Pathways for the movements of ions during calcium-free perfusion and the induction of the ‘calcium paradox’

J M o l Cell C a r d i o l 18, 2 4 1 - 2 5 4 (1986)

Pathways for the Movements of Ions During Calcium-free Perfusion and the Induction of the 'Calcium Paradox'* j. Tunstall, P. Busselen l, G. C. Rodrigo and R. A. Chapman Department of Physiology, The University, Leicester, LE1 7RH, UK and i Interdisciplinair Research Centrum, University of Leuven, Campus Kortr~ik, Belgium (Received 21 March 1985, acceptedin revisedform 11 June 1985) J. TUNSTALL,P. BUSSELEN,G. C. RODRIGO AND R. A. CHAPMAN. Pathways for the Movements of Ions during Calcium-free Perfusion and the Induction of the 'Calcium paradox' Journal of Molecular and Cellular Cardiology (1986) 18, 241-254. The intracellular sodium content of cardiac cells in fish and amphibia, measured with either an isotope technique or with sodium-sensitive micro-electrodes, rises steeply from around 15 mmol/l in calciumcontaining solution to as much as 70 mmol/l, during exposure to a Ca 2 +-free solution. This increase is associated with the development of spontaneous and prolonged action potentials so that the membrane may stabilise around --20 mV. O n reperfusion with calcium-containing medium the membrane repolarises before a strong contracture develops. Inhibition of the N a - p u m p increases both the sodium gain and the subsequent calcium re-admission tension. A number of agents e.g. divalent cations, anti-arrhythmic drugs, local anaesthetics and Ca-channel blockers are able to prevent the development of the contracture but only if they are present during the calcium-free perfusion. They also inhibit the development of spontaneous electrical activity and the rise in Na i . The calcium re-admission contracture can be blocked in amphibian preparations voltage clamped around the resting potential during low calcium perfusion. From the known pharmacological action of these agents and the voltage and time dependence of the calcium channel, it is concluded that during calcium depletion, the prolongation of the action potentials is associated with a sustained entry o f N a + via the Ca-channels which leads to the rise in Na i . Once Na i has risen, these agents with the exception of M n 2+, a known inhibitor of the Na/Ca exchange, are unable to prevent the development of the contracture. This suggests that the re-admission contracture follows calcium uptake by way of the Na/Ca exchange. KEY WORDS: Calcium; Calcium paradox; Calcium channels; Na +/Ca 2 + exchange; Calcium channel blockers; Intracellular sodium ; Divalent cations; Voltage clamp.

Introduction A number of studies have shown that a large reduction of the calcium concentration in a solution bathing heart muscle, causes a marked prolongation of the action potential [35, 48, 621 and leads to an elevation of the intracellular sodium concentration ([Na]i) [17, 27, 58]. This increase in [Na]i could occur by way of the Na/Ca exchange, but calcium ions are also known to have effects on ionic channels of a number of nerve and muscle cells. The effect of calcium deprivation upon the calcium channels, which then become permeable to monovalent cations may be of particular interest [2, 41, 43, 45, 49, 50, 53]. As the

rate of inactivation of these channels is slowed by calcium removal [24, 40], it is possible that in the absence of calcium a continuing passage of monovalent cations through the Cachannels, could account for both the prolongation of the action potential and the rise in Na i [17]. An elevated [Na]i has an inotropic action upon the tension generated by the heart [12, 20, 21, 26, 31, 32, 591, probably by favouring calcium uptake by way of the Na/Ca exchange. A mechanism involving an elevated [Na]i and the Na/Ca exchange in the aetiology of the calcium paradox has been recently suggested [11, 51, 57, 64]. An elevated [Nail

* This work was supported by grants from the Nuffield Foundation and SERC. 0022-2828/86/030241 + 14 $03.00/0

© 1986 Academic Press Inc. (London) Limited

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may have additional effects by reducing the calcium buffering capacity of t h e cells, because the metabolically dependent uptake of calcium by the mitochondria is opposed by a sodium dependent Calcium release [,13, 25]. Together these effects may produce a large rise in sarcoplasmic calcium and the development of a strong contracture when calcium ions are reintroduced to the solution bathing the heart, after a period of calcium deprivation. This contracture forms part of a sequence of usually damaging changes, which follow calcium removal and re-addition, known collectively as the calcium paradox [3,

66-68]. A number of agents, e.g. hypothermia [,41] the reduction of the bathing sodium concentration [3], the presence of divalent cations [-57] and certain calcium channel blockers [-37] are known to alleviate some features of the calcium paradox. I f a rise in [Na]i is indeed the necessary precondition for the calcium influx on calcium repletion, then these agents should act by either inhibiting the sodium uptake in low calcium solution or the subsequent calcium reuptake. Using a variety of techniques and preparations we set out to study the pathways for the sodium and calcium movements which follow calcium removal and re-addition. The initial findings of this study have already been published [,17].

Materials and Methods

Contracture experiments Atrial trabeculae of the frog Rana pipiens 20 to 100/Jm in diameter and 1 to 3 m m in length were used for this study. The methods of dissection, perfusion and for recording the tension generated by these preparations have been described before [,18]. Between observations the tissue was stimulated electrically at a rate of 6/min and maintained at 20°C, in a Ringer solution containing: in mmol/1, NaC1 115, sodium pyruvate 5, KC1 3 and CaCI 2 1, buffered t o p H 7.2 with 5 mmol/1 Tris HC1. The tension developed by the preparation was measured when this calcium-containing fluid was re-admitted to the experimental dish, following a period in a low calcium solution. The low calcium solutions were made by omitting

the CaC12 and adding a mixture of 4 mmol/1 C a E G T A and 4 mmol/1 E G T A in appropriate ratio. The free calcium concentration was calculated using a log binding constant for E G T A of 6.44/mol. As the majority of the experiments reported here were carried out over the range o f [ C a ] 0 10 nmol to 10 pmol/1, where the buffering of calcium by E G T A is optimal, the possible calcium contamination of the E G T A was ignored. Calcium concentrations above 10 pmol/1 were achieved by addition from a 1 mol/1 CaC12 stock without EGTA. Most drugs were added from concentrated aqueous stock solutions, however D600, verapamil, nifedipine and strophanthidin or ouabain were applied in ethanol when an equivalent concentration of alcohol was added to all the Ringer solutions.

22Na uptake experiments The ventricle was dissected from the heart of the trout Salmo gairdneri (Richardson) and placed in a Ringer solution at 20°C containing: in mmol/1, NaC1 137, KC1 5.4, CaC12 1.8, Glucose 5.0 and TrisHC1 10.0, at p H 7.2. Test solutions of differing calcium concentration were made by adding E G T A as in the experiments on amphibian preparations. For sodium uptake experiments 2ZNa (Amersham International) was added to the test solution to give a specific activity of 80 nCi/ml. In some experiments tritiated sucrose, at a specific activity of 400 nCi/ml in the test solution, was used to estimate the extracellular space which showed little change during the course of a typical experiment. Before being exposed to a radioactive low calcium solution, slices of ventricular muscle with a wet weight of approximately 40 mg, were placed in nonradioactive test solutions for 10 rains in order to equilibrate the calcium concentration in the extracellular spaces. At the end of the experiment the tissue was blotted between ashless filter paper and the wet weight determined. Control preparations were dried overnight in an oven at 90°C and weighed to give a dry weight. After overnight extraction in 3 ml of 0.15 N H N O 3 , 2 2 N a and 3H concentrations were determined using a dual tracer method, in a liquid scintillation counter (Packard Tri-earb). In order to derive the values for the intra-

Ions and ' C a l c i u m Paradox'

cellular sodium concentration given in the text, the total tissue concentration was corrected for the ions in the extracellular space as determined by the 3H sucrose measurements.

~ 5Ca experiments When the sodium stimulated efflux of calcium was measured the cells were first loaded with 45Ca for 60 mins in a sodium-free solution (lithium replacing sodium). The wash out of the 45Ca was then followed by placing the preparation into a series of tubes containing a low sodium solution. The tubes were changed every 5 mins. Sodium was added to the solution in the tubes after 1 h. 45Ca was measured by scintillation counting. At the end of the experiment the tissue was blotted, weighed and extracted as described above. The residual calcium was then determined and an effiux curve constructed.

Ion sensitive micro-electrode experiments In a number of experiments the intracellular sodium activity (a~a) of frog atrial trabeculae was measured, using microelectrodes filled with a sodium sensitive resin [60]. Details of the methods of construction and use of these i in frog cardiac electrodes to measure aNa muscle have been described before [21]. Values of the intracellular concentration of sodium ions were calculated from the measured aN,i assuming an activity coefficient of 0.746.

Voltage clamp experiments A double sucrose gap technique of the type described by Chapman and Leoty [16] was used. A 214 mmol/1 sucrose solution with a resistivity greater than 1 M ~ / c m / was used to produce the insulating gaps. Frog Ringer solutions with the compositions described above were passed over the preparation in the central compartment at a rate of 5 ml/min. The tension was monitored using a force transducer, which made a tangential contact with the preparation, and gave an indication of the contraction of the cells in the central compartment [64]. The apparatus allowed frog atrial preparations to be either current or voltage clamped. In current clamp, depolarising

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pulses were applied between the central and one of the lateral compartments and action potentials recorded between the central and the second lateral compartment. In voltage clamp, the holding membrane potential of the cells in the central compartment was set to - 8 0 m V and checked with an intracellular 3 mol/1 KC1 micro-electrode, all other changes in the membrane potential were made with reference to this level. These experiments presented some technical difficulty as spurious current transients often appeared on lowering the calcium concentration. The ability of the apparatus to maintain the membrane potential under these conditions was tested with a micro-electrode which measured the membrane voltage of cells in the central compartment. This showed that the duration of the loss of voltage control never exceeded 200 ms.

The experimental observations of both tension and membrane potential were recorded on an oscillograph (Rikadenki Type R. 10). Membrane currents were photographed from a cathode ray oscilloscope display and measured from projected negatives.

Results

The effect of calcium removal and re-addition When the ionised calcium in the solution bathing an isolated trabeculae is reduced to less than 1 /Jmol/l the cell membrane gradually depolarises and becomes spontaneously active (Fig. 1). The overshoot of the spontaneous action potentials progressively falls from around + 3 0 m V in normal Ringer, to - 1 0 m V after 10 mins of low calcium perfusion. This change is accompanied by a progressive failure of each action potential in the train to repolarise fully and a marked prolongation of the plateau phase as the membrane potential stabilises at - 2 0 to - 3 0 mV. The rate of development of these changes depends upon the rate ofperfusion and the free calcium concentration, so that in well perfused preparations ~he action potential may persist for the whole of the low calcium period. Initially the tissue may show some spontaneous contractile activity which probably corresponds to the development of the

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F I G U R E 1. The effect of calcium depletion and re-admission upon the contractile responses and membrane potential of frog atrial trabeculae. The bathing [Ca]o was reduced to 10 nmol/l as indicated by the arrows and electrical stimulation interrupted. The membrane becomes spontaneously active to give a series of action potentials which are progressively prolonged as the contractile responses decline. On the elevation of the [Ca]o to I mmol/1, the membrane rapidly repolarises to hyperpolarise during the development of a large calcium re-admission contracture. Electrical stimulation was re-started after the spontaneous relaxation of the contracture. The total duration of the low calcium perfusion was 10 mins. Four minutes of recording have been omitted.

spontaneous action potentials. The contractile behaviour is rapidly lost, however, when the tissue shows no mechanical response to electrical stimulation. Figure 1 shows that on return to normal calcium (I mmol/1) the prolonged action potentials are immediately curtailed as the membrane first rapidly repolarises to around - 7 0 mV and then hyperpolarises, as described by Bonvallet, Rougier and Tourneur [8], as a strong contracture develops. This contracture reaches a peak rapidly but then relaxes spontaneously with an exponential rate constant of around 0.03 __+0.005/s (mean + s.D.), i.e. at a rate close to that for the spontaneous relaxation of low sodium contractures in this tissue [20]. At the temperature at which these experiments were performed (20°C) relaxation was complete and the tissue regained its normal contractile activity after about 30 mins. The amplitude of ~the calcium re-admission contracture which follows a 10 min exposure to a calcium depleted fluid, shows a marked dependence upon the degree of the calcium reduction. As shown in Figure 2 (closed

symbols), if the bathing calcium concentration is greater than 1 #mol/1 during the period of low calcium perfusion, a contracture does

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the calcium re-admission contracture and the calciu'tn concentration in the pretreating solution. Tension is expressed as a percentage of the maximum response recorded when a frog atrial trabeculae is returned to a solution containing 1 mmol/l calcium after a period of 10 mins in a low calcium solution. Closed symbols represent untreated preparations, the open symbols the responses of preparations exposed to 1 #mol/1 strophanthidin during the low calcium period and during the development of the contracture. [Ca]o is expressed as pCa = - l o g [Ca]°. Bars represent standard deviations when n > 6.

Ions and 'Calcium Paradox'

not develop on calcium repletion. Following exposure to a calcium concentration less than 100 nmol/1 [Ca]o , however, a large contracture develops. The amplitude of the readmission contracture varies very steeply between these two values to be half-maximal following an exposure to an outside [Ca] of around 120 ± 50 nmol/1 (mean + S.D.). I f 1 #tool/1 strophanthidin is present in the calcium depleted fluid and when the contracture is induced, this relationship is moved to higher values of free calcium and the amplitude increased (Fig. 2 open symbols). The amplitude of any contracture which follows calcium re-admission depends upon the duration of the low calcium perfusion. An effect of reducing the calcium concentration to 10 nmol/1 is evident if the bathing calcium is raised after only 15 to 30 s of low calcium perfusion. This effect reaches approximately 50% of its final value after 1 rain, but the largest tension cannot be recorded until the preparation has been exposed to the low calcium fluid for about 5 mins. Longer exposure does not increase the amplitude further. Following the addition of 1 #mol/1 strophanthidin to the low calcium solution the magnitude of the response is increased and appears to develop more quickly, so that a half maximal effect is seen after only 30 to 45 s.

The effect of Ca deprivation on [Wa]i 22 Na experiments The intracellular concentration of sodium, estimated by allowing fish ventricle preparations to come into equilibrium with 22Na in a normal calcium containing fluid and with appropriate corrections for extracellular ions, was found to be 15 _+ 1.2 mmol/kg wet wt. ( m e a n _ S.D.). This value is substantially increased following exposure of the prepa]ation to a solution with a free [Ca] of 10 nmol/1, and comes to an equilibrium, with the normal bathing sodium concentration, at a mean value of 32 + 1.8 mmol/kg wet wt. after 40 rain [Fig. 3(a)]. In the presence ofouabain 10 #mol/1 this increase was much larger and appeared to rise more quickly to reach 70 ± 5 mmol/kg wet wt. after 80 rain. The magnitude of the increase in [Na]i following 60 mins in solutions with a reduced

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[Ca]o is illustrated in Figure 3(b). This shows ~ that a large uptake of sodium only occurs if the bathing calcium is reduced to less than 1 #mol/1 and that a gain equal to about half of the maximum recorded, follows exposure to a solution containing 100 nmol/1 calcium.

Ion-sensitive electrode experiments Measurement of the intracellular sodium activity made using ion sensitive electrodes in frog atrial trabeculae, show that when Ca o.is reduced the membrane depolarises and a~a rises with a time constant of around 30 s (see Fig. 1 [17]). In this tissue the steady level to which Na i may rise in different [Ca]o corresponds closely to those made with the isotope technique in fish heart, except that a more rapid and larger gain occurs. [Na]i was 18.75 + 0.67 mmol/1 (mean + s.E. in a number of measurements from four preparations). With the reduction of [Ca] o to 1 #mol/1, the steady state value of the Nai is doubled. With further reduction of the [Ca]o, the Na~ increases more steeply to reach a con-

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The effects of sodium reduction In a n u m b e r of experiments the effect of reducing the [ N a ] of the low calcium fluid upon the action potential duration, 22Na gain and calcium re-admission tension was studied (sodium replaced by either Tris or sucrose). It was noted that the reduction of the bathing [ N a ] to 50% of its normal value prevented the development of spontaneous electrical activity and the subsequent calcium readmission contracture. At [ N a ] o greater than this, the tension amplitude varies steeply so that a reduction of the m a x i m u m amplitude was evident for a reduction of [Na]o to 100 mmol/1. Radio-isotope estimations of [Na]i show that it is reduced by lowering [Na]o in normal calcium solutions. The relative increment of [Na]i when [Ca]° is reduced is the same irrespective of the bathing sodium concentration. In consequence in 50% [Na]o the rise in [Na]i , following calcium removal, returns [Na]i to a value similar to that recorded in normal Ringer solution.

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FIGURE 4. Dose response curves for the action of quinidine. (a) The effect on the calcium re-admission tension of frog atrial trabeculae, recorded when the bathing calcium concentration was raised to 1 mmol/l after 10 mins in a solution containing 10 nmol/1 calcium, with or without quinidine; (b) The effect on the gain of 22Na by fish ventricles exposed to a solution containing 10 nmol/l calcium for 60 mins. Bars represent standard deviation when n > 6.

The effect ofquinidine during low calciumperfusion T h e presence of 500 /.tmol/1 quinidine in the low calcium fluid, is able to block both the increase in [Na]t and the calcium readmission contracture. T h e dose response curves for the action of this agent, however, [Figs. 4(a) and (b)] show half inhibition of the response at around 22___ 16 #mol/1 (mean ± s.o.) for the contracture experiments or 30 ± 8 pmol/1 for sodium uptake. In a n u m b e r ofcontracture experiments the preparation was exposed to a solution containing 10 nmol/1 calcium, to induce a rise in [Na]i , and quinidine was applied at the time of calcium re-admission. These experiments showed that when applied in this way and at a concentration sufficient to block sodium uptake, quirt±dine does not inhibit the development of tension. Indeed at higher concentrations quinidine actually potentiates the contracture and slows its rate of relaxation. Quinidine therefore has an effect upon calcium re-admission tension similar to that

reported for the low sodium contracture in frog atrial preparations [22]. These observations suggest that quinidine does not block sodium m o v e m e n t in normal calcium containing solutions but that its action is specific to sodium uptake in calcium depleted solutions. This idea is supported by two sets of experiments. In the first, fish ventricles were exposed to a low sodium solution when [Na]i might be expected to fall [21, 33]. T h e preparations were then returned to a solution containing 22Na, with and without a relatively high concentration (500 pmol/1) of quinidine. T h e time course for the sodium uptake over the next 30 mins showed that in both cases the tissue rapidly regained its normal [Na]. In the second, it was seen that the loading of ventricles with 45Ca in a low sodium solution, was reduced in comparison to the controls by the presence of quinidine at 500 #mol/1, i.e. confirming the observation of C h a p m a n , Tunstall and Yates [22] for frog

Ions and ' C a l c i u m Paradox'

atria. I f quinidine, at this concentration, was added during the measurement of the 45Ca effiux, however, neither the rate of washout of the isotope nor the sodium stimulated calcium efflux was affected.

The effect of modifiers of membrane permeability on sodium gain and calcium re-admission tension The sodium channel Exposure to low calcium solutions containing T T X (1/~mol/1) fails to prevent the large gain in [Na]i or to block the calcium re-admission contracture.

The calcium channel A number of agents known to affect the Ca channel were tested including divalent cations, local anaesthetics, anti-arrhythmic drugs and the specific channel blockers-D600, verapamil, nifedipine. (a) Divalent cations. It is obvious from the data shown in Figure 3(b) that a calcium concentration greater than 10 pmot/1 will prevent sodium uptake into the cells. As shown in Figure 1 (a), however, the addition of Ca 2 + to the low calcium perfusate, so that the final concentration exceeds this value, causes the m e m b r a n e of frog atrial cells to re-polarise immediately. A similar result is observed when Mg z+ [see Fig. 7(a)] are added to the low calcium solution to give an extracellular Mg 2+ concentration greater than 100/~mol/1 suggesting that these ions are not as effective in preventing the prolongation of the action potential as calcium. If applied in the low calcium solution this Mg2 + concentration is sufficient to block the calcium re-admission contracture in frog atria, but does not prevent the development of tension once [Na]i has risen. For fish ventricle 22Na uptake was completely inhibited in 500 pmol/1 magnesium and reduced from the 32 mmol/1 seen in control preparations to 21 + 2 mmol/1 in 100 #tool/1 magnesium. At 50 pmol/1 manganese ions also inhibit the rise in [Na]i , in fish ventricle, but unlike Mg z+, at a higher concentration (2 mmol/1), they also block the calcium re-admission contracture, of frog atria if [Na]i has been allowed to rise.

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(b) Tetracaine. Tetracaine is known to inhibit the secondary inward current in the heart [16, 30]. Exposure of atrial preparations to this drug in low calcium fluid, blocks the calcium re-admission tension in a rather dramatic way suggesting threshold behaviour for the mechanism which underlies the contracture [Fig. 5(a)]. T h a t this is [Na]i seems confirmed by a study of the dose response curves of the effect of tetracaine upon the gain in [Na]i during low calcium perfusion [Fig. 5(b)]. These figures show that for both tension and sodium gain, the dose of tetracaine which half inhibits the measured response lies between 50 and 100 #mol/1. The very steep nature of the dose-response curve, which is also dependent upon the free divalent ions make a statistical evaluation very difficult. At these concentrations tetracaine was without effect on the calcium re-admission contracture if added to the perfusing solutions at the time of calcium repletion.

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F I G U R E 5. Dose response curves for the action of tetracaine. (a) The effect on the calcium re-admission tension recorded from frog atrial preparations; (b) The effect upon the 22Na uptake into fish ventricles. In both cases the tissue was exposed to a solution containing 10 nmol calcium with and without tetracaine for 10 mins in (a) and for 60 mins in (b).

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(c) Specific calcium channel blockers. A study of the effects of a number of specific blockers of the calcium channel upon the 22Na gain in fish ventricles, showed that 2 #mol/1 D600 or verapamil and 100 nmol/l nifedipine profoundly reduced the sodium uptake into hearts exposed to 10 nmol/1 calcium for 1 h. These drug concentrations were sufficient to inhibit the development of calcium readmission tension in frog atria. (d) The effect of membrane potential. Calcium channels are activated, in normal physiological media, by membrane voltages more positive than - 50 mV and inactivate over several ms [5, 55, 56, 61]. Measurements of the T T X insensitive slow inward current in our preparations showed that the activation curve is shifted by up to 10 mV in the negative direction by calcium reduction and the rate of inactivation of the current was slowed in agreement with the recent observations of Hess and Tsien [40]. The shift in the potential dependence of the current produced a large region of overlap between the activation and inactivation curves so that a sustained or window current could develop at potentials between --60 and + 10 mV. In consequence a considerable proportion of the calcium channels may remain activated at --20 mV, i.e. the level of depolarisation of the prolonged action potentials recorded in low calcium fluid. As, in the absence of calcium, these channels become permeable to a variety of monovalent ions [40] a large part of the current may be carried by sodium ions. I f this is so then the large rise in sodium during low calcium perfusion and the calcium readmission contracture may show a voltage dependence similar to that of the slow channel. Figure 6(a) shows calcium re-admission contractures recorded from preparations, voltage clamped to various membrane potentials during a 10 min period of low calcium perfusion, but returned to - 8 0 m V at the instant of calcium addition. The figure shows that the calcium re-admission tension is small in preparations clamped to a potential more negative than --50 m V i.e. the threshold potential for the activation of the slow inward current recorded for this preparation. At less negative voltages however, a large contracture is recorded. The figure also shows that in

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F I G U R E 6. (a) Calcium re-admission contractures recorded from frog atrial trabeculae when the bathing calcium concentration is raised to 1 mmol/1 after l0 mins in 10 nmol/1 calcium. The preparation was voltage clamped to a - 8 0 mV; b - 5 0 mV; c - 4 0 mV; d --20 mV and e 0 mV during the low calcium perfusion but returned to - 8 0 mV 5 s before the bathing calcium was raised. The figure also shows f the response of the preparation clamped to - 2 0 mV in the presence of 100 #mol/l Mg 2+ during the low calcium perfusion; (b) The tension recorded on calcium re-admission plotted against the membrane potential of the preparation during the low calcium perfusion. Tension is expressed as a percentage of the largest tension recorded which usually followed a voltage clamp between - 20 to 0 inV. The addition of 100 #mol/1 magnesium to the low calcium perfusate inhibits tension at all membrane potentials (open triangles). Points represent the mean _+ s.E. of 12 preparations.

comparison to the control responses, the tension recorded, if the low calcium solution contains 100 #mol/1 magnesium, is blocked at all membrane potentials. We noted that in the presence of magnesium the rate of inactivation of the slow inward current is not sig-

Ions and ' C a l c i u m Paradox'

nificantly different from that recorded in control preparations. The data for 12 preparations are replotted in Figure 6(b) which shows peak tension as a function of membrane potential, and that at membrane potentials more negative than approximately - 5 0 mV, the calcium readmission contracture is very small. At more positive potentials the contractures are larger and increase with depolarisation to reach a m a x i m u m - a t --20 mV. Above this potential there is some indication of a fall in the tension. In a few preparations this decline was quite marked.

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potentials have developed, however like Mg 2+ and tetracaine they inhibit the development of spontaneous action potentials if added before the reduction of the calcium concentration. Discussion

The experiments recorded here identify a sequence of cause and event, which m a y result in the development of a strong contracture when calcium ions are returned to a solution bathing cardiac muscle, after a period of low calcium perfusion. It seems clear from the close relationship between the rise in [Na]i and the degree to which [Ca]o must be The effect of agents which inhibit reduced in order that a calcium re-admission the increase in [ Na]i upon the contracture may develop, that a large rise in prolonged action potentials In Figure 7 the action of (a) Mg 2+ 100 pmol/1 [Na]i during the period of low calcium perfusion is the necessary precondition for the (b) tetracaine 500 pmol/1 and (c) 500/~mol/1 quinidine upon the spontaneous action poten- development of the subsequent tension [Figs 2 tials is illustrated. In each case the agent is and 3(b)]. This notion is further strengthened added after the spontaneous action potentials by the observation that agents which affect have developed, in consequence of the the sodium gain, either to increase it, e.g. reduction of the bathing calcium concentra- strophanthidin [Fig. 3(a)], or to inhibit it, tion to 10 nmol/1. The figure shows that the e.g. anti-arrhythmic drugs, divalent cations or response of the preparation to the addition or calcium channel blockers, also affect the removal of these agents is very similar. All calcium re-admission contracture, (Fig. 2) cause the prompt cessation of the train of and that the reduction of the sodium gradient action potentials which starts again once they during calcium depletion has direct effects are removed. Quinidine and D600 are more upon the amplitude of the following contracslow to act if added once spontaneous action ture. A number of workers have suggested that the elevation of [Na]i during the calcium free period could lead to the calcium paradox [11, 52, 58, 65]. This idea has gained support from oF the observation that the substantial rise in total cell sodium, which occurs in both mammalian and amphibian hearts exposed to low Co2+10nmol/I Ringer calcium solutions, may influence the contracMg 2+ Tetrocoine Quinidine 60s IO0/u.mol/I 500p.rnol/I ,500 ~mol/I tile response when calcium ions are readmitted [17, 58]. It is possible that the F I G U R E 7. The effects of (a) 100 ,umol/l Mg2+; (b) Na/Ca exchange may be responsible for the 500 #tool/1 tetracaine and (c) 500 #mol/1 quinidine upon sodium uptake [27]. The Na/Ca exchange is the prolonged action potentials recorded from a frog atrial preparation exposed to a Ringer solution containunlikely, however, to be the major pathway ing 10 nmol/l calcium. The preparation was exposed to for sodium gain because tetracaine, quinidine, this solution for 5 rains before the addition of M g 2+ and Mg 2+, D600 or verapamil and the dihywas not stimulated. Each agent was added and removed dropyridines block the calcium re-admission as indicated by the bars. In each case a prompt repolarisation of the prolonged action potential occurs and the tension and the rise in [Na]i. This occurs at a spontaneous activity stops. Upon removal of each agent concentration much below that which affects the preparation slowly depolarises before the spontaneous the spontaneous relaxation of the low sodium action potentials begin again. In the case of Mg 2+ the contracture, or phasic tension in isolated frog action potentials immediately lengthen, the effects of atrial trabeculae [19, 22]. tetracaine and quinidine however, are lost more slowly.

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The agents we have used to inhibit both sodium uptake and calcium re-admission tension, have a wide variety of actions upon ion transport in cardiac preparations. Quinidine inhibits calcium uptake by mitochondria with an IDso around 1 mmol/1 [4] and for this reason may affect the accumulation of 45Ca by frog atria exposed to low sodium solutions [23]. At concentrations greater than 50 #mol/1 it is reported to inhibit the Na/Ca exchange in dog heart sarcolemmal vesicles, but at 10 /~mol/1 may enhance Na/Ca exchanges [47]. Further, quinidine at 50 /~mol/l has marked effects as an inhibitor of the transmembrane currents in a variety of tissues, including molluscan neurones as well as mammalian and frog heart preparations [28, 38, 51]. Since quinidine is effective at 50 ktmol/1 in our experiments its effect upon ion channels may be important in inhibiting the rise in [Nail. The actions of D600, verapamil and the divalent cations upon ion exchanges are similarly confused. D600 and verapamil are known to affect the slow inward current [29, 38] but verapamil, at concentrations greater than 0.1 retool/1 may also inhibit the Na/Ca exchange in sarcolemmal vesicles [47]. At the ~;oncentrations which are effective in our experiments, however, an effect on the Na/Ca exchange is unlikely. The divalent cations which are able to replace the control by Ca I + of the slow inward current [2, 40], may also inhibit Na/Ca exchanges in dog sarcolemmal vesicles [63]. A number of divalent cations including M n z +, appear to have this effect as they are able to block low sodium contractures in frog atria [14]. C h a p m a n and Ellis [14] saw that a high concentration of Mg 2+, a potent inhibitor of calcium re-admission tension, but only if added during the time of calcium depletion, was without effect upon the tow sodium contracture recorded in normal calcium solutions. In the absence of extracellular sodium however, the tension produced by the elevation of calcium is weakly inhibited by the action of Mg z+ possibly indicating an effect upon the Na/Ca exchange [9]. The difference in the voltage dependence of the calcium re-admission contracture in the presence or absence of magnesium, illustrated in Figure 6, may reflect this action particularly at large negative voltages.

The actions of tetracaine and the specific calcium channel blocker nifedipine appear less confused. Tetracaine has no recorded affect upon the Na/Ca exchange yet like nifedipine has marked effects on the slow inward current of cardiac cells [1, 16, 30]. The results reported above would seem to rule out the Na/Ca exchange as the primary pathway for the influx of sodium during low calcium perfusion. It is noticeable that an ability to affect the calcium channel is a common feature of all the agents we have used to inhibit the rise in [Na]i and the calcium re-admission contracture. I f present before [Ca]o is lowered, these agents also prevent the development of prolonged action potentials or cause the repolarisation of the cells if added during low calcium perfusion (Fig. 7). Prolonged action potentials are known to develop in preparations perfused with low calcium solutions [35, 48, 62], and prolonged action potentials associated with a marked rise I"n aNa i have been described in ferret ventricular fibres [17]. Miller and Morchen [48] suggest that the prolongation of the action potential is a consequence of calcium depletion upon the K + conductance of the cells, because they found that D600 has no immediate effect upon the prolonged action potential. In our experiments D600 and the organic calcium channel blockers, whilst eventually causin~ the repolarisation of the prolonged actiori potentials, were less effective and more slow to act than either tetracaine, quinidine or Mg 2+. However if present before the reduction of the bathing calcium they prevented the development of prolonged action potentials. A similar result for the action of D600 upon the prolonged action potentials which develop in skeletal muscle fibres has been reported [50] suggesting that the mechanism which brings about prolongation of the action potentials is the same in both skeletal and cardiac preparations. T h a t sodium gain is inhibited by agents which prevent the development of prolonged action potentials, and reduced by lowering [Na]o suggests that it must occur by way of a voltage sensitive pathway activated by calcium removal. This conclusion is strongly supported by the voltage clamp experiments in that the potential for the onset and peak of the calcium re-admission contracture parallels

Ions and ' C a l c i u m Paradox'

~51

the voltage dependence of the inward current. passage of an outward current at this time, The fall in tension and inward current seen in which may arise from an exchange of more Some preparations at membrane potentials than two Na + for each Ca 2 +. more positive than --20 mV would further It was noted that, if added at the time of support this view. The pathway is unlikely to calcium repletion, a high concentration (1 be the sodium channel because T T X failed to mmol/1) of quinidine makes the re-admission prevent either the sodium gain or the calcium contracture larger and slows its rate of re-admission contracture. I n view of the recovery. It is probable that this action voltage and drug dependence of the sodium mirrors the effect of quinidine upon the spongain, the most likely pathway appears to be taneous relaxation of low sodium contracthe calcium Channel. The conclusion that in tures, i.e. to slow the recovery by an inhibition low calcium solutions the calcium channel ofmitochondrial calcium uptake [22]. may lose its ion specificitY, to become perThese results would suggest that an intermeable to monovalent cations, particularly pretation of the effects of a number of agents, sodium, has been arrived at for a number of such as quinidine, D600 and M g 2+ on preparations [1, 24, 40, 45, 56]. Under these calcium movements in isolated sarcolemmal conditions the maximum inward current is vesicles may be complicated by the effects increased and inactivation markedly slowed, these agents have upon the calcium channels. i.e. both factors will increase the ion flux This may be particularly relevant in experithrough the channel. ments where the vesicles are first loaded with The mechanism by which the re-addition of sodium, by exposure to low calcium, in order calcium ions leads to the development of to maximise Na/Ca exchange activity when tension m a y be more easily determined. It is calcium is re-introduced. The inhibitory effect clear that the re-introduction of calcium leads recorded may be due to a reduction of the to a massive uptake of this ion [3, 68] which sodium loading. may often be fatal to the cell. A number of The effects of calcium-deprivation and repathways have been considered for this admission on the tissues studied here show a uptake including passive diffusion across dis- number o f differences from the irreversible rupted cell membranes, the calcium channels changes seen in the intact mammalian heart and the N a / C a e x c h a n g e [36]. It is unlikely at 37°C [44, 52, 67, 68]. Principally the that the massive uptake of calcium results responses of our isolated tissues are reversible, from tissue disruption in our preparations, as with the Ca-readdition contracture returning the tension which develops subsequently to base-line, together with the recovery of relaxes and the preparations regain their electrical and mechanical activity and the resnormal contractile responses, it is also toration of ionic gradients (P. Busselen and J. unlikely that the slow channels are involved, Tunstall unpublished observations). Furtheras the prompt repolarisation of the tissue sug- more to produce these effects C a 2+ m u s t be gests that the re-addition of calcium causes a reduced to a ievel below that which is effective rapid inactivation and also that the agents, in the intact mammalian heart. The observawith the exception o f M n 2 +, which inhibit the tions listed below would seem to suggest that calcium re-addition tension if present during these differences are simply a matter of the low calcium perfusion, are ineffective i f degreel First: agents which alleviate the added at the time of calcium repletion. Man- severe effects of the calcium paradox in the ganese ions which are known to block the intact heart inhibit the rise in [Na]i and the sodium-withdrawal contracture in frog atria subsequent calcium-readdition contracture in [14] and Na/Ca exchanges in squid axons [6] our experiments. Second: isolated m a m m a was the only agent used in our experiments lian preparations from ferret ventricular and able to inhibit tension development if added sheep Purkinje fibres also give reversible after Nai had risen. This gives a strong indica- responses and show a similar sensitivity to tion of the activity of the Na/Ca exchange agents such as Mg 2+ [15, 17]. In Purkinje during calcium uptake. The hyperpolar- fibres the lethal after effects of calcium depriisation of the preparation during the develop- vation are generally only apparent when the ment of the tension would indicate the sodium-pump is also inhibited. This observa-

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t i o n m a y r e l a t e to t h e effects o f c a l c i u m d e p r i v a t i o n u p t h e N a +, K +, A T P a s e a c t i v i t y i n r a t h e a r t r e p o r t e d b y L a m e r s , Stinis a n d R u i g r o k [46]. T h e s e c o n s i d e r a t i o n s i n c l i n e us to t h e n o t i o n t h a t o u r d a t a a r e c o n c e r n e d w i t h t h o s e e v e n t s w h i c h p r e - d i s p o s e t h e h e a r t to severe c a l c i u m - l o a d i n g , t h e first s t a g e o f t h e calcium paradox. I n i s o l a t e d p r e p a r a t i o n s p r o b l e m s associ-

a t e d w i t h p e r f u s i o n , e q u i v a l e n t to t h o s e w h i c h follow c h a n g e s i n t h e c o r o n a r y c i r c u l a t i o n o f i n t a c t p r e p a r a t i o n s a r e u n l i k e l y to o c c u r . P r o f o u n d c h a n g e s i n flow r a t e t h r o u g h t h e c o r o n a r y vessels d u r i n g t h e i n d u c t i o n o f t h e calcium-paradox in intact heart have been n o t e d [44], a n d s i m i l a r i t i e s b e t w e e n i s c h a e m i c d a m a g e a n d t h e c a l c i u m - p a r a d o x a r e well d o c u m e n t e d [38].

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