Control of intracellular ionized calcium concentration by sarcolemmal and intracellular mechanisms

j Mol Cell Cardiol 16, 137-146 (1984)

Control o f Intracellular Ionized Calcium Concentration by S a r c o l e m m a l and Intracellular M e c h a n i s m s D. A. Eisner, C. H. O r c h a r d a n d D. G. Allen

Centrefor Cellular Cardiology, Department of Physiology, University CollegeLondon, Gower Street, London WC1E 6B T, UK

D. A. EISNER,C. H. ORCHARDAND D. G. ALLEN. Control of Intracellular Ionized Calcium Concentration by Sarcolemmal and Intracellular Mechanisms. Journalof MolecularandCellularCardiology(1984) 16, 137-146. The regulation of the resting intracellular ionized calcium concentration ([Ca2+]i) has been studied in ferret papillary muscle using the photoprotein aequorin to measure [Ca2+]i. Elevating [Ca2+]0 produced an initial rapid increase of [Ca 2+]i and tension which then decayed to a steady level. This secondary fall of [Ca 2+]i is attributed to a secondary decrease of Ca entry on N ~ C a exchange produced by the known fall of[Na+]i . Replacing external Na by K pro.duced a large transient increase of both [Ca2+]i and tension which then decayed spontaneously to near the resting level. IfNa was removed after metabolic inhibition with cyanide and deoxyglucose then neither tension nor [Ca 2+]i recovered. The addition of the mitochondrial uncoupler FCCP to a muscle in Na-free solution produced a gradual rise of tension but only elevated [Ca2+]i after a delay of many minutes. Similarly caffeine did not elevate [-Ca2+]i' These experiments do not support the hypothesis that the regulation of resting [Ca z +]i in Na-free solutions depends solely on intracellular sequestration of [Ca 2+]i. The first twitch elicited in Na-containing solutions after exposure to Na-free solution was much larger than control and was associated with a large Ca transient attributed to increased loading of the sarcoplasmic reticulum with Ca in the Na-free solution. The elevation of [Ca2+]i in Na-free solutions was accompanied by spontaneous fluctuations of both [-Ca2+]i and tension with a frequency of about 3 Hz. These fluctuations were abolished by drugs such as caffeine or ryanodine which interfere with sareoplasmic reticulum function. These results provide direct evidence for the spontaneous release of Ca from the sarcoplasmic reticulum inferred from previous, less direct, work. KEY "~u

: Intracellular calcium ; Cardiac muscle.

Introduction As in o t h e r e x c i t a b l e cells, the i n t r a c e l l u l a r resting ionized calcium concentration ([Ca2+]i) is m u c h less t h a n t h a t in p l a s m a ; [ Ca2+]i is a b o u t 200 nM [9, 19, 24] w h e r e a s the c a l c i u m c o n c e n t r a t i o n in p l a s m a is a b o u t 2 mM. T h i s c o n c e n t r a t i o n g r a d i e n t m u s t be m a i n t a i n e d in the p r e s e n c e o f passive leaks o f c a l c i u m i n t o the cell a n d t h e r e f o r e r e q u i r e s a c t i v e m e c h a n i s m s to r e m o v e c a l c i u m f r o m the c y t o p l a s m . H o w e v e r the m e c h a n i s m s responsible for this c o n t r o l of [ C a 2 +]i are still c o n t r o v e r s i a l : it c o u l d be a c h i e v e d e i t h e r by the surface m e m b r a n e or by i n t r a c e l l u l a r C a buffers. T w o systems m a y c o n t r i b u t e to i n t r a c e l l u lar C a b u f f e r i n g : m i t o c h o n d r i a a n d sarcop l a s m i c r e t i c u l u m (see [11] for a review). 0022-2828/84/020137 + 10 $03.00/0

A l t h o u g h the C a a c c u m u l a t i n g c a p a c i t y o f the f o r m e r is significant, it has b e e n suggested that, at i n t r a c e l l u l a r [-Mg2+], the affinity for C a is too low to p r o d u c e C a u p t a k e . S a r c o p l a s m i c r e t i c u l u m has a h i g h e r affinity for C a [11]. S u c h C a buffers can, of course, o n l y p r o d u c e t r a n s i e n t c h a n g e s of [ C a 2 +]i a n d , in the steady-state, C a m u s t be in e q u i l i b r i u m across the surface m e m b r a n e . However steady-state conditions may not apply under m a n y e x p e r i m e n t a l c o n d i t i o n s a n d it is possible that, for the d u r a t i o n o f a t y p i c a l experim e n t , a c o n s t a n t [ C a 2 + ] i m a y be m a i n t a i n e d by net C a u p t a k e into or release f r o m i n t r a c e l l u l a r buffers. T w o m e c h a n i s m s h a v e b e e n suggested for C a e x t r u s i o n by the surface m e m b r a n e : (i) an N a C a e x c h a n g e in w h i c h the free e n e r g y re9 1984 Academic Press Inc. (London) Limited

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leased by Na ions entering the cell down their electrochemical gradient is used to extrude Ca ions [6, 23] and (ii) a Ca ATPase in which the energy for Ca extrusion is provided directly by ATP hydrolysis [12]. Ca entry into the cell is generally attributed to entry through Ca channels such as the slow inward current [8]. This is activated by depolarization during the action potential. It is also possible that Ca can enter through other ionic channels as in the squid axon [7-J. It has also recently been suggested [20] that, under some conditions, the N a - C a exchange may produce a significant net Ca entry into the cell. The direction of Ca movement through the Na Ca exchange depends on the transmembrane electrochemical gradients of Na and Ca. There is evidence that the exchange may transport more than 2 Na ions per Ca ion [22]. In this case the rate of the exchange will depend not only on the concentration gradients but also on the membrane potential. This therefore provides another voltage-dependent pathway for Ca entry into the cell in addition to Ca channels. The above brief review gives some indication of the complexity of the control of [ Ca2 +]i. The purpose of the present paper is to provide experimental measures of [Ca2+]i under various conditions in order to characterize the contributions of the various mechanisms.

Methods

The experimental methods have been described elsewhere [5]. The experiments were performed on ferret papillary muscles (diameter 0.4 to 1.0 mm) dissected from the right ventricle. All experiments were performed at 30~ [Ca2+]i was measured by recording the light emission from aequorin which had been microinjected into 50 to 100 cells. Control experiments showed that none of the drugs used in the present paper affected either the quantum efficiency of aequorin or the light emission at a buffered [Ca 2+] of 4 x 10 7M.

Solutions The standard solution (used in the experiments of Figures 2, 3, 4, 6) contained (in retool/l): Na + 135, K + 5, Mg 2+ 1, Ca 2+ 2,

C1- 104, H C O 3 20, HPO,~ 1, acetate 20, glucose 10, insulin 4 x 10 -5 and was equilibrated with 95% 02/5% CO2 (pH 7.4). In experiments in which Na was replaced by K all the above Na salts were replaced by equimolar amounts of the corresponding K salts. In the experiments of Figures 1 and 5 a simple HEPES (N-2-HydroxyethylpiperazineN'-2-ethanesulphonic acid) buffered solution was used containing (in mmol/1): Na § 135, K + 5, Mg 2+ 1, Ca 2+ 2, CI- 141, H E P E S 5, glucose 10, insulin 4 x 10 -5 and was equilibrated with 100% O 1 (pH 7.4). Na-free solutions containing K or choline could be made simple by replacing NaC1 with KC1 or choline C1.

Results and Discussion

The effectsof changing [Ca 2 +]0 The simplest experiments involved elevating [ Ca2 § The experiment of Figure 1 was performed after inhibiting the N ~ K pump with strophanthidin although qualitatively similar but smaller effects were seen in the absence of this drug. Increasing [-Ca2+]0 from 2 to 8 mM produced an increase of [Ca 2 +]i as shown by aequorin light emission which then decayed to a steady-state level higher than control. On return to 2 mM [-Ca2+]0, [caa+]i fell and a subsequent increase to 8 mM produced a smaller and maintained increase of [Ca2+]i. The final exposure to 8 mM [Ca2+]0, obtained after a prolonged recovery in 2 mM [Ca2+]0, again shows an overshoot of [Ca2+]i . These changes of [Ca2+]i were accompanied by parallel tension changes. The rise of [CaZ+]i produced by elevating [caa+]0 could be produced by an extra Ca influx through (i) Ca channels or (ii) N a - C a exchange. In the absence of other information it seems likely that both these mechanisms will contribute. A plausible explanation for the secondary fall of [Ca 2+]i is that it is due to the fall of intracellular Na concentration ([Na+]i) known to be produced by elevating [Ca2+]0 [13]. In sheep cardiac Purkinje fibres this fall of [Na+]i has a similar time course to the secondary fall of tension [25] and such a decrease of [Na+]i will decrease Ca influx via

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FIGURE 1. The effects of increasing [Ca 2+]0 on aequorin light and tension. Traces show: top, aequorin light (1 s time constant filter) expressed as photomultiplier current; bottom, tension. The solution protocol is illustrated above the record. The bar marked by sc shows when the shutter in front of the photomultiplier was closed to establish the zero level of light. Strophanthidin ( 10 #M) was present throughout. N a - C a exchange. Therefore, in the presence of other Ca removal systems, [ C a 2 +]i will fall. T h e observation that a prolonged recovery in low [CaZ+]0 is required before exposure to high [ C a 2 +]0 gives a full tension response can be attributed to the slow rise o f [ N a + ] i produced by decreasing [CaZ+]0 . We have similarly found (not shown) that depolarization (produced by increasing [ K + ] 0 from 5 to 30 rag) produces a transient rise of [CaZ+]i similar to that produced by raising [ C a 2+]0. Again depolarization produces a fall of [ N a + ] i which accompanies the relaxation of tension [14]. These sorts of experiments therefore suggest that changes o f [ N a + ] i , by acting on the N a - C a exchange, can have significant effects on [ C a z +]i and tension.

The regulation o f [ C a 2 +]i in the absence of Na-Ca exchange The effects of Na removal. T h e previous experiment suggests a role for m e m b r a n e N a - C a exchange in mediating changes of [CaZ+]i. T h e experiment of Figure 2 was designed to eliminate this N a - C a exchange and then

examine the regulation o f [ C a 2 +]i- T h e preparation was initially in a control solution and was stimulated. T h e n external N a was replaced by K, resulting in a transient increase of both [Ca2+]i and tension. Both [ Ca2 +]i and tension then fell to a steady level. In this experiment this steady-state level of [ Ca2 +]i was slightly greater than the control (Figure 2B). Comparison of (A) and (B) shows that, although the contracture tension is less than the twitch, the peak aequorin light during the contracture is greater than that a c c o m p a n y i n g the twitch. A possible explanation for this is given below ('Spontaneous oscillations of [Ca2+]i ). [Ca2+]i in Nacontaining solutions is too low to be detected by aequorin and is p r o b a b l y less than 160 nM (our threshold for detection of [Ca2+]i [3]). In Na-free solutions the m e a n steady-state level of [ C a 2 +]i was estimated to be about 220 nM (see [.~ for further details). The rise of [ Ca2 +]i produced by removing N a is presumable due to a Ca influx on N a - C a exchange but the subsequent fall of [Ca2+]i to a low level requires some other active Ca extrusion process.

140

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F I G U R E 2. The effects of a Na-free solution on light and tension in an aequorin-injected papillary muscle. (A) Traces show: top, light; middle, filtered light (1 s time constant) ; bottom, tension. The solution protocol is illustrated above the record. The muscle was initially stimulated at 0.33 Hz and superfused with Na solution. The superfusate was changed to ONa(K) at the point indicated. To establish the zero level of light the shutter in front of the photomuhiplier tube (PMT) was closed for the period indicated by sc. (B) Traces show averaged (n = 32) records of light (top) and tension (bottom) obtained during the periods indicated on (A). The stimulus marker is shown below. (i) shows the light and tension records produced by stimulation in the Na solution. (ii) shows the light and tension achieved after prolonged exposure to Na-free solution, sc again indicates the PMT output when the shutter was closed. Taken from

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F I G U R E 3. Effects of metabolic inhibition on light and tension during Na-removal contractures. Traces show: top, filtered light (one second time constant) ; bottom, tension. The solution protocol is illustrated above the record. Na-free solutions were produced by replacing Na by K. (A) The effects of Na-removal in control conditions. The muscle was initially in the Na solution and, at the time indicated was transferred to Na-free solution. (B) The effects of Na-removal in the presence of C N - and 2-deoxyglucose. The muscle had been exposed to C N - plus deoxyglucose for a total of 45 min before the start of the record. Na was then removed and added back as shown. Note that the light record goes off-scale during the second period ofNa-removal. A lower gain record (not shown) indicated that it reached a value of about 350 nA during this period. Taken from [3].

[Ca2 +]i control

The effects of metabolic inhibition. T h e experim e n t shown in Figure 3 was designed to investigate whether the N a - i n d e p e n d e n t Ca extrusion system was sensitive to metabolic inhibition. A control exposure to Na-free solution is shown in Figure 3 (A). This was repeated i n Figure 3(B) except that the p r e p a r a t i o n had been exposed to cyanide (2 mM) a n d deoxyglucose (10 mM) to i n h i b i t respectively aerobic and anaerobic metabolism. T h e increases of both tension a n d rCa2+]i were m u c h larger than in the control and, after an initial partial recovery, [ C a 2 +]i continued to increase. [ Ca2 +]i could, however, be decreased to low levels by the addition of Na. Interestingly, tension does n o t relax suggesting that a large a m o u n t of this tension is due to the formation of rigor bridges due to lack of

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141

A T P (see below). This experiment therefore provides evidence for two processes controlling rCa2+]i : (i) N a - C a exchange; (ii) some metabolism d e p e n d e n t process which only becomes a p p a r e n t when N a - C a exchange is abolished. This latter system could represent either A T P - d e p e n d e n t Ca extrusion from the cell or energy d e p e n d e n t Ca buffering by intracellular organelles such as m i t o c h o n d r i a or sarcoplasmic reticulum.

The effects of agents which interfere with intracellular organelles. T o investigate the possible contribution of intracellular organelles to Ca regulation, we have examined the effects of drugs which release Ca ions from either the sarcoplasmic reticulum or mitochondria. I n the experiment illustrated in Figure 4 the muscle was first exposed to a Na-free solution

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FIGURE 4. The effects of FCCP and caffeine o n [Ca2+]i and tension. Traces show: top, low gain aequorin light; middle, filtered high gain aequorin light (time constant 1 s) ; bottom, tension. The solution protocol is shown above the record. The light record is calibrated in terms of fractional luminescencein order that some absolute estimate can be made of [Ca z+]i (see [19] for further details). The muscle was initially stimulated in Na solution before changing to a Na-free solution (K-substituted) as shown. To eliminate any residual Na gradient, strophanthidin (10 #~) was added to the perfusate 10 minutes after changing to the Na-free solution. The bars indicate when FCCP (1 #M) and caffeine (5 mM) were present. FCCP was added as a concentrated stock in dimethyl sulphoxide (DMSO). The final DMSO concentration was 0.01% (v/v). sc shows when the photomultiplier shutter was closed.

142

D . A . E i s n e r et ol.

resulting in the usual transient increase of tension and [Ca2+]i . When these had decayed to a steady state level FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was added. This drug, which has been shown to produce a rapid release of Ca from loaded mitochondria in many tissues [10] had no immediate effect on [Ca2+]i . Caffeine, w h i c h releases Ca ions from the sarcoplasmic reticulum [18], was then added and although it increased resting tension, no change of [Ca2+]i was observed. Using the aequorin calibration procedure described elsewhere [I] we have estimated that the level of [Ca 2 +]i in the presence of both caffeine and FCCP was less than 250 nM. This shows that a low [Ca2+]i can be maintained when mitochondrial and sarcoplasmic reticulum function are impaired and that these organelles either do not contain much Ca or that the released Ca can be removed from the cytoplasm by some other system. After sufficient exposure to FCCP, [Ca2+]i gradually began to rise. This rise is presumably occurring at a time at which [ A T P ] had fallen to very low levels, as the muscle had started to go into rigor, and is therefore (like Figure 3) only indicative of a metabolismsensitive component of Ca regulation. These results differ somewhat from the conclusions of previous work on the effects of substances which interfere with the functions of either mitochondria or the sarcoplasmic reticulum. For example the addition of C N - to cardiac muscle in Na-free solutions has been shown to produce a large increase of tension which was attributed to the release of Ca from mitdchondria [15]. Figures 3 and 4 show, however, that, in Na-free solutions, a large increase of resting tension can be produced by metabolic inhibition without any accompanying rise of [Ca2+]i . This suggests that the increase of tension is entirely due to decreased [ATP]. It is therefore possible that in the experiments of [15], the rise of tension produced by C N - was also mainly due to decreased [ A T P ] and cannot be taken to indicate an increase of [Ca 2 +]i. Similarly caffeine increases resting tension if applied in Na-free solutions [15]. This effect was suggested to represent an increase o f [ C a 2 +]i due to release from the sarcoplasmic reticuliam. However the present results show that the increase of

tension is not accompanied by a rise of [ Caz +]i. There are two possible explanations for a rise of tension produced by caffeine in the absence of increases of [Ca 2+]i. (i) Caffeine may increase the sensitivity of the contractile apparatus to [Ca2+]i [18]. (ii) Caffeine may synchronize contraction in the various cells in the preparation (see below for further discussion of this point).

The effects of Na-free solutions on sarcoplasmic reticulumfunction The recovery of the twitch. The above experiments failed to provide evidence for the presence of pharmacologically releasable Ca in the sarcoplasmic reticulum (SR). In an alternative approach, we have investigated whether the amount of Ca releasable by electrical stimulation is altered by Ca-loading in Na-free solutions. In Figure 5(A) the muscle was stimulated until a steady state was reached and then stimulation was discontinued for 6 min. When stimulation was recommenced the first twitch was small and then the twitch gradually increased to a steady level. Aequorin and tension records for both the first and steady state contractions are displayed below and show that the calcium transient also increased in parallel with the rise of tension. In the experiment of Figure 5(B) the same preparation was initially stimulated in an Na-containing solution and then stimulation was stopped and a Na-free solution applied. After 2 min Na was added back, and then stimulation was begun. The total period of rest was identical to that in (A). However the first twitch is now much larger than the steady-state although the subsequent ones are much smaller and tension only slowly recovers towards the steady level. The fact that there are only two or three large twitches on starting stimulation following the Na-free exposure is consistent with the hypothesis that the sarcoplasmic reticulum has been loaded with Ca by the elevated [Ca 2 +]i during the Na-free exposure. Alternative explanations such as the possibility that elevated [ Ca2+]i increases the calcium current across the sarcolemma cannot explain the observations since, on such models, the twitch should decay smoothly as a function of time after adding back Na. Two additional observations also support the hypothesis that

[Ca2 +]i control

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F I G U R E 5. The effects of prior exposure to Na-free solution on the twitch and accompanying light transient. In each part of the Figure, the upper panel shows an original tension trace and the lower panel shows: top, aequorin light; bottom, tension. (A) Control for the effects of rest. The muscle was initially stimulated at 3 Hz and then stimulation was discontinued for the period shown. The bottom panel shows aequorin light and tension records obtained at the points indicated on the top trace. (i) is a single record from the first twitch and (ii) is the average of 16 records in the steady-state. (B) The effects of exposure to Na-free solutions. The muscle was rested for 6 min and, during this period, was exposed for 2 rain to a Na-free solution (replaced by choline). The lower panel again shows specimen records obtained at the times indicated.

the sarcoplasmic reticulum contributes m u c h more to the first than the steady-state twitch. (i) I f the Na-free exposure is performed in the presence of caffeine the first twitch is smaller than the control and (ii) D600 which blocks Ca channels has much less effect on the first twitch than on the steady-state. T h e above results are consistent with the idea that a certain a m o u n t of the Ca which enters the cell during the Na-free contracture is buffered by the sarcoplasmic reticulum. T h e simplest way to reconcile this result with the lack of effect of caffeine on resting [Ca2+]i is to assume that, although the sarcoplasmic

reticulum is loaded by the rise of [ - C a 2 + ] i during the Na-free exposure, the rate at which it can be released by caffeine is less than the rate at which it can be removed from the cytoplasm by other mechanisms. This might suggest that some of the Ca entering the cell during the contracture has been extruded, perhaps by a Ca-ATPase. Unfortunately it is impossible to distinguish unequivocally between intracellular Ca buffering and net extrusion by measuring ionized levels of [ Caz +]i. To address this problem, it would be useful to repeat these experiments while measuring total cell Ca.

144

D . A . E i s n e r et al.

Spontaneous oscillations of [ C a 2 + ] i . T h e r e is also evidence that the sarcoplasmic reticulum m a y have more c o m p l i c a t e d effects on [ C a 2 + ] i . I n the e x p e r i m e n t of Figure 6 the p r e p a r a t i o n was again exposed to a Na-free solution resulting in an increase of both [ C a2 +]i and tension (AT). S a m p l e tension and light records are shown at a faster time base in (B). Oscillations of [CaZ+]i are evident with a frequency of a b o u t 3 H z a n d there are similar fluctuations of tension. F o u r i e r analysis of the light a n d tension signals shows that they b o t h have peaks at the same frequency [4, 21]. These oscillations of force are similar to those noted previously in cardiac cells in which [Ca2+]i had been increased and were a t t r i b u t e d to spontaneous release of Ca from the sarcoplasmic reticulum [16, 17]. T h e present e x p e r i m e n t shows that these fluctuations are, indeed, a c c o m p a n i e d by fluctua-

tions of [ C a 2 +]i- This hypothesis is s u p p o r t e d by the results of Figure 6(B) which also shows the results of a n o t h e r exposure to Na-free solution in the presence of r y a n o d i n e (2yM), an inhibitor of sarcoplasmic reticulum C a release. This abolishes the fluctuations of [ C a 2 + ] i . These fluctuations of [Ca2+]i have several i m p o r t a n t consequences. (i) I t becomes impossible to assign a unique value to [ C a 2 + l i since it is oscillating and therefore t h e r m o d y n a m i c analyses of the regulation of [Ca2+]i [241 become, at best, difficult to interpret. (ii) Since, at least over some ranges of [ C a 2 + l i , aequorin luminescence is prop o r t i o n a l to ([CaZ+]i)2: s [2] the presence of oscillations will m e a n that the m e a n aequorin luminescence will be greater than that which would be p r o d u c e d by. a steady [ C a 2 +]i with the same m e a n value. As discussed previously [31 it is likely that these fluctuations of

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[Ca2 +]i control [ C a 2 + ] i lead to an o v e r e s t i m a t e of the m e a n level o f [ C a 2 +]i m e a s u r e d in N a - r e m o v a l contractures. (iii) T h e m e a n tension d e v e l o p e d by the p r e p a r a t i o n will be less t h a n the m e a n of the i s o m e t r i c tensions of the i n d i v i d u a l cells. T h i s is because, if the ceils are c o n t r a c t i n g a s y n c h r o n o u s l y , the c o n t r a c t i n g cells will s h o r t e n against the c o m p l i a n c e of the n o n c o n t r a c t i n g ones a n d will therefore g e n e r a t e less force at these s h o r t e n e d lengths. C o n s i d e r ations (ii) a n d (iii) m e a n t h a t a n y m a n o e u v r e w h i c h decreases the a m p l i t u d e o f fluctuations w i t h o u t affection m e a n [ C a 2 +]i will d e c r e a s e the a e q u o r i n light b u t m a y increase tension. T h i s p r o v i d e s a n o t h e r e x p l a n a t i o n for the increase of resting tension p r o d u c e d by caffeine [15] in the absence o f a rise of a e q u o r i n light. A l t h o u g h a g e n u i n e increase of sensi-

145

tivity of the c o n t r a c t i l e proteins to [ C a 2 + ] i c a n n o t be e x c l u d e d (see a b o v e ) a d i m i n u t i o n o f the a m p l i t u d e o f C a f l u c t u a t i o n s c o u l d also e x p l a i n the c a f f e i n e - i n d u c e d increase o f tension. S i m i l a r l y the effects of oscillations on light a n d tension can e x p l a i n the large light signal associated w i t h little tension d u r i n g a c o n t r a c t u r e as c o m p a r e d to the t w i t c h [3, 21]. Acknowledgements

T h e w o r k d e s c r i b e d in this p a p e r was supp o r t e d by g r a n t s f r o m the British H e a r t F o u n d a t i o n a n d the M e d i c a l R e s e a r c h C o u n c i l . W e t h a n k D r J . R. Blinks for s u p p l y i n g us w i t h a e q u o r i n w h i c h was purified in his laboratory with support from NIH Grant HL 12186.

References 1 ALLEN,D. G., BL1NKS,J. R. The interpretation of light signals from aequorin injected skeletal and cardiac muscle cells--a new method of calibration. In: Detection and /Vleasurement of Free Calcium Ions in Cells. Ashley, C. C., Campbell, A. K. Eds, pp 159 174. Amsttwdam: Elsevier/North Holland (1979). 2

ALLEN, D. G., BLINKS,J. R., PRENDERGAST,F. G. Aequorin luminescence: relation of light emission to calcium

3

ALLEN, D. G., EISNER, D. A., LAB, M. J., ORCHARD, C. H. The effects of low Na solutions on intracellular Ca

4

ALLEN, D. G., EISNER, D. A., ORCHARD, C. H. Oscillations ofintracellular [Ca2+]i in resting ferret cardiac muscle.

5

ALLEN, D. G., ORCHARD, C. H. The effects of changes of pH on intracellular calcium transients in mammalian

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