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Ca uptake by heart cells: II. Most entering Ca appears to leave without mixing with the sarcoplasmic reticulum Ca pool
Cd/ Calcium (1998) 23(4), 199-205
Research
Q Hacourt
Brace 8 Co. Ltd 1998
Ca uptake by heart cells: Il. Most entering Ca appears to leave without mixina with the sarconlasmic reticulum Ca pool Robert A. Haworth, David Redon, Angela V. Biggs, Katherine T. Potter Department
of Anesthesiology,
University
of Wisconsin,
Madison,
Wisconsin,
USA
Summary The rate of verapamil-sensitive uptake of 45Ca by rat heart cells stimulated to beat in suspension with 0.2 mM Ca and isoproterenol was increased > 2-fold by cell loading with the chelator Quin-2. No effect of Quin-2 loading was observed on the rate of uptake of trace levels of 54Mn, present in addition to Ca, which was used as an index of Ca channel activity. Quin-2 loading also had little effect on the rate of 45Ca uptake by cells diluted into a high K/low Na medium, where Ca uptake was primarily by Na/Ca exchange. The fast chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was 3-fold more effective than the slow chelator EGTA at preventing Ca efflux. BAPTA loading also caused an increase in sarcoplasmic reticulum (SR) Ca content. These results suggest that chelator loading had little effect on the rate of Ca influx by Ca channels or by Na/Ca exchange, and that the increased rate of 45Ca uptake seen with Quin-2 loading was caused by an inhibition of Ca efflux, either directly by chelation or by increased Ca uptake by the SR or by other intracellular organelles. This further suggests that most of the Ca entering the cell without chelator leaves again within the same beat, and that this may result from Ca efflux from a kinetically limited Ca pool in or around the diad cleft.
INTRODUCTION
In the previous paper, we described the uptake of 45Caby isolated adult rat heart cells in suspension subjected to electric field stimulation [l]. We found an exponential increase in the extent of labelling of cellular Ca, and the initial rate of labelling was proportional to the rate of stimulation. We found a rate of cellular labelling of 5.25 pmol/mg/beat. This uptake was composed of several elements, the major stimulation-dependent one being the SR, which labelled at a rate of 3.87 + 0.17 pmol/mg/beat. Does the 5.25 pmol/mg/beat represent the rate of 45Ca Received
8 September
1997
influx, which on entry mixes with the cytosolic Ca pool, some of which is then accumulated by the SR? Or, does more labelled Ca than this enter, with most of it leaving again within the same beat, without mixing with most of the SR Ca? The latter scenario would be evidence for some kind of compartmentation of the cytosolic Ca pool. We sought to distinguish between these two possibilities by looking for evidence that the true rate of 45Ca entry was indeed greater than the measured rate of 45Ca uptake. Our approach was to load cells with Quin-2, a Ca chelator, in order to trap as much incoming Ca as possible. Our data suggest that the true rate of Ca influx is indeed significantly greater than the rate at which the Ca is retained, unless chelator is present.
Revised 20 January 1998 Accepted 20 January 1998 Correspondence to : Dr Robert A. Haworlh, Department of Surgery, University of Wisconsin Clinical Science Center, 600 Highland Avenue,
Cell isolation
Madison, WI 53792 USA Tel: +l 608 263 1339; Fax: +l 608 263 0575 E-mail: [email protected]
Heart cells were isolated from female retired breeder rats as in the previous paper [I]. 199
200
R A Haworth, D Redon, A V Biggs, K T Potter
Experimental medium
20
Experimental medium was based on a Krebs-Henseleit (basal K-HEPES) medium containing (mM): NaCl 118, KC1 4.8, HEPES 25, KH,PO, 1.2, MgSO, 1.2, glucose 11, and pH was adjusted to 7.4 with NaOH. For isotope uptake experiments, 0.2 mM CaCl,, 5 mM Na pyruvate, and 1 @I insulin were also present. For fluorescence experiments, 0.05 mM DTPA (diethylenetriamine pentaacetic acid) was included as a heavy metal scavenger, and CaCl, was increased to 0.25 mM. Suspensions were maintained aerobic by equilibration with air in a shaking incubator at 37°C.
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Labelling of cells with BAPTA, EGTA or Quin-2
Aliquots (1 ml) of cells in experimental medium (6.2 mg/ml) were incubated with the acetoxymethyl (AM) ester of chelators in amounts as described in the figures, for 20 ruin at 3Tc. The suspension was divided and centrifuged at 29 gfor 90 s and the cell pellet of each half resuspended to 0.5 ml in either experimental medium or assay medium (see below). Cells in experimental medium were exposed to isoproterenol (1 @VI) for 7 ruin, before measurement of 45Caand 54Mn uptake. Chelator assay
To measure the uptake of non-fluorescent chelators by cell suspensions incubated with the acetoxymethyl ester of the chelators, we developed an assay based upon chelator competition for Ca with the resin Chelex 100 (BioRad). Aliquots (0.5 ml, 6.2 mg/ml) of labelled cell suspension (or unlabelled controls) were centrifuged at 29 g for 90 s, the supernatant removed, and the pellet resuspended to 0.5 ml with assay buffer containing basal K-HEPES (see above) plus 0.2 mM CaCl,, 0.25 mCi 45Ca,5 u.M ruthenium red and 25 ug/ml digitonin. This caused release of chelator from the cytosol of the cells. After 2 min incubation at 37X, the cell suspension was centrifuged in a high speed bench centrifuge to remove cell debris. The supematant was added to 1 ml basal K-HEPES, and the mixture added to 63 mg Chelex 100 in a 9 x 50 mm acid washed glass tube. Standards were made by adding known amounts of chelator to 0.5 ml aliquots of assay buffer, then adding to 1 ml K-HEPES and Chelex as for cell extracts. Standards for EGTA were from a 0.1 M solution from Molecular Probes (Eugene, OR, USA). A standard BAPTA solution was prepared after drying solid BAPTA salt to constant weight [2]. The tubes were sealed with Parafilm and mixed by gentle inversion for 30 min at room temperature. During this time, the Chelex removes most of the Ca (and 45Ca) from the medium, leaving an amount proportional to the Cell Calcium
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Fig. 1 Measurement of BAPTA and EGTA by the chelator assay. Ordinate shows the concentration of Ca retained by the abscissa level of chelator, in competition with Chelex resin (see Materials and methods). Circles: EGTA; triangles: BAPTA.
chelator content of the medium. Samples were centrifuged to remove Chelex. Ca concentration remaining in the medium was determfned by liquid scintillation counting of aliquots of the sample supematants and of the assay buffer. Figure 1 shows standard curves for EGTA and for BAPTA. The assay has good linearity, and allows chelator contents to be accurately determined. To measure the uptake of fluorescent chelators, the Mn sensitive (* 0.2mM Mn) component of the fluorescence of a suspension of cells in basic K-HEPES buffer with 1 mM Ca plus 25 ug/ml digitonin and 10 uM DTPA was compared with that of a known concentration of chelator salt, in a cuvette measurement. Isotope flux measurements
Procedures were as in the previous paper [ 11.To measure uptake of 54Mn, M&l, containing 54Mn was added at time zero to give 2.5 uCi 54Mn/ml cell suspension and 10 @VIMnCl,. Ca (0.2mM) was still present in the medium. Ca uptake in high K medium
Cells (6.2 mg/ml) in experimental medium were loaded or not with Quin-2 as described above, except that isoproterenol (1 @I) was added 7 min before the cells were centrifuged. The cell pellet was resuspended by adding 0.1 ml experimental medium plus isoproterenol, to a final volume of 0.14 ml. To initiate 45Ca uptake, this was then added to 0.9 ml experimental medium with NaCl replaced by KC1 (where shown), 1 mM ouabain, 5 0 Harcouti
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Ca uptake by heart cells: II. Entering Ca does not mix with SR Ca pool 201
@vl ruthenium red, 1 @I isoproterenol, 0.2 mM CaCl, with 45Ca, and either 3H,0 or 10 pM MnCl, with 54Mn as above. 60 ~1 aliquots of cell suspension were removed at the times shown and added to 540 pl ice cold basal KHEPES medium with Na replaced by K, 0.5 mM EGTA, and 5 uM ruthenium red. The cells (0.5 ml aliquots) were then centrifuged after 2 min through 0.5 ml bromododecane into 0.1 ml 16% per&lo& acid as before [l]. Data analysis
Values shown are the mean and standard deviation of at least three experiments. Ca uptake by cells was fitted by non-linear least squares to the functions described in each figure. Lines on figures are these best fit curves, unless otherwise indicated.
Ca channel activity was unaffected. A nice feature of using Mn in this way is that 0.2 mM Ca is still present in the medium. Since we included 10 l.tM Mn in the 45Ca
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Ca chelators and their AM ester derivatives were obtained from Molecular Probes (Eugene, OR, USA).
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Since the condition of 0.2. mM Ca with isoproterenol allowed us to most easily measure the rate and extent of stimulation dependent cellular 45Ca fluxes [l], this continued to be our standard condition for measuring these parameters, for experiments described in this paper. To gain some measure of the amount of Ca entering the cell per beat, we loaded cells with the Ca chelator Quin-2 by incubation with 30 ).&I Quin-2/AM for 20 min, with the aim of trapping incoming Ca. This treatment resulted in a total uptake of 2.01 k 0.31 nmol Quin-2/mg. Quin-2 caused an increase in the initial rate of verapamil-sensitive Ca uptake by a factor of > 2 (Fig. 2A). The initial rate of verapamil sensitive Ca inilux per beat measured in the presence of Quin-2, calculated from the best fit curve in Figure 2A, was 14.73 pmol/mg/beat. This may be an underestimate of the rate of Ca influx, since this level of Quin-2 loading may not trap all incoming Ca. Is the Quin-2 effect on Ca uptake caused by an inhibition of Ca efflux or by a stimulation of Ca influx? To answer this we sought to measure the effect of Quin-2 loading on the rate of Ca inilux by each of the two known possible routes for Ca influx: Ca channels, and the Na/Ca exchanger. We have previously found that the rate of uptake of trace levels of in is an indicator of Ca channel activity, since Mn enters by Ca channels but not by Na/Ca exchange [3]. We, therefore, measured the effect of Quin-2 loading on the uptake of trace levels of Mn. We found that Mn uptake under these conditions was unaffected by Quin-2 loading (Fig. 2B), suggesting that 0 Harcouti
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time (s) Fig. 2 Effect of Quin-2 loading on uptake of %a and %Mn. (A) Ca uptake; (B) Mn uptake. Circles: no Quin-2 (Q); triangles: with Quin-2 (30 uM QuinG?/AM); filled symbols: no verapamil (V); open symbols: with 10 PM verapamil. Cells all with 1 uM isoproterenol, and all stimulated at 2 Hz. The dashed line in (A), shown for comparison, is the best fit line from [l] Figure 2, for the condition of no stimulation with verapamil. Ca uptake was fitted by a double exponential function as in the previous paper [l], using this line as the first exponential. Mn uptake was fit by a single exponential function.
Cell Calcium
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Fig. 3 Effect of Quin-2 loading on uptake of Ca and Mn on 1:9 dilution into high K medium. Circles: no Quin-2 (XI); triangles: with Quin-2 (+Q, 30 uM CUinGYAM). (A) Ca uptake: open symbols -to Na +verapamil, filled symbols-to K no verapamil. Data were fitted to a single exponential function. (B) Verapamil sensitive Mn uptake: open symbols - stimulation induced (the difference between the Mn uptake seen without and with verapamil in Fig. 28 (+S-V)-(+S+V)). Filled symbols - high K induced (the difference between Mn uptake by cells diluted to K -verapamil and that by cells diluted to Na +verapamil). Data were fitted to straight lines. See Materials and methods for experimental details.
uptake measurement in Figure 2A, the conditions for measuring 45Caand 54Mn uptake are chemically identical. Thus, we may conclude that the increased rate of Ca uptake seen with Quin-2 loading cannot be explained in terms of an increased rate of Ca entry by Ca channels. Ca can also enter the cell by Na/Ca exchange, and Quin-2 could potentially stimulate this [4]. To see whether or not we could find evidence for a stimulation of the rate of Ca influx by Qum-2, we measured the rate of 45Cauptake by unstimulated cells in which exchanger mediated Ca influx was stimulated and Ca efflux was inhibited, by diluting cells into medium in which all but 19 mM Na was replaced by K. Ouabain was also present in the dilution medium, to prevent energized efflux of cell Na. Under these conditions, intracellular and Cell Calcium (1998)
23(4),
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extracellular concentrations of Na and K are very similar, resulting in zero membrane potential. This will, in principle, favor Ca influx both by Ca channels and by Na/Ca exchange. Ca uptake under these conditions was fast and large, but Quin-2 loading increased the rate of uptake by only 16% (Fig. 3A; Table 1). Verapamilsensitive Mn uptake was low under these conditions (Fig. 3B; Table l), suggesting that most of the Ca uptake under these conditions was by Na/Ca exchange. This, therefore, suggests that Quin-2 loading under these conditions does not much stimulate Ca influx by Na/Ca exchange. Since 45Ca could also be carried into the cell by Ca/Ca exchange through the Na/Ca exchanger [5], we tested whether or not Quin-2 could be promoting this mode of entry. Cells under the conditions of Figure 3A were allowed to accumulate unlabelled Ca for 2 min, by which time Ca uptake, based on Figure 3A, was maximal. At that time, carrier-free 45Ca was added, and cells were centrifuged thereafter. The rate of 45Ca uptake was less than half that seen in Figure 3, and Quin-2 loading again only slightly increased the initial rate of 45Cauptake (data not shown). This suggests that there is little effect of the chelator on the rate of Ca influx through the exchanger in either mode. These results suggest that the increased rate of stimulation induced 45Ca uptake measured with Quin-2 is the result of trapping of incoming Ca rather than stimulation of the rate of influx. This further suggests that a significant fraction of the incoming Ca in the absence of Quin-2 is being extruded from the cell without mixing with most of the rapidly labelling cellular Ca pool. To gain further evidence for a kinetic separation between the cleft Ca pool and the rest of the cytosol, we have compared BAPTA loading and EGTA loading for their effectiveness at increasing the measured rate of 45Ca uptake. EGTA binds Ca much more slowly than BAF’TA, Table 1 Comparison of the effect of Quin-2 loading on initial of Ca uptake and verapamil sensitive Mn uptake induced by electric field stimulation and by high K Ca uptake (pmol/mg/s) Stimulation -Quin-2 +Quin-2 High
K -Quin-2 +Quin-2
rate
Mn uptake (pmol/mg/s)
rates
rate
at 2 Hz 13.78 29.45
* 1.86 * 2.34
0.388 0.392
f 0.016 f 0.013
44.20 51.32
f 5.30 2 6.60
0.050 0.046
f 0.011 zt 0.012
Values for initial rates of uptake were taken from the best fit curves shown in Figures 2 and 3. Ca uptake values are relative to the condition +stim +verapamil for stimulation induced uptake (Fig. 2A), and relative to the condition ‘to Na +verapamil’ for the high K induced uptake (Fig. 3A). Verapamil sensitive Mn uptake was as defined in Fig. 38.
63 Harcourf Brace & Co. Ltd 1998
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Fig. 4 Stimulation of Ca uptake by loading cells with BAPTA or EGTA. Circles: no chelator, with stimulation at 2 Hz (filled symbols) or without stimulation plus 10 uM verapamil (-S+V, open circles). (A) Cells incubated with 15 uM (triangles) or 45 uM (squares) BAPTA/AM for 20 min to give BAPTA uptakes of 1.472 f 0.431 and 3.445 * 0.177 nmol/mg, respectively. (6) Cells incubated with 35 uM (triangles) or 105 uM (squares) EGTAIAM for 20 min to give EGTA uptakes of 2.515 f 0.109 and 4.821 i0.183 nmollmg, respectively. Cells with chelator were also stimulated, at 2 Hz. All cells were treated with 1 ,uM isoproterenol.
6
Fig. 5 Dependence
of initial rate of Ca uptake on chelator content. Data from experiments like Figure 4 were fit to the sum of the linear rate (with no stimulation plus verapamil) and an exponential rate. The initial rate of the best fit exponential function for each condition from each experiment is shown, for five experiments. Each symbol is from a separate experiment. The lines are linear regressions for the data with BAPTA (filled symbols) and with EGTA (open symbols). Slopes of the regressions were 0.00870 for BAPTA and 0.00298 for EGTA.
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even though their affinities are similar [6]. BAPTA should thus be a much more effective chelator than EGTA at modulating processes which are governed by transient local gradients of Ca, such as may exist between the diad cleft and the cytosol. BAPTA has indeed been found to be more effective than EGTA at inhibiting the Ca dependent component of SR Ca release induced by t-tubule depolarization in skeletal muscle homogenates [7j. We, therefore, loaded cells with different levels of BAPTA and EGTA by incubation with different levels of their AM esters, and measured the rate of 45Cauptake as in Figure 2, along with the amount of chelator present in the cytosol of the cells. We found that EGTA was able to stimulate Ca uptake almost as much as BAFTA (Fig. 4). However, the amount of EGTA required to do this was greater by a factor of 3 (Fig. 5). To investigate how cell chelator loading may affect Ca uptake by the SR, we used the size of the caffeine releasable Ca pool as a measure of SR Ca content as described in the previous paper [l]. Cells were loaded with increasing levels of BAPTA by incubation with increasing levels of BAPTMAM, then stimulated to beat at 2 HZ for 2 min with 45Ca. We found that increasing levels of BAPTA loading resulted in increasing levels of caffeine-releasable Ca when the cells were diluted into EGTA with or without caffeine, though at the highest 0 Harcourt Brace & Co. Lfd 1998
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Fig. 6 Stimulation of SR Ca content by BAPTA. BAPTA loaded cells with isoproterenol and %a were stimulated at 2 Hz for 2 min before dilution into unlabelled medium with (filled symbols) or without (open symbols) 10 mM caffeine. Circles: no BAPTA/AM; upright triangles: 30 uM BAPTNAM; squares: 60 uM BAPTAfAM; inverted triangles: 120 uM BAPTAfAM; diamonds: 240 pM halfBAPTA/AM. The BAPTA content of cells was measured as described under Materials and methods.
Cell Calcium (1998) 23(4), 199-205
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level of BAPTA loading the magnitude of caffeine releasable Ca was again reduced (Fig. 6). The size of the SR Ca pools for each level of cell BAPTA measured is given in Table 2. Incubation with the AM ester of the non-chelating molecule half-BAPTA had no effect on SR pool size or Ca uptake (Fig. 6; Table Z), which suggests that, at these levels of ester, there is no artefactual effect of ester hydrolysis on cell Ca homeostasis. The stimulation of SR pool size seen with 60 @I BAF”IA/AM is, therefore, caused by the chelating property of BAPTA.
DISCUSSION
The major experimental finding of this paper is that the rate of Ca influx into heart cells stimulated to beat appears to be much greater than the measured rate of Ca uptake, unless intracellular chelators are present. This implies that most of the Ca entering the cell without Quin-2 is returned to the medium without mixing with most intracellular Ca stores and, in particular, the SR Ca store: since the SR is thought to re-accumulate > 90% of the Ca associated with a Ca transient [8], not all of the entering Ca can be reaching that Ca transient pool. This failure to mix suggests that kinetic factors impose a kind of compartmentation on the cytosolic Ca, even though the cytosol is spatially continuous. The above conclusion is unaffected by the observation that SR Ca content can be increased by chelators (Fig. 6). Even if all of the increase in Ca uptake seen with chelators is located in the SR, as opposed to being directly chelated by Quin-2, the cell can only retain this 45Caif the 45Cafirst enters the cell. Our data suggest that the Ca influx per beat is unaffected by the level of Quin-2 used here (Figs 2B & 3). If this is so, then in the absence of chelator most of this entering Ca must be leaving within the same beat, regardless of the mechanism by which it is retained in cells with chelator. The mechanism by which BAPTA loading increases SR Ca content is not yet known. There is evidence that chelators can substantially increase the rate of 45Ca accumulation by SRvesicles [9], and by either microsomes or permeabilized synaptosomes [lo]. Co-transported anions can strongly affect the magnitude of SR Ca content [l 11; however, no chelator accumulation was observed in microsomes [lo]. Labelling of rabbit (but not rat) heart cells with AM esters of chelators can result in chelator labelling of the SR [ 121. Since we used rat heart cells, we expected no SR labelling with chelator. However, such labelling may depend on the level of AM ester used, so this possibility cannot yet be ruled out. Even though chelator uptake into mitochondria and SR may occur under these conditions, such subcompartmentation does not affect our conclusion. Also, in the experiments Cell Calcium
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Table
2 Stimulation of SR Ca content by cell BAPTA loading
No addition 30 FM BAPTAIAM 60 FM BAPTAIAM 120 pM BAPTAIAM 240 FM half BAPTA/AM
SR Ca (nmol/mg)
BAPTA (nmol/mg)
0.132 0.363 0.420 0.238 0.152
0.523 zt 0.210 0.956 zt 0.278 1.798 zt.0.411
f f * f f
0.009 0.009 0.029 0.045 0.022
SR Ca is taken as the mean value of the difference between %a efflux with and without caffeine at each time in Figure 6.
reported here, the measured Quin-2 and BAPTA content was the amount of cytosolic dye, defined as the dye releasable by digitonin, which at the levels used here ruptures the sarcolemma while leaving the SR and mitochondrial membranes intact. Is the restricted compartment just the diad cleft, or could it extend to a subsarcolemmal space which could contain a visible amount of dye? Our data cannot distinguish between these two possibilities, though the difference we observed between EGTA and BAPTA is suggestive. Knowledge of the chelator content of cells (Fig. 5) allows an estimate to be made of the distance within which each chelator can be expected to buffer incoming Ca effectively. Free Ca will fall exponentially from its unbuffered value to its fully buffered value with a length scale given by [6] : 3L= (D/(Cfk+))“* where D is the Ca diffusion coefficient, C, is the concentration of free chelator, and k, is the association rate constant for Ca binding to chelator. In our chelator assay for BAETA and EGTA, we measured digitonin releasable chelator, which would be the cytosolic fraction, excluding any mitochondrial chelator. A chelator content of 3 nmol/mg (Fig. 5), in a volume of 3.384 pl/mg, of which a fraction 0.7 is non-mitochondrial [13], thus corresponds to a concentration of 1.27 mM in the cytosol. For a resting [Cal of 50 nM, this corresponds at 37°C and pH 74 to a free chelator concentration (C,) of 0.56 mM for EGTA and 0.91 mM for BAPTA, calculated with the MAXC program [2]. We could not find values of k+ for our condition of 37°C and pH 74 precisely but, from the data of Smith et al 1141, we estimate k, = 1.5 x lo7 M-Is-’ for EGTA and from the data of Lattanzio [ 151 k, = 1.5 x IO9 M-*s-l for BAPTA (using a value measured for Fura-2). Using D = 1O-gm2/s [6], we then find li = 345 mn for EGTA and 27 nm for BAF’TA. These distances will be less if the diffusion coefficient is less than the value used here, which is a value typical of free diffusion. Thus, within a diad cleft of radius 100 nm [ 161, EGTA under our loading conditions should have little buffering ability while BAPTA should be effective. Q Harcourt
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Our result of a factor of 3 difference between EGTA and BAPTA could mean that the size of the space in which chelator competes with cellular Ca efflux mechanisms is rather larger than a 100 mn cleft. This may, therefore, be more consistent with a more extended subsarcolemmal space than that found in the cleft. Evidence for the existence of such a space, kinetically separated from the bulk cytoplasm, has been found in rat ventricular cells, from the phase shift in signals reporting Ca concentrations as measured by Na/Ca exchange at the sarcolemma and by Ca sensitive dyes in the bulk cytoplasm [li’]. On the other hand, Berlin et al [ 181 have found that the rise of intracellular Ca concentration measured by Indo-l in thapsigargin treated rat ventricular cells correlated closely in time with the integrated Ca current from a 200 ms voltage clamp. Also, Cheng et al [ 191 found that the onset of the appearance of Ca visible by Fluo-3 was as fast in TG treated cells as in untreated cells, and was uniform across the cell. These observations suggest that, if there is a kinetic barrier between the diad cleft and a dye-containing pool, then it is measured in milliseconds rather than in tens or hundreds of milliseconds; alternatively, the subsarcolemmal pool might contain enough dye to make Ca in this pool visible. It is also likely that in these measurements [ 18,191 the dyes promoted Ca diffusion from the cleft, thereby decreasing Ca gradients present without them. Thus, in the absence of intracellular chelator, there may be a significantly greater kinetic separation between Ca in the diad cleft and Ca in the cytosol, and this separation may account for the effect of Quin-2 which we observe. This effect of Quin-2 is therefore evidence for kinetic subcompartmentation of Ca within the cytosol. ACKNOWLEDGEMENTS
We thank Dr Michael Stern for the suggestion that we compare BAPTA and EGTA loading (Figs 4 & 5). This work was supported by grant HL33652 from the National Heart Lung and Blood Institute, and by a Grant-in-aid from the University of Wisconsin Foundation. REFERENCES 1. Haworth R.A., Goknur A.B., Biggs A.V., Redon D., Potter K.T. Ca uptake by heart cells. I. Ca uptake by the sarcoplasmic reticulum of intact heart cells in suspension. Cell Calcium 1998; 23: 181-198. 2. Bers D.M., Patton C.W., Nuccitelli R. A practical guide to the preparation of Ca*+ buffers. Metkods Cell Bioll994; 40: 3-29.
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3. Haworth R.A., Goknur A.B., Berkoff H.A. Measurement of slow Ca channel activity of isolated heart cells using 54Mn. Arch Biockem Biopkys 1989; 268: 594-604. 4. Trosper T.L., Philipson K.D. Stimulatory effect of calcium chelators on Na/Ca exchange in cardiac sarcolemma vesicles. Cell Calcium 1984; 5: 2 1l-222. 5. Slaughter RX, Sutko J.L., Reeves J.P.Equilibrium cakiumcalcium exchange in cardiac sarcolemmal vesicles. / Biol Ckem 1983; 258: 3183-3190. 6. Stern M.D. Buffering of calcium in the vicinity of a channel pore. Cell Grlcium 1992; 13: 183-192. 7 Anderson K., Meissner G. T-tubule depolarization induced SR Ca release is controlled by dihydropyridine receptor- and Cadependent mechanisms in cell homogenates from rabbit skeletal muscle. J Gen Pkysiol 1995; 105: 363-383. 8. Bassani J.W.M., Bassani R.A., Bers D.M. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. JPkysiol (Land) 1994; 476: 279-293. 9. Berman M.C. Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N’,-tetraacetic acid. / Biol Ckem 1982; 257: 1953-1957 10. Moore J.E.,Abercrombie R.F. Calcium chelators enhance 45Ca accumulation in permeabilized synaptosomes and in microsomes. AmJPkysioZ 1996; 270: C628-C635. 11. Wimsatt D.K., Hohl C.M., Brierley G.P., Altschuld R.A. Calcium accumulation and release by the sarcoplasmic reticulum of digitonm-lysed adult mammalian ventricular cardiomyocytes. J Biol Ckem 1990; 265: 14849-14857. 12. Chen W., Steenbergen C., Levy L.A., Vance J., London R.E., Murphy E. Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6difluorophenoxy)ethane-N,N,N’,N’-tetraacetic acid by 19FNMR. J Biol Ckem 1996; 271: 7398-7403. 13. Barth E., Stammler G., Speiser B., Schaper J. Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. J Mol Cell Cardioll992; 24: 669-681. 14. Smith P.D., Liesegang G.W., Berger R.L., Czerlinski G., Podolsky RJ. A stopped flow investigation of calcium ion binding by ethylene glycol bis(beta-aminoethyl ether)-N,N’-tetraacetic acid. Anal Biockem 1984; 143: 188-195. 15. Lattanzio Jr F.A., Bar&chat D.K. The effect of pH on rateconstants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators. Biockem Biopkys Res Commun 1991; 177: 184-191. 16. Langer G.A., Peskoff A. Calcium concentration and movement in the diadic cleft space of the cardiac ventricular cell. Biopkys/ 1996; 70: 1169-1182. 17 Trafford A.W., Diaz M.E., O’Neill S.C., Eisner D.A. Comparison of subsarcolemmal and bulk calcium concentration during spontaneous calcium release in rat ventricular myocytes. J Pkysiol (Land) 1995; 488: 577-586. 18. Berlin J.R., Bassani J.W.M., Bers D.M. Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. BiopkysJ 1994; 67: 1775-l 787. 19. Cheng H., Cannel1 M.B., Lederer WJ. Propagation of excitationcontraction coupling into ventricular myocytes. Pftigen Arch 1994;
428: 4 15-4
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Cell Calcium
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Research
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Ca uptake by heart cells: Il. Most entering Ca appears to leave without mixina with the sarconlasmic reticulum Ca pool Robert A. Haworth, David Redon, Angela V. Biggs, Katherine T. Potter Department
of Anesthesiology,
University
of Wisconsin,
Madison,
Wisconsin,
USA
Summary The rate of verapamil-sensitive uptake of 45Ca by rat heart cells stimulated to beat in suspension with 0.2 mM Ca and isoproterenol was increased > 2-fold by cell loading with the chelator Quin-2. No effect of Quin-2 loading was observed on the rate of uptake of trace levels of 54Mn, present in addition to Ca, which was used as an index of Ca channel activity. Quin-2 loading also had little effect on the rate of 45Ca uptake by cells diluted into a high K/low Na medium, where Ca uptake was primarily by Na/Ca exchange. The fast chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was 3-fold more effective than the slow chelator EGTA at preventing Ca efflux. BAPTA loading also caused an increase in sarcoplasmic reticulum (SR) Ca content. These results suggest that chelator loading had little effect on the rate of Ca influx by Ca channels or by Na/Ca exchange, and that the increased rate of 45Ca uptake seen with Quin-2 loading was caused by an inhibition of Ca efflux, either directly by chelation or by increased Ca uptake by the SR or by other intracellular organelles. This further suggests that most of the Ca entering the cell without chelator leaves again within the same beat, and that this may result from Ca efflux from a kinetically limited Ca pool in or around the diad cleft.
INTRODUCTION
In the previous paper, we described the uptake of 45Caby isolated adult rat heart cells in suspension subjected to electric field stimulation [l]. We found an exponential increase in the extent of labelling of cellular Ca, and the initial rate of labelling was proportional to the rate of stimulation. We found a rate of cellular labelling of 5.25 pmol/mg/beat. This uptake was composed of several elements, the major stimulation-dependent one being the SR, which labelled at a rate of 3.87 + 0.17 pmol/mg/beat. Does the 5.25 pmol/mg/beat represent the rate of 45Ca Received
8 September
1997
influx, which on entry mixes with the cytosolic Ca pool, some of which is then accumulated by the SR? Or, does more labelled Ca than this enter, with most of it leaving again within the same beat, without mixing with most of the SR Ca? The latter scenario would be evidence for some kind of compartmentation of the cytosolic Ca pool. We sought to distinguish between these two possibilities by looking for evidence that the true rate of 45Ca entry was indeed greater than the measured rate of 45Ca uptake. Our approach was to load cells with Quin-2, a Ca chelator, in order to trap as much incoming Ca as possible. Our data suggest that the true rate of Ca influx is indeed significantly greater than the rate at which the Ca is retained, unless chelator is present.
Revised 20 January 1998 Accepted 20 January 1998 Correspondence to : Dr Robert A. Haworlh, Department of Surgery, University of Wisconsin Clinical Science Center, 600 Highland Avenue,
Cell isolation
Madison, WI 53792 USA Tel: +l 608 263 1339; Fax: +l 608 263 0575 E-mail: [email protected]
Heart cells were isolated from female retired breeder rats as in the previous paper [I]. 199
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Experimental medium
20
Experimental medium was based on a Krebs-Henseleit (basal K-HEPES) medium containing (mM): NaCl 118, KC1 4.8, HEPES 25, KH,PO, 1.2, MgSO, 1.2, glucose 11, and pH was adjusted to 7.4 with NaOH. For isotope uptake experiments, 0.2 mM CaCl,, 5 mM Na pyruvate, and 1 @I insulin were also present. For fluorescence experiments, 0.05 mM DTPA (diethylenetriamine pentaacetic acid) was included as a heavy metal scavenger, and CaCl, was increased to 0.25 mM. Suspensions were maintained aerobic by equilibration with air in a shaking incubator at 37°C.
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Labelling of cells with BAPTA, EGTA or Quin-2
Aliquots (1 ml) of cells in experimental medium (6.2 mg/ml) were incubated with the acetoxymethyl (AM) ester of chelators in amounts as described in the figures, for 20 ruin at 3Tc. The suspension was divided and centrifuged at 29 gfor 90 s and the cell pellet of each half resuspended to 0.5 ml in either experimental medium or assay medium (see below). Cells in experimental medium were exposed to isoproterenol (1 @VI) for 7 ruin, before measurement of 45Caand 54Mn uptake. Chelator assay
To measure the uptake of non-fluorescent chelators by cell suspensions incubated with the acetoxymethyl ester of the chelators, we developed an assay based upon chelator competition for Ca with the resin Chelex 100 (BioRad). Aliquots (0.5 ml, 6.2 mg/ml) of labelled cell suspension (or unlabelled controls) were centrifuged at 29 g for 90 s, the supernatant removed, and the pellet resuspended to 0.5 ml with assay buffer containing basal K-HEPES (see above) plus 0.2 mM CaCl,, 0.25 mCi 45Ca,5 u.M ruthenium red and 25 ug/ml digitonin. This caused release of chelator from the cytosol of the cells. After 2 min incubation at 37X, the cell suspension was centrifuged in a high speed bench centrifuge to remove cell debris. The supematant was added to 1 ml basal K-HEPES, and the mixture added to 63 mg Chelex 100 in a 9 x 50 mm acid washed glass tube. Standards were made by adding known amounts of chelator to 0.5 ml aliquots of assay buffer, then adding to 1 ml K-HEPES and Chelex as for cell extracts. Standards for EGTA were from a 0.1 M solution from Molecular Probes (Eugene, OR, USA). A standard BAPTA solution was prepared after drying solid BAPTA salt to constant weight [2]. The tubes were sealed with Parafilm and mixed by gentle inversion for 30 min at room temperature. During this time, the Chelex removes most of the Ca (and 45Ca) from the medium, leaving an amount proportional to the Cell Calcium
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Fig. 1 Measurement of BAPTA and EGTA by the chelator assay. Ordinate shows the concentration of Ca retained by the abscissa level of chelator, in competition with Chelex resin (see Materials and methods). Circles: EGTA; triangles: BAPTA.
chelator content of the medium. Samples were centrifuged to remove Chelex. Ca concentration remaining in the medium was determfned by liquid scintillation counting of aliquots of the sample supematants and of the assay buffer. Figure 1 shows standard curves for EGTA and for BAPTA. The assay has good linearity, and allows chelator contents to be accurately determined. To measure the uptake of fluorescent chelators, the Mn sensitive (* 0.2mM Mn) component of the fluorescence of a suspension of cells in basic K-HEPES buffer with 1 mM Ca plus 25 ug/ml digitonin and 10 uM DTPA was compared with that of a known concentration of chelator salt, in a cuvette measurement. Isotope flux measurements
Procedures were as in the previous paper [ 11.To measure uptake of 54Mn, M&l, containing 54Mn was added at time zero to give 2.5 uCi 54Mn/ml cell suspension and 10 @VIMnCl,. Ca (0.2mM) was still present in the medium. Ca uptake in high K medium
Cells (6.2 mg/ml) in experimental medium were loaded or not with Quin-2 as described above, except that isoproterenol (1 @I) was added 7 min before the cells were centrifuged. The cell pellet was resuspended by adding 0.1 ml experimental medium plus isoproterenol, to a final volume of 0.14 ml. To initiate 45Ca uptake, this was then added to 0.9 ml experimental medium with NaCl replaced by KC1 (where shown), 1 mM ouabain, 5 0 Harcouti
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@vl ruthenium red, 1 @I isoproterenol, 0.2 mM CaCl, with 45Ca, and either 3H,0 or 10 pM MnCl, with 54Mn as above. 60 ~1 aliquots of cell suspension were removed at the times shown and added to 540 pl ice cold basal KHEPES medium with Na replaced by K, 0.5 mM EGTA, and 5 uM ruthenium red. The cells (0.5 ml aliquots) were then centrifuged after 2 min through 0.5 ml bromododecane into 0.1 ml 16% per&lo& acid as before [l]. Data analysis
Values shown are the mean and standard deviation of at least three experiments. Ca uptake by cells was fitted by non-linear least squares to the functions described in each figure. Lines on figures are these best fit curves, unless otherwise indicated.
Ca channel activity was unaffected. A nice feature of using Mn in this way is that 0.2 mM Ca is still present in the medium. Since we included 10 l.tM Mn in the 45Ca
G
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Since the condition of 0.2. mM Ca with isoproterenol allowed us to most easily measure the rate and extent of stimulation dependent cellular 45Ca fluxes [l], this continued to be our standard condition for measuring these parameters, for experiments described in this paper. To gain some measure of the amount of Ca entering the cell per beat, we loaded cells with the Ca chelator Quin-2 by incubation with 30 ).&I Quin-2/AM for 20 min, with the aim of trapping incoming Ca. This treatment resulted in a total uptake of 2.01 k 0.31 nmol Quin-2/mg. Quin-2 caused an increase in the initial rate of verapamil-sensitive Ca uptake by a factor of > 2 (Fig. 2A). The initial rate of verapamil sensitive Ca inilux per beat measured in the presence of Quin-2, calculated from the best fit curve in Figure 2A, was 14.73 pmol/mg/beat. This may be an underestimate of the rate of Ca influx, since this level of Quin-2 loading may not trap all incoming Ca. Is the Quin-2 effect on Ca uptake caused by an inhibition of Ca efflux or by a stimulation of Ca influx? To answer this we sought to measure the effect of Quin-2 loading on the rate of Ca inilux by each of the two known possible routes for Ca influx: Ca channels, and the Na/Ca exchanger. We have previously found that the rate of uptake of trace levels of in is an indicator of Ca channel activity, since Mn enters by Ca channels but not by Na/Ca exchange [3]. We, therefore, measured the effect of Quin-2 loading on the uptake of trace levels of Mn. We found that Mn uptake under these conditions was unaffected by Quin-2 loading (Fig. 2B), suggesting that 0 Harcouti
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time (s) Fig. 2 Effect of Quin-2 loading on uptake of %a and %Mn. (A) Ca uptake; (B) Mn uptake. Circles: no Quin-2 (Q); triangles: with Quin-2 (30 uM QuinG?/AM); filled symbols: no verapamil (V); open symbols: with 10 PM verapamil. Cells all with 1 uM isoproterenol, and all stimulated at 2 Hz. The dashed line in (A), shown for comparison, is the best fit line from [l] Figure 2, for the condition of no stimulation with verapamil. Ca uptake was fitted by a double exponential function as in the previous paper [l], using this line as the first exponential. Mn uptake was fit by a single exponential function.
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1.81
-
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Fig. 3 Effect of Quin-2 loading on uptake of Ca and Mn on 1:9 dilution into high K medium. Circles: no Quin-2 (XI); triangles: with Quin-2 (+Q, 30 uM CUinGYAM). (A) Ca uptake: open symbols -to Na +verapamil, filled symbols-to K no verapamil. Data were fitted to a single exponential function. (B) Verapamil sensitive Mn uptake: open symbols - stimulation induced (the difference between the Mn uptake seen without and with verapamil in Fig. 28 (+S-V)-(+S+V)). Filled symbols - high K induced (the difference between Mn uptake by cells diluted to K -verapamil and that by cells diluted to Na +verapamil). Data were fitted to straight lines. See Materials and methods for experimental details.
uptake measurement in Figure 2A, the conditions for measuring 45Caand 54Mn uptake are chemically identical. Thus, we may conclude that the increased rate of Ca uptake seen with Quin-2 loading cannot be explained in terms of an increased rate of Ca entry by Ca channels. Ca can also enter the cell by Na/Ca exchange, and Quin-2 could potentially stimulate this [4]. To see whether or not we could find evidence for a stimulation of the rate of Ca influx by Qum-2, we measured the rate of 45Cauptake by unstimulated cells in which exchanger mediated Ca influx was stimulated and Ca efflux was inhibited, by diluting cells into medium in which all but 19 mM Na was replaced by K. Ouabain was also present in the dilution medium, to prevent energized efflux of cell Na. Under these conditions, intracellular and Cell Calcium (1998)
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extracellular concentrations of Na and K are very similar, resulting in zero membrane potential. This will, in principle, favor Ca influx both by Ca channels and by Na/Ca exchange. Ca uptake under these conditions was fast and large, but Quin-2 loading increased the rate of uptake by only 16% (Fig. 3A; Table 1). Verapamilsensitive Mn uptake was low under these conditions (Fig. 3B; Table l), suggesting that most of the Ca uptake under these conditions was by Na/Ca exchange. This, therefore, suggests that Quin-2 loading under these conditions does not much stimulate Ca influx by Na/Ca exchange. Since 45Ca could also be carried into the cell by Ca/Ca exchange through the Na/Ca exchanger [5], we tested whether or not Quin-2 could be promoting this mode of entry. Cells under the conditions of Figure 3A were allowed to accumulate unlabelled Ca for 2 min, by which time Ca uptake, based on Figure 3A, was maximal. At that time, carrier-free 45Ca was added, and cells were centrifuged thereafter. The rate of 45Ca uptake was less than half that seen in Figure 3, and Quin-2 loading again only slightly increased the initial rate of 45Cauptake (data not shown). This suggests that there is little effect of the chelator on the rate of Ca influx through the exchanger in either mode. These results suggest that the increased rate of stimulation induced 45Ca uptake measured with Quin-2 is the result of trapping of incoming Ca rather than stimulation of the rate of influx. This further suggests that a significant fraction of the incoming Ca in the absence of Quin-2 is being extruded from the cell without mixing with most of the rapidly labelling cellular Ca pool. To gain further evidence for a kinetic separation between the cleft Ca pool and the rest of the cytosol, we have compared BAPTA loading and EGTA loading for their effectiveness at increasing the measured rate of 45Ca uptake. EGTA binds Ca much more slowly than BAF’TA, Table 1 Comparison of the effect of Quin-2 loading on initial of Ca uptake and verapamil sensitive Mn uptake induced by electric field stimulation and by high K Ca uptake (pmol/mg/s) Stimulation -Quin-2 +Quin-2 High
K -Quin-2 +Quin-2
rate
Mn uptake (pmol/mg/s)
rates
rate
at 2 Hz 13.78 29.45
* 1.86 * 2.34
0.388 0.392
f 0.016 f 0.013
44.20 51.32
f 5.30 2 6.60
0.050 0.046
f 0.011 zt 0.012
Values for initial rates of uptake were taken from the best fit curves shown in Figures 2 and 3. Ca uptake values are relative to the condition +stim +verapamil for stimulation induced uptake (Fig. 2A), and relative to the condition ‘to Na +verapamil’ for the high K induced uptake (Fig. 3A). Verapamil sensitive Mn uptake was as defined in Fig. 38.
63 Harcourf Brace & Co. Ltd 1998
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Fig. 4 Stimulation of Ca uptake by loading cells with BAPTA or EGTA. Circles: no chelator, with stimulation at 2 Hz (filled symbols) or without stimulation plus 10 uM verapamil (-S+V, open circles). (A) Cells incubated with 15 uM (triangles) or 45 uM (squares) BAPTA/AM for 20 min to give BAPTA uptakes of 1.472 f 0.431 and 3.445 * 0.177 nmol/mg, respectively. (6) Cells incubated with 35 uM (triangles) or 105 uM (squares) EGTAIAM for 20 min to give EGTA uptakes of 2.515 f 0.109 and 4.821 i0.183 nmollmg, respectively. Cells with chelator were also stimulated, at 2 Hz. All cells were treated with 1 ,uM isoproterenol.
6
Fig. 5 Dependence
of initial rate of Ca uptake on chelator content. Data from experiments like Figure 4 were fit to the sum of the linear rate (with no stimulation plus verapamil) and an exponential rate. The initial rate of the best fit exponential function for each condition from each experiment is shown, for five experiments. Each symbol is from a separate experiment. The lines are linear regressions for the data with BAPTA (filled symbols) and with EGTA (open symbols). Slopes of the regressions were 0.00870 for BAPTA and 0.00298 for EGTA.
r
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even though their affinities are similar [6]. BAPTA should thus be a much more effective chelator than EGTA at modulating processes which are governed by transient local gradients of Ca, such as may exist between the diad cleft and the cytosol. BAPTA has indeed been found to be more effective than EGTA at inhibiting the Ca dependent component of SR Ca release induced by t-tubule depolarization in skeletal muscle homogenates [7j. We, therefore, loaded cells with different levels of BAPTA and EGTA by incubation with different levels of their AM esters, and measured the rate of 45Cauptake as in Figure 2, along with the amount of chelator present in the cytosol of the cells. We found that EGTA was able to stimulate Ca uptake almost as much as BAFTA (Fig. 4). However, the amount of EGTA required to do this was greater by a factor of 3 (Fig. 5). To investigate how cell chelator loading may affect Ca uptake by the SR, we used the size of the caffeine releasable Ca pool as a measure of SR Ca content as described in the previous paper [l]. Cells were loaded with increasing levels of BAPTA by incubation with increasing levels of BAPTMAM, then stimulated to beat at 2 HZ for 2 min with 45Ca. We found that increasing levels of BAPTA loading resulted in increasing levels of caffeine-releasable Ca when the cells were diluted into EGTA with or without caffeine, though at the highest 0 Harcourt Brace & Co. Lfd 1998
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Fig. 6 Stimulation of SR Ca content by BAPTA. BAPTA loaded cells with isoproterenol and %a were stimulated at 2 Hz for 2 min before dilution into unlabelled medium with (filled symbols) or without (open symbols) 10 mM caffeine. Circles: no BAPTA/AM; upright triangles: 30 uM BAPTNAM; squares: 60 uM BAPTAfAM; inverted triangles: 120 uM BAPTAfAM; diamonds: 240 pM halfBAPTA/AM. The BAPTA content of cells was measured as described under Materials and methods.
Cell Calcium (1998) 23(4), 199-205
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level of BAPTA loading the magnitude of caffeine releasable Ca was again reduced (Fig. 6). The size of the SR Ca pools for each level of cell BAPTA measured is given in Table 2. Incubation with the AM ester of the non-chelating molecule half-BAPTA had no effect on SR pool size or Ca uptake (Fig. 6; Table Z), which suggests that, at these levels of ester, there is no artefactual effect of ester hydrolysis on cell Ca homeostasis. The stimulation of SR pool size seen with 60 @I BAF”IA/AM is, therefore, caused by the chelating property of BAPTA.
DISCUSSION
The major experimental finding of this paper is that the rate of Ca influx into heart cells stimulated to beat appears to be much greater than the measured rate of Ca uptake, unless intracellular chelators are present. This implies that most of the Ca entering the cell without Quin-2 is returned to the medium without mixing with most intracellular Ca stores and, in particular, the SR Ca store: since the SR is thought to re-accumulate > 90% of the Ca associated with a Ca transient [8], not all of the entering Ca can be reaching that Ca transient pool. This failure to mix suggests that kinetic factors impose a kind of compartmentation on the cytosolic Ca, even though the cytosol is spatially continuous. The above conclusion is unaffected by the observation that SR Ca content can be increased by chelators (Fig. 6). Even if all of the increase in Ca uptake seen with chelators is located in the SR, as opposed to being directly chelated by Quin-2, the cell can only retain this 45Caif the 45Cafirst enters the cell. Our data suggest that the Ca influx per beat is unaffected by the level of Quin-2 used here (Figs 2B & 3). If this is so, then in the absence of chelator most of this entering Ca must be leaving within the same beat, regardless of the mechanism by which it is retained in cells with chelator. The mechanism by which BAPTA loading increases SR Ca content is not yet known. There is evidence that chelators can substantially increase the rate of 45Ca accumulation by SRvesicles [9], and by either microsomes or permeabilized synaptosomes [lo]. Co-transported anions can strongly affect the magnitude of SR Ca content [l 11; however, no chelator accumulation was observed in microsomes [lo]. Labelling of rabbit (but not rat) heart cells with AM esters of chelators can result in chelator labelling of the SR [ 121. Since we used rat heart cells, we expected no SR labelling with chelator. However, such labelling may depend on the level of AM ester used, so this possibility cannot yet be ruled out. Even though chelator uptake into mitochondria and SR may occur under these conditions, such subcompartmentation does not affect our conclusion. Also, in the experiments Cell Calcium
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Table
2 Stimulation of SR Ca content by cell BAPTA loading
No addition 30 FM BAPTAIAM 60 FM BAPTAIAM 120 pM BAPTAIAM 240 FM half BAPTA/AM
SR Ca (nmol/mg)
BAPTA (nmol/mg)
0.132 0.363 0.420 0.238 0.152
0.523 zt 0.210 0.956 zt 0.278 1.798 zt.0.411
f f * f f
0.009 0.009 0.029 0.045 0.022
SR Ca is taken as the mean value of the difference between %a efflux with and without caffeine at each time in Figure 6.
reported here, the measured Quin-2 and BAPTA content was the amount of cytosolic dye, defined as the dye releasable by digitonin, which at the levels used here ruptures the sarcolemma while leaving the SR and mitochondrial membranes intact. Is the restricted compartment just the diad cleft, or could it extend to a subsarcolemmal space which could contain a visible amount of dye? Our data cannot distinguish between these two possibilities, though the difference we observed between EGTA and BAPTA is suggestive. Knowledge of the chelator content of cells (Fig. 5) allows an estimate to be made of the distance within which each chelator can be expected to buffer incoming Ca effectively. Free Ca will fall exponentially from its unbuffered value to its fully buffered value with a length scale given by [6] : 3L= (D/(Cfk+))“* where D is the Ca diffusion coefficient, C, is the concentration of free chelator, and k, is the association rate constant for Ca binding to chelator. In our chelator assay for BAETA and EGTA, we measured digitonin releasable chelator, which would be the cytosolic fraction, excluding any mitochondrial chelator. A chelator content of 3 nmol/mg (Fig. 5), in a volume of 3.384 pl/mg, of which a fraction 0.7 is non-mitochondrial [13], thus corresponds to a concentration of 1.27 mM in the cytosol. For a resting [Cal of 50 nM, this corresponds at 37°C and pH 74 to a free chelator concentration (C,) of 0.56 mM for EGTA and 0.91 mM for BAPTA, calculated with the MAXC program [2]. We could not find values of k+ for our condition of 37°C and pH 74 precisely but, from the data of Smith et al 1141, we estimate k, = 1.5 x lo7 M-Is-’ for EGTA and from the data of Lattanzio [ 151 k, = 1.5 x IO9 M-*s-l for BAPTA (using a value measured for Fura-2). Using D = 1O-gm2/s [6], we then find li = 345 mn for EGTA and 27 nm for BAF’TA. These distances will be less if the diffusion coefficient is less than the value used here, which is a value typical of free diffusion. Thus, within a diad cleft of radius 100 nm [ 161, EGTA under our loading conditions should have little buffering ability while BAPTA should be effective. Q Harcourt
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Ca uptake by heart cells: II. Entering Ca does not mix with SR Ca pool 205
Our result of a factor of 3 difference between EGTA and BAPTA could mean that the size of the space in which chelator competes with cellular Ca efflux mechanisms is rather larger than a 100 mn cleft. This may, therefore, be more consistent with a more extended subsarcolemmal space than that found in the cleft. Evidence for the existence of such a space, kinetically separated from the bulk cytoplasm, has been found in rat ventricular cells, from the phase shift in signals reporting Ca concentrations as measured by Na/Ca exchange at the sarcolemma and by Ca sensitive dyes in the bulk cytoplasm [li’]. On the other hand, Berlin et al [ 181 have found that the rise of intracellular Ca concentration measured by Indo-l in thapsigargin treated rat ventricular cells correlated closely in time with the integrated Ca current from a 200 ms voltage clamp. Also, Cheng et al [ 191 found that the onset of the appearance of Ca visible by Fluo-3 was as fast in TG treated cells as in untreated cells, and was uniform across the cell. These observations suggest that, if there is a kinetic barrier between the diad cleft and a dye-containing pool, then it is measured in milliseconds rather than in tens or hundreds of milliseconds; alternatively, the subsarcolemmal pool might contain enough dye to make Ca in this pool visible. It is also likely that in these measurements [ 18,191 the dyes promoted Ca diffusion from the cleft, thereby decreasing Ca gradients present without them. Thus, in the absence of intracellular chelator, there may be a significantly greater kinetic separation between Ca in the diad cleft and Ca in the cytosol, and this separation may account for the effect of Quin-2 which we observe. This effect of Quin-2 is therefore evidence for kinetic subcompartmentation of Ca within the cytosol. ACKNOWLEDGEMENTS
We thank Dr Michael Stern for the suggestion that we compare BAPTA and EGTA loading (Figs 4 & 5). This work was supported by grant HL33652 from the National Heart Lung and Blood Institute, and by a Grant-in-aid from the University of Wisconsin Foundation. REFERENCES 1. Haworth R.A., Goknur A.B., Biggs A.V., Redon D., Potter K.T. Ca uptake by heart cells. I. Ca uptake by the sarcoplasmic reticulum of intact heart cells in suspension. Cell Calcium 1998; 23: 181-198. 2. Bers D.M., Patton C.W., Nuccitelli R. A practical guide to the preparation of Ca*+ buffers. Metkods Cell Bioll994; 40: 3-29.
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