209
J. Physiol. (1975), 247, pp. 209-226 With 9 text-figure8 Printed in Great Britain
A NOTE ON THE MECHANISM BY WHICH INHIBITORS OF THE SODIUM PUMP ACCELERATE SPONTANEOUS RELEASE OF TRANSMITTER FROM MOTOR NERVE TERMINALS
By P. F. BAKER AND A. C. CRAWFORD From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG
(Received 23 September 1974) SUMMARY
1. The actions of 0-1 mm ouabain and of K-free Ringer have been examined at the frog neuromuscular junction. 2. After a delay of more than 30 min, ouabain produces an increase in the miniature end-plate potential (m.e.p.p.) frequency. This increase occurs unchanged in Ca-free Ringer containing 1 mM-EGTA and is therefore unlikely to be due to an entry of Ca into the motor nerve terminals. 3. If the nerve to the preparation is stimulated repetitively in Ca-free Ringer containing 0-1 mM ouabain and 1 mM-EGTA the response of the m.e.p.p. frequency depends on the timing of the tetanus relative to the beginning of the ouabain treatment. 4. During the first 30 min of exposure to ouabain, the tetanus produces a small, transient increase in the m.e.p.p. frequency similar to that which occurs before ouabain is present. After about 30 min the same tetanus produces large, irreversible increases in the m.e.p.p. frequency. 5. Superfusion of an end-plate with K-free Ringer causes an immediate exponential rise in the m.e.p.p. frequency that is unaffected by the presence of external Ca ions. On replacing the normal K of the Ringer (2 mM) the m.e.p.p. frequency recovers quickly to its original value. 6. Late in an exposure to 0.1 mm ouabain the m.e.p.p. frequency becomes extremely sensitive to changes in the external Na concentration, [Na]0. Reducing [Na]0 increases the m.e.p.p. frequency. The sensitivity to [Na]0 is independent of external Ca ions or whether the isotonic substitute for NaCl is LiCl or sucrose. 7. It is suggested that the spontaneous release of transmitter is facilitated, in some way, by the changes in the monovalent cation content of the nerve terminals that result from blocking the Na-K exchange pump.
210
P. F. BAKER AND A. C. CRAWFORD
The Na sensitivity of the m.e.p.p. frequency that develops simultaneously can be explained if a Na-dependent Ca efflux system is present in the membrane of the presynaptic terminals. INTRODUCTION
When motoneurone terminals are exposed for long periods to cardiac glycosides both the mean quantal content of the end-plate potential (m) and the miniature end-plate potential frequency (f) slowly but progressively increase (Elmqvist & Feldman, 1965; Birks & Cohen, 1968a). There seems little doubt that the cardiac glycosides act by inhibiting the Na-K exchange pump, for the increase in evoked transmitter release also occurs when external K is omitted from the Ringer (Birks & Cohen, 1968 a). Moreover, the increases in both m andf seem to be secondary to redistribution of the sodium and potassium ion gradients of the motor nerve terminals, since replacement of part of the external Na of the Ringer by sucrose dramatically slows the growth of the end-plate potential and of the miniature end-plate potential frequency. Birks & Cohen proposed that a rise in the intracellular sodium concentration led to an increase in the resting Ca entry into the nerve terminals, as is known to occur in squid giant axons (Baker, Blaustein, Hodgkin & Steinhardt, 1969), and that the rise in intracellular Ca produced the increases in transmitter release. The starting point of this paper was the observation that the increase in f produced by ouabain occurred unimpeded in Ringer that lacked added Ca and included a Ca chelating agent to reduce the external Ca concentration to levels as low as 10-9 M. Any explanation of the glycoside effects in terms of a Ca influx (either due to depolarization or to Na-dependent transport) thus seems unlikely. The results are consistent with the release of Ca from internal stores in the motor nerve terminals. Evidence is also presented that Ca is extruded from the terminals by a Na-dependent transport mechanism similar to that described by Baker, Blaustein, Hodgkin & Steinhardt (1967) and by Blaustein & Hodgkin (1969) in squid giant axons. METHODS
Standard intracellular recording techniques were used to follow the miniature endplate potential (m.e.p.p.) frequency at superficial end-plates of frog (Rana temporaria) sartorius-sciatic preparations. Details of these techniques have been described elsewhere (Crawford, 1974).
Solutions Table 1 gives the composition of the major solutions used in these experiments. LiCl was obtained from Fisher Chemicals and neostigmine methylsulphate (Sigma)
Na PUMP AND TRANSMITTER RELEASE
211
10-6 gm/ml. was included in all solutions to increase the m.e.p.p. amplitude. Ouabain (B.D.H.), was made up as a stock solution at 10 mM in distilled water and stored at 40 C. The glycoside was added to the Ringer immediately before each experiment to give a final concentration of 0-1 mm. In a few experiments the m.e.p.p. frequency was followed in Ringer that contained no added K ('K-free Ringer', columns E and F of Table 1). Birks & Cohen (1968a) had some difficulty with sartorius preparations in reproducing the effects of glycosides with K-free Ringer, a fact that they attributed to a leakage of K out of the muscle and subsequent activation of the Na pump. Keynes (1954) found similar difficulties in his attempts to block the Na pump in sartorius muscle by the use of K-free solutions. These drawbacks were circumvented by the use of a 'superfusion' device similar to that introduced by Cooke & Quastel (1973). A 150 prm glass pipette was used to direct a jet of K-free Ringer at the end-plate once it had been located and the solution was sucked off continuously at a point distant from the site of recording. This method allowed Cooke & Quastel to raise the K concentration at an end-plate in less than 4 sec and in the present experiments proved an ideal method of washing the endplate region free of K ions. The behaviour of the muscle membrane potential when superfused with K-free Ringer was unpredictable. Either the muscle hyperpolarized slightly (in which case the experiment was continued) or it depolarized rapidly to as little as -50 mV. In the latter case the m.e.p.p. became too small to be distinguished reliably and another end-plate was chosen. The depolarization of muscles in K-free solutions has been noted previously (Hodgkin & Horowicz, 1959; Adrian & Slayman, 1966) and probably results from a reduction in the K conductance of the muscle fibre (Hodgkin & Horowicz, 1959; Adrian, 1964; Adrian & Slayman, 1966). The depolarization was particularly prevalent if the Ringer also contained EGTA and no added Ca. TABLE 1. Composition of the major solutions used in these experiments. All ion concentrations are millimolar. In addition the solutions contained neostigmine methylsulphate 10-6 g/l. Solution Ion Na+ Li+ K+ Ca2+ Mg2+ Tris EGTA
ClSucrose
A 114-5 2-0 0-4 3-2 2-5 126-2
B 114-5 2-0 3-6 2-5
1-0 122-2
C
D
114-5 2-0
57 2-0
3-6 2-5 1-0 122-2 -
3-6 2-5 1-0 68-7 115-0
E 116-5 0-4 3-2 2-5
F 116-5
3-6 2-5
1-0 126-2
122-2
RESULTS
External Ca ions and the rise in transmitter release caused by ouabain The time course of the increase in the miniature end-plate potential frequency during application of the cardiac glycoside, ouabain (0-1 mM), is illustrated in Fig. 1. Two features are striking: there is an initial delay of 60-80 min during which the spontaneous release rate remains
212 P. F. BAKER AND A. C. CRAWFORD stable at its original level, and then a period of 15 min when the m.e.p.p. frequency rises to levels that cannot be measured reliably using intracellular recording ( > 200 sec-1). Similar results have been reported by Birks & Cohen (1968a) using digoxin, strophanthidin and ouabain. Ouabain
>
200 r
0
0
0
A
U
1001-
0
'o.-
000
0000 0 0
0
0
00
30
0
0
0
60
ocP0 90
120
Ouabain
so
0
200 r B U
0
100
F
0 0 0
0 -0 o
0
0
30
o
oo
c)0V
60 Time (min)
90
120
Fig. 1. Ouabain accelerates the spontaneous release of transmitter after an initial delay, and the increase in the miniature end-plate potential frequency does not require external Ca ions. In both A and B ouabain was applied at a concentration of 0-1 mm as indicated by the arrows. The Ringer contained 0-4 mM-Ca2+, 3-2 mM-Mg2+, in A, and in B 3-6 mM-Mg2+, 1 mM-EGTA and no added Ca. Ordinate: m.e.p.p. frequency (f) sec-1, Abscissa: time (min).
The external Ca concentration [Ca]0, of the Ringer has little effect on the action of ouabain. In Fig. 1 two experiments are shown in which the Ringer contained either 0-4 mM-Ca2+, or no added Ca and 1 mM-EGTA. In the latter case the Ca-chelating agent should reduce the [Ca]0 to less than 10-9 M (Hubbard, Jones & Landau, 1968; Miledi & Thies, 1971). In both experiments the m.e.p.p. frequency accelerates in much the same way
213 Na PUMP AND TRANSMITTER RELEASE although the initial delay before the rise in spontaneous release is slightly shortened when external calcium ions are omitted. These observations seem to rule out any explanation of the glycoside effect in terms of an increased Ca influx. Two specific hypotheses are included in this category. Progressive depolarization of the motor nerve terminals, resulting from inhibition of the Na pump, is unlikely to be the cause of the rise in the m.e.p.p. frequency since increases in the spontaneous release rate induced by either K or electrical depolarization are abolished in EGTA Ringer (Landau, 1969; Cooke & Quastel, 1973; Cooke, Okamoto & Quastel, 1973). Similarly, although inhibition of the Na pump may lead to accumulation of Na inside the terminals this is also unlikely to increase the m.e.p.p. frequency by increasing a sodium-dependent Ca influx into the terminals (Baker et at. 1969; Baker, 1972) because the effects persist in EGTA Ringer. An explanation of the action of ouabain must be sought elsewhere than through mechanisms likely to cause Ca entry into the motor nerve terminals. 2K
150
'K-free'
2K
100 U
0
50
0 I
I
I
0
5
10 Time (min)
15
20
Fig. 2. The m.e.p.p. frequency rises when an end-plate is superfused with K-free Ringer and recovers when the K in the superfusing medium is replaced. The concentration of CaCl2 was 04 mm and the MgCl2 concentration 3-2 mm throughout. The Ringer contained 2 mM-KCl during the periods labelled '2K' and KC1 was replaced by NaCl in the K-free Ringer. Ordinate: m.e.p.p. frequency (see-); Abscissa: time (min). Temperature 19° C.
P. F. BAKER AND A. C. CRAWFORD
214
Comparison of ouabain with K-free Ringer One striking aspect of the action of ouabain is the long delay that precedes the rise in the m.e.p.p. frequency. Baker & Willis (1972) have shown that radioactive ouabain is taken up by squid nerve and enters the axoplasm for long periods of time after the Na pump has been completely inhibited. This observation raises the possibility that ouabain enters the motor nerve terminals and acts within them. Such an explanation seems 2K
100
K-free
.
0 0
10
I I
0
I
I
8
IU
16 Time (min)
I
I
24
I
I
I
32
Fig. 3. When an end-plate is superfused with K-free Ringer the m.e.p.p. frequency rises exponentially as soon as the external solution is changed even if the [Ca]0 is reduced to very low levels. Throughout the experiment no Ca was added to the Ringer and 1 mM-EGTA was present ([Mg]o was 3-6 mm throughout). Note the semilogarithmic co-ordinates. Ordinate: m.e.p.p. frequency (sec-). Abscissa: time (min). During the period labelled 2K the end-plate was superfused with a Ringer containing 2 mM-KCl, and in the period labelled 'K-free' K was omitted from the superfusing medium. Temperature 210 C.
21 215 Na PUMP AND TRANSMITTER RELEASE to be ruled out by the experiment illustrated in Fig. 2. When an end-plate is superfused (see Methods) with K-free Ringer the m.e.p.p. frequency increases with much the same time course as the increase seen with ouabain, but without the initial long delay. The omission of Ca from the Ringer and the addition of 1 mm-EGTA do not appear to alter this effect. In squid giant axons removal of external K ions blocks Na pumping as soon as the [K]0 can be lowered (Hodgkin & Keynes, 1955; Baker & Manil, 1968; Baker et al. 1969). The most. likely explanation for the long initial delay seen in Fig. 1 is that the Na pump is not 200
50
IV0 20
5
2
20 30 Time (min)
50
Fig. 4. The m.e.p.p. frequency recovers from a period in K-free Ringer when 2 mm-KCl is added to the Ringer. No added Ca and 1 mm-EGTA through. 3-6 mm in all solutions. The vertical lines indicate where the was changed. Initial and recovery solutions ('2K') contained 2 mm-KCI. Note the exponential rise and fall of the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Note the semilogarithmnic co-ordinates. Temperature 200 C. out.
[Mg],)
superfusion medium
inhibited significantly by Od1 mmw ouabain for about an hour after applying the glycoside. A similar, slow, Na-dependent binding of ouabain has been described by Baker & Willis (1972) in squid axons. The rise in the m.e.p.p. frequency that occurs on superfusion of an endplate with K-free Ringer bears a striking similarity to that which develops
P. F. BAKER AND A. C. CRA WFORD 216 when an end-plate is exposed to Ringer in which Li ions replace Na (Crawford, 1975). As shown in Fig. 3 the rise in the m.e.p.p. frequency in K-free Ringer occurs in the presence of 1 mM-EGTA and in the absence of added Ca. The time course of the rise in the m.e.p.p. frequency (as in Li Ringer) is at least initially a simple exponential with a time constant in five experiments similar to that of Fig. 3 of 4 7 + 1 1 (S.E.) min. On returning to 2 mM-K Ringer the m.e.p.p. frequency recovers to its initial value although the rate of recovery (as shown in Fig. 4) is clearly much faster than the rate of rise in K-free Ringer. In three experiments that followed the full time course of recovery from a period in K-free Ringer, the exponential time constant of recovery was 1x3 + 09 (s.E.) min. It is difficult to decide whether the change in intracellular Na or of intracellular K that would occur on inhibiting the Na pump of the nerve terminals is the most important factor that increases the m.e.p.p. frequency. The recovery of the m.e.p.p. frequency from K-free solutions might provide a way of answering this question, for the sodium pump is known to extrude Na ions from cells when activated externally by ions other than K (Solomon, 1952: Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt, 1969; Adrian & Slayman, 1966). Thus by activating the Na pump with either Rb+ or NH4+ instead of K+ the raised [Na]1 in the nerve terminals present after a period in K-free Ringer should fall but there should be no corresponding increase in the intracellular K concentration. Recovery of the m.e.p.p. frequency was followed in two experiments when the recovery Ringer contained 2 mM-RbCl instead of 2 mM-KCl, and in two experiments where 2 mM-NH4Cl replaced KC1. The results were consistent but ambiguous. The m.e.p.p. frequency recovered completely in the presence of Rb+ and apparently just as fast as in the presence of equimolar concentrations of K+ (time constants of the recovery were 1-3 and 09 min in the two experiments). In the experiments where NH4+ was used to reactivate the Na pump after a period in K-free Ringer, the m.e.p.p. frequency did not recover but was maintained at the level it had reached during the superfusion with K-free Ringer for at least 15 min. It does not seem possible on the basis of these observations to state whether the fall in [K]; or the rise in [Na]1 that would occur after the abolition of Na pumping is the important factor in producing the rise in the m.e.p.p. frequency.
Effects of repetitive stimulation If the action of ouabain and K-free Ringer is simply to block Na pumping and hence redistribute the Na and K gradients of the motor nerve terminals, then stimulating the preparation repetitively should accelerate the rise in the m.e.p.p. frequency by increasing Na entry and increasing the rate of K loss. This prediction is seen to be confirmed in the experiment of Fig. 5. The whole experiment was performed in EGTA Ringer where changes in the m.e.p.p. frequency due to Ca entry during the tetanus are negligible (Miledi & Thies, 1971). Two tetani were applied to the nerve at times T1 and T2 as indicated in Fig. 5. Each contained 1500 pulses at a frequency of 50 Hz. T1 falls before the application of 0-1 mM ouabain and causes a very small increase in the m.e.p.p. frequency (less than 0 4 extra
L
Na PUMP AND TRANSMITTER RELEASE 217 quanta/sec) that decays after the tetanus. The cardiac glycoside was then applied and the m.e.p.p. frequency allowed to rise by about an order of magnitude before applying the second tetanus (T2). This identical tetanus now has a very different effect. It accelerates the m.e.p.p. frequency to about 30 quanta/sec, an increment of more than 20 quanta/sec on the initial m.e.p.p. frequency before the tetanus, and there is no evidence of 1000 r
100
,, 10 10
I
0.1
Ouabain
F
0
30
60
0
30
60
90 90
Time (min) Fig. 5. A tetanus of 1500 pulses produces a different response of the m.e.p.p. frequency when placed before or after the application of ouabain. Tetanus frequency was 50 sec-1. Ouabain concentration 041 mm and presence as indicated by the arrows in the Figure. Throughout the experiment the Ringer contained no added Ca, 1 mM-EGTA and 3-6 mM-MgCl2. Note that the first tetanus (T1) produces a small transient response while the second (T2) produces a large step increase in the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Note the semilogarithmic co-ordinates. Temperature 230 C.
any recovery. The results are plotted on a semilogarithmic scale in Figs. 5 and 6 to display the small changes in the miniature end-plate potential frequency that occur in response to the tetanus at the beginning of a glycoside exposure,
P. F. BAKER AND A. C. CRAWFORD 218 The most striking difference between the increases in the m.e.p.p. frequency that occur in K-free Ringer and in the presence of ouabain, is the absence in the former case of any delay before the rise in spontaneous release. One possibility already mentioned is that ouabain takes a considerable time to inhibit the Na pump in the motor nerve terminals. This 1000 r-. Ouabain
N
100 1Ts5
0
10
1-
I-
4-
t 1 F
t
0.1 L I
I
0
20
-
40 Time (min)
60
80
Fig. 6. A standard tetanus applied to the nerve in EGTA Ringer, is only effective in producing a step increase in the m.e.p.p. frequency after more than 30 min in ouabain. Five tetani (T1-T5) were applied and their timings are indicated by the arrows in the Figure. T1 falls before the application of 0-1 mm ouabain. MgCl2 3-6 mm, EGTA 1 mm and no added Ca throughout. All tetani were of 600 pulses at a frequency of 20 sec-1. Note that T1 to T3 produce much the same small, reversible increase in the m.e.p.p. frequency. T4 and T5 produce step increases in the m.e.p.p. frequency. Note also the semilogarithmic co-ordinates. Ordinate: m.e.p.p. frequency (sec-1) Abscissa: time (min). Temperature 19.50 C.
interpretation would seem to be supported by the experiment illustrated in Fig. 6. Here five tetani (labelled T1 to T5 and indicated in the Figure by vertical arrows) each of 600 stimuli at a frequency of 20/sec were applied to the nerve at various times before and during an exposure to 0-1 mm
Na PUMP AND TRANSMITTER RELEASE 219 ouabain. The whole experiment was carried out in the presence of EGTA. The time courses of the early tetani are rather poorly defined because the m.e.p.p. frequency cannot be measured accurately when its mean value is low, but it is clear that the responses to the standard tetanus do not differ greatly when this is applied before exposure to ouabain or for up to 30 min after the glycoside is present. The final two tetani, T4 and T5, produce step increases in the m.e.p.p. frequency. In two similar experiments the behaviour of m.e.p.p. frequency was essentially similar; it is not until the release rate has begun to rise under the influence of the glycosides that an applied tetanus produces a sustained increase in the m.e.p.p. frequency. During the initial delay period monovalent ion movements associated with the action potential train produce no irreversible changes in the rate of quantal release. A dual effect of external Na ions during the action of ouabain Birks & Cohen (1968a, b) showed that if part of the Na of the Ringer was replaced by sucrose, the rise in the mean quantal content of the e.p.p. and of the m.e.p.p. frequency that develop in the presence of cardiac glycosides, did not occur. From this observation they proposed that changes in the intracellular concentrations of monovalent cations initiated the glycoside effects. Birks & Cohen did not follow the m.e.p.p. frequency in reduced Na-Ringer beyond times when the m.e.p.p. frequency in the presence of full Na would have reached its peak value. The possibility that the glycoside effects are only delayed by reducing [Na]o therefore cannot be eliminated. Since it is difficult, especially in EGTA Ringer, to follow the m.e.p.p. frequency at end-plates for periods of several hours, this problem was approached in another way. End-plates were exposed to ouabain in the presence of full (114 mm) external Na and the m.e.p.p. frequency allowed to rise. The [Na]o was then reduced by isotonic substitution with either sucrose or LiCl while the concentration of ouabain was left unaltered at 0.1 mm. Since the terminals will load with Na less quickly when [Na]o is reduced, the rise in the m.e.p.p. frequency ought to slow down in media of reduced Na content. In fact quite the opposite is seen. If [Na]o is reduced after the m.e.p.p. frequency has begun to rise spontaneous release of transmitter is accelerated. Thus reducing [Na]o can have different effects on the m.e.p.p. frequency depending on the time after application of glycoside that the external Na is lowered. If [Na]o is low from the beginning of the application of ouabain, the rise in the m.e.p.p. frequency can be inhibited whereas if [Na]o is reduced after the m.e.p.p. frequency has begun to rise spontaneous release is accelerated. Fig. 7 illustrates an experiment in which an end-plate was exposed to
P. F. BAKER AND A. C. CRAWFORD ouabain and the Na of the Ringer replaced isotonically by LiCl for 10 min periods at various times during the treatment with the glycoside. Lithium Ringer produces little change in the m.e.p.p. frequency over such a short period when ouabain is absent (Crawford, 1975). During the delay before the onset of the rise in the m.e.p.p. frequency caused by ouabain, the reduction of [Na]0 has little effect. Just at the point where the m.e.p.p. frequency begins to rise, however, the reduction in [Na]0 produces a 220
Ouabain Na
Na
Li
Li
Na
Li
Na
Li
Na
60
.40
20
0
140 80 100 120 60 Time (min) Fig. 7. The m.e.p.p. frequency becomes extremely sensitive to reduction of the external Na concentration late in an exposure to 0-1 mm ouabain Ringer. 0-4 mm CaCl2 and 3-2 mM-MgCl2 present throughout. The vertical boxes indicate periods in which the NaCl of the Ringer was replaced isotonically by LiCl. Ouabain was present as indicated by the horizontal arrow. Note that before applying ouabain and for the first 40 min of a ouabain treatment, the short period in Li-Ringer has little effect on the m.e.p.p. frequency. The last reduction in external Na produces a dramatic but reversible increase in the m.e.p.p. frequency followed by the rise normally seen at this time in ouabain (compare Fig. 1 A). Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 220 C. 0
20
40
dramatic rise in the spontaneous release rate that recovers on raising the [Na]0 to normal. The rise in the m.e.p.p. frequency produced by the glycoside continues unaffected by this procedure (compare Fig. 7 and Fig. 1). The experiment shown in Fig. 7 was carried out in the presence of 0-4 mM-Ca. One possible explanation for the acceleration of the m.e.p.p. frequency might be that a Na-dependent Ca influx of the type described by Baker et al. (1969) in squid giant axons might be activated by changing to Li Ringer. In squid axons it has been shown that the Na-dependent
Na PUMP AND TRANSMITTER RELEASE 221 Ca influx is highly dependent on the concentration of intracellular Na, the Ca influx increasing roughly as the square of the internal Na concentration. It follows that late in a ouabain exposure, when the nerve terminals are sodium loaded, the Na-dependent Ca influx might be much larger than at the beginning. 300
Li Na
Li
Na
Li Na Ouabain
Na
200 4I-
100
0
40
80
120
Time (min)
Fig. 8. The sensitivity of the m.e.p.p. frequency to external Na ions that develops late in an ouabain exposure is unaffected by removing external Ca ions. All Ringers contained no added Ca, 3-6 mM-MgCl2 and 1 mM-EGTA. Ouabain was present at 0.1 mm as indicated by the horizontal arrow. Vertical boxes labelled Li indicate periods where LiCl replaced the NaCl of the Ringer isosmotically. Between the boxes the [Na]0 was 112 mm. Note that only after the m.e.p.p. frequency has begun to rise under the influence of ouabain, does the reduction in [Na]0 produce an increase in the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 20° C.
This explanation of the results does not however appear to be correct, for essentially similar results are obtained if the whole experiment is performed in the absence of external calcium and in the presence of 1 mmEGTA. Fig. 8 illustrates such an experiment. Here an end-plate was exposed on three occasions to Li Ringer before and during treatment of the preparation with ouabain. Once the m.e.p.p. frequency has begun to rise under the influence of the glycoside the reduction in [Na]0 produces a striking acceleration of spontaneous release. It might be argued that the development of this sensitivity of the m.e.p.p. frequency to external sodium ions late in a ouabain exposure is a specific effect of Li and is not in fact due to reduction in the [Na]0. This does not seem to be the case. Reduction in the [Na]0 by isotonic replacement with sucrose produces essentially similar results to isotonic replacement of external Na by LiCl. Fig. 9 shows an experiment where an
222 P. F. BAKER AND A. C. CRAWFORD end-plate was changed to a Ringer containing only half the normal Na concentration (the period labelled ' Suc' in the Figure). The whole experiment was run in the presence of 1 mM-EGTA and no added Ca. Reductions of [Na]0 below 50 % by replacement with sucrose are difficult because the amplitude of the m.e.p.p. falls below the base line noise of the recording system, a difficulty that is not encountered with LiCl as a Na substitute (Onodera & Yamakawa, 1966; Kelly, 1968). In normal end-plates, reducing the [Na]0 by this amount produces a small rise in the m.e.p.p. frequency (Kelly, 1965, 1968) but nothing as dramatic as that seen when the [Na]0 is 120
I
90
Ouabain
s Suc.
Na
Na
60
30
0
0
20
40 Time (min)
60
80
Fig. 9. The acceleration of the m.e.p.p. frequency by reducing the external Na concentration late in an ouabain exposure occurs if the Na-substitute is sucrose instead of LiCl. Ouabain (0.1 mM) was present as indicated by the horizontal arrow. Throughout the experiment the Ringer contained no added Ca, 3-6 mM-MgCl2 and 1 mM-EGTA. During the vertical box labelled 'i Suc' half of the NaCl of Ringer (57 mM) was replaced isosmotically by sucrose. Note the rise of the m.e.p.p. frequency before the i-sucrose period caused by ouabain and the sudden increase that occurs during the i-sucrose period. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 210 C.
reduced late in an exposure to ouabain. The rise in the m.e.p.p. frequency in this experiment is perhaps not as sharp as those of Figs. 7 and 8 but the results are not directly comparable since in Figs. 7 and 8 all of the external Na was replaced by Li ions whereas in Fig. 9 only 50 % of the Na has been replaced by sucrose. It must be concluded that ouabain not only produces a gradual rise in
Na PUMP AND TRANSMITTER RELEASE
223 the m.e.p.p. frequency but also makes spontaneous release of transmitter extremely sensitive to changes in the external Na concentration. The sensitivity to external Na ions is independent of the presence or absence of external Ca. DISCUSSION
The rise in the m.e.p.p. frequency produced by ouabain or by K-free Ringer occurs unimpeded in the presence of enough EGTA to lower the external Ca concentration to very low levels. It seems unlikely that the increase in the spontaneous release rate is produced by Ca entry into the nerve terminals either resulting from depolarization or by activation of a Na-dependent Ca entry system. Rather similar observations have been made by Paton, Vizi & Zar (1971) and Vizi (1972) on the release of acetylcholine from parasympathetic ganglia and brain slices respectively. These authors suggest a direct involvement of the Na pump in the release mechanism but at the frog neuromuscular junction the present results do not support this conclusion. It seems not to be the inhibition of the Na pump per se that is important, but rather the redistribution that this brings about of the monovalent cation gradients within the nerve terminal. The most compelling piece of evidence for this conclusion is that hastening the dissipation of the ion gradients by repetitive stimulation of the nerve during a ouabain exposure accelerates spontaneous release irreversibly. The similarity of the time course of the rise in the m.e.p.p. frequency in K-free Ringer, to that which occurs when an end-plate is bathed in Li Ringer (Crawford, 1975), suggests that similar processes are occurring in the two situations. In both cases the nerve terminals are presumably losing intracellular K but in Li Ringer the [Na]1 is probably falling as Li ions accumulate in the terminals, while in K-free Ringer [Na]1 would be expected to rise when the sodium pump is inhibited. While it is possible that accumulation of either Na or Li ions may promote the observed increase in m.e.p.p. frequency, it is perhaps more attractive to suppose that the fall in the intracellular K concentration, which occurs in both solutions, in some way underlies the rise in the m.e.p.p. frequency. However, to maintain this view one must postulate that Rb ions can replace K in the process since the m.e.p.p. frequency recovers normally from a period in K-free Ringer, when 2 mM-RbCl is present in the recovery solution. The action of ouabain on the m.e.p.p. frequency depends on the Na concentration of the medium and two effects of external Na can be distinguished. The onset of the rise in m.e.p.p. frequency produced by ouabain is markedly slowed in the absence of external Na; but once the m.e.p.p. frequency has begun to rise replacement of external Na leads to a dramatic increase in spontaneous release. The Na-dependence of the onset of the 8
P HY
247
224 P. F. BAKER AND A. C. CRAWFORD glycoside effect may reflect a Na dependence of glycoside binding to the nerve terminal, similar to that which has been described in squid axons (Baker & Willis, 1972); whereas the stimulation of release induced by a reduction in external Na subsequent to glycoside binding may be related to changes in Na-dependent extrusion of Ca ions from the nerve terminals. End-plates become extremely sensitive to reduction of the external Na concentration late in an exposure to ouabain Ringer. In a normal end-plate, changes in the external Na concentration produce very small changes in the m.e.p.p. frequency. Replacing all of the external Na by Li ions produces no immediate change in the m.e.p.p. frequency (Onodera & Yamakawa, 1966; Kelly, 1968; Crawford, 1975). Exchanging Na ions for tris, methylammonium, dimethylammonium and guanidinium ions similarly has little initial effect on the spontaneous release rate (Kelly, 1968; Otsuka & Endo, 1960). Replacing part of the external Na by sucrose produces a small acceleration of the m.e.p.p. discharge (Kelly, 1965; Gage & Quastel, 1966). If the preparation is partially depolarized with an elevated K concentration in the Ringer the m.e.p.p. frequency becomes much more sensitive to changes in the external Na concentration (Gage & Quastel, 1966; Birks, Burstyn & Firth, 1968). In this situation there appears to be an antagonism between Ca and Na, for the sensitivity to [Na],, is much greater if the external Ca concentration is high (Gage & Quastel, 1966; Birks et al. 1968). Na sensitivity seen late in a period of treatment with ouabain would seem to be different from that of a K depolarized end-plate for it is independent of external Ca ions. This rather disparate set of observations on the Na sensitivity of the m.e.p.p. frequency can however be summarized in a rather simple way. When the m.e.p.p. frequency is high it is very sensitive to reduction of the external Na concentration, and when the m.e.p.p. frequency is normal it is relatively insensitive to changes in external Na.
Miledi (1973) has recently shown in squid giant synapses that intracellular iontophoresis of Ca into the presynaptic terminals will increase the frequency of miniature post-synaptic potentials. It is attractive to suppose that the conditions under which the m.e.p.p. frequency is Nasensitive are all situations where the intracellular Ca concentration of the nerve terminals is high. If under these conditions, an important factor in regulating the intracellular concentration of ionized Ca is membrane transport, the Na sensitivity of the m.e.p.p. frequency might be explained on the basis of a Ca efflux mechanism present in the nerve terminals that requires the presence of external Na ions. Such a system has been described in squid axons and other nerves (see Baker, 1972) and it is possible that if the internal free Ca has been elevated in some way, it can be raised further by inhibiting the extra Ca efflux that would normally develop under these conditions. Such a situation would seem to explain the results obtained here for ouabain and perhaps also the results from potassium-depolarized preparations. With regard to the increase in the m.e.p.p. frequency produced by ouabain, at least two plausible explanations can be put forward. The changes in monovalent cation concentration resulting from inhibition of the Na pump
225 Na PUMP AND TRANSMITTER RELEASE could (1) release Ca from internal stores or (2) make internal Ca more effective in releasing transmitter, perhaps by increasing the affinity for Ca of the sites that bind Ca in the release process. It is difficult to choose at present between these two explanations but an elevated internal Ca concentration is certainly consistent with the Na sensitivity of the m.e.p.p. frequency that develops during the application of ouabain. At first sight an alternative explanation seems attractive. If there is a slow release of Ca from intracellular binding sites the maintenance of a low internal ionized Ca may be very dependent on membrane transport by a presumably Na-dependent mechanism. In these circumstances a rise in [Na]1 or fall in [K]i might inhibit calcium extrusion and lead to a rise in [Ca]i and transmitter release. Subsequent reduction in [Na]0 would slow extrusion further and give the observed increment in release. This simple explanation seems unable to account for the alteration in response to lithium during exposure to ouabain. In each test of Li, total replacement of [Na]0 by Li should inhibit Ca extrusion to a similar extent and should, if there is a steady leak of Ca from some internal store, produce a constant increment in [Ca]i. The observation that the response to Li grows during exposure to ouabain argues strongly that either the relation between [Ca]1 and transmitter release is non-linear (but see Crawford 1974), or that the rate at which Ca is leaking from an internal store is increasing during the exposure to glycoside. This work was performed while A. C. C. was in receipt of an M.R.C. Scholarship and was supported by a grant to P. F. B. from the Medical Research Council. REFERENCES
ADRIAN, R. H. (1964). The rubidium and potassium permeability of frog muscle membrane. J. Physiol. 175, 135-159. ADRIAN, R. H. & SLAYMAN, C. L. (1966). Membrane potential and conductance during transport of sodium, potassium and rubidium in frog muscle. J. Phy8iol. 184, 970-1014. BAKER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophy8. molec. Biol. 24, 179-223. BAKER, P. F. BLAUSTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1967). The effect of sodium concentration on calcium movements in giant axons of Loligo forbesi. J. Physiol. 192, 43P. BAR PR, P. F., BLAUSTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux from squid giant axons. J. Physiol. 200, 431-459. BA1RR, P. F., BLAUSTEIN, M. P., KEYNES, R. D., MANIL, JACQUELINE, SHAW, T. I. & STEINHARDT, R. A. (1969). The ouabain-sensitive fluxes of sodium and potassium in squid axons. J. Physiol. 200, 459-496. BAKER, P. F., HODGKIN, A. L. & RIDGwAY, E. B. (1971). Depolarization and calcium entry in squid axons. J. Physiol. 218, 709-755. BAKER, P. F. & MANIL, J. (1968). The rates of action of K+ and ouabain on the sodium pump in squid giant axons. Biochim. biophy8. Acta 150, 328-330. BAKER, P. F. & WILLIS, J. S. (1972). Inhibition of the sodium pump in squid giant axons by cardiac glycosides; dependence on extracellular ions and metabolism. J. Physiol. 224, 463-475. BIRnS, R. I., BURSTYN, P. G. R. & FIRTH, D. R. (1968). The form of sodiumcalcium competition at the frog neuromuscular junction. J. gen. Physiol. 52,887-908. 8-2
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BIRKS, R. I. & COHEN, M. W. (1968a). The action of sodium pump inhibitors on neuromuscular transmission. Proc. Roy. Soc. B 170, 381-399. BIRKS, R. I. & COHEN, M. W. (1968b). The influence of internal sodium on the behaviour of motor nerve endings. Proc. Roy. Soc. B 170, 401-421. BLAUSTEIN, M. P. & HODGKIN, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. 200, 497-529. COOKE, J. D., OKAMOTO, K. & QUASTEL, D. M. J. (1973). The role of calcium in depolarization-secretion coupling at the motor nerve terminal. J. Physiol. 228, 459-497. COOKE, J. D. & QUASTEL, D. M. J. (1973). Transmitter release by mammalian motor nerve terminals in response to focal polarization. J. Physiol. 228, 377-405. CRAWFORD, A. C. (1974). The dependence of evoked transmitter release on external calcium ions at very low mean quantal contents. J. Physiol. 240, 255-278. CRAWFORD, A. C. (1975). Lithium ions and the release of transmitter at the frog neuromuscular junction. J. Physiol. 246, 109-142. ELMQVIST, D. & FELDMAN, D. S. (1965). Effects of sodium pump inhibitors on spontaneous acetylcholine release at the neuromuscular junction. J. Physiol. 181, 498-505. GAGE, P. W. & QUASTEL, D. M. J. (1966). Competition between sodium and calcium ions in transmitter release at a mammalian neuromuscular junction. J. Physiol. 185, 95-123. HODGKIN, A. L. & HOROWICZ, P. (1959). The influence of potassium and chloride ions on membrane potential of single muscle fibres. J. Physiol. 148, 127-160. HODGKIN, A. L. & KEYNES, R. D. (1955). Active transport of cations in giant axons of Sepia and Loligo. J. Physiol. 128, 28-60. HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1968). On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals. J. Physiol. 194, 381-407. KELLY, J. S. (1965). Antagonism between Na+ and Ca2+ at the neuromuscular junction. Nature, Lond. 205, 296-297. KELLY, J. S. (1968). The antagonism of Ca2+ by Na+ and other monovalent ions at the frog neuromuscular junction. Q. JZ exp. Physiol. 53, 239-249. KEYNES, R. D. (1954). The ionic fluxes in frog muscle. Proc. Roy. Soc. B 142, 359-382. LANDAU, E. M. (1969). The interaction of presynaptic polarization with calcium and magnesium in modifying spontaneous transmitter release from mammalian motor nerve terminals. J. Physiol 203, 281-299. MILEDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. Roy. Soc. B 183, 421-425. MILEDI, R. & THIES, R. E. (1971). Tetanic and post-tetanic rise in the frequency of minature end-plate potentials in low calcium solutions. J. Physiol. 212, 245-257. ONODERA, K. & YAMAKAWA, K. (1966). The effects of lithium on the neuromuscular junction of the frog. Jap. J. Physiol. 16, 541-550. OTSUKA, M. & ENDO, M. (1960). The effect of guanidine on neuromuscular transmission. J. Pharmac. exp. Ther. 128, 273-282. PATON, W. D. M., Vizi, E. S. & ZAR, M. ABoo (1971). The mechanism of acetylcholine release from parasympathetic nerves. J. Physiol. 215, 819-848. SOLOMON, A. K. (1952). The permeability of the human erythrocyte to sodium and potassium. J. gen. Physiol. 36, 57-110. Vizi, E. S. (1972). Stimulation by inhibition of (Na+-K+-Mg2+)-activated ATPase of acetylcholine release in cortical slices from rat brain. J. Physotl. 226, 95-117.
J. Physiol. (1975), 247, pp. 209-226 With 9 text-figure8 Printed in Great Britain
A NOTE ON THE MECHANISM BY WHICH INHIBITORS OF THE SODIUM PUMP ACCELERATE SPONTANEOUS RELEASE OF TRANSMITTER FROM MOTOR NERVE TERMINALS
By P. F. BAKER AND A. C. CRAWFORD From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG
(Received 23 September 1974) SUMMARY
1. The actions of 0-1 mm ouabain and of K-free Ringer have been examined at the frog neuromuscular junction. 2. After a delay of more than 30 min, ouabain produces an increase in the miniature end-plate potential (m.e.p.p.) frequency. This increase occurs unchanged in Ca-free Ringer containing 1 mM-EGTA and is therefore unlikely to be due to an entry of Ca into the motor nerve terminals. 3. If the nerve to the preparation is stimulated repetitively in Ca-free Ringer containing 0-1 mM ouabain and 1 mM-EGTA the response of the m.e.p.p. frequency depends on the timing of the tetanus relative to the beginning of the ouabain treatment. 4. During the first 30 min of exposure to ouabain, the tetanus produces a small, transient increase in the m.e.p.p. frequency similar to that which occurs before ouabain is present. After about 30 min the same tetanus produces large, irreversible increases in the m.e.p.p. frequency. 5. Superfusion of an end-plate with K-free Ringer causes an immediate exponential rise in the m.e.p.p. frequency that is unaffected by the presence of external Ca ions. On replacing the normal K of the Ringer (2 mM) the m.e.p.p. frequency recovers quickly to its original value. 6. Late in an exposure to 0.1 mm ouabain the m.e.p.p. frequency becomes extremely sensitive to changes in the external Na concentration, [Na]0. Reducing [Na]0 increases the m.e.p.p. frequency. The sensitivity to [Na]0 is independent of external Ca ions or whether the isotonic substitute for NaCl is LiCl or sucrose. 7. It is suggested that the spontaneous release of transmitter is facilitated, in some way, by the changes in the monovalent cation content of the nerve terminals that result from blocking the Na-K exchange pump.
210
P. F. BAKER AND A. C. CRAWFORD
The Na sensitivity of the m.e.p.p. frequency that develops simultaneously can be explained if a Na-dependent Ca efflux system is present in the membrane of the presynaptic terminals. INTRODUCTION
When motoneurone terminals are exposed for long periods to cardiac glycosides both the mean quantal content of the end-plate potential (m) and the miniature end-plate potential frequency (f) slowly but progressively increase (Elmqvist & Feldman, 1965; Birks & Cohen, 1968a). There seems little doubt that the cardiac glycosides act by inhibiting the Na-K exchange pump, for the increase in evoked transmitter release also occurs when external K is omitted from the Ringer (Birks & Cohen, 1968 a). Moreover, the increases in both m andf seem to be secondary to redistribution of the sodium and potassium ion gradients of the motor nerve terminals, since replacement of part of the external Na of the Ringer by sucrose dramatically slows the growth of the end-plate potential and of the miniature end-plate potential frequency. Birks & Cohen proposed that a rise in the intracellular sodium concentration led to an increase in the resting Ca entry into the nerve terminals, as is known to occur in squid giant axons (Baker, Blaustein, Hodgkin & Steinhardt, 1969), and that the rise in intracellular Ca produced the increases in transmitter release. The starting point of this paper was the observation that the increase in f produced by ouabain occurred unimpeded in Ringer that lacked added Ca and included a Ca chelating agent to reduce the external Ca concentration to levels as low as 10-9 M. Any explanation of the glycoside effects in terms of a Ca influx (either due to depolarization or to Na-dependent transport) thus seems unlikely. The results are consistent with the release of Ca from internal stores in the motor nerve terminals. Evidence is also presented that Ca is extruded from the terminals by a Na-dependent transport mechanism similar to that described by Baker, Blaustein, Hodgkin & Steinhardt (1967) and by Blaustein & Hodgkin (1969) in squid giant axons. METHODS
Standard intracellular recording techniques were used to follow the miniature endplate potential (m.e.p.p.) frequency at superficial end-plates of frog (Rana temporaria) sartorius-sciatic preparations. Details of these techniques have been described elsewhere (Crawford, 1974).
Solutions Table 1 gives the composition of the major solutions used in these experiments. LiCl was obtained from Fisher Chemicals and neostigmine methylsulphate (Sigma)
Na PUMP AND TRANSMITTER RELEASE
211
10-6 gm/ml. was included in all solutions to increase the m.e.p.p. amplitude. Ouabain (B.D.H.), was made up as a stock solution at 10 mM in distilled water and stored at 40 C. The glycoside was added to the Ringer immediately before each experiment to give a final concentration of 0-1 mm. In a few experiments the m.e.p.p. frequency was followed in Ringer that contained no added K ('K-free Ringer', columns E and F of Table 1). Birks & Cohen (1968a) had some difficulty with sartorius preparations in reproducing the effects of glycosides with K-free Ringer, a fact that they attributed to a leakage of K out of the muscle and subsequent activation of the Na pump. Keynes (1954) found similar difficulties in his attempts to block the Na pump in sartorius muscle by the use of K-free solutions. These drawbacks were circumvented by the use of a 'superfusion' device similar to that introduced by Cooke & Quastel (1973). A 150 prm glass pipette was used to direct a jet of K-free Ringer at the end-plate once it had been located and the solution was sucked off continuously at a point distant from the site of recording. This method allowed Cooke & Quastel to raise the K concentration at an end-plate in less than 4 sec and in the present experiments proved an ideal method of washing the endplate region free of K ions. The behaviour of the muscle membrane potential when superfused with K-free Ringer was unpredictable. Either the muscle hyperpolarized slightly (in which case the experiment was continued) or it depolarized rapidly to as little as -50 mV. In the latter case the m.e.p.p. became too small to be distinguished reliably and another end-plate was chosen. The depolarization of muscles in K-free solutions has been noted previously (Hodgkin & Horowicz, 1959; Adrian & Slayman, 1966) and probably results from a reduction in the K conductance of the muscle fibre (Hodgkin & Horowicz, 1959; Adrian, 1964; Adrian & Slayman, 1966). The depolarization was particularly prevalent if the Ringer also contained EGTA and no added Ca. TABLE 1. Composition of the major solutions used in these experiments. All ion concentrations are millimolar. In addition the solutions contained neostigmine methylsulphate 10-6 g/l. Solution Ion Na+ Li+ K+ Ca2+ Mg2+ Tris EGTA
ClSucrose
A 114-5 2-0 0-4 3-2 2-5 126-2
B 114-5 2-0 3-6 2-5
1-0 122-2
C
D
114-5 2-0
57 2-0
3-6 2-5 1-0 122-2 -
3-6 2-5 1-0 68-7 115-0
E 116-5 0-4 3-2 2-5
F 116-5
3-6 2-5
1-0 126-2
122-2
RESULTS
External Ca ions and the rise in transmitter release caused by ouabain The time course of the increase in the miniature end-plate potential frequency during application of the cardiac glycoside, ouabain (0-1 mM), is illustrated in Fig. 1. Two features are striking: there is an initial delay of 60-80 min during which the spontaneous release rate remains
212 P. F. BAKER AND A. C. CRAWFORD stable at its original level, and then a period of 15 min when the m.e.p.p. frequency rises to levels that cannot be measured reliably using intracellular recording ( > 200 sec-1). Similar results have been reported by Birks & Cohen (1968a) using digoxin, strophanthidin and ouabain. Ouabain
>
200 r
0
0
0
A
U
1001-
0
'o.-
000
0000 0 0
0
0
00
30
0
0
0
60
ocP0 90
120
Ouabain
so
0
200 r B U
0
100
F
0 0 0
0 -0 o
0
0
30
o
oo
c)0V
60 Time (min)
90
120
Fig. 1. Ouabain accelerates the spontaneous release of transmitter after an initial delay, and the increase in the miniature end-plate potential frequency does not require external Ca ions. In both A and B ouabain was applied at a concentration of 0-1 mm as indicated by the arrows. The Ringer contained 0-4 mM-Ca2+, 3-2 mM-Mg2+, in A, and in B 3-6 mM-Mg2+, 1 mM-EGTA and no added Ca. Ordinate: m.e.p.p. frequency (f) sec-1, Abscissa: time (min).
The external Ca concentration [Ca]0, of the Ringer has little effect on the action of ouabain. In Fig. 1 two experiments are shown in which the Ringer contained either 0-4 mM-Ca2+, or no added Ca and 1 mM-EGTA. In the latter case the Ca-chelating agent should reduce the [Ca]0 to less than 10-9 M (Hubbard, Jones & Landau, 1968; Miledi & Thies, 1971). In both experiments the m.e.p.p. frequency accelerates in much the same way
213 Na PUMP AND TRANSMITTER RELEASE although the initial delay before the rise in spontaneous release is slightly shortened when external calcium ions are omitted. These observations seem to rule out any explanation of the glycoside effect in terms of an increased Ca influx. Two specific hypotheses are included in this category. Progressive depolarization of the motor nerve terminals, resulting from inhibition of the Na pump, is unlikely to be the cause of the rise in the m.e.p.p. frequency since increases in the spontaneous release rate induced by either K or electrical depolarization are abolished in EGTA Ringer (Landau, 1969; Cooke & Quastel, 1973; Cooke, Okamoto & Quastel, 1973). Similarly, although inhibition of the Na pump may lead to accumulation of Na inside the terminals this is also unlikely to increase the m.e.p.p. frequency by increasing a sodium-dependent Ca influx into the terminals (Baker et at. 1969; Baker, 1972) because the effects persist in EGTA Ringer. An explanation of the action of ouabain must be sought elsewhere than through mechanisms likely to cause Ca entry into the motor nerve terminals. 2K
150
'K-free'
2K
100 U
0
50
0 I
I
I
0
5
10 Time (min)
15
20
Fig. 2. The m.e.p.p. frequency rises when an end-plate is superfused with K-free Ringer and recovers when the K in the superfusing medium is replaced. The concentration of CaCl2 was 04 mm and the MgCl2 concentration 3-2 mm throughout. The Ringer contained 2 mM-KCl during the periods labelled '2K' and KC1 was replaced by NaCl in the K-free Ringer. Ordinate: m.e.p.p. frequency (see-); Abscissa: time (min). Temperature 19° C.
P. F. BAKER AND A. C. CRAWFORD
214
Comparison of ouabain with K-free Ringer One striking aspect of the action of ouabain is the long delay that precedes the rise in the m.e.p.p. frequency. Baker & Willis (1972) have shown that radioactive ouabain is taken up by squid nerve and enters the axoplasm for long periods of time after the Na pump has been completely inhibited. This observation raises the possibility that ouabain enters the motor nerve terminals and acts within them. Such an explanation seems 2K
100
K-free
.
0 0
10
I I
0
I
I
8
IU
16 Time (min)
I
I
24
I
I
I
32
Fig. 3. When an end-plate is superfused with K-free Ringer the m.e.p.p. frequency rises exponentially as soon as the external solution is changed even if the [Ca]0 is reduced to very low levels. Throughout the experiment no Ca was added to the Ringer and 1 mM-EGTA was present ([Mg]o was 3-6 mm throughout). Note the semilogarithmic co-ordinates. Ordinate: m.e.p.p. frequency (sec-). Abscissa: time (min). During the period labelled 2K the end-plate was superfused with a Ringer containing 2 mM-KCl, and in the period labelled 'K-free' K was omitted from the superfusing medium. Temperature 210 C.
21 215 Na PUMP AND TRANSMITTER RELEASE to be ruled out by the experiment illustrated in Fig. 2. When an end-plate is superfused (see Methods) with K-free Ringer the m.e.p.p. frequency increases with much the same time course as the increase seen with ouabain, but without the initial long delay. The omission of Ca from the Ringer and the addition of 1 mm-EGTA do not appear to alter this effect. In squid giant axons removal of external K ions blocks Na pumping as soon as the [K]0 can be lowered (Hodgkin & Keynes, 1955; Baker & Manil, 1968; Baker et al. 1969). The most. likely explanation for the long initial delay seen in Fig. 1 is that the Na pump is not 200
50
IV0 20
5
2
20 30 Time (min)
50
Fig. 4. The m.e.p.p. frequency recovers from a period in K-free Ringer when 2 mm-KCl is added to the Ringer. No added Ca and 1 mm-EGTA through. 3-6 mm in all solutions. The vertical lines indicate where the was changed. Initial and recovery solutions ('2K') contained 2 mm-KCI. Note the exponential rise and fall of the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Note the semilogarithmnic co-ordinates. Temperature 200 C. out.
[Mg],)
superfusion medium
inhibited significantly by Od1 mmw ouabain for about an hour after applying the glycoside. A similar, slow, Na-dependent binding of ouabain has been described by Baker & Willis (1972) in squid axons. The rise in the m.e.p.p. frequency that occurs on superfusion of an endplate with K-free Ringer bears a striking similarity to that which develops
P. F. BAKER AND A. C. CRA WFORD 216 when an end-plate is exposed to Ringer in which Li ions replace Na (Crawford, 1975). As shown in Fig. 3 the rise in the m.e.p.p. frequency in K-free Ringer occurs in the presence of 1 mM-EGTA and in the absence of added Ca. The time course of the rise in the m.e.p.p. frequency (as in Li Ringer) is at least initially a simple exponential with a time constant in five experiments similar to that of Fig. 3 of 4 7 + 1 1 (S.E.) min. On returning to 2 mM-K Ringer the m.e.p.p. frequency recovers to its initial value although the rate of recovery (as shown in Fig. 4) is clearly much faster than the rate of rise in K-free Ringer. In three experiments that followed the full time course of recovery from a period in K-free Ringer, the exponential time constant of recovery was 1x3 + 09 (s.E.) min. It is difficult to decide whether the change in intracellular Na or of intracellular K that would occur on inhibiting the Na pump of the nerve terminals is the most important factor that increases the m.e.p.p. frequency. The recovery of the m.e.p.p. frequency from K-free solutions might provide a way of answering this question, for the sodium pump is known to extrude Na ions from cells when activated externally by ions other than K (Solomon, 1952: Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt, 1969; Adrian & Slayman, 1966). Thus by activating the Na pump with either Rb+ or NH4+ instead of K+ the raised [Na]1 in the nerve terminals present after a period in K-free Ringer should fall but there should be no corresponding increase in the intracellular K concentration. Recovery of the m.e.p.p. frequency was followed in two experiments when the recovery Ringer contained 2 mM-RbCl instead of 2 mM-KCl, and in two experiments where 2 mM-NH4Cl replaced KC1. The results were consistent but ambiguous. The m.e.p.p. frequency recovered completely in the presence of Rb+ and apparently just as fast as in the presence of equimolar concentrations of K+ (time constants of the recovery were 1-3 and 09 min in the two experiments). In the experiments where NH4+ was used to reactivate the Na pump after a period in K-free Ringer, the m.e.p.p. frequency did not recover but was maintained at the level it had reached during the superfusion with K-free Ringer for at least 15 min. It does not seem possible on the basis of these observations to state whether the fall in [K]; or the rise in [Na]1 that would occur after the abolition of Na pumping is the important factor in producing the rise in the m.e.p.p. frequency.
Effects of repetitive stimulation If the action of ouabain and K-free Ringer is simply to block Na pumping and hence redistribute the Na and K gradients of the motor nerve terminals, then stimulating the preparation repetitively should accelerate the rise in the m.e.p.p. frequency by increasing Na entry and increasing the rate of K loss. This prediction is seen to be confirmed in the experiment of Fig. 5. The whole experiment was performed in EGTA Ringer where changes in the m.e.p.p. frequency due to Ca entry during the tetanus are negligible (Miledi & Thies, 1971). Two tetani were applied to the nerve at times T1 and T2 as indicated in Fig. 5. Each contained 1500 pulses at a frequency of 50 Hz. T1 falls before the application of 0-1 mM ouabain and causes a very small increase in the m.e.p.p. frequency (less than 0 4 extra
L
Na PUMP AND TRANSMITTER RELEASE 217 quanta/sec) that decays after the tetanus. The cardiac glycoside was then applied and the m.e.p.p. frequency allowed to rise by about an order of magnitude before applying the second tetanus (T2). This identical tetanus now has a very different effect. It accelerates the m.e.p.p. frequency to about 30 quanta/sec, an increment of more than 20 quanta/sec on the initial m.e.p.p. frequency before the tetanus, and there is no evidence of 1000 r
100
,, 10 10
I
0.1
Ouabain
F
0
30
60
0
30
60
90 90
Time (min) Fig. 5. A tetanus of 1500 pulses produces a different response of the m.e.p.p. frequency when placed before or after the application of ouabain. Tetanus frequency was 50 sec-1. Ouabain concentration 041 mm and presence as indicated by the arrows in the Figure. Throughout the experiment the Ringer contained no added Ca, 1 mM-EGTA and 3-6 mM-MgCl2. Note that the first tetanus (T1) produces a small transient response while the second (T2) produces a large step increase in the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Note the semilogarithmic co-ordinates. Temperature 230 C.
any recovery. The results are plotted on a semilogarithmic scale in Figs. 5 and 6 to display the small changes in the miniature end-plate potential frequency that occur in response to the tetanus at the beginning of a glycoside exposure,
P. F. BAKER AND A. C. CRAWFORD 218 The most striking difference between the increases in the m.e.p.p. frequency that occur in K-free Ringer and in the presence of ouabain, is the absence in the former case of any delay before the rise in spontaneous release. One possibility already mentioned is that ouabain takes a considerable time to inhibit the Na pump in the motor nerve terminals. This 1000 r-. Ouabain
N
100 1Ts5
0
10
1-
I-
4-
t 1 F
t
0.1 L I
I
0
20
-
40 Time (min)
60
80
Fig. 6. A standard tetanus applied to the nerve in EGTA Ringer, is only effective in producing a step increase in the m.e.p.p. frequency after more than 30 min in ouabain. Five tetani (T1-T5) were applied and their timings are indicated by the arrows in the Figure. T1 falls before the application of 0-1 mm ouabain. MgCl2 3-6 mm, EGTA 1 mm and no added Ca throughout. All tetani were of 600 pulses at a frequency of 20 sec-1. Note that T1 to T3 produce much the same small, reversible increase in the m.e.p.p. frequency. T4 and T5 produce step increases in the m.e.p.p. frequency. Note also the semilogarithmic co-ordinates. Ordinate: m.e.p.p. frequency (sec-1) Abscissa: time (min). Temperature 19.50 C.
interpretation would seem to be supported by the experiment illustrated in Fig. 6. Here five tetani (labelled T1 to T5 and indicated in the Figure by vertical arrows) each of 600 stimuli at a frequency of 20/sec were applied to the nerve at various times before and during an exposure to 0-1 mm
Na PUMP AND TRANSMITTER RELEASE 219 ouabain. The whole experiment was carried out in the presence of EGTA. The time courses of the early tetani are rather poorly defined because the m.e.p.p. frequency cannot be measured accurately when its mean value is low, but it is clear that the responses to the standard tetanus do not differ greatly when this is applied before exposure to ouabain or for up to 30 min after the glycoside is present. The final two tetani, T4 and T5, produce step increases in the m.e.p.p. frequency. In two similar experiments the behaviour of m.e.p.p. frequency was essentially similar; it is not until the release rate has begun to rise under the influence of the glycosides that an applied tetanus produces a sustained increase in the m.e.p.p. frequency. During the initial delay period monovalent ion movements associated with the action potential train produce no irreversible changes in the rate of quantal release. A dual effect of external Na ions during the action of ouabain Birks & Cohen (1968a, b) showed that if part of the Na of the Ringer was replaced by sucrose, the rise in the mean quantal content of the e.p.p. and of the m.e.p.p. frequency that develop in the presence of cardiac glycosides, did not occur. From this observation they proposed that changes in the intracellular concentrations of monovalent cations initiated the glycoside effects. Birks & Cohen did not follow the m.e.p.p. frequency in reduced Na-Ringer beyond times when the m.e.p.p. frequency in the presence of full Na would have reached its peak value. The possibility that the glycoside effects are only delayed by reducing [Na]o therefore cannot be eliminated. Since it is difficult, especially in EGTA Ringer, to follow the m.e.p.p. frequency at end-plates for periods of several hours, this problem was approached in another way. End-plates were exposed to ouabain in the presence of full (114 mm) external Na and the m.e.p.p. frequency allowed to rise. The [Na]o was then reduced by isotonic substitution with either sucrose or LiCl while the concentration of ouabain was left unaltered at 0.1 mm. Since the terminals will load with Na less quickly when [Na]o is reduced, the rise in the m.e.p.p. frequency ought to slow down in media of reduced Na content. In fact quite the opposite is seen. If [Na]o is reduced after the m.e.p.p. frequency has begun to rise spontaneous release of transmitter is accelerated. Thus reducing [Na]o can have different effects on the m.e.p.p. frequency depending on the time after application of glycoside that the external Na is lowered. If [Na]o is low from the beginning of the application of ouabain, the rise in the m.e.p.p. frequency can be inhibited whereas if [Na]o is reduced after the m.e.p.p. frequency has begun to rise spontaneous release is accelerated. Fig. 7 illustrates an experiment in which an end-plate was exposed to
P. F. BAKER AND A. C. CRAWFORD ouabain and the Na of the Ringer replaced isotonically by LiCl for 10 min periods at various times during the treatment with the glycoside. Lithium Ringer produces little change in the m.e.p.p. frequency over such a short period when ouabain is absent (Crawford, 1975). During the delay before the onset of the rise in the m.e.p.p. frequency caused by ouabain, the reduction of [Na]0 has little effect. Just at the point where the m.e.p.p. frequency begins to rise, however, the reduction in [Na]0 produces a 220
Ouabain Na
Na
Li
Li
Na
Li
Na
Li
Na
60
.40
20
0
140 80 100 120 60 Time (min) Fig. 7. The m.e.p.p. frequency becomes extremely sensitive to reduction of the external Na concentration late in an exposure to 0-1 mm ouabain Ringer. 0-4 mm CaCl2 and 3-2 mM-MgCl2 present throughout. The vertical boxes indicate periods in which the NaCl of the Ringer was replaced isotonically by LiCl. Ouabain was present as indicated by the horizontal arrow. Note that before applying ouabain and for the first 40 min of a ouabain treatment, the short period in Li-Ringer has little effect on the m.e.p.p. frequency. The last reduction in external Na produces a dramatic but reversible increase in the m.e.p.p. frequency followed by the rise normally seen at this time in ouabain (compare Fig. 1 A). Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 220 C. 0
20
40
dramatic rise in the spontaneous release rate that recovers on raising the [Na]0 to normal. The rise in the m.e.p.p. frequency produced by the glycoside continues unaffected by this procedure (compare Fig. 7 and Fig. 1). The experiment shown in Fig. 7 was carried out in the presence of 0-4 mM-Ca. One possible explanation for the acceleration of the m.e.p.p. frequency might be that a Na-dependent Ca influx of the type described by Baker et al. (1969) in squid giant axons might be activated by changing to Li Ringer. In squid axons it has been shown that the Na-dependent
Na PUMP AND TRANSMITTER RELEASE 221 Ca influx is highly dependent on the concentration of intracellular Na, the Ca influx increasing roughly as the square of the internal Na concentration. It follows that late in a ouabain exposure, when the nerve terminals are sodium loaded, the Na-dependent Ca influx might be much larger than at the beginning. 300
Li Na
Li
Na
Li Na Ouabain
Na
200 4I-
100
0
40
80
120
Time (min)
Fig. 8. The sensitivity of the m.e.p.p. frequency to external Na ions that develops late in an ouabain exposure is unaffected by removing external Ca ions. All Ringers contained no added Ca, 3-6 mM-MgCl2 and 1 mM-EGTA. Ouabain was present at 0.1 mm as indicated by the horizontal arrow. Vertical boxes labelled Li indicate periods where LiCl replaced the NaCl of the Ringer isosmotically. Between the boxes the [Na]0 was 112 mm. Note that only after the m.e.p.p. frequency has begun to rise under the influence of ouabain, does the reduction in [Na]0 produce an increase in the m.e.p.p. frequency. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 20° C.
This explanation of the results does not however appear to be correct, for essentially similar results are obtained if the whole experiment is performed in the absence of external calcium and in the presence of 1 mmEGTA. Fig. 8 illustrates such an experiment. Here an end-plate was exposed on three occasions to Li Ringer before and during treatment of the preparation with ouabain. Once the m.e.p.p. frequency has begun to rise under the influence of the glycoside the reduction in [Na]0 produces a striking acceleration of spontaneous release. It might be argued that the development of this sensitivity of the m.e.p.p. frequency to external sodium ions late in a ouabain exposure is a specific effect of Li and is not in fact due to reduction in the [Na]0. This does not seem to be the case. Reduction in the [Na]0 by isotonic replacement with sucrose produces essentially similar results to isotonic replacement of external Na by LiCl. Fig. 9 shows an experiment where an
222 P. F. BAKER AND A. C. CRAWFORD end-plate was changed to a Ringer containing only half the normal Na concentration (the period labelled ' Suc' in the Figure). The whole experiment was run in the presence of 1 mM-EGTA and no added Ca. Reductions of [Na]0 below 50 % by replacement with sucrose are difficult because the amplitude of the m.e.p.p. falls below the base line noise of the recording system, a difficulty that is not encountered with LiCl as a Na substitute (Onodera & Yamakawa, 1966; Kelly, 1968). In normal end-plates, reducing the [Na]0 by this amount produces a small rise in the m.e.p.p. frequency (Kelly, 1965, 1968) but nothing as dramatic as that seen when the [Na]0 is 120
I
90
Ouabain
s Suc.
Na
Na
60
30
0
0
20
40 Time (min)
60
80
Fig. 9. The acceleration of the m.e.p.p. frequency by reducing the external Na concentration late in an ouabain exposure occurs if the Na-substitute is sucrose instead of LiCl. Ouabain (0.1 mM) was present as indicated by the horizontal arrow. Throughout the experiment the Ringer contained no added Ca, 3-6 mM-MgCl2 and 1 mM-EGTA. During the vertical box labelled 'i Suc' half of the NaCl of Ringer (57 mM) was replaced isosmotically by sucrose. Note the rise of the m.e.p.p. frequency before the i-sucrose period caused by ouabain and the sudden increase that occurs during the i-sucrose period. Ordinate: m.e.p.p. frequency (sec-1). Abscissa: time (min). Temperature 210 C.
reduced late in an exposure to ouabain. The rise in the m.e.p.p. frequency in this experiment is perhaps not as sharp as those of Figs. 7 and 8 but the results are not directly comparable since in Figs. 7 and 8 all of the external Na was replaced by Li ions whereas in Fig. 9 only 50 % of the Na has been replaced by sucrose. It must be concluded that ouabain not only produces a gradual rise in
Na PUMP AND TRANSMITTER RELEASE
223 the m.e.p.p. frequency but also makes spontaneous release of transmitter extremely sensitive to changes in the external Na concentration. The sensitivity to external Na ions is independent of the presence or absence of external Ca. DISCUSSION
The rise in the m.e.p.p. frequency produced by ouabain or by K-free Ringer occurs unimpeded in the presence of enough EGTA to lower the external Ca concentration to very low levels. It seems unlikely that the increase in the spontaneous release rate is produced by Ca entry into the nerve terminals either resulting from depolarization or by activation of a Na-dependent Ca entry system. Rather similar observations have been made by Paton, Vizi & Zar (1971) and Vizi (1972) on the release of acetylcholine from parasympathetic ganglia and brain slices respectively. These authors suggest a direct involvement of the Na pump in the release mechanism but at the frog neuromuscular junction the present results do not support this conclusion. It seems not to be the inhibition of the Na pump per se that is important, but rather the redistribution that this brings about of the monovalent cation gradients within the nerve terminal. The most compelling piece of evidence for this conclusion is that hastening the dissipation of the ion gradients by repetitive stimulation of the nerve during a ouabain exposure accelerates spontaneous release irreversibly. The similarity of the time course of the rise in the m.e.p.p. frequency in K-free Ringer, to that which occurs when an end-plate is bathed in Li Ringer (Crawford, 1975), suggests that similar processes are occurring in the two situations. In both cases the nerve terminals are presumably losing intracellular K but in Li Ringer the [Na]1 is probably falling as Li ions accumulate in the terminals, while in K-free Ringer [Na]1 would be expected to rise when the sodium pump is inhibited. While it is possible that accumulation of either Na or Li ions may promote the observed increase in m.e.p.p. frequency, it is perhaps more attractive to suppose that the fall in the intracellular K concentration, which occurs in both solutions, in some way underlies the rise in the m.e.p.p. frequency. However, to maintain this view one must postulate that Rb ions can replace K in the process since the m.e.p.p. frequency recovers normally from a period in K-free Ringer, when 2 mM-RbCl is present in the recovery solution. The action of ouabain on the m.e.p.p. frequency depends on the Na concentration of the medium and two effects of external Na can be distinguished. The onset of the rise in m.e.p.p. frequency produced by ouabain is markedly slowed in the absence of external Na; but once the m.e.p.p. frequency has begun to rise replacement of external Na leads to a dramatic increase in spontaneous release. The Na-dependence of the onset of the 8
P HY
247
224 P. F. BAKER AND A. C. CRAWFORD glycoside effect may reflect a Na dependence of glycoside binding to the nerve terminal, similar to that which has been described in squid axons (Baker & Willis, 1972); whereas the stimulation of release induced by a reduction in external Na subsequent to glycoside binding may be related to changes in Na-dependent extrusion of Ca ions from the nerve terminals. End-plates become extremely sensitive to reduction of the external Na concentration late in an exposure to ouabain Ringer. In a normal end-plate, changes in the external Na concentration produce very small changes in the m.e.p.p. frequency. Replacing all of the external Na by Li ions produces no immediate change in the m.e.p.p. frequency (Onodera & Yamakawa, 1966; Kelly, 1968; Crawford, 1975). Exchanging Na ions for tris, methylammonium, dimethylammonium and guanidinium ions similarly has little initial effect on the spontaneous release rate (Kelly, 1968; Otsuka & Endo, 1960). Replacing part of the external Na by sucrose produces a small acceleration of the m.e.p.p. discharge (Kelly, 1965; Gage & Quastel, 1966). If the preparation is partially depolarized with an elevated K concentration in the Ringer the m.e.p.p. frequency becomes much more sensitive to changes in the external Na concentration (Gage & Quastel, 1966; Birks, Burstyn & Firth, 1968). In this situation there appears to be an antagonism between Ca and Na, for the sensitivity to [Na],, is much greater if the external Ca concentration is high (Gage & Quastel, 1966; Birks et al. 1968). Na sensitivity seen late in a period of treatment with ouabain would seem to be different from that of a K depolarized end-plate for it is independent of external Ca ions. This rather disparate set of observations on the Na sensitivity of the m.e.p.p. frequency can however be summarized in a rather simple way. When the m.e.p.p. frequency is high it is very sensitive to reduction of the external Na concentration, and when the m.e.p.p. frequency is normal it is relatively insensitive to changes in external Na.
Miledi (1973) has recently shown in squid giant synapses that intracellular iontophoresis of Ca into the presynaptic terminals will increase the frequency of miniature post-synaptic potentials. It is attractive to suppose that the conditions under which the m.e.p.p. frequency is Nasensitive are all situations where the intracellular Ca concentration of the nerve terminals is high. If under these conditions, an important factor in regulating the intracellular concentration of ionized Ca is membrane transport, the Na sensitivity of the m.e.p.p. frequency might be explained on the basis of a Ca efflux mechanism present in the nerve terminals that requires the presence of external Na ions. Such a system has been described in squid axons and other nerves (see Baker, 1972) and it is possible that if the internal free Ca has been elevated in some way, it can be raised further by inhibiting the extra Ca efflux that would normally develop under these conditions. Such a situation would seem to explain the results obtained here for ouabain and perhaps also the results from potassium-depolarized preparations. With regard to the increase in the m.e.p.p. frequency produced by ouabain, at least two plausible explanations can be put forward. The changes in monovalent cation concentration resulting from inhibition of the Na pump
225 Na PUMP AND TRANSMITTER RELEASE could (1) release Ca from internal stores or (2) make internal Ca more effective in releasing transmitter, perhaps by increasing the affinity for Ca of the sites that bind Ca in the release process. It is difficult to choose at present between these two explanations but an elevated internal Ca concentration is certainly consistent with the Na sensitivity of the m.e.p.p. frequency that develops during the application of ouabain. At first sight an alternative explanation seems attractive. If there is a slow release of Ca from intracellular binding sites the maintenance of a low internal ionized Ca may be very dependent on membrane transport by a presumably Na-dependent mechanism. In these circumstances a rise in [Na]1 or fall in [K]i might inhibit calcium extrusion and lead to a rise in [Ca]i and transmitter release. Subsequent reduction in [Na]0 would slow extrusion further and give the observed increment in release. This simple explanation seems unable to account for the alteration in response to lithium during exposure to ouabain. In each test of Li, total replacement of [Na]0 by Li should inhibit Ca extrusion to a similar extent and should, if there is a steady leak of Ca from some internal store, produce a constant increment in [Ca]i. The observation that the response to Li grows during exposure to ouabain argues strongly that either the relation between [Ca]1 and transmitter release is non-linear (but see Crawford 1974), or that the rate at which Ca is leaking from an internal store is increasing during the exposure to glycoside. This work was performed while A. C. C. was in receipt of an M.R.C. Scholarship and was supported by a grant to P. F. B. from the Medical Research Council. REFERENCES
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BIRKS, R. I. & COHEN, M. W. (1968a). The action of sodium pump inhibitors on neuromuscular transmission. Proc. Roy. Soc. B 170, 381-399. BIRKS, R. I. & COHEN, M. W. (1968b). The influence of internal sodium on the behaviour of motor nerve endings. Proc. Roy. Soc. B 170, 401-421. BLAUSTEIN, M. P. & HODGKIN, A. L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. 200, 497-529. COOKE, J. D., OKAMOTO, K. & QUASTEL, D. M. J. (1973). The role of calcium in depolarization-secretion coupling at the motor nerve terminal. J. Physiol. 228, 459-497. COOKE, J. D. & QUASTEL, D. M. J. (1973). Transmitter release by mammalian motor nerve terminals in response to focal polarization. J. Physiol. 228, 377-405. CRAWFORD, A. C. (1974). The dependence of evoked transmitter release on external calcium ions at very low mean quantal contents. J. Physiol. 240, 255-278. CRAWFORD, A. C. (1975). Lithium ions and the release of transmitter at the frog neuromuscular junction. J. Physiol. 246, 109-142. ELMQVIST, D. & FELDMAN, D. S. (1965). Effects of sodium pump inhibitors on spontaneous acetylcholine release at the neuromuscular junction. J. Physiol. 181, 498-505. GAGE, P. W. & QUASTEL, D. M. J. (1966). Competition between sodium and calcium ions in transmitter release at a mammalian neuromuscular junction. J. Physiol. 185, 95-123. HODGKIN, A. L. & HOROWICZ, P. (1959). The influence of potassium and chloride ions on membrane potential of single muscle fibres. J. Physiol. 148, 127-160. HODGKIN, A. L. & KEYNES, R. D. (1955). Active transport of cations in giant axons of Sepia and Loligo. J. Physiol. 128, 28-60. HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1968). On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals. J. Physiol. 194, 381-407. KELLY, J. S. (1965). Antagonism between Na+ and Ca2+ at the neuromuscular junction. Nature, Lond. 205, 296-297. KELLY, J. S. (1968). The antagonism of Ca2+ by Na+ and other monovalent ions at the frog neuromuscular junction. Q. JZ exp. Physiol. 53, 239-249. KEYNES, R. D. (1954). The ionic fluxes in frog muscle. Proc. Roy. Soc. B 142, 359-382. LANDAU, E. M. (1969). The interaction of presynaptic polarization with calcium and magnesium in modifying spontaneous transmitter release from mammalian motor nerve terminals. J. Physiol 203, 281-299. MILEDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. Roy. Soc. B 183, 421-425. MILEDI, R. & THIES, R. E. (1971). Tetanic and post-tetanic rise in the frequency of minature end-plate potentials in low calcium solutions. J. Physiol. 212, 245-257. ONODERA, K. & YAMAKAWA, K. (1966). The effects of lithium on the neuromuscular junction of the frog. Jap. J. Physiol. 16, 541-550. OTSUKA, M. & ENDO, M. (1960). The effect of guanidine on neuromuscular transmission. J. Pharmac. exp. Ther. 128, 273-282. PATON, W. D. M., Vizi, E. S. & ZAR, M. ABoo (1971). The mechanism of acetylcholine release from parasympathetic nerves. J. Physiol. 215, 819-848. SOLOMON, A. K. (1952). The permeability of the human erythrocyte to sodium and potassium. J. gen. Physiol. 36, 57-110. Vizi, E. S. (1972). Stimulation by inhibition of (Na+-K+-Mg2+)-activated ATPase of acetylcholine release in cortical slices from rat brain. J. Physotl. 226, 95-117.
