J. Physiol. (1977), 268, pp. 319-333 With 7 text-figurem Printed in Great Britain

319

INTERACTING EFFECTS OF TEMPERATURE AND EXTRACELLULAR CALCIUM ON THE SPONTANEOUS RELEASE OF TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION

By C. J. DUNCAN AND HELEN E. STATHAM From the Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX

(Received 26 July 1976) SUMMARY

1. Temperature has a characteristic effect on the frequency of m.e.p.p.s at the frog neuromuscular junction; the spontaneous release of transmitter is not affected by temperature changes below 100 C whereas the system is highly temperature-sensitive above 200 C. 2. A very similar result is obtained when the experiment is repeated in saline containing Ca2+ buffered at 5 x 10-7 M, suggesting that it is unlikely that the major action of temperature is to cause an increase in Ca2+ influx. 3. It is suggested that the main effect of temperature at the presynaptic terminals is a modification of [Ca2+]i by an action on intracellular Ca2+ stores. 4. The interacting effects of theophylline and the divalent cation ionophore A23187 on m.e.p.p. frequency suggest that intracellular Ca2+ stores, in addition to the mitochondria, may well be of importance in controlling

[Ca2+]1. 5. Changes in [Ca2+]0 produce a modification of m.e.p.p. frequency, but the details of the response are dependent on temperature. The spontaneous release of transmitter is most sensitive to an increase in [Ca2+]. at 230 C, whereas the greater effect is found at 130 C when [Ca2+]o is lowered. 6. It is suggested (i) that m.e.p.p. frequency is primarily determined by [Ca2+] at the presynaptic terminals, (ii) that the presynaptic terminals are normally able to maintain [Ca2+]1 almost constant in spite of increases in Ca influx associated with ionophore treatment or with a rise in [Ca2+]0. However, if the steady-state position of [Ca2+], is previously raised by an increased efflux from intracellular stores (produced by elevated temperature or theophylline pre-treatment), increased influx causes a rise in both [Ca2+]1 and in m.e.p.p. frequency.

320

C. J. DUNCAN AND H. E. STATHAM INTRODUCTION

It is almost certain that the arrival of a nervous impulse at the presynaptic terminals of the frog neuromuscular junction causes a transient increase in the Ca2+ permeability of the plasma membrane and that the associated entry of Ca2+ triggers the synchronized release of quanta of transmitter. The magnitude of the evoked response (the e.p.p.) is therefore primarily dependent on [Ca2+]. (Crawford, 1974). Evidence is now accumulating that the frequency of the spontaneous release of quanta of transmitter (the m.e.p.p.s) is largely determined, on the other hand, by [Ca2+], (Baker, 1972; Alnaes & Rahamimoff, 1975; Statham & Duncan, 1975; Duncan & Statham, 1977). A variety of treatments are able to affect m.e.p.p. frequency and, as will be discussed later, many of these may act via a modification of [Ca2+]O. However, under certain conditions, the frog presynaptic terminals are apparently able to maintain [Ca2+]1 almost constant, even when Ca2+ influx is markedly increased; thus application of the divalent cation ionophore A23187, which accelerates Ca2+ entry, has little effect on spontaneous transmitter release at 170 C, but markedly augments m.e.p.p. frequency at temperatures above 200 C (Statham & Duncan, 1975; Statham, Duncan & Smith, 1976; Kita, Madden & Van der Kloot, 1975). In the present paper, the interacting effects of temperature and [Ca2+]O on m.e.p.p. frequency at the frog neuromuscular junction are considered and, in the light of the results obtained, an hypothesis is presented concerning the factors governing [Ca2+], in the presynaptic terminals. Such an analysis can serve to explain the probable modes of action of many agents that modify m.e.p.p. frequency. Temperature is known to increase markedly the frequency of m.e.p.p.s at the frog neuromuscular junction, with a Q10"-20' C of 4-0 (Fatt & Katz, 1952; Takeuchi, 1958). M.e.p.p. frequency at mammalian synapses shows an unusual response to temperature, with a positive Q10 at 5-14° C, a negative Q10 at 14-.28' C and a positive Q10 at 28-39° C (Hubbard, Jones & Landau, 1971; Ward, Crowley & Johns, 1972); temperature clearly has a complicated effect on the release system of the mammalian neuromuscular junction. In contrast, the effect on m.e.p.p. frequency of changing [Ca2+]o is reported as being relatively small at the mammalian neuromuscular junction, there being a fivefold increase in frequency for a 1000-fold increase in [Ca2+]0 (Boyd & Martin, 1956; Hubbard, 1961), although Ca2+-free solutions are known to depress the rate of release markedly (Elmqvist & Feldmnan, 1965). A rather greater sensitivity to extracellular Ca2+ has been described for the m.e.p.p. frequency recorded in the frog preparations

321 FROG NEUROMUSCULAR JUNCTION (Mambrini & Benoit, 1964; Birks, Burstyn & Firth, 1968). Changes in [Ca2+]0 will modify Ca2+ influx and the frog presynaptic terminals have a different sensitivity to changes in Ca2+ influx at temperatures above and below 200 C (Statham & Duncan, 1975); the effect of changing [Ca2+]o on m.e.p.p. frequency at the frog neuromuscular junction has therefore been studied at 13 and 230 C. METHODS

Electrophysiological recordings were made from the isolated cutaneous pectoris nerve muscle preparation of the frog Rana temporaria. Frogs were maintained in the laboratory at 50 C. All salines in which the preparations were bathed contained NaCl 115 mM, KCl 2*5 mm, Na2HPO4 2*15 mm and NaH2PO4 0-85 mm at pH 7-1. Normal saline contained 1.8 mm-CaCl2, but in most salines, however, [Ca2+]0 was maintained constant by the use of Ca2+-EGTA buffers, in which pH was maintained at 741 and 0 5 mM EGTA was added, together with the appropriate volume of£AnalaR standard volumetric solution of CaCl2. Free Ca2+ concentrations were calculated following the method of Portzehl, Caldwell & Rfegg (1964). Solutions containing theophylline were prepared by dissolving it in the appropriate saline immediately before use. The ionophore A23187 was dissolved in ethanol so that the stock solution contained 1 mg A23187 ml-'. The final concentration of A23187 used in experiments was 5 jug ml.-' saline and the concentration of ethanol was then 5 t1l. ml-". Control saline for A23187 experiments contained the same concentration of ethanol. The muscle was excised and equilibrated in the appropriate control saline for 45 min at 100 C. It was then pinned out in the experimental bath, the microelectrode inserted in the end-plate region and the temperature adjusted to the experimental value. Records of m.e.p.p.s began after a further 10 min. Electrophysiological recordings were from the muscle by the use of conventional glass electrodes filled with 3 m-KCl; the temperature of the bath was controlled (± 0.50 C) by a Peltier device. Potentials were fed through a cathode follower to a Tektronix 502A oscilloscope. M.e.p.p.s were recorded on moving film and counted. In any one experiment, the m.e.p.p.s were monitored in a single fibre at the intervals shown in the Figures and at least 100 m.e.p.p.s were measured. M.e.p.p. frequencies were compared using Student's t test. In experiments in which [Ca2+]. was varied, the new Ca2+-buffered saline was run into the bottom of the experimental bath whilst the level was maintained constant by continuous aspiration from the upper surface. The efficacy of this exchange system was checked by a series of preliminary experiments in which the Ca2+ content of the bath saline was checked by murexide after addition of standard volumes of saline. Colour development was read in a spectrophotometer (SP600) at 542 nm. Exchange was completed after the addition of 100 ml. but 500 ml. was used in all experiments. All inorganic salts used were AnalaR grade. Theophylline, EGTA and cyclic nucleotides were obtained from Sigma Chemical Co., St Louis, U.S.A. A23187 was a gift of Lilly Research Centre, Windlesham, Surrey. RESULTS

Effect of temperature on m.e.p.p. frequency The control m.e.p.p. frequency varied between different preparations, although only small oscillations (less than 10 % of the initial rate) were

322 C. J. DUNCAN AND H. E. STATHAM recorded in any one preparation. In most figures, therefore, comparisons of the effect of treatments are shown by expressing m.e.p.p. frequency as a ratio of the control frequency (expressed as F1/F0, where F0 is the control frequency and F1 the frequency after treatment).

40

_LC_3020

10

0 5

10

20 15 Temperature ('C)

25

Fig. 1. Effect of temperature on m.e.p.p. frequency at the frog neuromuscular junction at normal extracellular Ca2+. Ordinate: m.e.p.p. frequency (P'1) as a ratio of control frequency at 10° C (FO). [Ca2+]0 = 1.8 x 1O-3 M. Points = mean of thirteen separate experiments; vertical bars indicate ± s.E. of mean where this exceeds the diameter of the points.

The effect on m.e.p.p. frequency of changing temperature in thirteen separate preparations maintained in normal saline ([Ca2+]o = 1-8 x 10-3 M) is shown in Fig. 1; it is clear that the rate of spontaneous release is not affected by temperature changes below 10° C, whereas the system is particularly sensitive above 200 C. It was noteworthy that the m.e.p.p. frequency was more variable at 22-5 and 25° C, resulting in a much higher S.E. of mean. These experiments were also carried out on winter and summer frogs; no significant difference between the results was found in

323 FROG NEUROMUSCULAR JUNCTION these preparations, although the plots for winter animals were slightly shifted to the right. If, as has been suggested, m.e.p.p. frequency is primarily determined by [Ca2+]1 at the presynaptic terminals, these findings suggest that [Ca2+], rises markedly with increasing temperature. An accelerated Ca2+ influx with rising temperature would serve to explain this phenomenon and this possibility was tested by repeating the experiments in saline in which [Ca2+]0 was buffered at 5 x 10-7 M. The results are shown in Fig. 2 and, 50

40

30 U-

20

10

0

5

10

15 20 25 Temperature (0C) Fig. 2. Effect of temperature on m.e.p.p. frequency at low extracellular Ca2+. [Ca2+]. buffered at 5 x 1O-7 M with a Ca2+-EGTA buffer. Points = mean of seven separate experiments; vertical bars indicate ± s.E. of mean where this exceeds the diameter of the points. Other details as Fig. 1.

although m.e.p.p. frequency is always depressed at low [Ca2+]0, as shown below, the overall plot is essentially the same as that obtained at normal [Ca2+]0. Again m.e.p.p. frequency was more variable at the higher temperatures. Thus, although there are small differences between the plots obtained at 18 x 103 M [Ca2+]0 and 5 x 1O-7 M [Ca2+]0, their basic similarity suggests that it is unlikely that rising temperature causes a marked influx of Ca2+ and a consequent rise in m.e.p.p. frequency.

324

C. J. DUNCAN AND H. E. STATHAM

Role of intracellular Ca2+ stores As we have shown previously (Statham & Duncan, 1975, 1976), release of Ca2+ from intracellular binding sites is a major determinant in the elevation of [Ca2+], at the frog presynaptic terminals. For example, the divalent cation ionophore A23187 has a marked effect on m.e.p.p. frequency TABLE 1. Action of theophylline (10-3 M) on m.e.p.p. frequency at the frog neuromuscular junction at different values of [Ca2+]. Time

Expt.

[Ca2+]

no. 1 2 3 4 5

(M) 1-8 x 10-3 5X 10-7 5 X 10-7 0 0

,

0-5 1.84

5 2.10

1*33 1-06

2*12

1-67 0-57

(min) 15 2.03

2*50

10 2*19 1.95 1-95 2.48

2*77

2-44

2.51

3.25

1.61

2418 2.16

20 1-75 2.53 2.29 2.93 2-62

- I 30 1-98 1-63 1-92 2*43 2-39

Results expressed as a ratio of control frequency for each experiment at times stated after addition of theophylline. For [Ca2+]i = 0, saline contained no added Ca2+ and no EGTA and was prepared in deionized water and stored in plastic bottles. 5 x 10-7 M [Ca2+]0 maintained by Ca2+-EGTA buffered saline. Temperature = 17° C. All results for 5-30 min significantly different from controls (P < 0.001).

only at temperatures above 200 C, a response that can be suppressed by the application of Dantrolene sodium (DaNa) which apparently operates at intracellular Ca2+ stores. The inter-relationships between temperature and such stores in the regulation of [Ca2+], was investigated in the following experiments which supplemented previous studies. Theophylline has been shown to produce a small but significant rise in m.e.p.p. frequency and its action is probably a liberation of Ca2+ from intracellular stores (Statham & Duncan, 1976). It is unlikely that it modifies Ca2+ influx since the same effect (an approximate doubling in rate) is seen with 10-3 M theophylline in 1-8 x 10-3 M [Ca2+]O, 5 X 10-7 M [Ca2+]. and in saline in which Ca2+ is omitted but no EGTA is added (Table 1). The effect is reversible, m.e.p.p. frequency falling 10 min after the theophylline is washed off. Caffeine also increases m.e.p.p. frequency, even in the absence of external Ca2+, in frog (Onodera, 1973) and mammalian motor nerve terminals (Elmqvist & Feldman, 1965). These methyl xanthines have two main pharmacological actions: they are phosphodiesterase inhibitors (and so raise the intracellular levels of cyclic nucleotides) and they can free bound Ca2+ from intracellular membranes (Bianchi, 1961; Statham & Duncan, 1976). A series of experiments with dibutyryl cAMP 10-6 to 10-5 M) showed that this cyclic nucleotide did not increase m.e.p.p.

325 FROG NEUROMUSCULAR JUNCTION frequency at thefrog neuromuscular junction. Furthermore, a small decrease in the rate of spontaneous release was recorded at 10-4 to 10-3 M cAMP. Addition of 1O-5M cAMP plus 10-3 M theophylline has the same effect as 10-3 M theophylline alone, an approximate doubling in m.e.p.p. frequency. Such experiments agree with previous studies that show that cyclic nucleotides do not have an important role in triggering transmitter release either 4

3 U-

2

I

0

10

30 20 Time (min)

40

50

Fig. 3. Interacting effects of A23187 and theophylline. Ordinate: m.e.p.p. frequency (FE) as a ratio of control frequency before addition of theophylline (FO). Abscissa: time after addition of theophylline; A23187 added 20 min later at arrow, still in the presence of theophylline. A23187 = 5 ,ug ml.-'; theophylline = 1O-3 M, [Ca2+]0 = 1*8 x 10-3 M, temp. = 170 C. Time course of five separate experiments shown.

at motor nerve terminals under normal conditions (Wilson, 1974; Miyamoto & Breckenridge, 1974) or at the mammalian adrenal medulla

(Jaanus & Rubin, 1974). It is therefore probable that theophylline acts at the presynaptic terminals of the frog neuromuscular junction primarily by facilitating the release of Ca2+ from intracellular storage sites. The divalent cation ionophore A23187 has little effect on m.e.p.p. frequency at 170 C whereas it causes a marked and progressive increase in the rate of spontaneous release at 25° C (Statham & Duncan, 1975). The preparation was therefore pretreated with theophylline (10-3 M) at

326 C. J. DUNCAN AND H. E. STATHAM 170 C, thereby liberating Ca 2+ from intracellular stores and causing a small but significant rise in m.e.p.p. frequency. Addition of A23187 (5 ,ug ml.-'), still in the presence of theophylline and at 170 C, now causes an immediate and significant further rise in the rate of spontaneous release in most preparations (see Fig. 3); the effect is generally maintained over the next 30 min, but not invariably, and can be compared with the continuing response with theophylline alone (Statham & Duncan, 1976) and with the lack of response to A23187 alone at 170 C (Statham & Duncan, 1975).

Effect of raising [Ca2+]0 Raising [Ca2+]o above normal values at either

13 or 230 C causes a rise in m.e.p.p. frequency, with a sigmoidal relationship when the mean rate is plotted against [Ca2+]0. However, the details of the response are different at these two temperatures. The four separate experiments at 230 C (control frequency at 1P8 x 10-3 M [Ca2+]_- 0*7 to 2.9 m.e.p.p.s sec-', mean = 1-8 m.e.p.p.s sec'-) show a generally clear positive correlation between [Ca2+]. and m.e.p.p. frequency and the calculated individual regression lines in Fig. 4, in which log frequency is plotted against pCa, have similar slopes, with a mean value of 1- 16. As can be seen in the upper part of this Figure, where mean values for the change in m.e.p.p. frequency as a ratio of the control frequency when [Ca2+]o = 1.8 x 10-3 M have been calculated, there is only an approximately linear relationship between log mean F1/F0 andpCa and the graph is probably more nearlysigmoidal, as described for the neuromuscular junction of the mouse (Cooke, Okamoto & Quastel, 1973). The effect of increasing [Ca2+]0 at 130 C is less pronounced and much more variable results were obtained. Control frequencies at 1'8 x 10-3 M [Ca2+]o recorded at this temperature were 0-28-0-62 m.e.p.p.s sec1(mean = 0 47 m.e.p.p.s sec-'). Fig. 5 shows that in one experiment a fall in m.e.p.p. frequency was recorded with increasing [Ca2+]0 and this is reflected in the plot of log mean F11Fo vs. pCa; the fit to the calculated regression line is poor, the S.E.s are greater, and the slope of the line (0 64) is only half the value obtained at 230 C (see upper part of Fig. 5). In these experiments, in which [Ca2+]. was varied in the range 1-8 to 7.3 x 10-3 M, the m.e.p.p. frequency changed smoothly and equilibration to the new [Ca2+]O was accomplished in 20 min, both when [Ca2+]o was raised and also when it returned to the control value of 1'8 x 10-3 M Ca2+.

Effect of reducing [Ca2+]0 When [Ca2+]. is reduced the m.e.p.p. frequency falls at both 23 and 130 C, but under these conditions (in contrast with the effect of raising [Ca2+]O) the greatest effects are found at the lower temperature. The mean results from eight (at 230 C) and five (at 130 C) separate experiments are

FROG NEUROMUSCULAR JUNCTION 327 shown in Figs. 6 and 7. At 230 C, reducing [Ca2+]0 from 1-8 x 10-3 to 10-4 M had little effect, with mean m.e.p.p. frequency showing a small rise that was not statistically significant. Changing [Ca2+]0 to 5 x 10-5 M produced the maximum effect, with F1/F0 falling to 05, and further reductions in [Ca2+]0 produced little additional alteration in m.e.p.p. rate. It is clear that, at 230 C, a reduction in [Ca2+]0 is not a major factor in determining the frequency of spontaneous release (Fig. 6). At 130 C, however, reductions

ii~~3

/

/ /

10

LZ.

L? U_~~~~~~~~

1 0~~~~~~~~~~~~

0.~~~~~~~~~~0

02

3

*

0-2

04 Log

016

II

0-8

(Cal+]o (mM) Fig. 4

1-0

0-2

04 Log

0-6

0-8

1-0

[Ca2i]o (mM)

Fig. 5 Fig. 4. Effect of increasing [Ca2+J0 on m.e.p.p. frequency at 230 C. Abscissa: log [Ca2+]0 in range 1P8-7o3 x 1O-3 M. Lower part of figure (left-hand ordinate): results from four separate experiments with m.e.p.p. frequency (quanta sec-1) plotted on a log scale. Calculated regression lines shown. Upper part of figure (right-hand ordinate): mean of the four experiments, m.e.p.p. frequency (F1) as a ratio of control frequency (Fo) at 1o8 x 1O-3 M [Ca2+]0, log scale (±+5.E. of mean). Calculated regression line shown, although inspection of the points suggests that the relationship is prob. ably sigmoidal rather than linear. Fig. 5. Effect of increasing [Ca2+]0 on m.e.p.p. frequency at 130 C. Five separate experiments and their mean shown. Other details as Fig. 4. Note greater variability of results and smaller slope of mean results (upper part of Figure) when compared with results at 230 C (Fig. 4).

328

C. J. DUNCAN AND H. E. STATHAM --I--

1

03 LE LZ

0-1l

-//7A

. I

0

7

6

pCa

5

3

4

Fig. 6. Effect of decreasing [Ca2+]0 on m.e.p.p. frequency at 230 C. [Ca2+], was maintained by Ca2+-EGTA buffer in the range 8-7 x 10-8 M to 1-8 x 10-3 M; results for zero [Ca2+]0 (0.5 mM-EGTA) also shown ('0'). Ordinate: m.e.p.p. frequency (F1) as a ratio of control frequency (Fo) at 1-8 x 10-3 M[Ca2+]0, log scale. Abscissa: pCa. Points = mean of eight separate experiments + s.E. of mean.

o*3 Le.

0.1

0.03

0

6 pCa

5

4

3

Fig. 7. Effect of decreasing [Ca2+]0 on m.e.p.p. frequency at 130 C. Points = mean of five separate experiments + S.E. of mean. Other details as Fig. 6.

FROG NEUROMUSCULAR JUNCTION 329 in [Ca2+]0 had a more pronounced effect on the m.e.p.p. frequency, which fell at 10-4 M and at all lower concentrations to a level significantly different from the control rate at 1-8 x 10-3 M [Ca2+]0 (Fig. 7). An additional feature was also seen at 130 C; when all Ca2+ is removed, leaving 0 5 mMEGTA in the bathing saline, a significant rise in m.e.p.p. frequency was found, when compared with the results obtained at 5 x 10-7 M and 8 x 1f-8 M [Ca2+]0. Reductions in [Ca2+]0 produced a characteristic pattern of equilibration that was in contrast with the results obtained when [Ca2+]0 was raised. In a typical experiment, changing from 18 x 10-3 M [Ca2+]0 to 5 x 10-7 M [Ca2+]0 caused a transient doubling in m.e.p.p. frequency 1 min after exchange of salines, which fell below the control rate after a further 1 min. Equilibration was then completed after 14 min. Returning from 5 x 10-7 M to 1*8 x 10-3 M [Ca2+]0 produced a much slower rate of equilibration and some 50 min were required for the m.e.p.p.s to return to a steady rate. DISCUSSION

1. Control of [Ca2+]i We have suggested previously that m.e.p.p. frequency is dependent on [Ca2+], (Statham & Duncan, 1975, 1976). The systems that probably control [Ca2+]1 in the frog presynaptic terminals are as follows, (i) Passive movements of Ca2+ across the plasma membrane determined by the activities and the passive permeability for the ion. Passive influx can be modified experimentally at the frog presynaptic terminals by changes in [Ca2+]0 or by application of the divalent cation ionophore A23187 (Statham & Duncan, 1975, 1976; Kita et al. 1975). (ii) Active efflux of Ca2+ by membrane pumps. (iii) Active uptake by, and leakage from, the mitochondria which have been suggested to have an important regulatory role in a variety of cells (Borle & Anderson,1976; van Rossum, Smith & Beeton, 1976). (iv) There is evidence for the presence of additional freely exchangeable Ca2+ stores within the presynaptic terminals. Caffeine and theophylline probably act by displacing or accelerating the release of Ca2+ from such stores, whilst DaNa has the opposite effect (Statham & Duncan, 1976). [Ca2+]1 is established in such a system as the resultant steady-state position of these interacting fluxes; any one treatment may have opposing actions on the systems (i)-(iv) above and various treatments may interact with one another. Thus, a temperature rise is potentially able to modify all these opposing Ca2+-fluxes, accelerating both influx into the cytosol on the one hand and active efflux and uptake by the mitochondria on the other.

330

3C. J. DUNCAN AND H. E. STATHAM

2. The effect of temperature Fig. 1 shows the characteristic effect of temperature on the m.e.p.p. frequency at the frog neuromuscular junction ([Ca2+]0 = 1-8 x 1O-3 M), with a high temperature-sensitivity above 200 C. The major action of temperature could be either to increase [Ca2+]1 or to modify directly the release system and the mechanism of exocytosis. In the light of the interacting effects of temperature with other agents known to modify Ca2+ fluxes (see below), it seems most probable that the main effect of temperature is to modify [Ca2+],. Since essentially the same plot is obtained at low [Ca 2+]o (5 x 10-7 M), see Fig. 2, it is unlikely that rising temperature markedly increases Ca2+ influx by a modification of the Ca2+ permeability of the plasma membrane. We therefore conclude that the major effects of temperature on the systems that control [Ca2+]i at the frog presynaptic terminals are (i) to raise [Ca2+], by an action on intracellular Ca2+ stores, promoting efflux from either the mitochondria, or from other sites, (ii) to stimulate the Ca2+ pumps of the plasma membrane, a factor that will serve to oppose the effects of the mobilization of intracellular Ca2+ and hence to reduce m.e.p.p. frequency. These conflicting effects are seen most clearly at the mammalian neuromuscular junction (Hubbard et al. 1971; Ward et al. 1972). Whereas the over-all effect on the rat phrenic nerve-diaphragm preparation of raising the temperature from 0 to 370 C is a marked increase in m.e.p.p. frequency, the rate of spontaneous release falls over the intermediate temperature range of 150 C to about 280 C. The effect of temperature on the Ca2+ATPase of mammalian synaptosomes has been described elsewhere (Duncan, 1976) and this enzyme (and presumably Ca2+ transport) is almost completely inhibited at temperatures below 130 C. We have suggested that, on raising the temperature from 00 C, the Ca2+-transport systems begin to operate at 130 C, lowering [Ca2+]1, and m.e.p.p. frequency falls. Above about 280 C, however, Ca2+ mobilization exceeds the efficacy of the Ca2+ pumps, and both the equilibrium position of [Ca2+]i and the rate of spontaneous release rise progressively (Duncan & Statham, 1977). This complicated effect of temperature is not seen at the neuromuscular junction of the poikilothermic frog (Figs. 1. 2) and we conclude that, over the lower temperature range below 120 C, the Ca2+ pump of the plasma membrane, perhaps working in concert with the Ca2+ uptake mechanism of the mitochondria, operates effectively, maintaining [Ca2+]1 almost constant. At higher temperatures, mobilization of Ca2+ from intracellular stores comes to have the dominant effect and the steady-state level of [Ca2+], rises progressively.

FROG NEUROMUSCULAR JUNCTION

331

3. Modifications of Ca2+ influx by changes in [Ca2+]0 The results summarized in Figs. 4-7 agree with the general pattern described for frog (Mambrini & Benoit, 1964) and mammal (Cooke et al. 1973), although Birks et al. (1968) describe a fall in m.e.p.p. frequency in the frog preparation when [Ca2+]0 is raised from 1O0 to 4 0 mM. An increase in [Ca2+]0 will cause an increased Ca2+ influx which will be partially compensated by a rise in Ca2+ efflux associated with increased activity of the Ca2+ pump. Figs. 4 and 5 show that, as predicted, an increase in [Ca2+]0 causes a rise in m.e.p.p. frequency associated with an increase in Ca2+ influx, but it is clear that the magnitude of the response differs at 13 and 230 C, the system being most sensitive at the higher temperature. M.e.p.p. frequency is also sensitive to reductions in [Ca2+]4, and again the magnitude of the response differs at 13 and 230 C, although the effect is now greatest at the lower temperature. We believe that clues to these paradoxical effects may be obtained from the results of the following experiments. (i) Treatment of the frog neuromuscular junction with the divalent cation ionophore A23187 has little effect at 170 C, but causes a marked and progressive rise in m.e.p.p. frequency at 250 C (Statham & Duncan, 1975). (ii) M.e.p.p. frequency is sensitive to A23187 at 170 C, however, if the preparation is pretreated with theophylline, as shown in the present study

(Fig. 3).

(iii) The neuromuscular junction also becomes sensitive to A23187 at 170 C if the preparation is pretreated with Ni2+ (Kita et al. 1975). (iv) The stimulatory effect of A23187 at 250 C can be prevented by pretreatment with Dantrolene (Statham & Duncan, 1976). (v) The sensitivity of the mouse neuromuscular junction to changes in [Ca2+]0 is dependent on depolarization. The relationship between log m.e.p.p. frequency and log [Ca2+]0 was sigmoidal; depolarization caused a progressive shift of the curve upward and to the left. Thus, at 5 mm[K+]o m.e.p.p. frequency was relatively insensitive to changes in [Ca2+]1; raising [K+]0 to 30 mm caused a marked rise in frequency (associated with a change in PCa consequent upon the depolarization) whilst the slope of the response was very much greater (Cooke et al. 1973). Of these treatments, elevated temperature, depolarization (by increasing PCa), Ni2+ (see Kita et al. 1975) and theophylline (probably by interacting with intracellular stores, see above and Statham & Duncan, 1975, 1976) will all serve to raise [Ca2+], and under these conditions m.e.p.p. frequency is most sensitive to increased Ca2+ influx produced either by A23187 or by a rise in [Ca2+]0. It appears that the Ca2+ pump and the intracellular Ca2+ uptake systems are able to maintain [Ca2+],

C. J. DUNCAN AND H. E. STATHAM 332 constant in spite of a marked increase in Ca2+ influx, except when [Ca2+]1 is already raised by one of the treatments listed above. Conversely, the severe effects of high temperature combined with A23187 can be suppressed by pre-treatment with Dantrolene which probably acts here by suppressing Ca2+ release from intracellular stores in a way that is analogous to its mode of action at the sarcoplasmic reticulum in skeletal muscle. We suggest that these observations may serve to explain the effects of changing [Ca2+]0, Figs. 4-7. At 230 C (when [Ca2+]0 = 1-8 x 10-3 M), the initial equilibrium for [Ca2+], will be high, due to efflux from intracellular sites at this temperature and an increase in influx associated with a rise in [Ca2+]0 will have a considerable effect on [Ca2+], (Fig. 4). In contrast, at 130 C, when the system is in steady state at a low [Ca2+]1, the Ca2+ pump will more easily be able to compensate for the rise in Ca2+ influx associated with an increase in [Ca2+]0 (Fig. 5). However, a reduction in Ca2+ influx (associated with a fall in [Ca2+]0) at 130 C will be reflected in a fall in [Ca2+], and consequently in m.e.p.p. frequency. As [Ca2+]0 is progressively reduced, [Ca2+], will eventually fall below the operating range of the Ca2+ pump and [Ca2+]0 would come to have the major influence on [Ca2+]i and m.e.p.p. frequency (Fig. 7). Conversely, the presynaptic terminals will be less sensitive to reductions in [Ca2+]O at 230 C; at this temperature Ca2+ leakage from the intracellular storage sites will dominate the control of [Ca2+]1, maintaining it at a high level. Changes in Ca2+ influx, associated with a reduction in [Ca2+]0, would be of secondary importance and m.e.p.p. frequency never falls to very low levels at this temperature (see Fig. 6) although, clearly, a reduction in Ca2+ influx would assist the Ca2+ pump in reducing [Ca2+]1. Thus, an explanation for the different changes in m.e.p.p. frequency observed when [Ca2+]0 is altered at 13 and 230 C can be proposed and which depends on the rise in [Ca2+]1 at higher temperatures. REFERENCES ALNAES, E. & RAHAMIMOFF, R. (1975). On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol. 248, 285-306. BAKER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. molec. Biol. 24, 177-223. BIANCHI, C. P. (1961). The effect of caffeine on radiocalcium movement in frog sartorius. J. gen. Physiol. 44, 845-858. BIRKS, R. I., BURSTYN, P. G. R. & FIRTH, D. R. (1968). The form of sodium-calcium competition at the frog myoneural junction. J. gen. Physiol. 52, 887-907. BORLE, A. B. & ANDERSON, J. H. (1976). A cybernetic view of cell calcium metabolism. In Calcitum in Biological Systems, S.E.B. Symposia, vol. 30, ed. DUNCAN, C. J., pp. 141-160. Cambridge: Cambridge University Press. Bov-n, I. A. & MARTIN, A. R. (1956). The end-plate potential in mammalian muscle. J. Physiol. 132, 74-91.

FROG NEUROMUSCULAR JUNCTION

333

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. 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. DUNCAN, C. J. (1976). Properties ofthe Ca2+-ATPase activity of mammalian synaptic membrane preparations. J. Neurochem. 27, 1277-1279. DUNCAN, C. J. & STATHAM, H. E. (1977). The effect of temperature on spontaneous release at the mammalian neuromuscular junction. J. therm. Biol. 2, 23-25. ELMQVIST, D. & FELDMAN, D. S. (1965). Calcium dependence of spontaneous acetylcholine release at mammalian motor nerve terminals. J. Phyaiol. 181, 487-497. FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. HUBBARD, J. I. (1961). The effects of calcium and magnesium on the spontaneous release of transmitter from mammalian motor nerve endings. J. Physiol. 159, 507-517. HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1971). The effect of temperature change upon transmitter release, facilitation and post-tetanic potentiation. J. Physiol. 216, 591-609. JAANUS, S. D. & RUBIN, R. P. (1974). Analysis of the role of cyclic adenosine 3',5'monophosphate in catecholamine release. J. Physiol. 237, 465-476. KITA, H., MADDEN, K. & VAN DER KLOOT, W. (1975). Effects of the 'calcium ionophore' A-23187 on transmitter release at the frog neuromuscular junction. Life Sci. 17, 1837-1842. MAMBRINI, J. & BENOIT, P. R. (1964). Action du calcium sur la jonction neuromusculaire chez la Grenouille. C. r. Seanc. Soc. Biol. 158, 1454-1458. MIYAMOTO, M. D. & BRECKENRIDGE, B.McL. (1974). A cyclic adenosine monophosphate link in the catecholamine enhancement of transmitter release at the neuromuscular junction. J. gen. Physiol. 63, 609-624. ONODERA, K. (1973). Effect of caffeine on the neuromuscular junction of the frog and its relation to external calcium concentration. Jap. J. Physiol. 23, 587-597. PORTZEHL, H., CALDWELL, P. C. & RiYEGG, J. C. (1964). The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591. VAN RossUM, G. D. V., SMITH, K. P. & BEETON, P. (1976). Role of mitochondria in control of calcium content of liver slices. Nature, Lond. 260, 335-337. STATHAM, H. E. & DUNCAN, C. J. (1975). The action of ionophores at the frog neuromuscular junction. Life Sci. 17, 1401-1406. STATHAM, H. E. & DUNCAN, C. J. (1976). Dantrolene and the neuromuscular junction: evidence for intracellular calcium stores. Eur. J. Pharmac. 39, 143-152. STATHAM, H. E., DUNCAN, C. J. & SMITH, J. L. (1976). The effect of the ionophore A23187 on the ultrastructure and electrophysiological properties of frog skeletal muscle. Cell Tis8. Res. 173, 193-209. TAKEUCHI, N. (1958). The effect of temperature on the neuromuscular junction of the frog. Jap. J. Physiol. 8, 391-404. WARD, D., CROWLEY, W. J. & JOHNS, T. R. (1972). Effect of temperature at the neuromuscular junction. Am. J. Physiol. 222, 216-219. WILSON, D. F. (1974). The effects of dibutyryl cyclic adenosine 3',5'-monophosphate, theophylline and aminophylline on neuromuscular transmission in the rat. J. Pharmac. exp. Ther. 188, 447-452.

13

P11Y 268