229
J. Physiol. (1975), 246, pp. 229-253 With 11 text-ftigure8 Printed in Great Britain
THE EFFECT OF SUBSTANCES RELEASING INTRACELLULAR CALCIUM IONS ON SODIUM-DEPENDENT CALCIUM EFFLUX FROM GUINEA-PIG AURICLES
BY H. JUNDT, H. PORZIG, H. REUTER* AND J. W. STUCKI From the Department of Pharmacology, University of Bern, Friedbihlstrasse 49, 3010 Bern, Switzerland (Received 25 July 1974) SUMMARY
1. 45Ca efflux and resting tension were measured in isolated guinea-pig auricles under conditions known to change the intracellular free Ca ion concentration. 2. In the presence of [Na]o, caffeine (2 mM) increases 45Ca efflux, but does not produce a contracture, while in the absence of [Na]0 and [Ca]o caffeine causes a contracture without increasing 45Ca efflux. Adrenaline (10-5 - 10-4 M) with or without theophylline (0.5-1 0 mM) has no effect on either 45Ca efflux or resting tension. 3. In the presence of caffeine the rate of net efflux of Ca depends on [Na]2. Caffeine contractures of muscles in Na-free solution relax upon the addition of [Na]o. Relaxation is correlated with the increase in net efflux of Ca. 4. Cyanide (2 mM) produces a variable increase in 45Ca efflux without a concomitant contracture in Na-containing solutions, but in Na,Ca-free solutions a large contracture occurs without significant increase in 45Ca efflux. 5. A large increase in 45Ca efflux and a contracture were observed with the 'Ca-ionophore' X 537A. 6. Changes in membrane potential (K-depolarization) in hypertonic solutions have no significant effect on Na-dependent 45Ca efflux, which is in agreement with an electroneutral 2: 1 Na-Ca exchange. 7. Cyanide and X 537A both cause a considerable release of Ca ions from isolated guinea-pig heart mitochondria, while caffeine has no effect. * Send reprint requests to Prof. H. Reuter. Supported by grants from the Swiss National Science Foundation (grant numbers 3-734-72 and 3-0510-73).
230 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI 8. The results suggest a powerful role of the Na-Ca exchange system in reducing the intracellular Ca concentration after Ca release from intracellular stores. INTRODUCTION
In most biological tissues outward transport of Ca ions is an energyrequiring process since it occurs against a large electrochemical gradient. However, the energy required for Ca extrusion is derived from different sources in different tissues. Schatzmann (1966) was the first to show coupling between Ca transport and hydrolysis of ATP in red cells, whereas the Na gradient across the cell membrane seems to be a major energy source for Ca extrusion in cardiac muscle (Reuter & Seitz, 1968; Glitsch, Reuter & Scholz, 1970), squid axons (Baker, Blaustein, Hodgkin & Steinhardt, 1969; Blaustein & Hodgkin, 1969; Di Polo, 1973) and other excitable tissues (for review see Baker, 1972). However, in many excitable tissues the extremely low intracellular Ca ion concentration is assumed to be regulated by intracellular stores (mitochondria, sarcoplasmic reticulum) rather than by Ca transport across the surface membrane. For example, release of Ca ions from mitochondria into the axoplasm by cyanide leads to a large increase in Na- and Ca-dependent Ca efflux from squid axons (Blaustein & Hodgkin, 1969; Di Polo, 1973). In order to establish the mechanism and functional role of Na-dependent Ca efflux in cardiac muscle further, it was of interest to investigate Ca efflux under various conditions known to cause a release of Ca ions from intracellular stores. Therefore, in the present study metabolic inhibitors and pharmacological agents were used to change the intracellular free Ca ion concentration in guinea-pig auricles. Ca release from intracellular stores was established by measuring 45Ca efflux and tension development and by demonstrating Ca release from isolated mitochondria. The data suggest that Na-dependent Ca efflux (Na-Ca exchange) can play a powerful role in the maintenance of low intracellular Ca ion concentrations. 45Ca efflux
METHODS
The method for measuring simultaneously 45Ca efflux and tension in resting guinea-pig auricles has been described in detail by Reuter & Seitz (1968). Briefly, left auricles were dissected from guinea-pigs weighing 200-400 g. The auricles were loaded with 45Ca for 60 min in radioactive Tyrode solution. During the loading period the preparations were stimulated at a frequency of 2/sec. After loading with 45Ca the muscles were attached to a force displacement transducer and transferred through a series of glass tubes containing inactive solutions of various compositions (Table 1). In all experiments the first hour of 45Ca efflux was in Ca-free Tyrode solution and the second hour in normal Tyrode solution. These first 2 hr of 45Ca
INTERNAL Ca2+ AND Ca EFFLUX
231
efflux are not graphed in the Figures. The radioactivity lost during each collection period from the muscle into the inactive solutions was counted in a liquid scintillation counter. The radioactivity in the muscle was determined at the end of the experiment. 4iCa efflux is expressed as rate coefficients (cf. Reuter & Seitz. 1968), i.e. as fraction of 45Ca lost per minute from the muscle into the inactive efflux media. This rate coefficient, k (min-), was calculated for each sample by k (min-1)
=
c.p.m. min-' in sample mean c.p.m. in muscle
The solutions used as efflux media are listed in Table 1. The use of Tris buffer instead of bicarbonate-phosphate buffer had no effect on 45Ca efflux or tension development. Drugs used were caffeine, theophylline, tetracaine hydrochloride, (-) adrenaline bitartrate, NaCN or KCN, X 537A (structure, see Levy, Cohen & Inesi, 1973). The antibiotic X 537A was kindly supplied by Hoffmann-La Roche AG, Basle. Whenever possible k values for 45Ca efflux are given as means + S.E. For tension traces in Figs. 1, 2, 6-9 a single representative experiment of each set was redrawn and adjusted to the time scale of presentation of the 45Ca efflux curves.
Mitochondria Guinea-pig heart mitochondria were isolated according to the method by Carafoli & Lehninger (1971) with the exception that the mitochondria were washed twice with 0-25 M mannitol - 0 07 M sucrose solution (Graven, Lardy & Rutter, 1966) after the first sedimentation. Washed mitochondria obtained from 1 g original heart weight were suspended in 1 ml. of this medium. The mitochondrial suspensions were incubated in Erlenmeyer flasks in a shaking water bath at 370 C in a medium containing 6-6 mM K phosphate buffer (pH 7.4), 6-6 mm triethanolamine buffer (pH 7.4), 5 mi-MgSO4, 10 mm pyruvate and 10 mM malate. The medium was adjusted to the final volume with 0-25 M irnannitol 0 07 M sucrose solution. The millipore filter technique was used for the separation of the mitochondria from the incubation mixture (Stucki, Brawand & Walter, 1972). Total Ca of the mitochondrial pellet on the filters was extracted and assayed by atomic absorption spectrometry as described by Carafoli & Lehninger (1971). The calculation of the calcium content of the samples was based on the recovery of internal standards added during the extraction of the mitochondria. The blanks resulting from the calcium contamination of the millipore filters and of the hydrochloric acid used for the extraction were subtracted. Changes in the concentrations of Ca ions, H ions and oxygen in the incubation medium were followed simultaneously with suitable electrodes in a thermostated incubation chamber at 37° C (Stucki & Ineichen, 1974). The solution in the chamber was agitated by magnetic stirring. A Ca ion sensitive didecylphosphoric acid/ dioctylphenylphosphonate liquid ion exchange electrode (Philips IS 560 Ca) was used for the measurement of Ca ions. The signal from this electrode was amplified with an ion-activity meter (Philips PW 9413). pH changes were measured with a glass electrode and a pH meter (Pye model 251). For these two electrodes one common saturated calomel electrode with a 0-1 MNaNO3 salt bridge was used as a reference. Oxygen was measured with a Clark-type electrode (Yellow Springs Instruments, Ohio, U.S.A.). The traces of the signals were recorded with a Watanabe multichannel MC 611 strip chart recorder.
~
232 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI
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.INTERNAL Ca2+ AND Ca EFFLUX23 233 RESULTS The effect of caffeine The effect of caffeine (2 mm) on 45Ca efflux from resting guinea-pig auricles in normal Tyrode solution (solution 1 in Table 1) is shown in Fig. 1. Caffeine was added to the efflux medium when the rate of 45Ca efflux was almost constant, i.e. after preceding efflux periods of 1 hr in Ca-free Tyrode solution and 1-5 hr in normal Tyrode solution. The initial 2 hr of 45Ca efflux are not documented in Fig. 1. Caffeine produced a marked rise in 45Ca efflux which reached its peak within 10 min. This effect was reversible when the drug was washed out. The caffeine effect on 45Ca efflux is in agreement with results obtained in skeletal muscle (Bianchi, 1961;
0.5
30
0 137
0
1
60min 137
2
I
I
137
0
nm-CaCI2
r
nm-NaCI fnm caffeine
I
Fig. 1. Effect of caffeine on 450a efflux from six guinea-pig auricles (means ± 5sE.). The upper part of the Figure indicates that resting tension, T (redrawn from, a single experiment; preload 0.5 g), was not changed by caffeine; the lower part shows rate coefficients (k, min-"; ordinate) of 450a efflux plotted against time (abscissa). 2 mm caffeine was added during efflux period b to normal Tyrode solution.
234 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI
Feinstein, 1963; Caidwell, 1970) and toad heart (Nayler, 1963). However, in contrast to skeletal muscle no change in resting tension was observed with caffeine in Na-containing solution even with caffeine concentrations as high as 10 mm. Replacement of NaCl in Tris-buffered Tyrode solution by LiCl and simultaneous reduction of CaCl2 from 0-45 to 0 mm (solution 3) caused a rapid decline in the rate of 45Ca efflux and the muscle developed a small contracture (Fig. 2). The average rate coefficient of 45Ca efflux fell from 0-014 min-' during the last two efflux periods in Na,Ca-containing solution to 0-005 min-' after 25 min in Na,Ca-free solution. This result agrees with those obtained by Reuter & Seitz (1968). The addition of 2 mm caffeine to the Na,Ca-free solution caused a rapid contracture without a concomitant rise in 45Ca efflux. However, replacement of LiCl by NaCl
0.5I
c
d
0 0 137 2
0 137
b
a
0-03 T
0.0
0.02
60
30
0 0.45 137 0 0
0 0
137 0
90 0 2
120 min
mm-CaCI2 mm-NaCI mm-LiCI mm caffeine
Fig. 2. Effect of caffeine (2 m ) on resting tension, T (upper part: preload 0.5 g), and 45Ca efflux (lower part). Rate coefficients (k, min-"; ordinate) of 465Ca efflux represent means + S.E. of six guinea-pig auricles, while T is redrawn from a single representative experiment. Changes in composition of solutions during the efflux periods a-d are indicated below the graph.
235 INTERNAL Ca2+ AND Ca EFFLUX promptly increased 45Ca efflux about twofold and the muscles relaxed partially. The increase in 45Ca efflux after readmission of NaCl is a net efflux of Ca since no CaCl2 was in the solution (in some experiments 0-1-0-5 mM-EGTA was present), and therefore Ca-Ca exchange (Reuter & Seitz, 1968) was negligible. When CaCl2 was added to the Na-containing solution there was a further slight increase in 45Ca efflux and the muscle became rapidly excitable again. When CaCl2 (0.1-0.5 mM) was added to the Na,Ca-free, caffeine-containing medium 45Ca efflux increased (Ca-Ca exchange), but resting tension was markedly enhanced. In two experiments Tyrode solution (solution 2) was replaced by a choline Cl-containing, Ca-free bathing medium (solution 4) at a time when caffeine (5 mM) had produced a maximal increase in 45Ca efflux. The Na,Ca-free solution caused a rapid drop of 45Ca efflux to about 15 % of the preceding period and a large increase in tension occurred (for a similar experiment with cyanide see Fig. 6). When Tyrode solution (solution 2) was readmitted in the presence of caffeine, 45Ca efflux increased greatly and the muscle relaxed partially. These results suggest that the increase in 45Ca efflux by caffeine is dependent on [Na]o and [Ca]o. In the absence of [Na]o net efflux of Ca is inhibited (Reuter & Seitz, 1968) and the release of intracellular Ca ions by caffeine (Weber & Herz, 1968; Kerrick & Best, 1974) is sufficient to cause a contracture. A quantitative relation between net efflux of Ca from auricles in the presence of 2 mm caffeine and [Na]o could be established by gradual replacement of LiCI by NaCl in the absence of [Ca].. The experimental procedure was the same as shown in Fig. 2, except that 17-1, 34-25 or 68-5 (solutions 5-7) instead of 137 mm-LiCl were replaced by NaCl. The changes in Ca efflux resulting from the increase in [Na]o are plotted in Fig. 3. The curve relating maximal changes in rate coefficients (Ak) of 45Ca efflux and [Na]0 is non-hyperbolic and hence does not follow simple saturation kinetics. However, the data could be fitted by a straight line in a reciprocal plot (Dixon & Webb, 1958) if [Na]2 instead of [Na]0 was used in the graph shown in Fig. 4b. The linearity of the plot in Fig. 4b implies that the results follow a function of the form of the Hill equation
_a[Na]l
Ak =b +[Na]2
where
a
and b
are
constants.
Although
a
molecular
interpretation is not possible, on the basis of such a function it indicates that two extracellular Na ions may exchange for one intracellular Ca ion. Similarly, Glitsch et al. (1970) found in the same preparation that two intracellular Na ions appear to exchange for one extracellular Ca ion and Reuter & Seitz (1968) showed competition of two Na ions and one Ca ion for transport sites (carriers) at the external surface of the membrane. Therefore, these data are consistent with the concept of an electroneutral
236 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI Na-Ca exchange system. The apparent Km for [Na]o in this countertransport is 34 mM. The functional significance of the Na-dependent Ca efflux (Na-Ca exchange) was substantiated by the finding that caffeine-treated auricles produced contractures only in Na-free solution and that the addition of [Na]o caused relaxation (Fig. 2). In Fig. 5 the extent of relaxation (AT) is plotted against the change in the rate of Na-dependent Ca efflux (Ak, data from Fig. 3). The maximal change in the rate of net efflux of Ca and maximal relaxation in 137 mM-[Na]. were taken as 100 %. The plot in 0 01
I
1~~~~~~~~(6)
T ini6
T
E 0-005
/i~~~~~~(7)
J. Physiol. (1975), 246, pp. 229-253 With 11 text-ftigure8 Printed in Great Britain
THE EFFECT OF SUBSTANCES RELEASING INTRACELLULAR CALCIUM IONS ON SODIUM-DEPENDENT CALCIUM EFFLUX FROM GUINEA-PIG AURICLES
BY H. JUNDT, H. PORZIG, H. REUTER* AND J. W. STUCKI From the Department of Pharmacology, University of Bern, Friedbihlstrasse 49, 3010 Bern, Switzerland (Received 25 July 1974) SUMMARY
1. 45Ca efflux and resting tension were measured in isolated guinea-pig auricles under conditions known to change the intracellular free Ca ion concentration. 2. In the presence of [Na]o, caffeine (2 mM) increases 45Ca efflux, but does not produce a contracture, while in the absence of [Na]0 and [Ca]o caffeine causes a contracture without increasing 45Ca efflux. Adrenaline (10-5 - 10-4 M) with or without theophylline (0.5-1 0 mM) has no effect on either 45Ca efflux or resting tension. 3. In the presence of caffeine the rate of net efflux of Ca depends on [Na]2. Caffeine contractures of muscles in Na-free solution relax upon the addition of [Na]o. Relaxation is correlated with the increase in net efflux of Ca. 4. Cyanide (2 mM) produces a variable increase in 45Ca efflux without a concomitant contracture in Na-containing solutions, but in Na,Ca-free solutions a large contracture occurs without significant increase in 45Ca efflux. 5. A large increase in 45Ca efflux and a contracture were observed with the 'Ca-ionophore' X 537A. 6. Changes in membrane potential (K-depolarization) in hypertonic solutions have no significant effect on Na-dependent 45Ca efflux, which is in agreement with an electroneutral 2: 1 Na-Ca exchange. 7. Cyanide and X 537A both cause a considerable release of Ca ions from isolated guinea-pig heart mitochondria, while caffeine has no effect. * Send reprint requests to Prof. H. Reuter. Supported by grants from the Swiss National Science Foundation (grant numbers 3-734-72 and 3-0510-73).
230 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI 8. The results suggest a powerful role of the Na-Ca exchange system in reducing the intracellular Ca concentration after Ca release from intracellular stores. INTRODUCTION
In most biological tissues outward transport of Ca ions is an energyrequiring process since it occurs against a large electrochemical gradient. However, the energy required for Ca extrusion is derived from different sources in different tissues. Schatzmann (1966) was the first to show coupling between Ca transport and hydrolysis of ATP in red cells, whereas the Na gradient across the cell membrane seems to be a major energy source for Ca extrusion in cardiac muscle (Reuter & Seitz, 1968; Glitsch, Reuter & Scholz, 1970), squid axons (Baker, Blaustein, Hodgkin & Steinhardt, 1969; Blaustein & Hodgkin, 1969; Di Polo, 1973) and other excitable tissues (for review see Baker, 1972). However, in many excitable tissues the extremely low intracellular Ca ion concentration is assumed to be regulated by intracellular stores (mitochondria, sarcoplasmic reticulum) rather than by Ca transport across the surface membrane. For example, release of Ca ions from mitochondria into the axoplasm by cyanide leads to a large increase in Na- and Ca-dependent Ca efflux from squid axons (Blaustein & Hodgkin, 1969; Di Polo, 1973). In order to establish the mechanism and functional role of Na-dependent Ca efflux in cardiac muscle further, it was of interest to investigate Ca efflux under various conditions known to cause a release of Ca ions from intracellular stores. Therefore, in the present study metabolic inhibitors and pharmacological agents were used to change the intracellular free Ca ion concentration in guinea-pig auricles. Ca release from intracellular stores was established by measuring 45Ca efflux and tension development and by demonstrating Ca release from isolated mitochondria. The data suggest that Na-dependent Ca efflux (Na-Ca exchange) can play a powerful role in the maintenance of low intracellular Ca ion concentrations. 45Ca efflux
METHODS
The method for measuring simultaneously 45Ca efflux and tension in resting guinea-pig auricles has been described in detail by Reuter & Seitz (1968). Briefly, left auricles were dissected from guinea-pigs weighing 200-400 g. The auricles were loaded with 45Ca for 60 min in radioactive Tyrode solution. During the loading period the preparations were stimulated at a frequency of 2/sec. After loading with 45Ca the muscles were attached to a force displacement transducer and transferred through a series of glass tubes containing inactive solutions of various compositions (Table 1). In all experiments the first hour of 45Ca efflux was in Ca-free Tyrode solution and the second hour in normal Tyrode solution. These first 2 hr of 45Ca
INTERNAL Ca2+ AND Ca EFFLUX
231
efflux are not graphed in the Figures. The radioactivity lost during each collection period from the muscle into the inactive solutions was counted in a liquid scintillation counter. The radioactivity in the muscle was determined at the end of the experiment. 4iCa efflux is expressed as rate coefficients (cf. Reuter & Seitz. 1968), i.e. as fraction of 45Ca lost per minute from the muscle into the inactive efflux media. This rate coefficient, k (min-), was calculated for each sample by k (min-1)
=
c.p.m. min-' in sample mean c.p.m. in muscle
The solutions used as efflux media are listed in Table 1. The use of Tris buffer instead of bicarbonate-phosphate buffer had no effect on 45Ca efflux or tension development. Drugs used were caffeine, theophylline, tetracaine hydrochloride, (-) adrenaline bitartrate, NaCN or KCN, X 537A (structure, see Levy, Cohen & Inesi, 1973). The antibiotic X 537A was kindly supplied by Hoffmann-La Roche AG, Basle. Whenever possible k values for 45Ca efflux are given as means + S.E. For tension traces in Figs. 1, 2, 6-9 a single representative experiment of each set was redrawn and adjusted to the time scale of presentation of the 45Ca efflux curves.
Mitochondria Guinea-pig heart mitochondria were isolated according to the method by Carafoli & Lehninger (1971) with the exception that the mitochondria were washed twice with 0-25 M mannitol - 0 07 M sucrose solution (Graven, Lardy & Rutter, 1966) after the first sedimentation. Washed mitochondria obtained from 1 g original heart weight were suspended in 1 ml. of this medium. The mitochondrial suspensions were incubated in Erlenmeyer flasks in a shaking water bath at 370 C in a medium containing 6-6 mM K phosphate buffer (pH 7.4), 6-6 mm triethanolamine buffer (pH 7.4), 5 mi-MgSO4, 10 mm pyruvate and 10 mM malate. The medium was adjusted to the final volume with 0-25 M irnannitol 0 07 M sucrose solution. The millipore filter technique was used for the separation of the mitochondria from the incubation mixture (Stucki, Brawand & Walter, 1972). Total Ca of the mitochondrial pellet on the filters was extracted and assayed by atomic absorption spectrometry as described by Carafoli & Lehninger (1971). The calculation of the calcium content of the samples was based on the recovery of internal standards added during the extraction of the mitochondria. The blanks resulting from the calcium contamination of the millipore filters and of the hydrochloric acid used for the extraction were subtracted. Changes in the concentrations of Ca ions, H ions and oxygen in the incubation medium were followed simultaneously with suitable electrodes in a thermostated incubation chamber at 37° C (Stucki & Ineichen, 1974). The solution in the chamber was agitated by magnetic stirring. A Ca ion sensitive didecylphosphoric acid/ dioctylphenylphosphonate liquid ion exchange electrode (Philips IS 560 Ca) was used for the measurement of Ca ions. The signal from this electrode was amplified with an ion-activity meter (Philips PW 9413). pH changes were measured with a glass electrode and a pH meter (Pye model 251). For these two electrodes one common saturated calomel electrode with a 0-1 MNaNO3 salt bridge was used as a reference. Oxygen was measured with a Clark-type electrode (Yellow Springs Instruments, Ohio, U.S.A.). The traces of the signals were recorded with a Watanabe multichannel MC 611 strip chart recorder.
~
232 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI
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6
C)
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.INTERNAL Ca2+ AND Ca EFFLUX23 233 RESULTS The effect of caffeine The effect of caffeine (2 mm) on 45Ca efflux from resting guinea-pig auricles in normal Tyrode solution (solution 1 in Table 1) is shown in Fig. 1. Caffeine was added to the efflux medium when the rate of 45Ca efflux was almost constant, i.e. after preceding efflux periods of 1 hr in Ca-free Tyrode solution and 1-5 hr in normal Tyrode solution. The initial 2 hr of 45Ca efflux are not documented in Fig. 1. Caffeine produced a marked rise in 45Ca efflux which reached its peak within 10 min. This effect was reversible when the drug was washed out. The caffeine effect on 45Ca efflux is in agreement with results obtained in skeletal muscle (Bianchi, 1961;
0.5
30
0 137
0
1
60min 137
2
I
I
137
0
nm-CaCI2
r
nm-NaCI fnm caffeine
I
Fig. 1. Effect of caffeine on 450a efflux from six guinea-pig auricles (means ± 5sE.). The upper part of the Figure indicates that resting tension, T (redrawn from, a single experiment; preload 0.5 g), was not changed by caffeine; the lower part shows rate coefficients (k, min-"; ordinate) of 450a efflux plotted against time (abscissa). 2 mm caffeine was added during efflux period b to normal Tyrode solution.
234 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI
Feinstein, 1963; Caidwell, 1970) and toad heart (Nayler, 1963). However, in contrast to skeletal muscle no change in resting tension was observed with caffeine in Na-containing solution even with caffeine concentrations as high as 10 mm. Replacement of NaCl in Tris-buffered Tyrode solution by LiCl and simultaneous reduction of CaCl2 from 0-45 to 0 mm (solution 3) caused a rapid decline in the rate of 45Ca efflux and the muscle developed a small contracture (Fig. 2). The average rate coefficient of 45Ca efflux fell from 0-014 min-' during the last two efflux periods in Na,Ca-containing solution to 0-005 min-' after 25 min in Na,Ca-free solution. This result agrees with those obtained by Reuter & Seitz (1968). The addition of 2 mm caffeine to the Na,Ca-free solution caused a rapid contracture without a concomitant rise in 45Ca efflux. However, replacement of LiCl by NaCl
0.5I
c
d
0 0 137 2
0 137
b
a
0-03 T
0.0
0.02
60
30
0 0.45 137 0 0
0 0
137 0
90 0 2
120 min
mm-CaCI2 mm-NaCI mm-LiCI mm caffeine
Fig. 2. Effect of caffeine (2 m ) on resting tension, T (upper part: preload 0.5 g), and 45Ca efflux (lower part). Rate coefficients (k, min-"; ordinate) of 465Ca efflux represent means + S.E. of six guinea-pig auricles, while T is redrawn from a single representative experiment. Changes in composition of solutions during the efflux periods a-d are indicated below the graph.
235 INTERNAL Ca2+ AND Ca EFFLUX promptly increased 45Ca efflux about twofold and the muscles relaxed partially. The increase in 45Ca efflux after readmission of NaCl is a net efflux of Ca since no CaCl2 was in the solution (in some experiments 0-1-0-5 mM-EGTA was present), and therefore Ca-Ca exchange (Reuter & Seitz, 1968) was negligible. When CaCl2 was added to the Na-containing solution there was a further slight increase in 45Ca efflux and the muscle became rapidly excitable again. When CaCl2 (0.1-0.5 mM) was added to the Na,Ca-free, caffeine-containing medium 45Ca efflux increased (Ca-Ca exchange), but resting tension was markedly enhanced. In two experiments Tyrode solution (solution 2) was replaced by a choline Cl-containing, Ca-free bathing medium (solution 4) at a time when caffeine (5 mM) had produced a maximal increase in 45Ca efflux. The Na,Ca-free solution caused a rapid drop of 45Ca efflux to about 15 % of the preceding period and a large increase in tension occurred (for a similar experiment with cyanide see Fig. 6). When Tyrode solution (solution 2) was readmitted in the presence of caffeine, 45Ca efflux increased greatly and the muscle relaxed partially. These results suggest that the increase in 45Ca efflux by caffeine is dependent on [Na]o and [Ca]o. In the absence of [Na]o net efflux of Ca is inhibited (Reuter & Seitz, 1968) and the release of intracellular Ca ions by caffeine (Weber & Herz, 1968; Kerrick & Best, 1974) is sufficient to cause a contracture. A quantitative relation between net efflux of Ca from auricles in the presence of 2 mm caffeine and [Na]o could be established by gradual replacement of LiCI by NaCl in the absence of [Ca].. The experimental procedure was the same as shown in Fig. 2, except that 17-1, 34-25 or 68-5 (solutions 5-7) instead of 137 mm-LiCl were replaced by NaCl. The changes in Ca efflux resulting from the increase in [Na]o are plotted in Fig. 3. The curve relating maximal changes in rate coefficients (Ak) of 45Ca efflux and [Na]0 is non-hyperbolic and hence does not follow simple saturation kinetics. However, the data could be fitted by a straight line in a reciprocal plot (Dixon & Webb, 1958) if [Na]2 instead of [Na]0 was used in the graph shown in Fig. 4b. The linearity of the plot in Fig. 4b implies that the results follow a function of the form of the Hill equation
_a[Na]l
Ak =b +[Na]2
where
a
and b
are
constants.
Although
a
molecular
interpretation is not possible, on the basis of such a function it indicates that two extracellular Na ions may exchange for one intracellular Ca ion. Similarly, Glitsch et al. (1970) found in the same preparation that two intracellular Na ions appear to exchange for one extracellular Ca ion and Reuter & Seitz (1968) showed competition of two Na ions and one Ca ion for transport sites (carriers) at the external surface of the membrane. Therefore, these data are consistent with the concept of an electroneutral
236 H. JUNDT, H. PORZIG, H. REUTER AND J. W. STUCKI Na-Ca exchange system. The apparent Km for [Na]o in this countertransport is 34 mM. The functional significance of the Na-dependent Ca efflux (Na-Ca exchange) was substantiated by the finding that caffeine-treated auricles produced contractures only in Na-free solution and that the addition of [Na]o caused relaxation (Fig. 2). In Fig. 5 the extent of relaxation (AT) is plotted against the change in the rate of Na-dependent Ca efflux (Ak, data from Fig. 3). The maximal change in the rate of net efflux of Ca and maximal relaxation in 137 mM-[Na]. were taken as 100 %. The plot in 0 01
I
1~~~~~~~~(6)
T ini6
T
E 0-005
/i~~~~~~(7)
