257

Journal of Physiology (1991), 442, pp. 257-276 With 11 figures Printed in Great Britain

CYTOSOLIC FREE Ca2+ DURING OPERATION OF SODIUM-CALCIUM EXCHANGE IN GUINEA-PIG HEART CELLS

BY A. NOMA*, T. SHIOYA*, L. F. C. PAVER, V. W. TWIST AND T. POWELLt From the University Laboratory of Physiology, Parks Road, Oxford OXI 3PT

(Received 14 January 1991) SUMMARY

1. Membrane current generated by the Na+-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltageclamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa Ca) was measured from records of the 2 mM-Ni2+-sensitive current and used in an attempt to clamp [Ca2+ ]i at a level determined by the ionic compositions of the external and pipette solutions. 2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa ca expected for a 3Na+: ICa2+ exchanger. The measured value of ENa ca was more positive than the theoretical value when the membrane potential was held positive to ENa ca and the opposite was true when the holding potential was more negative than the expected ENa Ca. 3. As Indo-l diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly. 4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+. 5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette was reduced from 30 to 1 mm, an increase in [Ca2+]i was observed during a depolarizing ramp pulse. The Ca2+ influx estimated by integrating the 2 mw-Ni2+-sensitive current during the pulse correlated with the * Present address: Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. t To whom correspondence should be sent.

MS 9074 9

PHY 442

258

A. NOMA AND OTHERS

increase in [Ca2+]i estimated from Indo- 1 using the extracellular calibration curve, but the values of the influx determined directly from Indo- 1 fluorescence were always larger than those calculated from the exchanger current. 6. Hyperpolarization to -80 mV from a holding potential of -40 mV decreased [Ca2+]i, while the membrane slope conductance for outward Na+-Ca2+ exchange current increased rapidly. The opposite was true when the membrane was depolarized. These effects were reversed by clamping back to -40 mV. Membrane conductance for the inward exchanger current did not change markedly during these manoeuvres. 7. These observations suggest that when Indo-1 is applied through a dialysing patch pipette the resulting intracellular fluorescence can be calibrated to give a satisfactory estimate of [Ca2+]i, and that changes in membrane potential produce marked variations in [Ca2+]i via modulation of Na+-Ca2+ exchange. INTRODUCTION

Quantitative measurement of the cytosolic free Ca2+ concentration ([Ca2+]i) is obviously essential for studying the role of Na+-Ca2+ exchange in controlling [Ca2+]i in the heart (Crespo, Grantham & Cannell, 1990). The tetracarboxylate fluorescent Ca2+ indicators Fura-2 and Indo-1 (Grynkiewicz, Poenie & Tsien, 1985) have been used frequently to monitor [Ca2+]i in cardiac muscle (Barcenas-Ruiz & Wier, 1987; Cannell, Berlin & Lederer, 1987; Wier, Cannell, Berlin, Marban & Lederer, 1987; Beukelmann & Wier, 1988; Callewaert, Cleemann & Morad, 1988; Bers, Lederer & Berlin, 1990; Crespo et al. 1990; LeBlanc & Hume, 1990). Ih many studies, calibration of the fluorescent signals has been achieved through the use of extracellular calibration solutions, under the assumption that the Caa2+ indicator is distributed uniformly within the cytoplasm with properties identical to those observed during the calibration procedures. As noted in some of the reports cited above, there is now much evidence to suggest that such an assumption is tenuous under a variety of experimental conditions. In skeletal muscle, a number of Ca2+ indicators, including Fura-2, are bound to myoplasmic proteins which results in alteration of their spectral and kinetic properties (see, for example, Baylor & Hollingworth, 1988; Kao & Tsien, 1988; Konishi, Olson, Hollingworth & Baylor, 1988; Hirota, Chandler, Southwick & Waggoner, 1989). Such factors result in uncertainty in the estimation of [Ca2+]i, especially during rapid [Ca2+]i transients (Klein, Simon, Szucz & Schneider, 1988) and on theoretical grounds this may also be the case in the heart (Noble & Powell, 1990). In experiments using single cardiac cells, particularly when the membrane-permeant forms of Fura-2 or Indo- 1 are used, similar problems arise, including accumulation of the probe in intracellular organelles (Powell, Twist & Yamaoka, 1988; Spurgeon, Stern, Baartz, Raffaeli, Hansford, Talo, Lakatta & Capogrossi, 1990). Recently, Blatter & Wier (1990) have shown that, under their experimental conditions, approximately one-third of either Fura-2 or Indo-1 was not diffusible in the myoplasm of guinea-pig ventricular myocytes when loaded using the acetoxymethyl ester forms of the indicators. The paper by Blatter & Wier (1990) should be consulted for a detailed description and analysis of these problems. In the present study, we have loaded Indo-1 into single cardiac myocytes through

259 CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART a perfused patch-pipette used to record Na+-Ca2' exchange current (Kimura, Noma & Irisawa, 1986; Kimura, Miyamae & Noma, 1987). We attempted to clamp [Ca2+]i at the level of Ca21 in the dialysing pipette by holding the cell under voltage-clamp at the expected equilibrium potential for a 3Na+: ICa2+ exchanger (Ehara, Matsuoka & Noma, 1989) and have compared Indo-1 emitted fluorescence under these conditions with that obtained extracellularly from solutions identical in composition to those in the patch pipette. Using this approach, we were able to demonstrate quantitatively the differences between in vivo and in vitro calibration curves for determining [Ca2+]i using Indo- 1. We conclude that under our experimental conditions ([Ca2+]i usually > 500 nM) Indo-1 can be used with an in vitro calibration curve to obtain quantitative estimates of [Ca2+]i in the heart, but that near resting [Ca2+]i (- 100 nM) a combination of intracellular and extracellular calibration constants is required to adequately described cellular fluorescence. METHODS

Myocyte preparation Single ventricular cells were dissociated from guinea-pig hearts, obtained from animals killed by cervical dislocation after stunning, by digestion with collagenase and protease using established techniques described elsewhere (Powell & Twist, 1976; Powell, Terrar & Twist, 1980). Isolated myocytes were stored at room temperature in Dulbecco's modified Eagle's medium (MEM) solution, supplemented with 20 mM-HEPES and 5 % (v/v) of a serum substitute (Ultroser G, GIBCO, UK). Aliquots of cells were transferred to a bath on the stage of an inverted microscope (Nikon Diaphot) and superfused with Tyrode solution containing (mM): NaCl, 140; KCI, 5-4; MgCl2, 0 5; CaCl2, 1-8; NaH2PO4, 0 33; glucose, 11; HEPES, 5 (pH adjusted to 7-4 at 22-24 °C with NaOH). Bath temperature was controlled via a thermocouple feedback circuit and the fluid level was regulated by an Akers level-sensing system (Cannell & Lederer, 1986). The experiments reported here were carried out at temperatures of 34-36 'C.

Electrophysiology The whole-cell tight-seal voltage-clamp technique was used for electrophysiological recording (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Patch electrodes were fabricated from glass capillaries (WPI, TW150-4) on a puller (Narishige PP83) and were used without fire-polishing or coating with Sylgard. In order to facilitate equilibration of the intracellular medium with the pipette solution, glass electrodes having large tip diameters were used. The tip resistances ranged from 1-2 MQ when filled with Tyrode solution. After forming a tight seal on the cell surface with Tyrode in the pipette, the pipette solution was exchanged for the appropriate internal solution using a perfusion device (Matsuda & Noma, 1984). Brief strong suction was then applied to the pipette interior to rupture the membrane patch. Membrane current and voltage were recorded using a patch-clamp amplifier (Axopatch 1C, Axon Instruments, USA). The current-voltage (I-V) relation was measured by applying a triangular ramp pulse of 1 V s-, first by depolarizing to + 70 mV followed by a hyperpolarization to -120 mV. The I-V curve was measured from the negative-going limb of the ramp pulse. This protocol was repeated at an interval of 5 s with both current and voltage monitored on an oscilloscope and chart recorder. The cell input capacitance was measured from the jump in membrane current recorded at the positive peak of the ramp pulse and in the experiments reported here typical input capacitance measured in a sample of cells was 141 + 35 pF (mean + S.D.; n = 35). Current and voltage were digitized (CED 1401, Cambridge, TJK) and then stored in a computer (IBM-AT) for subsequent analysis. Capacitative current has been subtracted from all of the records shown here, with the exception of those in Fig. 9.

Fluorescence measurements The optical system used in the experiments described here was developed over the last four years in collaboration with Nikon Instruments (Telford, UK) and all of the components are commercially available. A cell was illuminated using a 75 W xenon arc lamp via a quartz epi-fluorescence 9-2

~~~

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260

attachment, with the light passing through a 360 nm band-pass filter (10 nm bandwidth, Omega Optical, Brattleboro, USA) and directed on to the specimen by a Nikon 380 nm dichroic mirror through a Fluor 40 x dry objective (N.A. 0-85; Nikon, UK). The lamp-house containing the xenon source was modified so that an electronic shutter (Vincent Associates, Rochester, USA) could be A J

0 5~

P.

[

405n

\

>

480 nm

~~~~~~~~E

100 ms

B 405nm a

480 nm

a

_

a a

>

4 min

Fig. 1. Experimental protocol. A, records of fluorescent signals at 405 and 480 nm used for computer analysis. The shutter for the excitation light source (360 nm) was opened for a period of 300 ms, indicated by the horizontal bar. Each signal was filtered by a low-pass filter at 500 Hz and averaged over a period of 200 ms, once a steady state had been reached. Following closure of the shutter another average of the base-line level was calculated and the amplitude of the Indo- 1 signal taken as the difference between the two mean values. B, records obtained from three representative cells. In each panel the letter 'a' indicates when exchange of the pipette solution began from Tyrode to one containing 50 4uM-Indo- 1. Whole-cell recording commenced at the arrow. Brief displacements of the baseline were caused when the experimental enclosure door was opened to change the pipette solution. The current-to-voltage converter for each photomultiplier tube had an overall gain of 1 mV/nA. attached and this shutter was opened for 300 ms every 5 s, in synchrony with the voltage-clamp commands. Fluorescent signals were directed through the side-port of the microscope to an adjustable rectangular aperture and thence to a Microflex AFX-DX camera attachment, which had an ocular in order that a cell could be positioned correctly in a suitable rectangular field of view. The light path then entered a beam-splitter containing a Nikon 455 nm dichroic mirror, the outputs of which were directed to two photomultiplier tubes (9635B, Thorn-EMI, UK) through two Nikon band-pass filters at 405 nm (10 nm bandwidth) and 470-490 nm, respectively. Signals from the photomultiplier tubes were amplified by a two-channel current-to-voltage converter (Applegarth Electronics, Oxford, UK) filtered at 500 Hz and stored in an IBM-AT computer via the CED 1401 used to collect the voltage-clamp data. Experimental protocols and data collection were controlled by this computer through software written by T. Shioya and A. Noma. Sample fluorescence records are shown in Fig. 1A. The signals reached a steady level within

CYTOSOLIC Ca2+ AND Nat_Ca2+ EXCHANGE IN HEART

261

20 ms after opening the excitation shutter. When measuring the time course of the fluorescence change (Figs 7 and 11), each fluorescence signal was averaged over the period when the shutter was open and from this was subtracted a similar average when the shutter was closed. The experimental protocol used for the fluorescence measurements is illustrated by reference to Fig. 1 B. In this chart recording, the baseline indicates the signal during the period when the shutter was closed and the TABLE 1. Composition of internal solutions (mM unless indicated otherwise) 803 600 600 803 803 300 300 153 153 153 17-4 15-5 15-5 10 17-4 12-3 10 10 12-3 10 [Na'] 0 0 0 0 0 0 0 0 140 0 [K+] 30 1 30 1 1 30 1 BAPTA 30 10 30 24-1 0-8 4-4 22-6 075 241 06 044 18-2 13-2 CaC12 7-4 7-4 0 5-5 5-5 2-32 0 0 2-32 NaCl 0 1-8 1P8 1-8 1-8 18 1-8 1-8 11-8 1-8 1-8 MgCl2 42 71 42 42 71 71 42 42 71 42 Aspartate To these solutions were added (mM): MgATP, 10; Na2CrP, 5; TEA-Cl, 5. The pH was adjusted to 7-4 with CsOH. In the K+-containing solution, BAPTA and ATP were added as potassium salts and the pH was adjusted with KOH.

[Ca2"] (nM)

peak value when the shutter was open. Fluorescent signals from the region of a cell defined by the rectangular aperture (see above) were first measured after a gigaseal was formed. Subsequent exchange of the pipette solution from the normal Tyrode to the appropriate internal solution containing 50 /SM-Indo-1 (starting at a in Fig. 1B) increased the fluorescent signals to new steady levels, due to reflection from the recording electrode. After this, the patch membrane was ruptured (at arrows in Fig. 1B) to establish whole-cell voltage clamp and to allow diffusion of the pipette contents into the cell. Figure 1B shows three representative records. Considering the 480 nm signal, which in our system responds more markedly to changes in [Ca2+] than that at 405 nm (cf. Fig. 7A and B), then in the left-hand panel the fluorescence intensity increases by approximately 12 % on changing the Tyrode solution for one containing Indo- 1, followed by a marked increase after membrane rupture. In another cell (Fig. 1B, middle panel), the initial background fluorescence was larger (probably due to a large viewing aperture) and the introduction of indicator in the electrode increased this signal by some 25 %. In some experiments the electrode could contribute as much as 50% of the total background signal (Fig. 1B, right panel), with intracellular loading of Indo-1 proceeding at a much slower rate. We observed no obvious correlation of these effects with initial electrode resistance, although access resistance was not monitored in these experiments. Most of the cells studied exhibited characteristics similar to those shown in Fig. 1A and B, but whatever the time-course of Indo- 1 loading we saw no evidence of the fluorescence signals reaching equilibration, even after 30-45 min of recording. Solutions8 The composition of the external solution was (in mM): NaCl, 140; CaCl2, 2; MgCl2, 2; BaCl2, 2; HEPES, 5, and the pH was adjusted to 7-4 with NaOH. Nicardipine (2 /SM) was added to block Ca2+ channels and ouabain (50 ,UM) to block Na+-K+ pump current. The internal solutions were similar to those used previously (Ehara et al. 1989; see Table 1). When perfused over long periods with internal solutions containing 803 nM [Ca2+]i, cells were more contracted than with lower [Ca2+]i solutions, but showed no obvious signs of damage. The free Ca2+ concentrations ([Ca2+]) were calculated for each solution by using the equations of Fabiato & Fabiato (1979), with the correction by Tsien & Rink (1980) and with the dissociation constants for BAPTA given by Tsien & Rink (1980). Indo-1 (potassium salt) was obtained from either Molecular Probes (Eugene, Oregon, USA) or Calbiochem (UK). In this paper, the equilibrium potential (EN., Ca) for a 3Na+: 1Ca2+ exchanger was calculated from:

ENa ca = 3ENa-2Eca

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262

where ENS and EC. are the equilibrium potentials of Na+ and Ca2+ predicted by the Nernst equation (Mullins, 1977). As indicated in the text, results are presented as mean'+ S.D., with the number of observations in parenthesis. The product-moment correlation coefficient (r) was calculated from the data in Fig. 10 using standard methods (Sokal & Rohlf, 1969), as were the principal axis and 95 % confidence limits. 20

-

OK+

10mM-K+ 0K+

lOmM-K+

0 _

0

E 3 -20 -40

* -

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0 mM

[K+lJ 140 mM

-60

Fig. 2. Comparison of the measured EN, Ca at different K+ concentrations. Data points on the left were obtained with the 153 nM-Ca2 , 10 mM-Na4, 0 K4 internal solution and 2 mM [Ca2+]0 (upper group) or 5 mm [Ca2+]O (lower group). The data points on the right were obtained using an 153 nM-Ca2+, 10 mM-Na+, 140 mM-K+ internal solution with 2 mm [Ca2+]0. Consecutive measurements on the same cell are joined together. RESULTS

Previous studies using isolated ventricular cells have demonstrated that the Na+-Ca2+ exchange current, when activated by increasing external [Ca2+] or [Na+], decays with time. This relaxation was suggested to be due to a continuous ion flux through the exchanger, which caused time-dependent changes in the intracellular ion concentrations (Kimura et al. 1986). To avoid this continuous ion flux through the exchanger, the holding potential was set to the expected ENa Ca immediately before the application of Ca2+ or Na+ (Ehara et al. 1989). Since a major aim of the present study was to ensure that [Ca2+]i was close to that in the patch-pipette, the predicted ENa Ca must be correctly calculated, and to achieve this it must be confirmed that only Ca2+ and Na+ are involved in the exchange process. To address this point, the Ni2+-sensitive current was measured in the presence and absence of both external and internal K+. In Fig. 2 are summarized the results of thirteen experiments. The data points in the left part of the figure were obtained using the 153 nM-Ca2+, 10 mM-Nat internal solution, in order to compare the reversal potential of the 2 mM-Ni2+-sensitive current with either no added external K+ or 10 mm [K+]o. The right portion of the figure shows results using the same protocol, but with an internal solution containing 140 mM-K+ instead of Cs2+. Points joined together in Fig. 2 indicate responses from the same cell. It is clear that the results support the contention that varying membrane K+ concentration gradients does not affect the Exa ca of Na+-Ca2+ exchange. This latter finding is in agreement with reports published recently (Yasui & Kimura, 1990; Crespo et al. 1990). These data contrast with those obtained from studies of Na+-Ca2+ exchange in the outer segment of retinal rod cells, which indicated a stoichiometry of

CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART

263

4Na+: ICa2+, 1K+ for this preparation (Cervetto, Lagnado, Perry, Robinson & McNaughton, 1989). Measurements of the reversal potential using different holding potentials Given that only Ca2+ and Na+ are involved directly in the exchange process, setting the holding potential to the value of ENa Ca calculated from the compositions of the extracellular and internal solutions provides estimates of [Ca2+]i and [Na+]i, B

A -100 mVl ,, I,

I,, I,

-100 mV

I

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I

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l

{-0.4 nA

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-0*2nA

-04 nA

Fig. 3. Effects of varying the holding potential on the reversal potential of the Ni2+sensitive current. Averages of three recordings of the I-V curves using ramp pulses were obtained both before (0) and after (@) the application of 2 mM-Ni2+. The thick arrows indicate the holding potential and each thin arrow indicates the measured E,Cac. The internal solution in A contained 153 nM-Ca2 , 10 mM-Na+ (see Table 1); and 803 nM-Ca2t 10 mM-Na+ in B.

since equilibration should have been achieved with the dialysing pipette. We have examined this effect systematically by measuring ENa.ca while using a range of holding potentials. In the experiment shown in Fig. 3A, the concentrations of Ca2+ and Na+ in both the external and internal solutions were set to give a calculated ENa,ca Of- 38-6 mV for a 3Na+: lCa2+ exchange. The Na+-Ca2+ exchange current was measured as the 2 mM-Ni2+-sensitive current by applying Ni2+ after recording stable control I-V curves at a holding potential of -40 mV. The holding potential was then altered to a new value, resulting in a gradual change in the current during the ramp pulse, until a new steady level was reached within 2-3 min. Then, 2 mM-Ni2+ was applied again for about 1 min to inhibit the Na+-Ca2+ exchange current. An average of the ramp currents was obtained from two to four consecutive pulses before and

A. NOMA AND OTHERS

264

after the application of Ni2+ and the two averages have been superimposed in Fig. 3 in order to estimate ENa, Ca The value of ENa, Ca measured with a holding potential of 0 mV was more positive than the theoretical value of - 38'6 mV, while it was more negative when the holding potential was -80 mV. When the holding potential was set to -40 mV, ENa Ca was close to the equilibrium potential for a 3Na+: 1Ca2+ exchange elicited by 2 mm [Ca2+1. = 153 nM 0

-20

2 mM-Ca2+ 9 7

A

-60

-

2 mM-Ca2+

5 mM-Ca2+

20 0

-

E

[Ca2+] = 803 nM

10 mM-Ca2+

AoLtAJ* * -20

&-40

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Fig. 4. Relationship between the measured EN, Ca (thin arrows) and the holding potential (horizontal lines). Arrows on the same line were obtained in the same experiment. The thick arrows indicate the theoretical equilibrium potential for a 3Na+: 1Ca2+ exchanger. Data were obtained with 2 or 10 mm [Ca2+]0 in the left panel and 2 or 5 mM [Ca2+]t in the right panel.

[Ca2+]0. Essentially the same finding was obtained when the holding potential was changed in the continuous presence of 10 mm [Ca2+]. and 153 nM [Ca2+]i. ENa Ca was also measured with a [Ca2+]i of 803 nm (Fig. 3B). In this experiment, the equilibrium potential of the 3Na+: 1Ca2+ exchange was set to + 5-3 mV for a [Ca2+]o of 2 mm. The ENa Ca obtained experimentally was about + 3 mV when the holding potential was 0 mV and it became progressively more negative as the holding potential was changed to -40 mV and then to -70 mV in the presence of 2 mm [Ca2+]0 (cf. Fig. 3B). Results from a series of experiments are summarized in Fig. 4. The thick arrows indicate the level of the equilibrium potential for - a 3Na+: 1Ca2+ exchanger, determined from the composition of the internal and external solutions. The holding potentials are indicated by the horizontal lines and the measured reversal potentials are indicated by the vertical arrows. It is evident that the experimental ENa, Ca are affected by varying the holding potential, but that the values tend to converge on the theoretical level for 3Na+: Ca 2+ exchange. Reversal potential measured during exchange of the internal solution As a further test of the assumption that the contents of the dialysing pipette equilibrate with the intracellular milieu, the pipette solution was changed for one in which the concentrations of both free Ca2+ and Na+ in the internal solution were

CYTOSOLIC Ca2+ AND Na_Ca2+ EXCHANGE IN HEART 265 increased, but in such a manner that ENa, Ca remained unaltered at - 38-6 mV, with a [Ca2+]0 of 2 mm (Fig. 5). Ramp pulses were applied throughout the period of equilibration with the new internal solution. The time course of equilibration was examined by measuring the slope conductance of each I-V curve. Since the I-V A 0-4 nA

A 111111

I11 II

B

-

+

-100 mV II

C a

I

I

0m8 nA

I

I

I II

v I

0

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0 c 0

0

10 Time (min)

5

15 -100 mV I

I

I

I,I

/

I

+ I,

,/ I)I

I

I

4@nA ~~~~~-0

Fig. 5. The left panel shows the time course of conductance changes during internal dialysis with 153 nM-Ca2+, 10 mm-Na+ solution, followed by dialysis with the 803 nM-Ca2 , 17-4 mM-Na+ solution. The I-V curve was divided into five segments of a constant voltage range (35-6 mV) and the slope conductance was measured by calculating regression lines for each segment. The top curve was obtained from the most positive segment and the bottom curve from the most negative segment of the I-V curve. Application of 2 mM-Ni2+ is indicated by the thick bars and zero time on the abscissa indicates the start of the whole-cell clamp combined with internal dialysis. The external solution contained 2 mMCa2+ and the input capacitance of the cell was 174 pF. The I-V curves in A, B and C were obtained at the corresponding times indicated in the left panel. The I-V curves in B were sampled at an interval of 1.5 min during the process of equilibration with the 803 nM-Ca2+ solution. The thin arrow in B indicates the direction of the time-dependent change in the I-V curves and in A, B and C the thick arrows indicate the holding potential (- 38-6 mV).

curves were non-linear (cf. Fig. 3), the slope conductance was measured by calculating regression lines for five 35-6 mV segments of each experimental trace. The slope conductance was smallest for the most negative segment and increased at more positive potentials, as shown in the left panel of Fig. 5. In this figure, zero time indicates the start of the whole-cell recording. The slope conductance increased on dialysis with the 153 nm-Ca2+, 10 mm-Na+ pipette solution and reached a new steady value 3 min after the start of internal dialysis. The application of 2 mM-Ni2+

A. NOMA AND OTHERS

266

reversibly suppressed this increase in membrane conductance. After switching the pipette solution to one containing 803 nM-Ca2" and 17-4 mM-Na+, the slope conductance increased markedly, accompanied by slow oscillations. Application of 2 mM-Ni2+ again suppressed the membrane conductance. Oscillations in membrane slope conductance at a frequency of 1-2 min-1 were often observed when using internal solutions containing 803 nM-Ca21, but we have no clear explanation of their origin. 50

0

>

0 co

o

d-50_

-100 L 0.1

1

10

[Ca2+]o (mM) Fig. 6. Comparison of the values of ENa,Ca obtained with a 153 nM-Ca2+, 10 mM-Nat internal solution (M) with one containing 803 nM-Ca2+ and 17-4 mM-Na+ (0). The squares indicate the reversal potential obtained during the process of equilibration after switching to a new internal solution. The line indicates the theoretical ENaCa for 3Na+: ICa2+ exchange as a function of [Ca2+]0.

Records from which values for ENa, Ca were determined at times A, B and C in the left-hand panel of Fig. 5 are shown in the panels on the right in this figure. The 2 mMNi2+-sensitive current measured at A, after equilibration with a pipette solution containing 153 nM-Ca2+ (10 mM-Na'), provided a measure of ENa ca for the exchange current which coincided with the theoretical value of - 38-6 mV (panel A). After switching the pipette solution from 153 nM-Ca2l to 803 nM-Ca2+ and 17-4 mM-Na+ the membrane conductance gradually increased, but the I-V curves measured during the time course of equilibration also intersected at around -39 mV (panel B). After steady-state was attained, application of 2 mM-Ni2+ again resulted in an experimental ENa Ca for the Na+-Ca2+ exchange current which was close to that predicted theoretically. Results obtained from thirteen experiments are summarized in Fig. 6. In these experiments, [Ca2+]o was varied in the range 0-2-10 mm and the reversal potentials of the Ni2+-sensitive current for 153 and 803 nm [Ca2+]i solutions are indicated by closed and open circles, respectively, whereas those determined during the process of equilibration with the new internal solution are shown as squares. These data include experiments in which the order of dialysing solutions was reversed from that shown in Fig. 5. The theoretical relation between ENa ca for a 3Na+: lCa2+ exchanger with [Ca2+]o is indicated by the line, and it is evident that there is a good fit with the experimental observations. The slope conductance of the Ni2+-sensitive current was

CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART

267

A Ni2+

0*75

0 mV

-40

1

-80~~~ 0

O

480 nm

a)

C.)

a)C.) U)

Ratio

a) 0

/~

_

405 nm

---------- ----- ------- ---- -- ------

I

I

10

a

20

30

Nil+N2+

mV -40~0 mV

I

I

40

B 0.751

0 L.

a1)

0

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20 Time (min)

30

Fig. 7. Fluorescence recordings from two cells, obtained using the protocol shown in Fig. 1. In A the pipette contained 300 nM-Ca2+, 12-3 mM-Na+ and in B 803 nM-Ca2+, 17-4 mMNa+ (see Table 1). The top trace is membrane potential and the horizontal bars indicate the periods when 2 mM-Ni2+ was applied; the two thin middle traces are fluorescence intensities (in arbitrary units) for 405 and 480 nm, as indicated, with the interrupted lines showing the base-line for each wavelength and the arrows indicating when whole-cell recording commenced. The ordinate gives the ratio of the 405/480 signals, after background subtraction, and the time-course is shown as the thick trace in each panel.

measured near ENa, after equilibration with the new internal solution, and on membrane slope conductance increased by a factor of 3.5 + 1-4 (mean + S.D.; n = 13) when [Ca2+]i was changed from 153 to 803 nm. We conclude from these series of experiments that [Ca2+]i and [Na+]i should be close to the [Ca2+] and [Na+] in the pipette solution when the holding potential is set to the theoretical value for ENa Ca determined from the ionic compositions of the bathing and pipette solutions. Ca

average

268

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[Ca2+]i measured by Indo-1 The changes in ENa Ca described above could arise from changes in [Ca2+]i when the holding potential is varied. This was tested by using the fluorescent probe Indo-1, applied intracellularly through the whole-cell voltage-clamp electrode. Figure 7 shows records of the fluorescent signals obtained during two typical experiments. The patch membrane was ruptured at the time indicated by the arrow and this was followed by an increase in the intensities of the emitted fluorescence measured at both 405 and 480 nm. The [Ca2+] in the pipette was 300 nm in Fig. 7A and 803 nM in Fig. 7B. The initial slight rise in recorded fluorescence before the rupture of the patch membrane was due to exchange of the pipette solution from the control Tyrode to the internal solution containing 50 /LM-Indo-1, indicating that in these cases reflection from the patch pipette was relatively small (cf. Fig. 1). The steady fluorescence levels before the rupture of the patch membrane, indicated by the interrupted lines in Fig. 7, were defined as the background signals for each fluorescence wavelength. The ratio of the fluorescence intensity at 405 nm to that at 480 nm was determined after the background signals were subtracted from each light signal. As can be seen in Fig. 7A, this ratio reached a steady level within 5 min after the start of cell dialysis, even though the fluorescence intensities at both wavelengths continued to increase. In Fig. 7 it is also shown that hyperpolarization of the membrane from -40 to -80 mV decreased the value of this ratio and depolarization increased it, indicating [Ca2+]i decreased and increased, respectively. These effects were more marked when the pipette solution contained 803 nM [Ca2+] (Fig. 7B). Near the end of the record in Fig. 7B, the membrane potential was again varied, but this time in the continuous presence extracellularly of 2 mM-Ni2+. The changes in the fluorescence ratio observed previously were completely abolished, suggesting that these changes in the fluorescence ratio are due to activation of the Na+-Ca2+ exchanger. Comparison of the cellular fluorescence signal with that obtained extracellularly In an attempt to calibrate the Indo-1 signals the measurements shown in Fig. 7 were repeated with pipette solutions containing [Ca2+] of 0, 153, 300, 600 and 803 nM, buffered with 30 mM-BAPTA and each containing the appropriate level of Na+ to give an ENa Ca of-40 mV at a [Ca2+]o of 2 mm. When zero [Ca2+]i was required, Ca2+_ free external solution was perfused and the pipette solution contained no added Ca2 The steady-state value of the fluorescence ratio was determined, as indicated by the open circles in Fig. 8. Since, for pipette solutions containing Ca2 the holding potential (-40 mV) was equal to the theoretical ENa Ca the cellular [Ca2+]i should be very close to that in the pipette. This assumption was tested by measuring directly the fluorescent signals from pipette solutions, a manoeuvre also designed to investigate whether there might be any evidence of differences between Indo-1 signals recorded in vitro and those detected in vivo (see Introduction). To achieve this, a constant volume of each pipette solution was put in the recording chamber on the microscope stage and the ratios measured are shown as crosses in Fig. 8. Similar results were obtained when these solutions were perfused through the recording chamber under conditions identical to those used for cells. The smooth curve in Fig. 8 is the best fit to the extracellular data points using an .

,

269 CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART equation derived from the theoretical relationship for a dual-wavelength indicator between [Ca2+]i and the fluorescence ratio, R, at wavelengths of 405 and 480 nm (Grynkiewicz et al. 1985), R = {(Rmax [Ca2+]i) + (K* Rmin)}/([Ca2+]i +K*), (1) where K* = KD(Sf2/Sb2), with KD the dissociation constant of Indo-1 and Ca2 , Sf2/Sb2 the ratio of intensities at 480 nm when Indo- 1 is either unbound or saturated with Ca2+, respectively. Rmin is the value of R at zero [Ca2+] and Rmax the maximum ratio at saturating [Ca2+]. From experiments using extracellular solutions containing Indo-1, Rmax was found to be 087+0-04 (means+.D.; n = 7) and Rmin had a mean value of 0-039 + 0 003 (n = 11). Using these values, the best fit curve shown in Fig. 8 was obtained from the extracellular calibration points using KD = 288 nm and Sf2/Sb2= 3-47. The values of the fluorescence ratios determined within myocytes were close to those determined extracellularly at 600 and 803 nm [Ca2+], whereas those measured using internal solutions containing 0, 153 and 300 nm [Ca2+] were larger than the values obtained extracellularly (see Fig. 8). 1.0

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1000 500 Free [Ca2+] (nM) Fig. 8. Comparison of the ratio (R) of fluorescence intensities at 405 to 480 nm obtained in cells (0) or extracellularly ( * ), with the number of observations shown in parentheses. The abscissa is free Ca2l concentration and the extracellular measurements were made with solutions identical to those used in the dialysing pipette. The 300 and 600 nM-Ca2+ solutions contained 12-3 and 15-5 mM-Na+, respectively, to give a -40 mV ENa,Ca for a 3Ca2+: lNa+ exchanger with a [Ca2+]. of 2 mm (see Table 1). The dashed horizontal line indicates the value for RmaX' obtained extracellularly in a solution containing 2 mM-free Ca2+. From the extracellular measurements, the ratio of intensities at 480 nm for unbound and Ca2+-saturated Indo-1 (Sf2/Sb2) was calculated as 3*47 and KD determined for each extracellular point using eqn (1). The continuous line was drawn using all the extracellular calibration constants, together with the mean value of KD (288 nM). If the value of Rm,m obtained in cells was substituted for that determined extracellularly, then the interrupted line was obtained.

The deviations of the in vivo data points at low [Ca21] from the calibration curve derived from the in vitro measurements might be explained in two ways. Firstly,

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270

since the data points at zero [Ca2+]i were obtained in the absence of Ca2+ in both internal and external solution, the cellular fluorescence ratio under these conditions might be considered as an appropriate value of Rmin for Indo-1, which might have modified characteristics within the cell (see Introduction). The interrupted curve in B

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250 500 0 250 500 Time (ms) Time (ms) Fig. 9. Records obtained with pipette solutions containing 803 nM-Ca2+; 1 mM-BAPTA was included in A and 30 mm in B. In each panel, the top trace is membrane potential, the middle traces membrane current before (0) and after (0) application of 2 mM-Ni2+, and the lower trace [Ca2+]i determined using Indo- 1. In these experiments the fluorescence signals were not averaged over the period when the excitation shutter was open (see Fig. 1), but an average taken of three successive pulses and used to determined [Ca2+]i from the extracellular calibration curve shown in Fig. 8. Note the different scales for [Ca2+]i in the two panels.

Fig. 8 was drawn by using this value of Rmin (04113 + 0-008; mean + S.D.; n = 4) in eqn (1), together with the remaining calibration values determined extracellularly. With this arbitrary procedure, we obtained a good fit with the intracellular data points. Alternatively, it might be assumed that the cellular -Ca2+ at the beginning of dialysis became bound to Indo-1 and BAPTA and was trapped intracellularly to keep a residual level of [Ca2+]i, even in the absence of extracellular Ca2+. If this is so, then from the extracellular calibration curve shown in Fig. 8, this level is about 80 nm

[Ca2+i]. Changes in [Ca2+]i caused by Na+-Ca2+ exchange In the pipette solution, 30 mM-BAPTA was usually included to provide a large buffering capacity for [Ca2+]i. Under these conditions, the fluorescence ratio did not change during short ramp pulses, as shown in Fig. 9B. However, when the [Ca2+] of the pipette solution was buffered using only 1 mM-BAPTA, the fluorescence ratio and

CYTOSOLIC Ca2+ AND Na -Ca2+ EXCHANGE IN HEART 271 therefore [Ca2+]i varied during the ramp pulse (Fig. 9A). This finding was used in an attempt to test two assumptions; that the 2 mm-Ni2+-sensitive current is the Na+-Ca2+ exchange current and that the calibration curve obtained extracellularly can be used to convert cellular fluorescence ratios to [Ca2+]i. 600

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(A[Ca2+], )iN,Ca (nM) Fig. 10. Changes in [Ca2+]i (A[Ca2+]i) during depolarizing ramp pulses. In these experiments, the internal solutions contained 1 mM-BAPTA. A[Ca2+]i was determined

((A[Ca2+]i)IddO-l) or by integration of the Ni2+-sensitive membrane current ((A[Ca2+]i)Na c), as described in the text. The solid line is the principal axis of the bivariate scattergram drawn from the equation = 1-34(A[Ca c. +20-5. with 95% confidence limits for the slope of 1-63 and 1-12, respectively (Sokal & Rohlf, 1969). The interrupted line indicates the relation (A[Ca2+]i)IdO l = (A[Ca2+]i)iN Ca. directly using Indo-1

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Figure 9A shows a representative experiment, where the top trace is membrane potential, the middle traces the control current (0) together with the membrane current recorded after suppression of Na+-Ca2+ exchange with 2 mM-Ni2+ (M), and the bottom record is [Ca2+]i determined using the extracellular calibration curve shown in Fig. 8. The total amount of intracellular Ca2+ before the pulse was determined from the steady level of the fluorescence ratio and by assuming a BAPTA concentration of 1 mm (this should be a good approximation, since the total concentration of intracellular Ca2+ buffers in non-dialysed cells is of the order 041-0-2 mM; see Fabiato (1983) and Hilgemann & Noble 1987). The influx of Ca2+ through the exchanger was calculated by integrating the Ni2+-sensitive current assuming a 3Na+:lCa2+ stoichiometry. This amount of Ca2+ pumped in by the exchanger was added to the total Ca2+ and the change in [Ca2+]i (A[Ca2+]i) was calculated, using a cell volume determined from the measured input capacitance assuming a specific membrane capacitance of 1 ,sF cm-2 and a rectangular cell geometry with a thickness of 6 ,am (cf. Powell et al. 1980). This increase in [Ca2+]i was compared with that measured directly by Indo-1, as shown in Fig. 10. It is evident that there is a good correlation between the two estimates of the changes in [Ca2+]i (r = 0 97, n = 12; P < 0-01), but that the A[Ca2+]i estimated directly using Indo-1 was always larger than that determined from integration of the exchanger current.

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272

Membrane conductance change and [Ca2+]i The experimental results shown in Fig. 5 suggest that Na+-Ca2+ exchange is enhanced when [Ca2±]i or [Na+]i is increased. This view was tested by measuring the membrane slope conductance when [Ca2+]i was changed by varying the membrane 0 mv

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Time (min) Fig. 11. The effects of membrane potential on membrane slope conductance and [Ca2+]i. The holding potential under voltage-clamp (top trace) was altered as indicated, fluorescence ratios were converted to [Ca2+]i (middle trace) using the extracellular calibration curve, and membrane slope conductance (bottom traces) are shown for the regions -80 to -50 mV (a) and -5 to +25 mV (b).

potential. The slope conductance for the outward current was measured over the potential range -5 to + 25 mV, and for the inward current using a range from -80 to -50 mV (Fig. 11). The pipette solution contained 803 nm [Ca2"] and 17A4 mm [Na+]. The transient application of 2 mm-Ni2+ almost completely blocked the membrane conductance increase evoked by internal dialysis, indicating that the conductance is due mostly to Na+-Ca2+ exchange. Hyperpolarization to -80 mV from the holding potential gradually decreased [Ca2+]i, but the membrane

CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART 273 conductance for the outward current quickly increased. This effect was reversed by clamping back to -40 mV. A subsequent depolarization increased [Ca2+]i and the membrane conductance for the outward current decreased. The slope conductance for the inward current showed only minor changes during these manoeuvres. Essentially the same findings were obtained in two other experiments. These results suggest that an increase in [Ca21]i is not the primary cause of the increase in membrane conductance observed in experiments of the type shown in Fig. 5. DISCUSSION

The present study has shown that the intracellular free Ca2+ concentration in guinea-pig ventricular myocytes is strongly influenced by the Na+-Ca2+ exchange mechanism, even in the presence of 30 mM-Ca2+ buffer in the pipette solution. It is clear that [Ca2+]i is changed by varying the membrane potential through altered activity of the Na+-Ca2+ exchanger. We used this observation in an attempt to calibrate the emitted Indo-1 fluorescence from cells. To facilitate equilibration of the intracellular medium with the pipette solution, the holding potential was set at the equilibrium potential for a 3Na+: 1Ca2+ exchange, calculated using the ionic compositions of both the extracellular and pipette solutions. Under these conditions, the ratio of the cellular fluorescent signals at 600 and 803 nm [Ca2+]i showed good agreement with those obtained extracellularly (Fig. 8), but at lower concentrations of Ca21 (153 and 300 nM) the fluorescence intensity ratios measured within myocytes were always larger than those determined extracellularly. Thus, under our experiment conditions, it is clear that the differences between the in vivo and in vitro fluorescence intensity ratios of Indo-1 could result in errors in determining resting levels of [Ca2+]i (- 100 nM). Although the theoretical curve using the cellular Rmin produced a good fit with the experimental data (Fig. 8), we can think of no rational reason to combine this cellular value of Rmin with the remaining extracellular calibration constants. This contrasts with the approach adopted by Crespo et al. (1990), who used Rmin and KD obtained in vitro with an Rmax determined inside the cell. To be consistent, we adopted the extracellular calibration curve as appropriate for conversion of cellular fluorescence ratios to [Ca2+]1. Deviations of the in vivo data points from the in vitro calibration curve will not seriously effect the measurements of [Ca2+]i shown in Figs 9 and 11, since in these cases [Ca2+]i was at levels (> 500 nM) where such deviations are minimal (cf. Fig. 8). The validity of adopting the in vitro calibration curve for determining [Ca2+]i was tested by using it in calculating the increase in [Ca2+]i during a depolarizing ramp pulse and comparing this value with that estimated from integrating the Na+-Ca2+ exchange current. Although the two estimates varied in proportion to each other, values obtained directly from the fluorescence data were larger than those calculated from the current measurements (Fig. 10). This difference can be accounted for by assuming that the concentration of the Ca2+ buffer, BAPTA, was lower than the 1 mm in the pipette. Such an assumption is consistent with the observation that the cellular fluorescence signal continuously increased throughout the course of an experiment (Fig. 7), indicating that the intracellular concentration of Indo-1 and therefore also of BAPTA, which is of similar chemical structure, did not equilibrate

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with the pipette solution. If the intracellular concentration of BAPTA reached 90 % of the dialysing solution (1 mM), it can be shown that a given influx of Ca2+ through the Na+-Ca2+ exchanger would cause an increase in [Ca2+]i about 40 % above the estimate based on equilibration, thus accounting for the difference shown in Fig. 10. This effect would be time dependent as loading of intracellular BAPTA proceeded, but the scatter of the data points precluded further quantitative analysis of this problem. Although it is not known how cell dialysis will affect the concentrations of native intracellular Ca2+ buffers, Ca2+ transport by the sarcoplasmic reticulum (SR) would obviously be important, if present. However, the twitch contraction induced by a voltage-clamp pulse completely disappears in single cardiac myocytes when the pipette solution contains more than 0-5 mM-EGTA. This observation indicates that release of Ca2+ from the SR is almost completely buffered under these conditions. We assumed that release of Ca2+ from the SR in our experiments was minimal, since the dialysing solutions contained 30 or 1 mM-BAPTA. This assumption is supported by the finding that [Ca2+]i rises or falls with an apparent latency of about 100 ms after the Na+-Ca2+ exchange current changes polarity, even when the BAPTA concentration was 1 mm in the pipette (Fig. 9). Such a time course for the [Ca21]i change might be explained by the sigmoidal rise of [Ca2+]i estimated by integrating the Ca2+ influx induced by the ramp pulse. The deviation of the intracellular calibration data points from those obtained extracellularly at 0 mm [Ca2+]i in Fig. 8 could also arise through a residual amount of Ca2+ within the cell (see Results). Another source of error might be a Na+ leak, for if a significant amount of Na+ were continuously entering the cell, this would be exchanged for extracellular Ca2+ to give a higher value of [Ca2+]i than in the pipette solution. The measured ENa, Ca should still be equal to the holding potential, since the shift in ENa compensates the deviation of Eca to give a constant ENa Ca. This assumption is attractive because it is then unnecessary to assume altered characteristics of Indo-1 within the cell. Measurements of [Na+]i will be required to investigate this problem further. If confirmed, this would show that our assumption that holding the membrane potential at the theoretical ENa Ca determines [Ca2+]i is not correct. Nevertheless, it is also possible that under the conditions of our experiments Indo1 does exhibit modified characteristics intracellularly. For example, Konishi et al. (1988) demonstrated a decrease in the KD of Fura-2 in skeletal muscle cytoplasm. However, our results show that the cellular fluorescence signals could be fitted to the theoretical calibration curve using the cellular value for R min and a KD which was obtained from extracellular measurements (Fig. 8). This experimental value of KD (288 nM) is close to the 250 nm determined for Indo-1 by Grynkiewicz et al. (1985) and the 300 nm assumed by Bers et al. (1990). In addition, Benham (1989) determined a value for K* of 952 nm for Indo-1 and calculated a KD of 213 nM. A stoichiometry of 3Na+: lCa2+ for Na+-Ca2+ exchange was recently tested by measuring [Ca2+]i during whole-cell voltage-clamp of guinea-pig ventricular myocytes (Crespo et al. 1990). These authors observed good agreement between theoretical and experimental increases in [Ca2+]i at around 0 mV. Their results were consistent with a 3Na+: 1Ca2+ stoichiometry by assuming extrusion of Ca2+ through

CYTOSOLIC Ca2+ AND Na+-Ca2+ EXCHANGE IN HEART 275 a sarcolemmal Ca2+-ATPase at very positive potentials and a calcium leak flux at negative potentials. Our experiments may give additional direct support for such a stoichiometry, since we measured simultaneously the Ni2+-sensitive current, to estimate Na+-Ca2+ exchange current, together with the fluorescence signals from Indo- 1. The increase in the membrane conductance for the Ni2+-sensitive outward current on hyperpolarization (Figs 3 and 11) is similar to that induced by increasing both intracellular Na+ and Ca2+ (Fig. 5). However, the fluorescence measurements indicated that [Ca2+]i decreased as the membrane was hyperpolarized (Figs 7 and 11), so that the increase in membrane conductance must be attributed to changes in [Na+]i. Although we did not measure [Na+]i, it should increase via exchange of intracellular Ca2+ for external Na+ during hyperpolarization, in accord with the theoretical prediction that the outward exchanger current is a function of [Na+]iS at constant [Ca21]0 (DiFrancesco & Noble, 1985; Noble, 1986). This work was supported by grants from the British Council, the British Heart Foundation and a Programme Grant from the MRC. L. F. C. P. is an MRC Scholar. REFERENCES

BARCENAS-RuIz, L. & WIER, W. G. (1987). Voltage dependence of intracellular [Ca21]i transients in guinea pig ventricular myocytes. Circulation Research 61, 148-154. BAYLOR, S. M. & HOLLINGWORTH, S. (1988). Fura-2 calcium transients in frog skeletal muscle fibres. Journal of Physiology 403, 151-192. BENHAM, C. D. (1989). Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp. Journal of Physiology 415, 143-158. BERS D. M., LEDERER, W. J. & BERLIN, J. R. (1990). Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling. American Journal of Physiology 258, C944-954. BEUKELMANN, D. J. & WIER, W. G. (1988). Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. Journal of Physiology 405, 233-255. BLATTER, L. A. & WIER, W. G. (1990). Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators Indo-1 and Fura-2. Biophysical Journal 58, 1491-1499. CALLEWAERT, G., CLEEMANN, L. & MORAD, M. (1988). Epinephrine enhances Ca2+ currentregulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes. Proceedings of the National Academy of Sciences of the USA 82, 2009-2013. CANNELL, M. B., BERLIN, J. R. & LEDERER, W. J. (1987). Effect of membrane potential changes on the calcium transient in single rat cardiac cells. Science 238, 1419-1423. CANNELL, M. B. & LEDERER, W. J. (1986). A novel experimental chamber for single-cell voltageclamp and patch-clamp applications with low electrical noise and excellent temperature control. Pflugers Archiv 406, 536-539. CERVETTO, L., LAGNADO, L., PERRY, R. J., ROBINSON, D. W. & MCNAUGHTON, P. A. (1989). Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients. Nature 337, 740-743. CRESPO, L. M., GRANTHAM, C. J. & CANNELL, M. B. (1990). Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes. Nature 345, 618-621. DIFRANCESCO, D. & NOBLE, D. (1985). A model of cardiac electrical activity incorporating ionic pumps and concentration changes. Philosophical Transactions of the Royal Society B 307, 353-398. EHARA, T., MATSUOKA, S. & NOMA, A. (1989). Measurement of reversal potential of Na+-Ca2+ exchange current in single guinea-pig ventricular cells. Journal of Physiology 410, 227-249. FABIATO, A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. American Journal of Physiology 245, C1-14.

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FABIATO, A. & FABIATO, F. (1979). Calculator programs for computing the composition of solutions containing multiple metals and ligands used for experiments in skinned muscle cells. Journal de Physiologie 90, 463-505. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2+ probes with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, J. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluigers Archiv 391, 85-100. HILGEMANN, D. W. & NOBLE, D. (1987). Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: Reconstruction of basic cellular mechanisms. Proceedings of the Royal Society B 230, 163-205. HIROTA, A. W., CHANDLER, P. L., SOUTHWICK, P. L. & WAGGONER, A. S. (1989). Calcium transients recorded from two new purpurate indicators inside frog cut twitch fibres. Journal of General Physiology 94, 597-631. KAo, J. P. Y. & TsIEN R. Y. (1988). Ca2' binding kinetics of Fura-2 and Diazo-1 from temperaturejump relaxation measurements. Biophysical Journal 53, 635-639. KIMURA, J., MIYAMAE, S. & NOMA, A (1987). Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig. Journal of Physiology 384, 199-222. KIMURA, J., NOMA, A. & IRISAWA, H. (1986). Na-Ca exchange current in mammalian heart cells. Nature 319, 596-597. KLEIN, M. G., SIMON, B. J., Szucz, G. & SCHNEIDER, M. F. (1988). Simultaneous recordings of calcium transients in skeletal muscle using high- and low-affinity calcium indicators. Biophysical Journal 53, 971-988. KONISHI, M., OLSON, A., HOLLINGWORTH, S. & BAYLOR, S. M. (1988). Myoplasmic binding of Fura2 investigated by steady-state fluorescence and absorbance measurements. Biophysical Journal 54, 1089-1104. LEBLANC, N. & HUME, J. R. (1990). Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science 248, 372-375. MATSUDA, H. & NOMA, A. (1984). Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig. Journal of Physiology 257, 553-573. MULLINS, L. J. (1977). A mechanism for Na/Ca transport. Journal of General Physiology 70, 681-695. NOBLE, D. (1986). Sodium-calcium exchange and its role in generating electric current. In Cardiac Muwcle: The Regulation of Excitation and Contraction ed. NATHAN, D., pp. 171-200. Academic Press, New York. NOBLE, D. & POWELL, T. (1990). The attenuation and slowing of calcium signals in cardiac muscle by fluorescent indicators. Journal of Physiology 425, 54P. POWELL, T., TERRAR, D. A. & TWIST, V. W. (1980) Electrical properties of individual cells isolated from adult rat ventricular myocardium. Journal of Physiology 302, 131-153. POWELL, T. & TWIST, V. W. (1976). A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium. Biochemical and Biophysical Research Communications 72, 327-333. POWELL, T., TWIST, V. W. & YAMAOKA, K. (1988). Intracellular Ca transients in single guinea-pig ventricular myocytes monitored using Indo-1 fluorescence. Journal of Physiology 406, 105P. SOKAL, R. R. & ROHLF, F. J. (1969). Biometry. The Principles and Practice of Statisti in Biological Research, 1st edn. W. H. Freeman and Company, San Francisco. SPURGEON, H. A., STERN, M. D., BAARTZ, G., RAFFAELI, S., HANSFORD, R. G., TALO, A., LAKATTA, E. G. & CAPOGROSSI, M. C. (1990). Simultaneous measurement of Ca2+, contraction and potential in cardiac myocytes. American Journal of Physiology 258, H574-586. TSIEN, R. Y. & RINK, T. J. (1980). Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium. Biochimica et Biophysica Acta 599, 623638. WIER, W. G., CANNELL, M. B., BERLIN, J. R., MARBAN, E. & LEDERER, W. J. (1987). Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura-2. Science 235, 325-328. YASUI, K. & KIMURA, J. (1990). Is potassium co-transported by the cardiac Na-Ca exchange? Pfliugers Archiv 415, 513-515.