition are given in parentheses: * significantly different from basal [Ca2+Jj (P < 0.02); ** significantly different from basal [Ca2+]i (P < 0.01).
[Ca2+]i (nM)
Control medium Caffeine, ryanodine
Low-Ca2+/high-Mg2+/La3+ medium
Basal
MgATP
69+ 5 (n= 17) 76+6 (n = 6) 47 + 3 (n = 4)
150+9** (n= 17) 140+ I1** (n = 6) 66+7* (n = 4)
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Fig. 1. Effect of MgATP on ICa2+li of single cardiac cells (a) A single quiescent indo-1-loaded cell was superfused with 10 ,tM-MgATP. (b) A single cell was electrically stimulated at a frequency of 1 Hz and superfused with 10 ,sM-MgATP. Data acquisition was performed at a frequency of 10 Hz. The inset shows more details of the same effect with data acquisition performed at 100 Hz. The right scale indicates the ratio of fluorescence at the two emitted wavelengths. [Ca2J], values shown on the left-hand scale were determined as described in the Materials and methods section.
Vol. 274
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channels. This was studied by preventing the influx of Ca 2+ ions through these channels. Bathing the cells in a solution containing 0.1 mM-Ca2+, 3.4 mm-Mg2+ and 1 pM-La3+ slightly decreased [Ca2+]1, and Ca2+ release from the sarcoplasmic reticulum by caffeine was also less (Fig. 4a). The further application of ATP still induced a Ca2+ transient, but of a low amplitude (Table 1). This Ca2+ transient could not be related to membrane depolarization, since the addition of 30 mM-KCI was ineffective (Fig. 4a). This latter experiment indicated that Ca2+ ions should be released from an internal Ca2+ store, and that this store is not the sarcoplasmic reticulum. To examine whether internal Ca2+ release, whatever its origin, could be the trigger for the large Ca2+ spikes, we loaded cells for 30 min with 25 /M of the fast Ca2+ chelator BAPTA. Under these conditions, as illustrated in Fig. 4(b), ATP did not increase [Ca2+]i; the application of caffeine was also ineffective, indicating that intracellular Ca2+ release was well buffered by BAPTA. Only a depolarization induced by 30 mM-KCI was able to increase [Ca2+]i slightly, and [Ca2+], returned to its basal value when KCI was washed. Relationship between ICa2+1j and pH; We previously showed that ATP induces a transient acidification by activating the HCO3 /Cl- exchanger [28]. The effects (a)
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Fig. 2. Effect of caffeine on Ca2l transient induced by MgATP in single cardiac cell (a) The rapid application of 10 mM-caffeine to a single indo-l-loaded cell induced release of Ca2l from the sarcoplasmic reticulum. The cell was then superfused with 10 uM-MgATP. (b) The cell was superfused with 10 1sM-MgATP; when [Ca2l], had reached a plateau, 10 mM-caffeine was rapidly applied to the myocyte. Similar results were obtained with four other cells in each case.
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Fig. 4. Effects of MgATP on JCa2+jj of a single cardiac cell superfused with a low-Ca2+/high-Mg2+ medium and on ICa2+li of a BAPTA-loaded cell
Fig. 3. Effect of MgATP on the fluorescence of an indo-l-loaded single cell superfused with a Ca2l-free buffer containing MnCl2 A single indo- 1-loaded cell was superfused with a Ca2"-free medium (no Ca2" added) containing mM-MnCl2. The changes in fluorescence were recorded at the two emitted wavelengths when 10 mMcaffeine and 10 ,sM-MgATP were successively applied to the myocyte. The right-hand scale indicates the change in fluorescence recorded by the photomultipliers (in mV). Similar results were obtained with three other cells.
(a) A single indo- 1-loaded cardiac cell was superfused with a lowCa2+ (0.1 mM)/high-Mg2" (3.4 mM) medium containing 1 /uM-La3+. After 10 mM-caffeine was transiently applied to the cell to deplete the sarcoplasmic-reticulum Ca2+ load, 10 ,uM-MgATP and 30 mMKCI were successively added to the cell. (b) Indo- 1-loaded myocytes were further incubated for 30 min in the presence of 25 4MBAPTA/AM. After 1 h to allow the hydrolysis of the ester into the cell, a single myocyte was successively superfused with a Hepesbuffered solution (1.8 mM-CaCl2) containing 1O ,/M-MgATP, 1O mmcaffeine or 30 mM-KCl. Similar results four cells.
were
obtained with at least
1991
ATP-induced rise in HI+ Ca2' and depolarization (a)
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30s Fig. 5. Effects of ATP on single-cardiac-cell pH1 and [Ca2+]i (a) A single snarf-l-loaded cell was superfused with a Mg2+-free Hepes buffer; 10 1zM-ATP was then applied to the cell. After switching to the usual solution containing 1.7 mM-MgCl2, the change in pH1 was recorded in response to 10 /LM-MgATP. The right-hand scale indicates the ratio of snarf-1 fluorescence at the two emitted
wavelengths. The left-hand scale shows values of pHi determined as described in the Materials and methods section. (b) A single indo-1loaded cell was superfused with a Mg2+-free medium; 10 ,M-ATP was then added to the cell. After switching to a medium containing 1.7 mM-MgCl2, the changes in [Ca21]i were recorded in response to 1O #M-MgATP. Similar results were obtained in two other experiments in both cases.
of ATP on [Ca2+], share the same characteristics as the ATPinduced acidification. Figs. 5(a) and 5(b) show that omitting Mg2+ ions from the bathing solution prevented both the acidification and the Ca2' rise elicited by ATP, whereas the nucleotide became effective after re-admission of Mg2 The hydrolysis-resistant analogues adenosine 5'-[y-thio]triphosphate (ATP[S]), adenosine 5'-[/Jy-methylene]triphosphate, adenosine 5'-[a,-methylene]triphosphate and adenosine 5'-[/?yimido]triphosphate in the presence of Mg2+ were able to reproduce the effects of ATP on ionic movements when used at 100 or 500 ZM. Other nucleotides, ADP (in the presence of hexokinase to prevent the formation of ATP by ecto-kinases, as previously reported in Ehrlich ascites-tumour cells [10]) and AMP, as well as GTP, ITP, CTP, UTP, all used at 100 /M, were ineffective. Some examples are illustrated in Fig. 6. No acidification was induced when ATP was applied in a Cl--free medium, nor was [Ca2+]1 changed. However, the nucleotide was effective when Clions were re-admitted (Figs. 7a and 7b). Preincubation of myocytes for 30 min in the presence of 200 ZM of the stilbene derivative DIDS or with 100 ,uM-probenecid (two inhibitors of the HCO3-/CI- exchanger) prevented both the acidification and the Ca2+ rise on ATP application (Figs. 7c and 7d), but the caffeine-induced Ca2+ release from sarcoplasmic reticulum was not altered by the drug (Fig. 7d). To examine whether the Ca2+ rise could be responsible for the acidification, as suggested in several tissues following agonist stimulation [39,40], we investigated the effects of ATP on pHi .
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Fig. 6. Effects of different nucleotides on single-cardiac-cell jCa2l;j and pH; (a) Changes in [Ca2ll] were recorded on a single indo-l-loaded cell successively superfused with 1000,M-MgGTP and lO0 sMMgATP[SJ; 10 mM-caffeine was then applied to the myocyte to stop the Ca2"-induced release of Ca2" from the sarcoplasmic reticulum. (b) Changes in cell [Ca2+]i were recorded in response to 100 ,sM-MgADP in the presence of 10 units of hexokinase/ml and then to 10 1uMMgATP. (c) Changes in pHi were recorded in the presence of 10 /SMEIPA on a single snarf-l-loaded cell successively superfused with 100/lM-MgADP in the presence of 10 units of hexokinase/ml, 100 ,#M-MgGTP and 10 ,uM-MgATP. Similar results were obtained in two other experiments for [Ca2"], and pH,.
after the influx of Ca2l had been prevented (in a Ca2l-free medium containing 50 /eM-EGTA, or in a low-Ca2l high-Mg2+ solution containing La3+). Under these experimental conditions, the nucleotide triggered an acidification of the same magnitude as in medium containing CaCl2 (Table 2). ATP was also as potent when, furthermore, the cells were loaded with BAPTA to buffer the release of Ca2+ from intracellular stores (Fig. 8). Effects of nigericin on pH1 and [Ca2+I1 In order to investigate whether the acidification induced by ATP could be responsible for the increase in [Ca2+]1, cell acidification was induced by the K+-H+ ionophore nigericin. A low dose (2 ,g/ml) of nigericin decreased pH, by the same magnitude as 10,uM-ATP did (Fig. 9a). A similar application of the ionophore to an indo- 1-loaded cell triggered some Ca2+ spikes followed by a rapid Ca2+ transient. The [Ca2+], reached 142+16 mM (basal [Ca2+]1 was 62+ 12 nm in this series; n = 5).
Effects of ATP on membrane potential The membrane potential was monitored with the fluorescent dye bis-oxonol. Fig. 10(a) shows that ATP added to a cell suspension (final concn. 10 uM) induced a depolarization (26+ 2.5 mV; n = 12). When cells were bathed in a Ca2+-free medium,
60
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Fig. 7. Effects of MgATP on cardiac-cell pH; and ICa2]i; in a Cl--free buffer or after preincubation of the cells in the presence of DIDS (a) A snarf- 1-loaded cell was superfused with a Cl--free medium (sodium gluconate replaced NaCl, and KCl, CaCl2 and MgCl2 were respectively replaced by KHCO3, CaCO3 and MgSO4) added with 10 ,sM-EIPA and 10 ,uM-bumetanide; the increase in pHi is due to the influx of HCO3- ions in exchange for Cl- efflux. When pHi had reached a steady state, 10 /StM-MgATP was applied to the cell without effect. The cell was then switched to a medium containing 125 mM-Cl-, 10 ,#M-EIPA and 10 ,tM-bumetanide; when pHi had returned to its basal value, 10 /LM-MgATP was added to the cell. (b) An indo- 1-loaded cell was superfused with a Cl--free medium [same composition as in (a)] for at least 2 min before application of 10 uM-MgATP. After switching to a medium containing 125 mM-Cl-, the changes in [Ca21]i were recorded in response to 10 /LM-MgATP. (c) MgATP (10 ,UM) was applied in the presence of 10 /LM-EIPA to a single snarf-l-loaded cell preincubated for 30 min with 200 /LM-DIDS. (d) MgATP (10 /,M) and 10 mM-caffeine were successively added to a single indo- 1-loaded cell preincubated for 30 min with 200 ,#M-DIDS. Experiments shown in (a)H(d) were performed on four different cells. Similar results were obtained in each experimental condition with at least three cells. Table 2. Effects of MgATP EIPA
on
single-cardiac-cell pH; in the presence of
7.1
Single snarf-l-loaded cells were superfused with 1O ,zM-MgATP in the presence of 10 #uM-EIPA in control medium or either in a lowCa2" (0.1 mM)/high-Mg2" (3.4 mM) medium containing 1 ,M La3" or
in
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7.0
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Basal
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Control medium
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Ca2"-free medium
(n = 8) 6.92+0.06 (n = 9)
6.71 +0.13** (n = 8) 6.63 +0.05**
(n
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buffer added with 50 ,tM-EGTA. These two last
experimental conditions gave similar results (Ca2"-free + EGTA, 3 cells; low-Ca2+/high-Mg2+/La3+, 6 cells). They were pooled in the second line of the Table (Ca2`-free medium). pH, was determined as described in the Materials and methods section. Numbers of experiments performed in each experimental condition are given in parentheses: ** significantly different from basal pH, (P < 0.01).
MgATP
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Fig. 8. Effect of MgATP on pH. of a single cardiac cell loaded with BAPTA Snarf- 1-loaded cells were incubated for 30 min in the presence of 25 /tM-BAPTA/AM. After 1 h to allow the hydrolysis of the ester, the change in pH, of a single cell was recorded in response to 10 /LMMgATP in a Ca2"-free buffer added with 50 1zM-EGTA and 10 /uMEIPA. Similar results were obtained in two other experiments.
hand, under each of these experimental conditions KCl (20 mM) still able to depolarize the myocytes, as it did in control conditions (Fig. 10b).
9)
was
ATP still triggered a depolarization of similar amplitude (results not shown). The effects of ATP on the membrane potential
were
tested under some of the experimental conditions in which ATP failed to trigger both acidification and Ca2+ transient. Thus, when cells were suspended in a Mg2+-free or a Cl--free medium, ATP did not alter the membrane potential. The nucleotide was also unable to induce a depolarization in the presence of probenecid (100,UM, cell suspension preincubated for 30 min). Pretreatment of cells in the presence of DIDS (200,uM) also suppressed the depolarization elicited by ATP. On the other
DISCUSSION The major experimental findings are that extracellular ATP induces simultaneous changes in intracellular Ca2 pH and membrane potential. These confirm and extend previous results. Furthermore, the principal aim of this work was to investigate the relationships between these changes and to propose a mechanism by which ATP triggers the variations in [Ca2+]i. The rapid depolarization, acidification and Ca2+ rise were elicited by ,
1991
ATP-induced rise in H+, Ca2+ and depolarization (a)
61
the same agonists (ATP and its poorly hydrolysable analogues). They required the presence of extracellular Mg2", ruling out a non-selective membrane-permeabilization effect of ATP such as occurs, for example, in mast cells [41]. The ionic movements mediated by the nucleotide appeared with similar apparent kinetics; their maximal value was reached within a few tens of
Nigericin
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0.9 15s
(b) 250 pI
Nigericin
3.5
seconds. Our results provide evidence that acidification, following activation of the HCO3 /C1- exchanger [28], is the primary event induced by MgATP. Experimental conditions designed to suppress acidification (Cl--free medium, DIDS, probenecid) also prevented the transient increase in [Ca2+], and the depolarization. In contrast, the conditions used to suppress the rapid rise in [Ca2+]1 by buffering the intracellular Ca2+ release with BAPTA and/or by preventing the Ca2+ influx both through the Ca2+ conductance and the exchange (Ca2+-free or low-Ca2+ medium still allow MgATP to trigger containing high-Mg2+ an acidification with the same magnitude. This acidification is associated with a small but consistent increase in [Ca2+], even when Ca2+ entry was prevented in a lowCa2+ high-Mg2+ medium containing La3+, as indicated by the rise (Fig. 4a) and when the inability of KCI to induce a sarcoplasmic reticulum was unloaded by a high concentration of caffeine. This result suggests that MgATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum. It is proposed that Ca2+ ions are displaced by protons from cation-binding sites on, or close to, the sarcolemma. It is generally agreed that H+ and Ca2+ ions compete for the same binding sites [42-44]. An exchange of Ca2+ for H+ at common binding sites other than sarcoplasmic reticulum has already been reported to be in part responsible for the Ca2+ rise triggered by acidification [45]. The experiment using the ionophore nigericin, showing that transient, is in an acidification could also induce a rapid good agreement with these data. Furthermore, it has been demonstrated that a decrease in pHi facilitates the spontaneous Ca2+ release from the sarcoplasmic reticulum [46]. The existence of such a localized release of Ca2+ from the junctional sarcoplasmic reticulum close to the sarcolemma by the MgATPinduced acidification was tentatively excluded, since a single bolus application of 10 mM-caffeine was proved to be sufficient to deplete fully the sarcoplasmic-reticulum Ca2+ load. This localized increase in Ca2+ ions can trigger a transient inward current [23], owing to an increase in a non-selective
Na+/Ca2+
i 1501-
C4
a
c
3.0
"
C.)
Ca2+
2.5
501L 15s Fig. 9. Effects of a low dose of nigericin on a single-cell pH1 and (a) Variations in pHi of a single cell loaded with snarf-l/AM were recorded in response to 2,ug of nigericin/ml. (b) Changes in [Ca2+]i
ICaz+li
of a single cell loaded with indo- 1/AM were monitored in response to 2 jug of nigericin/ml. Similar results were obtained in four other
experiments.
Ca2+
(a) MgATP
0 L~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ C 0)
I~ ~~~~I 0
La3+)
c.)
0
1U
2min
m11I (b)
a)0
KCI
,IN
PIh1
|
c
i
(04)
C.)
0 0
2min
Fig. 10. Effect of MgATP on membrane potential of cardiac cells The fluorescent indicator bis-oxonol (20 nM) was added to 1 ml of the suspension of cells bathed in normal Hepes buffer as described in the Materials and methods section. (a) Changes in fluorescence were recorded in response to 10 ,uM-MgATP. The increase in fluorescence giving an upward deflection indicates a depolarization. (b) Changes in fluorescence induced by adding KCI (final concn. 20 mM) allowed for calibration (see the Materials and methods section). The right-hand scales indicate fluorescence changes in arbitrary units (a.u.) and estimated membrane potential changes in mV.
Vol. 274
cationic conductance. This was first observed in cardiac cells and reported to be sensitive to Ca2+ concentration as low as 100 nm [47,48]. The opening of this conductance could lead to depolarization, as indicated by the potential-sensitive dye bis-oxonol (Fig. 10) above the action-potential threshold. The depolarization thus induced triggers the opening of the voltage-dependent Ca2+ channels and will lead to automaticity and arrhythmias. During these spikes Ca2+ ions accumulate and add to those released by acidification. This hypothesis is well supported by the 'Mn quench' experiments. Indeed, the application of MnCl2 alone to the cell did not affect the indo-1 fluorescence, whereas a rapid quenching occurred when MgATP was applied. These observations are in full agreement with the facts first that Mn2+ ions do not passively enter the cell and secondly that they pass through voltage-dependent Ca2+ channels [49]. On the other hand, when the localized increase in Ca2+ was buffered by loading the cells with BAPTA, MgATP was unable to trigger the initial Ca2+ spikes. MgATP transiently increased the cellular Ca2+ concentration from 70 to 150 nm in a single cell. Ryanodine and caffeine, two compounds which prevent Ca2+ release from the sarcoplasmic reticulum, did not affect the magnitude of the initial rapid Ca2+ rise induced by MgATP, but significantly shortened the Ca 21
M. Puceat and others
62 plateau by preventing the cluster of Ca2' oscillations. Moreover, further application of caffeine during the Ca2+ plateau after MgATP addition led to a rapid decrease in [Ca2+]i. These data confirm that the sarcoplasmic reticulum is not the initial source of Ca2+ mobilized by MgATP as suggested above. They also indicate that the nucleotide mobilizes Ca2+ ions from this intracellular compartment by triggering the Ca2+-induced Ca2+ release. It should be taken into account that such a release would be facilitated by the simultaneous increase in InsP3. It has been demonstrated that ATP increases turnover of phosphoinositides [9] and that InsP3 strongly potentiated the spontaneous release of Ca2+ which occurs when the sarcoplasmic reticulum is overloaded with Ca2+ [50]. In conclusion, adenine nucleotides have pharmacological effects which are generally attributable to stimulation of the P1or P2-purinergic receptors on a variety of tissues. On the other hand, in our study, ionic movements mediated by MgATP appear to be highly specific to the nucleotide and to its hydrolysisresistant analogues; moreover, they require the presence of Mg2+ ions. These observations are inconsistent with the pharmacology of the P1- and P2-purinergic receptors as previously described [51]. Our results are in favour of the existence of a novel purinergic receptor with specific agonists and biochemical pathways. The Ca2+ rise as previously reported by others [7,24,26,27] and during the present work has at least three origins. (1) The increase in H+, induced by ATP activation of the HCO3 /Cl1 exchanger, displaces Ca2+ ions from internal binding sites. (2) These Ca2+ ions open a non-specific membrane conductance which, by depolarizing the cell, triggers spiking and thus opening of voltage-gated Ca2+ channels. This leads to the initial large rise in [Ca2+]i. (3) This increase in [Ca2+]1, together with the facilitation owing to the InsP3 increase, induces repetitive Ca2+ release from the sarcoplasmic reticulum which determines the plateau Ca2+ rise. The physiological consequences of MgATP released by nerve stimulation or during circulatory shock can thus vary from vasodilatation [52] and increase in myocardial contractility [7-9] to the deleterious ventricular arrhythmias expected from the above results. We thank Dr. B. Berthon for discussion and critical reading of previous manuscripts, P. Lechene for skilful computer programming and D. Angelini for secretarial assistance.
REFERENCES 1. Burnstock, G. (1981) J. Physiol. (London) 313, 1-35 2. Forrester, T. & Williams, C. A. (1977) J. Physiol. (London) 268, 371-390 3. Mohabir, R., Lee, H.-C. & Clusin, W. T. (1988) J. Gen. Physiol. 92, 48a (Abstr.) 4. Burnstock, G. & Meghji, P. (1983) J. Pharmacol. 79, 211-218 5. Kurachi, Y., Nakajima, T. & Sugimoto, T. (1986) Pfluigers Arch. 407, 264-274 6. Bellardinelli, L. & Isenberg, G. (1983) Am. J. Physiol. 244, H734H737 7. Danziger, R. S., Rafffaeli, S., Moreno-Sanchez, R., Sakai, M., Capogrossi, M., Spurgeon, H. A., Hansford, R. G. & Lakatta, E. G. (1988) Cell Calcium 9, 193-199 8. Scamps, F., Legssyer, A., Mayoux, E. & Vassort, G. (1990) Circ. Res. 67, 1007-1016 9. Legssyer, A., Poggioli, J., Renard, D. & Vassort, G. (1988) J. Physiol. (London) 401, 185-199 10. Dubyak, G. R. & De Young, M. B. (1985) J. Biol. Chem. 260, 10653-10661 11. Phaneuf, S., Berta, P., Casanova, J. & Cavadore, J. C. (1987) Biochem. Biophys. Res. Commun. 143, 454-460
12. Kuroki, M., Takeshige, K. & Minakami, S. (1989) Biochim. Biophys. Acta 1012, 103-106 13. Kitazono, T., Takeshige, K., Cragoe, E. J., Jr. & Minakami, S. (1989) Biochim. Biophys. Acta 1013, 152-158 14. El Moatassim, C., Maurice, T., Mani, J. C. & Dornand, J. (1989) FEBS Lett. 242, 391-396 15. Sung, S.-S. J., Young, J. D.-E., Origlio, A.-M., Heiple, J. M., Kaback, H. R. & Silverstein, S. C. (1985) J. Biol. Chem. 260, 13442-13449 16. Hume, R. I. & Thomas, S. A. (1988) J. Physiol. (London) 406, 503-524 17. Igusa, Y. (1988) J. Physiol. (London) 405, 169-185 18. Eriksson, H. & Heilbronn, E. (1989) Biochem. Biophys. Res. Commun. 159, 878-885 19. Benham, C. D. & Tsien, R. W. (1987) Nature (London) 328, 275-278 20. Honore, C., Martin, C., Mironneau, C. & Mironneau, J. (1989) Am. J. Physiol. 257, C297-C305 21. Sasaki, T. & Gallacher, D. V. (1990) FEBS Lett. 264, 130-134 22. Friel, D. D. & Bean, B. P. (1988) J. Gen. Physiol. 91, 1-27 23. Scamps, F. & Vassort, G. (1990) Pfluigers Arch., in the press 24. Sharma, V. K. & Sheu, S.-S. (1986) Biophys. J. 49, 351a (abstr.) 25. De Young, M. B. & Scarpa, A. (1987) FEBS Lett. 223, 53-58 26. De Young, M. B. & Scarpa, A. (1989) Am. J. Physiol. 257,
C750-C758 27. Bjornsson, 0. G., Monck, J. R. & Williamson, J. R. (1989) Eur. J. Biochem. 186, 395-404 28. Puceat, M., Clement, 0. & Vassort, G. (1990) J. Physiol. (London) 429, 25P (abstr.) 29. Wallert, M. A. & Fr6hlich, 0. (1989) Biophys. J. 55, 287a (abstr.) 30. Puceat, M., Clment, O., Lechene, P., Pelosin, J. M., VenturaClapier, R. & Vassort, G. (1990) Circ. Res. 67, 517-524 31. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 32. Poenie, M., Alderton, J., Steinhardt, R. & Tsien, R. (1986) Science 233, 886-889 33. Thomas, J. A., Buchsbaum, R. N., Zimmiak, A. & Racker, E. (1979) Biochemistry 18, 2210-2218 34. Li, Q., Altschuld, R. A. & Stokes, B. T. (1987) Biochem. Biophys. Res. Commun. 147, 120-126 35. Smith, J. C., Russ, P., Cooperman, B. S. & Chance, B. (1976) Biochemistry 15, 5094-5105 36. Meier, C. F., Jr., Briggs, G. M. & Claycomb, W. C. (1986) Am. J. Physiol. 250, H731-H735 37. Fabiato, A. & Fabiato, F. (1975) J. Physiol. (London) 249, 469495 38. Hallam, T. J. & Rink, T. J. (1985) FEBS Lett. 186, 175-179 39. Zavoico, G. B., Cragoe, E. J. & Feinstein, M. B. (1986) J. Biol. Chem. 261, 13160-13167 40. Berck, B. C., Brock, T. A., Gimbrone, M. A., Jr. & Alexander, R. (1987) J. Biol. Chem. 262, 5065-5072 41. Cockcroft, S. & Gomperts, B. D. (1979) Nature (London) 279, 541-542 42. Ellis, D., Deitmer, J. W. & Bers, D. M. (1981) in Progress in Enzyme and Ion-Selective Electrodes (Lubbers, D. W., Acker, H., Buck, R. P., Eisenman, G., Kessler, M. & Simon, W., eds.), pp. 148-155, Springer Verlag, Heidelberg 43. Vaughan-Jones, R. D., Lederer, W. J. & Eisner, D. A. (1983) Nature (London) 301, 522-524 44. Mullins, L. J., Tiffert, T., Vassort, G. & Whittemburry, J. (1983) J. Physiol. (London) 338, 295-319 45. Kohmoto, O., Spitzer, K. W., Movsesian, M. A. & Barry, W. H. (1990) Circ. Res. 66, 622-632 46. Orchard, C. H., Houser, S. R., Kort, A. A., Bahinski, A., Capogrossi, M. C. & Lakatta, E. G. (1987) J. Gen. Physiol. 90, 145-165 47. Colquhoun, D., Neher, E., Reuter, H. & Stevens, C. F. (1981) Nature (London) 294, 752-754 48. Hill, J. A., Jr., Coronado, R. & Strauss, H. C. (1988) Circ. Res. 62, 411-415 49. Anderson, M. (1983) J. Gen. Physiol. 81, 805-807 50. Fabiato, A. (1986) Biophys. J. 49, 190a (abstr.) 51. Burnstock, G. (1978) in Cell Membrane Receptors for Drugs and Hormones: a Multidisciplinary Approach (Straub, R. W. & Bolis, L., eds.), pp. 107-118, Raven Press, New York -52. Gordon, J. L. (1986) Biochem. J. 233, 309-319
Received 24 August 1990/29 October 1990; accepted 30 October 1990
1991
55
Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes Michel PUCEAT, Odile CLEMENT, Frederique SCAMPS and Guy VASSORT Laboratoire de Physiologie Cellulaire Cardiaque, INSERM U-241, Universite Paris-Sud, Bat. 443, 91405 Orsay Cedex, France
The origin of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) induced by extracellular ATP was investigated in single isolated cardiac myocytes loaded with indo- 1. The nucleotide added at a concentration of 10 uM triggers a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations, increasing [Ca2+]1 to around 200 nm from a basal value of 70 nm. Neither caffeine nor ryanodine affects the magnitude of the Ca2+ transient, but both shorten it by preventing the Ca2+ oscillations. This indicates that the latter must be related to the release of Ca2+ from the sarcoplasmic reticulum. Since ATP also induces cell depolarization (as shown by experiments using the potential sensitive dye bis-oxonol), the initial Ca2+ spikes were attributed to the opening of voltage-dependent Ca2+ channels. A small Ca2+ transient still remains under experimental conditions designed to prevent Ca2+ influx from external medium (low-Ca2+ high-Mg2+ medium containing La3+) and after depletion of the sarcoplasmic-reticulum Ca2+ load with caffeine. Under these conditions, when this Ca2+ transient was buffered by 1,2-bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid, ATP was unable to trigger the initial Ca2+ spikes. These results indicate that ATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum and that this Ca2+ release is responsible for the depolarization. The effects of ATP on [Ca2+]i share the same characteristics as the acidification simultaneously induced by the nucleotide (as shown by experiments using the pH-sensitive probe snarf-1). These ionic variations are highly specific to ATP and its hydrolysis-resistant analogues. They both require the presence of Mg2+ and Cl- ions in the extracellular medium, and they are prevented by pretreatment of the cells with 4,4'-di-isothiocyanostilbene or probenecid. These results suggest that: (1) the ATP-induced acidification leads to displacement of Ca2+ ions from or close to the internal face of sarcolemma; (2) the Ca2+ ions activate a nonspecific membrane conductance responsible for the depolarization of the cells; (3) the depolarization leads to a Ca2+ influx, owing to the opening of the voltage-dependent Ca2+ channels; (4) this increase in Ca2+ triggers the release of Ca2+ from the sarcoplasmic reticulum, which is facilitated by the increase in inositol trisphosphate following P2-purinergic stimulation.
INTRODUCTION ATP has been reported to be a neurotransmitter released by sympathetic nerves [1]. Its concentration can also increase significantly in the coronary vessels during cardiac hypoxia [2], and could be responsible for the early increase in intracellular Ca2+ during ischaemia [3]. The effects of purinergic agonists on cardiac contractility are well described. Adenosine and ATP decrease mechanical activity of the atria, probably by stimulating P,-purinergic receptors coupled via G-proteins to both adenylate cyclase and K+ conductance [4-6]. ATP also induces a positive
found only a slow rise in intracellular Ca2". More recently, ATP demonstrated to trigger a membrane depolarization associated with the rapid Ca2+ transient [27]. These effects of ATP on [Ca2+]i were reported in cells in suspension, and the mechanism by which ATP induces this Ca2+ transient remains unclear. We previously showed that ATP applied to a single cardiac cell induced a transient acidification, owing to the activation of the HCO3-/Cl- exchanger [28], which is followed by an alkalinization due to the activation of the Na+/H+ antiport [28,29]. The purpose
inotropic effect on the rat ventricle [7,8] by activation of P2purinergic receptors leading to the hydrolysis of phospho-
a
inositides [9]. -Several effects of ATP were reported in various tissues. ATP was shown to release Ca2+ from intracellular stores sensitive to InsP3 [10-12]. This rise in [Ca21]i is associated with biphasic changes in cytoplasmic pH in endothelial cells [13]. Furthermore, in the same study, it was shown that the nucleotide also increased membrane ionic permeabilities as well as it does in thymocytes [14] and in macrophages [15]. More particularly, ATP activates a cationic conductance in cultured muscle cells [16-18], in smooth-muscle cells [19,20] and in lacrimal acinar cells [21]. Some of these effects of ATP were also reported in isolated cardiac ventricular myocytes. Thus the nucleotide also induces a depolarization due to a non-specific inward current in cardiac cells [22,23]. Moreover, Sharma & Sheu [24] and De Young & Scarpa [25,26] showed that ATP triggered a large Ca2+ transient, followed by a weaker sustained increase, whereas others [7]
was
of this study was to characterize the effects of ATP on [Ca2+]i in single cardiac cell and to examine the relationship between
changes in
pH, and [Ca2+]1. We propose a mechanism by which the ATP-induced acidification triggers the release of Ca2+ from intracellular stores other than the sarcoplasmic reticulum. This Ca2+ release leads to the opening of a non-specific conductance responsible for cell depolarization and the subsequent large Ca2+ transient.
MATERIALS AND METHODS Preparation of cardiac cells Cardiac ventricular cells were isolated from 200 g male Wistar rats by the method previously described [30].
Measurement of [Ca2+1i and pHi in single cells The experiments on a single cardiac myocyte were performed with an epifluorescence Nikon Diaphot microscope. The cells were loaded in a Hepes buffer containing (in mM) NaCl 117,
Abbreviations used: [Ca2"]i, free cytosolic calcium concentration; pHp, intracellular pH; BAPTA, 1,2-bis-(O-aminophenoxy)ethane-NNNN''-tetraacetic acid;,CCCP, earbonyl cyanide m-chflorophenylhydrazone; DIDS, 4,4'-di-isothiocyanostilbene-2,2'-disulphonate; EIPA, ethylisopropylamiloride.
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M. Puceat and others
KCl 5.7, MgCl2 1.7, NaH2PO4 1.2, NaHCO3 4.4, CaC12 1.8, Hepes 20, glucose 10 and 0.5 % BSA at pH 7.4, either with 4 uM-indo1/AM [31] for 15 min or with 5 juM-snarf-I/AM for 25 min at room temperature. The fluorescent probes were prepared in accordance with ref. [32], except that serum was omitted. After at least 1 h to allow for hydrolysis of the ester, the myocytes were placed in a small bath (500 ,l) on the stage of the microscope. The bath was then continuously washed at 1.5 ml/min with the different solutions. All experiments on single cells were performed at 32 °C in the same buffer as that used during the indicator loading procedure, except that BSA was omitted. A 100 W xenon lamp provided the illumination; the excitation intensity was, however, attenuated by two neutral-density filters to decrease photobleaching of the dyes. After passing through the excitation filter (360 nm for indo- 1 or 514 nm for snarf- 1), the light was reflected by a dichroic mirror (DM 400 for indo- 1 or DM 565 for snarf-l) and was focused on to the cell by a CF Fluor immersion x40 Nikon objective. The emitted light of longer wavelengths was transmitted by the dichroic mirror and reflected by a second one (DM 455 for indo- 1 or DM 605 for snarf- 1) to separate the two emission wavelengths with two filters (405 nm and 470-490 nm for indo- 1 or 580 nm and 640 nm for snarf-1) before they reached two photomultiplier tubes. The basal fluorescence of the medium and the autofluorescence of the cell were negligible; they were not detected by the photomultiplier tubes, owing to the low excitation intensity. The signals from the photomultiplier tubes were filtered at a frequency of 10 Hz, processed by a Compaq computer and simultaneously displayed on a video tape and a chart recorder. The computer program allowed us to calculate on line the ratio of the two wavelengths (405/480 nm for [Ca2ll1 measurement, or 640/580 nm for pH1 measurement) and to plot this ratio as a function of the time. Photographs of the indo- 1- and snarf- I-loaded cells revealed a quite homogeneous distribution of the probes into the cells. Calibration of pH; pHi was calibrated by the nigericin method [33]. The cell was superfused with Mes buffer at pH 5.5 or with Hepes buffer at pH 8.5 containing 140 mM-KCl, 1.7 mM-MgCl2, 1.2 mM-KH2PO4 and 10/lM-nigericin. The fluorescence ratio R (F640/F580) was calculated at each pH; pHi was then determined from the
equation:
pH, = PKa + log [(R - Rmin.)/(Rmax - R)] + log ,# Rmin and Rmax are respectively the fluorescence ratios measured at pH 5.5 and 8.5, and /3 is the ratio of fluorescence yield of the H-free/H-bound indicator at 640 nm. We assumed that the PKa of snarf is 7.6.
Calibration of ICa2+jj At the end of the experiment, the bath was washed with the Hepes solution without glucose, and 3 mM-amytal and 5, MCCCP were added to deplete the cellular ATP pool as described in [34]. When the cell became squared (rigor contracture), 5 /tMionomycin was added to the bath to obtain the maximum fluorescence ratio (Rmax ). The minimum fluorescence ratio (Rmin.) was obtained by bathing some cells for 1 h with 5 mM-EGTA and 5 /M-ionomycin. The Ca2+ concentration was calculated from the following
equation:
[Ca2]1i = Kd 3 (R-Rmin.)/(Rmax. - R) , is the ratio of fluorescence yield of the Ca2+-free/Ca2+-bound indicator at 480 nm, and K, is 250 nM. The absolute values of [Ca21], must be taken with caution because of the possible incomplete hydrolysis of the indo- 1 ester.
However, these values allowed us to compare the effects of ATP under different experimental conditions. Cell stimulation The cells were externally stimulated with a Teflon-coated 60 um tungsten electrode. A pulse of 2 V and 2 ms duration was applied to the cell at a frequency of 1 Hz. Measurement of membrane potential in cell suspension The membrane potential was recorded in a cell suspension with a Jobin Yvon JY3D fluorimeter by using the fluorescent dye bis-oxonol [35]. The cells were bathed in the same solution used during the loading protocol for indo-1 and snarf-1, except that BSA was omitted; 20 nm of the dye was added to the fluorimeter cuvette containing 4 x 105 myocytes. Experiments began after only 5 min of equilibration and redistribution of oxonol between intra- and extra-cellular medium. Monochromator wavelengths used were 540 nm for excitation and 580 nm for emission (both 4 nm slit width). Membrane potential was calibrated by adding KCI to the cell suspension from a 3 M stock solution to give different final concentrations. The potential was calculated as a function of extracellular KCI concentration by the Nernst equation, assuming an intracellular concentration of K+ of 140 mm [36]. Variations of fluorescence appeared to be a linear function of potential. These experiments were performed at 37 °C.
Materials DIDS, probenecid and bumetanide were obtained from Sigma. Indo-1/AM, BAPTA/AM, nigericin and ionomycin were purchased from Calbiochem, and snarf-1/AM and bis-oxonol were from Molecular Probes. Collagenase and hexokinase were obtained from Boehringer. All other chemicals were obtained from commercial sources. Statistical analysis Data are presented as means+S.E.M. Statistical significances were estimated by Student's t test. RESULTS General effects of ATP on ICa2l;i Under resting conditions, the [Ca2"], of single isolated cardiac cells was 69 + 5 nM (n = 17) (Table 1). Fig. 1 (a) shows that a fast application to the cell of a Hepes-buffered solution containing 10,uM-ATP first induced a few Ca2+ spikes, followed by a cluster of Ca2+ oscillations. The damped oscillations formed a plateau at which [Ca2l], reached about 200 nm. The plateau was maintained even after removal of ATP. Then [Ca2+]i decreased within 1 min to 150 nm, a value significantly higher than the resting one, and recovered the basal value within 3-5 min. Similar results were obtained on electrically driven cells (1 Hz). After ATP application, the cell ceased to follow the stimulation and developed a few Ca2+ spikes at its own rhythm; then [Ca2+]i slowly decreased to 120 nm (Fig. lb). Similar effects of ATP on [Ca2+]i were observed when cells were superfused with a Krebs-Henseleit solution gassed with 02/CO2 (19: 1), with 20 mM-Hepes replaced by 20 mM-NaHCO3 (results not shown). Sources of Ca2+ mobilized by ATP The increase in [Ca2+]i could originate from various intracellular compartments and from Ca2+ entry. We first measured how much the sarcoplasmic reticulum could contribute, since ATP increases InsP3 [9], which in many tissues is reported to release Ca2+ from intracellular stores. When cells were incubated
1991
ATP-induced rise in HI, Ca2+ and depolarization
57
ICa2"Ii
with 10 M-ryanodine to prevent Ca2+ release from the sarcoplasmic reticulum, or when 10 mM-caffeine was applied to deplete the sarcoplasmic-reticulum Ca2+ load fully (as indicated by the ineffectiveness of a second application of the drug), ATP still triggered some Ca2` spikes, and the magnitude of the Ca2+ transient was unchanged (Table 1). However, under these conditions [Ca2+]1 rapidly returned to its basal value (Fig. 2a). This last result suggested that the cluster of Ca2+ oscillations which maintained a high [Ca2+]1 value during the plateau could be related to the release of Ca2+ from the sarcoplasmic reticulum [37]. To test this hypothesis further, in the experiment shown in Fig. 2(b) we applied 10 mM-caffeine to a cell after [Ca2+1] had reached the plateau after ATP application. Caffeine induced a large rise in [Ca2+]1, after which Ca2+ oscillations stopped and [Ca2+]1 rapidly fell back to its basal concentration. To reinforce the view that MgATP triggered a Ca2+ influx from the external medium, we used the 'Mn2+-quench' technique [38]. Fig. 3 shows that application of ATP to a single cell in a Ca2+-free solution containing 1 mM-MnCl2 induced a quenching of the indo-1 fluorescence at the two emission wavelengths. The superfusion of MnCl2 alone did not significantly alter the fluorescence, nor did it alter the caffeine-induced release of Ca2+ from intracellular stores. It might be expected that the initial large Ca2+ spikes triggered by ATP were related to the activation of voltage-dependent Ca2+
Table 1. Effects of MgATP on single-cardiac-cell
Single indo- l-loaded cells were rapidly superfused with 10 ztMgATP in control medium (first line), in the presence of 10 UMryanodine after preincubation for 30 min or of 10 mM-caffeine (second line), or after superfusion with a low-Ca2" (0.1 mM)/highMg2+ (3.4 mM) medium containing 1 ,sM-La3" after Ca2+ depletion ofthe sarcoplasmic reticulum with 10 mM-caffeine (third line). [Ca2+]i was determined as described in the Materials and methods section. Since after MgATP application [Ca2+], was not at a steady-state level, the magnitude of the Ca2+ transient was arbitrarily estimated at the top of the oscillations during the plateau or at the top of the spikes for ryanodine and caffeine experiments. Numbers of experiments performed in each experimental condition are given in parentheses: * significantly different from basal [Ca2+Jj (P < 0.02); ** significantly different from basal [Ca2+]i (P < 0.01).
[Ca2+]i (nM)
Control medium Caffeine, ryanodine
Low-Ca2+/high-Mg2+/La3+ medium
Basal
MgATP
69+ 5 (n= 17) 76+6 (n = 6) 47 + 3 (n = 4)
150+9** (n= 17) 140+ I1** (n = 6) 66+7* (n = 4)
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.,,
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Il 2.5
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Fig. 1. Effect of MgATP on ICa2+li of single cardiac cells (a) A single quiescent indo-1-loaded cell was superfused with 10 ,tM-MgATP. (b) A single cell was electrically stimulated at a frequency of 1 Hz and superfused with 10 ,sM-MgATP. Data acquisition was performed at a frequency of 10 Hz. The inset shows more details of the same effect with data acquisition performed at 100 Hz. The right scale indicates the ratio of fluorescence at the two emitted wavelengths. [Ca2J], values shown on the left-hand scale were determined as described in the Materials and methods section.
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_~ ~
M. Puceat and others
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LI-
C.)
channels. This was studied by preventing the influx of Ca 2+ ions through these channels. Bathing the cells in a solution containing 0.1 mM-Ca2+, 3.4 mm-Mg2+ and 1 pM-La3+ slightly decreased [Ca2+]1, and Ca2+ release from the sarcoplasmic reticulum by caffeine was also less (Fig. 4a). The further application of ATP still induced a Ca2+ transient, but of a low amplitude (Table 1). This Ca2+ transient could not be related to membrane depolarization, since the addition of 30 mM-KCI was ineffective (Fig. 4a). This latter experiment indicated that Ca2+ ions should be released from an internal Ca2+ store, and that this store is not the sarcoplasmic reticulum. To examine whether internal Ca2+ release, whatever its origin, could be the trigger for the large Ca2+ spikes, we loaded cells for 30 min with 25 /M of the fast Ca2+ chelator BAPTA. Under these conditions, as illustrated in Fig. 4(b), ATP did not increase [Ca2+]i; the application of caffeine was also ineffective, indicating that intracellular Ca2+ release was well buffered by BAPTA. Only a depolarization induced by 30 mM-KCI was able to increase [Ca2+]i slightly, and [Ca2+], returned to its basal value when KCI was washed. Relationship between ICa2+1j and pH; We previously showed that ATP induces a transient acidification by activating the HCO3 /Cl- exchanger [28]. The effects (a)
2.5
70 F
150w
Caffeine
1 0s
Fig. 2. Effect of caffeine on Ca2l transient induced by MgATP in single cardiac cell (a) The rapid application of 10 mM-caffeine to a single indo-l-loaded cell induced release of Ca2l from the sarcoplasmic reticulum. The cell was then superfused with 10 uM-MgATP. (b) The cell was superfused with 10 1sM-MgATP; when [Ca2l], had reached a plateau, 10 mM-caffeine was rapidly applied to the myocyte. Similar results were obtained with four other cells in each case.
c C4 N
[
70
KCI
MgATP
3.0
co LL
2.5
50
MnCI2
Caffeine 480
nm
MgATP
30s (b)
(U) c ()
Caffeine
0 (U
KCI
MgATP
|E Ho
0
30s 405
nm
)20
1.54
l1 min
Fig. 4. Effects of MgATP on JCa2+jj of a single cardiac cell superfused with a low-Ca2+/high-Mg2+ medium and on ICa2+li of a BAPTA-loaded cell
Fig. 3. Effect of MgATP on the fluorescence of an indo-l-loaded single cell superfused with a Ca2l-free buffer containing MnCl2 A single indo- 1-loaded cell was superfused with a Ca2"-free medium (no Ca2" added) containing mM-MnCl2. The changes in fluorescence were recorded at the two emitted wavelengths when 10 mMcaffeine and 10 ,sM-MgATP were successively applied to the myocyte. The right-hand scale indicates the change in fluorescence recorded by the photomultipliers (in mV). Similar results were obtained with three other cells.
(a) A single indo- 1-loaded cardiac cell was superfused with a lowCa2+ (0.1 mM)/high-Mg2" (3.4 mM) medium containing 1 /uM-La3+. After 10 mM-caffeine was transiently applied to the cell to deplete the sarcoplasmic-reticulum Ca2+ load, 10 ,uM-MgATP and 30 mMKCI were successively added to the cell. (b) Indo- 1-loaded myocytes were further incubated for 30 min in the presence of 25 4MBAPTA/AM. After 1 h to allow the hydrolysis of the ester into the cell, a single myocyte was successively superfused with a Hepesbuffered solution (1.8 mM-CaCl2) containing 1O ,/M-MgATP, 1O mmcaffeine or 30 mM-KCl. Similar results four cells.
were
obtained with at least
1991
ATP-induced rise in HI+ Ca2' and depolarization (a)
59 180 r (a)
Mg-free medium
MgGTP MgATP[S] 7.0
Ix
ATP
MgATP
1.0
.
6.8
1501.
3.0
2~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A
UY
LA.l
L.)
6.6
1
min
0.8
2.5
15s (b) Mg-free medium 250
ATP
10r(b)
3.5
I:-
+
rC.
MgAT3.
MgADP
5
MgATP
3.0-
150
I0
la
ii
70
iSs
2.5
L
L.J
1LA
1.0
30s Fig. 5. Effects of ATP on single-cardiac-cell pH1 and [Ca2+]i (a) A single snarf-l-loaded cell was superfused with a Mg2+-free Hepes buffer; 10 1zM-ATP was then applied to the cell. After switching to the usual solution containing 1.7 mM-MgCl2, the change in pH1 was recorded in response to 10 /LM-MgATP. The right-hand scale indicates the ratio of snarf-1 fluorescence at the two emitted
wavelengths. The left-hand scale shows values of pHi determined as described in the Materials and methods section. (b) A single indo-1loaded cell was superfused with a Mg2+-free medium; 10 ,M-ATP was then added to the cell. After switching to a medium containing 1.7 mM-MgCl2, the changes in [Ca21]i were recorded in response to 1O #M-MgATP. Similar results were obtained in two other experiments in both cases.
of ATP on [Ca2+], share the same characteristics as the ATPinduced acidification. Figs. 5(a) and 5(b) show that omitting Mg2+ ions from the bathing solution prevented both the acidification and the Ca2' rise elicited by ATP, whereas the nucleotide became effective after re-admission of Mg2 The hydrolysis-resistant analogues adenosine 5'-[y-thio]triphosphate (ATP[S]), adenosine 5'-[/Jy-methylene]triphosphate, adenosine 5'-[a,-methylene]triphosphate and adenosine 5'-[/?yimido]triphosphate in the presence of Mg2+ were able to reproduce the effects of ATP on ionic movements when used at 100 or 500 ZM. Other nucleotides, ADP (in the presence of hexokinase to prevent the formation of ATP by ecto-kinases, as previously reported in Ehrlich ascites-tumour cells [10]) and AMP, as well as GTP, ITP, CTP, UTP, all used at 100 /M, were ineffective. Some examples are illustrated in Fig. 6. No acidification was induced when ATP was applied in a Cl--free medium, nor was [Ca2+]1 changed. However, the nucleotide was effective when Clions were re-admitted (Figs. 7a and 7b). Preincubation of myocytes for 30 min in the presence of 200 ZM of the stilbene derivative DIDS or with 100 ,uM-probenecid (two inhibitors of the HCO3-/CI- exchanger) prevented both the acidification and the Ca2+ rise on ATP application (Figs. 7c and 7d), but the caffeine-induced Ca2+ release from sarcoplasmic reticulum was not altered by the drug (Fig. 7d). To examine whether the Ca2+ rise could be responsible for the acidification, as suggested in several tissues following agonist stimulation [39,40], we investigated the effects of ATP on pHi .
Vol. 274
0).
0.98 LA.
-0.8
Fig. 6. Effects of different nucleotides on single-cardiac-cell jCa2l;j and pH; (a) Changes in [Ca2ll] were recorded on a single indo-l-loaded cell successively superfused with 1000,M-MgGTP and lO0 sMMgATP[SJ; 10 mM-caffeine was then applied to the myocyte to stop the Ca2"-induced release of Ca2" from the sarcoplasmic reticulum. (b) Changes in cell [Ca2+]i were recorded in response to 100 ,sM-MgADP in the presence of 10 units of hexokinase/ml and then to 10 1uMMgATP. (c) Changes in pHi were recorded in the presence of 10 /SMEIPA on a single snarf-l-loaded cell successively superfused with 100/lM-MgADP in the presence of 10 units of hexokinase/ml, 100 ,#M-MgGTP and 10 ,uM-MgATP. Similar results were obtained in two other experiments for [Ca2"], and pH,.
after the influx of Ca2l had been prevented (in a Ca2l-free medium containing 50 /eM-EGTA, or in a low-Ca2l high-Mg2+ solution containing La3+). Under these experimental conditions, the nucleotide triggered an acidification of the same magnitude as in medium containing CaCl2 (Table 2). ATP was also as potent when, furthermore, the cells were loaded with BAPTA to buffer the release of Ca2+ from intracellular stores (Fig. 8). Effects of nigericin on pH1 and [Ca2+I1 In order to investigate whether the acidification induced by ATP could be responsible for the increase in [Ca2+]1, cell acidification was induced by the K+-H+ ionophore nigericin. A low dose (2 ,g/ml) of nigericin decreased pH, by the same magnitude as 10,uM-ATP did (Fig. 9a). A similar application of the ionophore to an indo- 1-loaded cell triggered some Ca2+ spikes followed by a rapid Ca2+ transient. The [Ca2+], reached 142+16 mM (basal [Ca2+]1 was 62+ 12 nm in this series; n = 5).
Effects of ATP on membrane potential The membrane potential was monitored with the fluorescent dye bis-oxonol. Fig. 10(a) shows that ATP added to a cell suspension (final concn. 10 uM) induced a depolarization (26+ 2.5 mV; n = 12). When cells were bathed in a Ca2+-free medium,
60
M. Puceat and others
(a)
(c)
Cl-free medium MgATP
MgATP MgATP
1.1
Q
0.8
6.6 1 min
(b)
(d)
Cl-free mAdium
250
Caffeine
MgATP
r
MgATP Caffeine
3.5
-
C
O
I-I
- 150
3.0 X
MgATP
N
ilMq
?,-.ii
r
70 L
2.5 30s
Fig. 7. Effects of MgATP on cardiac-cell pH; and ICa2]i; in a Cl--free buffer or after preincubation of the cells in the presence of DIDS (a) A snarf- 1-loaded cell was superfused with a Cl--free medium (sodium gluconate replaced NaCl, and KCl, CaCl2 and MgCl2 were respectively replaced by KHCO3, CaCO3 and MgSO4) added with 10 ,sM-EIPA and 10 ,uM-bumetanide; the increase in pHi is due to the influx of HCO3- ions in exchange for Cl- efflux. When pHi had reached a steady state, 10 /StM-MgATP was applied to the cell without effect. The cell was then switched to a medium containing 125 mM-Cl-, 10 ,#M-EIPA and 10 ,tM-bumetanide; when pHi had returned to its basal value, 10 /LM-MgATP was added to the cell. (b) An indo- 1-loaded cell was superfused with a Cl--free medium [same composition as in (a)] for at least 2 min before application of 10 uM-MgATP. After switching to a medium containing 125 mM-Cl-, the changes in [Ca21]i were recorded in response to 10 /LM-MgATP. (c) MgATP (10 ,UM) was applied in the presence of 10 /LM-EIPA to a single snarf-l-loaded cell preincubated for 30 min with 200 /LM-DIDS. (d) MgATP (10 /,M) and 10 mM-caffeine were successively added to a single indo- 1-loaded cell preincubated for 30 min with 200 ,#M-DIDS. Experiments shown in (a)H(d) were performed on four different cells. Similar results were obtained in each experimental condition with at least three cells. Table 2. Effects of MgATP EIPA
on
single-cardiac-cell pH; in the presence of
7.1
Single snarf-l-loaded cells were superfused with 1O ,zM-MgATP in the presence of 10 #uM-EIPA in control medium or either in a lowCa2" (0.1 mM)/high-Mg2" (3.4 mM) medium containing 1 ,M La3" or
in
a
Ca2"-free
7.0
pHi
Basal
MgATP
Control medium
7.02 + 0.07
Ca2"-free medium
(n = 8) 6.92+0.06 (n = 9)
6.71 +0.13** (n = 8) 6.63 +0.05**
(n
=
1.0 co
I
6.9 .
buffer added with 50 ,tM-EGTA. These two last
experimental conditions gave similar results (Ca2"-free + EGTA, 3 cells; low-Ca2+/high-Mg2+/La3+, 6 cells). They were pooled in the second line of the Table (Ca2`-free medium). pH, was determined as described in the Materials and methods section. Numbers of experiments performed in each experimental condition are given in parentheses: ** significantly different from basal pH, (P < 0.01).
MgATP
6.8 L 15s
Fig. 8. Effect of MgATP on pH. of a single cardiac cell loaded with BAPTA Snarf- 1-loaded cells were incubated for 30 min in the presence of 25 /tM-BAPTA/AM. After 1 h to allow the hydrolysis of the ester, the change in pH, of a single cell was recorded in response to 10 /LMMgATP in a Ca2"-free buffer added with 50 1zM-EGTA and 10 /uMEIPA. Similar results were obtained in two other experiments.
hand, under each of these experimental conditions KCl (20 mM) still able to depolarize the myocytes, as it did in control conditions (Fig. 10b).
9)
was
ATP still triggered a depolarization of similar amplitude (results not shown). The effects of ATP on the membrane potential
were
tested under some of the experimental conditions in which ATP failed to trigger both acidification and Ca2+ transient. Thus, when cells were suspended in a Mg2+-free or a Cl--free medium, ATP did not alter the membrane potential. The nucleotide was also unable to induce a depolarization in the presence of probenecid (100,UM, cell suspension preincubated for 30 min). Pretreatment of cells in the presence of DIDS (200,uM) also suppressed the depolarization elicited by ATP. On the other
DISCUSSION The major experimental findings are that extracellular ATP induces simultaneous changes in intracellular Ca2 pH and membrane potential. These confirm and extend previous results. Furthermore, the principal aim of this work was to investigate the relationships between these changes and to propose a mechanism by which ATP triggers the variations in [Ca2+]i. The rapid depolarization, acidification and Ca2+ rise were elicited by ,
1991
ATP-induced rise in H+, Ca2+ and depolarization (a)
61
the same agonists (ATP and its poorly hydrolysable analogues). They required the presence of extracellular Mg2", ruling out a non-selective membrane-permeabilization effect of ATP such as occurs, for example, in mast cells [41]. The ionic movements mediated by the nucleotide appeared with similar apparent kinetics; their maximal value was reached within a few tens of
Nigericin
1.1
I a.
1.0
7.0
6.8 L
14!
0.9 15s
(b) 250 pI
Nigericin
3.5
seconds. Our results provide evidence that acidification, following activation of the HCO3 /C1- exchanger [28], is the primary event induced by MgATP. Experimental conditions designed to suppress acidification (Cl--free medium, DIDS, probenecid) also prevented the transient increase in [Ca2+], and the depolarization. In contrast, the conditions used to suppress the rapid rise in [Ca2+]1 by buffering the intracellular Ca2+ release with BAPTA and/or by preventing the Ca2+ influx both through the Ca2+ conductance and the exchange (Ca2+-free or low-Ca2+ medium still allow MgATP to trigger containing high-Mg2+ an acidification with the same magnitude. This acidification is associated with a small but consistent increase in [Ca2+], even when Ca2+ entry was prevented in a lowCa2+ high-Mg2+ medium containing La3+, as indicated by the rise (Fig. 4a) and when the inability of KCI to induce a sarcoplasmic reticulum was unloaded by a high concentration of caffeine. This result suggests that MgATP mobilizes Ca2+ ions from an intracellular pool other than the sarcoplasmic reticulum. It is proposed that Ca2+ ions are displaced by protons from cation-binding sites on, or close to, the sarcolemma. It is generally agreed that H+ and Ca2+ ions compete for the same binding sites [42-44]. An exchange of Ca2+ for H+ at common binding sites other than sarcoplasmic reticulum has already been reported to be in part responsible for the Ca2+ rise triggered by acidification [45]. The experiment using the ionophore nigericin, showing that transient, is in an acidification could also induce a rapid good agreement with these data. Furthermore, it has been demonstrated that a decrease in pHi facilitates the spontaneous Ca2+ release from the sarcoplasmic reticulum [46]. The existence of such a localized release of Ca2+ from the junctional sarcoplasmic reticulum close to the sarcolemma by the MgATPinduced acidification was tentatively excluded, since a single bolus application of 10 mM-caffeine was proved to be sufficient to deplete fully the sarcoplasmic-reticulum Ca2+ load. This localized increase in Ca2+ ions can trigger a transient inward current [23], owing to an increase in a non-selective
Na+/Ca2+
i 1501-
C4
a
c
3.0
"
C.)
Ca2+
2.5
501L 15s Fig. 9. Effects of a low dose of nigericin on a single-cell pH1 and (a) Variations in pHi of a single cell loaded with snarf-l/AM were recorded in response to 2,ug of nigericin/ml. (b) Changes in [Ca2+]i
ICaz+li
of a single cell loaded with indo- 1/AM were monitored in response to 2 jug of nigericin/ml. Similar results were obtained in four other
experiments.
Ca2+
(a) MgATP
0 L~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ C 0)
I~ ~~~~I 0
La3+)
c.)
0
1U
2min
m11I (b)
a)0
KCI
,IN
PIh1
|
c
i
(04)
C.)
0 0
2min
Fig. 10. Effect of MgATP on membrane potential of cardiac cells The fluorescent indicator bis-oxonol (20 nM) was added to 1 ml of the suspension of cells bathed in normal Hepes buffer as described in the Materials and methods section. (a) Changes in fluorescence were recorded in response to 10 ,uM-MgATP. The increase in fluorescence giving an upward deflection indicates a depolarization. (b) Changes in fluorescence induced by adding KCI (final concn. 20 mM) allowed for calibration (see the Materials and methods section). The right-hand scales indicate fluorescence changes in arbitrary units (a.u.) and estimated membrane potential changes in mV.
Vol. 274
cationic conductance. This was first observed in cardiac cells and reported to be sensitive to Ca2+ concentration as low as 100 nm [47,48]. The opening of this conductance could lead to depolarization, as indicated by the potential-sensitive dye bis-oxonol (Fig. 10) above the action-potential threshold. The depolarization thus induced triggers the opening of the voltage-dependent Ca2+ channels and will lead to automaticity and arrhythmias. During these spikes Ca2+ ions accumulate and add to those released by acidification. This hypothesis is well supported by the 'Mn quench' experiments. Indeed, the application of MnCl2 alone to the cell did not affect the indo-1 fluorescence, whereas a rapid quenching occurred when MgATP was applied. These observations are in full agreement with the facts first that Mn2+ ions do not passively enter the cell and secondly that they pass through voltage-dependent Ca2+ channels [49]. On the other hand, when the localized increase in Ca2+ was buffered by loading the cells with BAPTA, MgATP was unable to trigger the initial Ca2+ spikes. MgATP transiently increased the cellular Ca2+ concentration from 70 to 150 nm in a single cell. Ryanodine and caffeine, two compounds which prevent Ca2+ release from the sarcoplasmic reticulum, did not affect the magnitude of the initial rapid Ca2+ rise induced by MgATP, but significantly shortened the Ca 21
M. Puceat and others
62 plateau by preventing the cluster of Ca2' oscillations. Moreover, further application of caffeine during the Ca2+ plateau after MgATP addition led to a rapid decrease in [Ca2+]i. These data confirm that the sarcoplasmic reticulum is not the initial source of Ca2+ mobilized by MgATP as suggested above. They also indicate that the nucleotide mobilizes Ca2+ ions from this intracellular compartment by triggering the Ca2+-induced Ca2+ release. It should be taken into account that such a release would be facilitated by the simultaneous increase in InsP3. It has been demonstrated that ATP increases turnover of phosphoinositides [9] and that InsP3 strongly potentiated the spontaneous release of Ca2+ which occurs when the sarcoplasmic reticulum is overloaded with Ca2+ [50]. In conclusion, adenine nucleotides have pharmacological effects which are generally attributable to stimulation of the P1or P2-purinergic receptors on a variety of tissues. On the other hand, in our study, ionic movements mediated by MgATP appear to be highly specific to the nucleotide and to its hydrolysisresistant analogues; moreover, they require the presence of Mg2+ ions. These observations are inconsistent with the pharmacology of the P1- and P2-purinergic receptors as previously described [51]. Our results are in favour of the existence of a novel purinergic receptor with specific agonists and biochemical pathways. The Ca2+ rise as previously reported by others [7,24,26,27] and during the present work has at least three origins. (1) The increase in H+, induced by ATP activation of the HCO3 /Cl1 exchanger, displaces Ca2+ ions from internal binding sites. (2) These Ca2+ ions open a non-specific membrane conductance which, by depolarizing the cell, triggers spiking and thus opening of voltage-gated Ca2+ channels. This leads to the initial large rise in [Ca2+]i. (3) This increase in [Ca2+]1, together with the facilitation owing to the InsP3 increase, induces repetitive Ca2+ release from the sarcoplasmic reticulum which determines the plateau Ca2+ rise. The physiological consequences of MgATP released by nerve stimulation or during circulatory shock can thus vary from vasodilatation [52] and increase in myocardial contractility [7-9] to the deleterious ventricular arrhythmias expected from the above results. We thank Dr. B. Berthon for discussion and critical reading of previous manuscripts, P. Lechene for skilful computer programming and D. Angelini for secretarial assistance.
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Received 24 August 1990/29 October 1990; accepted 30 October 1990
1991
