J. Physiol. (1976), 255, pp. 575-586 Wi& 5 text-figure" Printed in Great Britain
575
THE EFFECT OF EXTERNAL CALCIUM AND MAGNESIUM IONS ON THE RESPONSE OF DENERVATED MUSCLE TO ACETYLCHOLINE
BY TESSA GORDON From the Department of Phy8iology, Medical School, Birmingham B15 2TJ
(Received 6 November 1974) SUIMMARY
1. The effect of external Ca and Mg on the membrane depolarization and contracture of rat denervated muscle in response to acetylcholine, ACh, was studied. 2. Raising external Ca concentration reduced the rate of rise and the amplitude of the ACh contracture, and prolonged its time course. 3. Increasing external Ca reduced the membrane depolarization in response to ACh. The dose increment required to elicit depolarization in high Ca concentration increased with external Ca, and was greater for depolarization than for contracture. 4. External Mg was less effective than Ca in reducing ACh depolarization but was more effective in reducing contracture. In contrast to Ca, Mg did not alter the time course of relaxation. 5. It is concluded that external Ca has two opposing effects on the ACh contracture: one of stabilizing the membrane and the other of increasing intracellular Ca concentration. External Mg may interfere with Ca influx and hence reduce contractility. INTRODUCTION
The external concentration of Ca is known to influence the ability of muscle to contract, but it is still unclear whether the influence is exerted on the contractile process directly or indirectly as a consequence of an action on the surface membrane. In skeletal muscle, extracellular Ca ions can influence contractility by affecting the electrical properties of the membrane. Thus, the failure of the K-contracture in Ca-free solutions can be accounted for, not by loss of Ca required for activation of the contractile proteins, but rather by the combined effect of the lowered membrane potential (Edman & Grieve, 1961; Jenden & Reger, 1963), and decreased contractile threshold (Costantin, 1968). On the other hand, there is evidence to suggest that the influx of 19-2
TESSA GORDON extracellular Ca ions across the chemically excited membrane of skeletal muscle may be more directly involved in activation ofthe contractile proteins: in denervated rat muscle which has been completely depolarized by K+, acetylcholine (ACh) causes an increased Ca influx and a contracture which varies in amplitude with the extracellular Ca concentration (Jenkinson & Nicholls, 1961). Under these experimental conditions, ACh does not change the membrane potential and presumably depolarization-induced Ca release does not occur. The effects of high Ca on the muscle membrane may, however, influence the contracture since there is evidence from studies on normally innervated frog muscle that high concentrations of Ca reduce carbachol depolarizations (Nastuk & Liu, 1966; see also Taylor, 1973). If high concentrations of Ca similarly reduce the ACh depolarization of the chemosensitive membrane of denervated muscle, then the enhancing action of Ca on the contracture may well be opposed by the effect on the membrane. It therefore seemed of interest to study the effects of changes in external Ca and Mg on depolarization and contracture responses of mammalian denervated muscle to ACh. 576
METHODS All experiments were performed in vitro on rat soleus muscles at room temperature. Adult albino rats of between 150 and 200 g body weight were used. The sciatic nerve was sectioned unilaterally or bilaterally in the thigh under ether anaesthesia, using aseptic precautions; 3-13 days later, the denervated soleus muscles were excised and secured in an organ bath for membrane potential or isometric tension recording. Membrane potential was recorded intracellularly with conventional 3 m-KCl-filled glass micropipettes (10-20 Ml resistance) connected by a short Ag-AgCl wire to a negative capacitance amplifier ELS-ELSA-2 and displayed on an oscilloscope (Tektronix 502) and with a pen recorder (Devices Ltd M2). Isometric tension was measured with Statham strain gauges (4 oz.) and recorded on a Devices M2 pen recorder. The muscles set up for potential recording were perfused with oxygenated Krebs-Henseleit solution at a rate of 4-8 ml./min. Fibres were impaled at a discrete site outside the former end-plate region but not in the myotendinous junctions, and doses of ACh or carbachol were given at regular intervals. Although the depolarization was maximal within 9-15 sec in all the solutions used, the muscles were exposed to the drug for 30-60 see; the exposure time was constant throughout each experiment. In the experiments in which ACh contractures were studied, the drug was in contact with the muscle until the maximum tension was attained. It was then washed out. Solutions. The normal Krebs-Henseleit solution used contained (mM): KCl, 4-7; NaCl, 118-4; CaCl2, 2-5; MgSO4, 1-2; KH2PO4, 1-11; NaHCO3, 25; glucose, 5-53. The solutions were aerated continuously with 95 % 02 and 5 % CO2 to attain a pH of 7-3. In the solutions which contained high concentrations of Cat+ or Mg2+, NaCl was replaced isomotically by CaCl2 or MgSO4, respectively. In the solution containing high concentrations of Ca, the Mg concentration remained at 1-2 mM; similarly in solutions containing high Mg concentrations 2-5 mm-Ca was present. The concentration of Ca could not be raised further than 12-5 mm without precipitating the Ca since the solubility coefficient of Ca2(PO4)3 is low. In the experiments in which Ca-
Ca-ACh INTERACTION IN SKELETAL MUSCLE
577
free solutions were used, 1-2 mM chelating agent, ethyleneglycol bis (aminoethylether)-N,N tetra-acetic acid (EGTA), was included in the bathing solution. The pH of the solution was adjusted to 7-3 by adding NaOH. The drugs used in these experiments were acetylcholine chloride (Laboratoire Lematte et Boinot Ltd), and carbachol (B.D.H. Ltd). All dilutions were made up in the same solution as that bathing the muscle. The dose of the drug was expressed in FuM for ACh and mm for carbachol. RESULTS
Parallel experiments were done in which ACh depolarization or contractures were measured. In solutions containing 2-5 mM-Ca, first a measurable contracture was recorded in response to doses of ACh between 0-55 and 1-0 /SM. The maximum tension and maximum rate of rise of tension increased with the dose until maximal responses were obtained at doses greater than 100 /2M (Fig. 2). The dose to elicit a measurable depolarization in single fibres was the same as for threshold ACh contractures and the amplitude of the response increased with ACh over the same dose range in the lower 50 % of the dose-response curve (Fig. 4). It was impossible to record depolarizations larger than 35 mV because of movement of the muscle.
Effect of Ca on ACh contracture The ACh contracture was reduced by raising the external concentration of Ca. Fig. 1 shows that the maximum tension developed by a muscle in response to ACh was less in the solution containing 12-5 mM-Ca. In 2-5 mM-Ca solution, muscles develop maximal tension in 3-7 sec, and then relax spontaneously. In solutions containing high concentrations of Ca, the time taken to reach peak tension is greatly prolonged and the muscles then relax very slowly, if at all. ACh would be expected to have diffused beyond the surface fibres after 7 sec but its effect on deeper fibres would normally be masked by the spontaneous relaxation of those at the surface. It is thus likely that, because the fibres do not relax, more became activated by ACh applied in solutions containing high concentrations of Ca. It is interesting in this respect that lowering the temperature of the muscle from 22 to 120 C mimics the effect of a fivefold increase in external Ca in reducing the rate of spontaneous relaxation and thereby prolonging the time course of contracture. Dose-response curves were plotted for each muscle in solutions containing the normal and raised Ca concentrations. The maximal tension and the maximum rate of rise of tension were measured. The tension developed by the muscle in solutions containing raised Ca concentrations was also measured at the time when that muscle reached peak tension in the normal solution. This latter tension has been used to plot the dose-response curves
TESSA GORDON 578 in Fig. 2, which shows that the curve is shifted to the right by raising the external Ca concentration while the slope of the curve and the maximum response are unaltered. The effect of raised Ca concentration is rapid and reversible. The ACh contractures in the experiment shown in Fig. 2 were reduced within 2-3 min of raising the Ca concentration, and this effect was reversible in the same time. 2*5mMCa
12-5 mmCa
r
-A
_ -1.1 ,uM
JN~~~~~~~~~~~~~W ~~2.7 PM
+
+
12z it
~~~5-5 FUM ACh 10 sec
Fig. 1. The effect of a fivefold increase in external Ca concentration on ACh contractures of a 4-day-denervated muscle.
As the Ca concentration was raised in steps from 2-5 to 12-5 mm, the dose-response curves were shifted progressively further to the right. Since the curves were parallel, the dose increment to elicit the same response in the high Ca solution was the same for different response amplitudes. The dose increment was expressed as the ratio of the doses of ACh which produced the same response in solutions containing high and normal concentrations of Ca. The ratio for three different responses was averaged for each experiment (Jenkinson, 1960) and plotted against the rise in Ca concentration which was expressed as the ratio of the high tQ the normal concentration. The plot shown in Fig. 3B summarizes the results from
Ca-ACI INTERACTION IN SKELETAL MUSCLE 579 twenty-four experiments. If the response was measured as the maximal rate of rise of tension, the dose increments were identical with those shown in the Figure. When the maximum tension was used as the parameter of response irrespective of the time at which it occurred, the dose increment was less. 5
4A
/
C
0
E
21 A
I
I
I
I
I
I
I
I
I
100 1000 Am ACh dose Fig. 2. Dose-response curves relating tension to ACh dose are plotted on 10
semilogarithmic scale for 13-day-denervated muscle in solutions containing 2*5 (@) and 12*5 mm-Ca (A). The tension recorded in 2-5 mm-Ca after being in high Ca solution is shown by open circles (0).
Effect of Ca on membrane potential changes produced by ACh Depolarization of superficial fibres was recorded in response to a range of ACh doses in solutions containing the normal and raised Ca concentrations. As the external Ca was raised two to five times above the normal concentration, ACh depolarization was reduced. This is shown by the shift of the dose-response curve for depolarization farther to the right with increasing Ca concentration (Fig. 4). The effects of raised Ca concentrations were rapid and reversible. The mean resting potential was 66 mV in solutions containing 2-5 mmCa and increased to 69 mV in 12-5 mM-Ca solutions (Table 1). The relationship between ACh sensitivity and external Ca is summarized in Fig. 3A. The plot shows that the increase in ACh dose required to depolarize the membrane is proportional to the rise in external Ca
580 TESSA GORDON concentration. The dose increment was the same whether ACh or carbachol was applied so that it is unlikely that Ca reduces sensitivity by altering cholinesterase activity. The slope of the curve obtained for the contractile response was 0 5 (Fig. 3B) as compared with 1P0 (Fig. 3A) for the depolarization response. If the contractile response to ACh was dependent only on voltage dependent release of internal Ca, the two plots might be expected
4E 63
1
2
3
4
5
6
1
[Caln /[Ca], 12 10
:2
4
2
3
4
5
6
10
12
[Ca]n /[Cal,
:I~~a:-
" /S
46 6
8
1
1
2
.8
10
12
2
6
-
2 2
4
6
[Mg] n /[Mg],
4
6
8
[Mg]n /[MgJ,
Fig. 3. The ACh dose increment, [ACh]/[ACh], to elicit response in high, and normal concentrations of Ca or Mg is plotted against the increase in Ca or Mg concentration, [Ca],, and [Mg],,, above normal, [Ca]1 and [Mg],. The dose increments in A and C are for depolarization response. In B and D the dose increments are to elicit contracture. The interrupted lines in each graph show the relationship between ACh dose and Ca or Mg concentration for contracture A and C, and depolarization, B and D.
to be similar. However, the depolarization was more reduced than the contracture by an increase in Ca, presumably because the enhancing effect of external Ca on the contracture is countered by the reduced depolarization of the surface membrane. The depolarization response of the fibres to ACh was unaltered in Ca-free solutions. It is probable that the diffusion of Ca from the membranes of fibres in the muscle
Ca-ACh INTERACTION IN SKELETAL MUSCLE
581
centre could replace Ca released from the superficial fibres in which potential was recorded. When the chelating agent was included in the bathing solution to help remove Ca from the membrane, the muscle fibres became depolarized. A small and variable drop of potential was measured in solutions containing 1 mM-EGTA; in these fibres there was a small increase in ACh depolarization. In solutions containing 2 mm-EGTA, resting membrane potential dropped significantly and ACh depolarization was depressed.
A
B
0
A
mV
30/ /
20 10
1|
2
C
|10120 mVL 110 10 sec
~
mV
p
20 20
10
1
D / 40 * |-
0//_
-
v
so1:0
10
1
PM
1
10 JAM
Fig. 4. Dose-response curves are plotted for depolarization in response to ACh. Responses in 2-5 mm-Ca are shown by filled circles. Responses recorded in solutions with raised Ca concentration are shown by filled triangles. A, 5-0 mM-Ca; B, 7-5 mM-Ca; C, 10 mi-Ca; D, 12-5 mM-Ca. Responses in solutions containing 2-5 mm-Ca, after muscles had been in high Ca solutions, are shown by open circles. Sample records of depolarizations recorded in presence of maintained concentrations of A, 1-67; B, 5-0; C and D, 2-7 j/M-ACh are shown in normal and high Ca solutions. The drug was administered at the beginning of each trace.
The effect of external Mg on ACh depolarization Raising the external Mg concentration also reduced the depolarization response of denervated muscle but Mg was less effective than Ca. Increasing the Mg concentration tenfold had the same effect on ACh depolarization as raising the Ca concentration twofold. The resting potential was not significantly altered by raising the Mg concentration (Table 1).
TESSA GORDON
582
TABLE 1. Resting potentials in normal and high external Ca and Mg bathing solutions Divalent ion concentration (mM)
Ca
Mg
5*0
1*2
7*5
1P2
10.0
1P2
12-5
1*2
2-5
6-0
2-5
12*0
Membrane potential (mY)
66*6+3-3, n = 20 (normal 66-6 ± 3 5, = 20 (normal 66-7 ± 3 9, 67-8+4-3, n = 20 (normal 66-7 ± 4*7, 69*0 ± 4*2, n = 30 (normal 66-2 ± 3-6, 66-5±3-7, n = 20 (normal 66*4 ± 3-2, 66-8±3-3, n = 20
n = 30)
67'2±2-8 n
(normal 66-9±441,
n = 40) n = 20) n =
50)
n =
20)
n =
20)
Values represent means and S.D. of observation; the normal solution contained 2*5 mm-Ca and 12 mm-Mg.
Mg and the ACh contracture If Mg ions in the extracellular medium influence only the muscle membrane during the action of ACh, it might be expected that the effect of raising the concentration on the contractile response to ACh would be as small as that on depolarization. However, Mg had a rather marked depressant effect on the contractile response. The peak tension developed in response to ACh was immediately reduced by raising the external concentration of Mg. The effect of Mg was concentration dependent, as shown in Fig. 5. It can be seen that raised Mg concentrations reduced the tension developed and the rate of rise of tension without altering the time course of the response. In all Mg solutions, the time taken for the muscle to reach peak tension in response to a dose of ACh varied between 6 and 9 see which contrast with 15-40 sec taken by muscles in solutions containing 12-5 mM-Ca. Moreover, the tension fell spontaneously after the peak tension: the fall of tension followed an exponential time course characteristic of the normal ACh contracture. The effect of the raising external Mg concentrations on the depolarization and contractile response was evaluated in the same way as in the experiments with increased external Ca. The shift of the dose-response curve to the right by raising Mg concentration was again measured by averaging the dose increment for three responses. The effects of Mg on ACh depolarizations and contractures are compared with the effects of Ca on these responses in Fig. 3. For both responses, the dose increment
583 Ca-ACh INTERACTION IN SKELETAL MUSCLE increased with the rise in the Ca or Mg concentration. With increasing Ca concentration, the dose increment for depolarization was twice that for
contracture (Fig. 3A, B). With Mg, on the other hand, the dose increment for contracture was five times that for depolarization.
1.2 mmMg
12 mmMgp
5.5 isM
]]
u
,M 55~
10 sec ACh Fig. 5. The effect of Mg on tension developed by denervated muscle in response to doses of ACh. High concentrations of Mg reduce the rate of rise and amplitude of ACh contractures without affecting the relaxation of the muscle.
DISCUSSION
It is known that ACh depolarizes chemosensitive membranes by increasing conductance to Na and K ions (Takeuchi & Takeuchi, 1960). Ca conductance is also increased so that Ca ions flow into the sarcoplasm (Jenkinson & Nicholls, 1961). At the end-plate region of innervated muscle, this Ca influx is small and it is only when the Ca concentration is raised to 80 mm that a localized contractile response can be observed on application of ACh (Takeuchi, 1963). Manthey (1974) has recently measured these localized contractions in response to carbachol in frog muscles which have been depolarized by high K solutions. He showed that the degree of contraction at the end-plate was closely related to the extracellular Ca concentration and excluded the possibility that carbachol could directly mobilize sequestered intracellular stores of Ca as suggested by Marco, Mikiten & Nastuk ( 1969). When Ca influx occurs along the entire length of the chemo-sensitive membrane of denervated muscle, ACh contractures can be elicited even when the membrane is completely depolarized by K ions (Jenkinson & Nicholls, 1961; Lorkovic, 1970). The results of the
TESSA GORDON present study show that the ACh contracture is diminished when external Ca concentration is raised, although it is known that the Ca influx into the muscle increases with the external concentration. It appears that in a muscle which is not depolarized but has a resting membrane potential, external Ca contributes only a small part of the Ca for activation of contraction and internal sources of Ca must also be involved. Since depolarization of the membrane is known to control the release of Ca ions from the sarcoplasmic reticulum (Hodgkin & Horowicz, 1960; Ashley & Ridgeway, 1970) the effect of external Ca in reducing ACh contractures could be accounted for by the stabilizing effect of Ca on the membrane. Ca has a well established effect on the electrical excitability of membranes: Na and K conductance of resting and active nerve and muscle membranes depends on the external Ca concentration (Brink, 1954; Shanes, 1958; Manery, 1966; Koketsu, 1969). To account for the finding that changes in external Ca concentration have similar effects to changes in membrane potential on the systems that control Na and K movements through nerve membranes, Frankenhaeuser & Hodgkin (1957) suggested that Ca is adsorbed on to the outer surface of the membrane and, by neutralizing negative fixed charges, sets up a local positive potential which would increase the electrical field within the membrane. A change in potential across the membrane would therefore alter the adsorption of the ions and thereby allow monovalent ions to pass through the membrane more readily. The effect of raising the external Ca concentration on the chemically excitable membrane is to reduce the depolarization in response to ACh. It could be that following interaction of ACh with the receptor, a conformational change would alter the binding of Ca to a site on or near the receptor, and allow Na and K to pass through the membrane readily: on this basis, it could be imagined that Ca ions would be less readily displaced in solutions containing high Ca concentrations, so that the ACh depolarization is reduced. The experiments of Taylor (1973) provide evidence that the cholinergic receptor at the mammalian end-plate is normally occupied by Ca and Mg ions and that these are displaced by quaternary ammonium agonists in an ion-exchange process. Since two monovalent carbachol ions exchange for one Mg or Ca ion, Taylor suggests that receptors are located in pairs that are bridged by a divalent cation and that the displacement of the cation leads to structural rearrangement which precedes and leads to 584
depolarization. The effect of external Ca in reducing ACh contracture was consistent with the reduced ACh depolarization, but the finding that external Ca reduced the contracture less than membrane depolarization indicated that Ca influenced the contractile response to ACh in opposing ways. By
585 Ca-ACh INTERACTION IN SKELETAL MUSCLE reducing the membrane depolarization, Ca depresses the voltage dependent release of Ca from internal stores and so reduces the contractile ability of the muscle. At the same time, external Ca enters the fibre to enhance the contracture and this Ca influx increases with the external concentration. The balance between the two opposing effects remains constant over a range of Ca concentrations. The finding of Jenkinson & Nicholls (1969) that the tension developed by denervated muscles to ACh was very much less when the membrane was completely depolarized also suggests that Ca influx normally contributes only a part of the Ca required for contraction. The Ca that enters from the extracellular medium may enhance contracture by increasing the release of Ca from internal stores (Endo, Tanaka & Ogawa, 1970; Ford & Podolsky, 1970, 1972). Ca influx into the sarcoplasm could also disturb the balance betwen release and uptake by the reticular system and it is in this way that high concentrations of Ca may interfere with the spontaneous relaxation following ACh depolarization. In contrast to Ca, high external Mg markedly inhibited the ACh contracture at concentrations which have little effect on ACh depolarization. There is evidence that the Ca influx at the end-plate is reduced by raising extracellular Mg concentration (Evans, 1974). Extracellular Mg could also act at sites other than the surface membrane. Since ACh increases membrane permeability to all cations, Mg ions may enter the cell and inhibit the contractile proteins directly. Experiments using isolated myofibrillar preparations (Portzehl, Zaoralek & Gaudin, 1969) and skinned muscle fibres (Kerrick-& Donaldson, 1972) have shown that more Ca is required for contraction when the intracellular Mg concentration is raised. I would like to thank Dr Gertra Vrbova for her continuous support and helpful advice during this investigation and I am grateful to the Wellcome Trust for their support. Thanks are due also to Dr Rosemary Jones with whom I collaborated in some of the experiments. A preliminary report of some of this work has been published with Dr Jones in this Journal (Gordon & Jones, 1971). This work forms part of a thesis accepted by Birmingham University for the degree of Ph.D. REFERENCES Asui Y, C. P. C. & RIDGEWAY, E. B. (1970). On the relationships between potential calcium transient and tension in single barnacle muscle fibres. J. Phy8iol. 209,
105-130. BRINK, F. (1954). The role of calcium in neural processes. Pharmac. Rev. 6, 233-298. COSTANTIN, L. L. (1968). The effect of calcium on contraction and conductance thresholds in frog skeletal muscle. J. Physiol. 195, 119-132. EDXAN, K. A. P. & GRIEVE, D. W. (1961). The role of calcium and zinc in the electrical and mechanical responses of frog sartorius muscle. Experientia 17, 557558.
ENDO, M., TANSxA, M. & OGAWA, Y. (1970). Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature, Lond. 228, 34-36.
586
TESSA GORDON
EvANs, R. H. (1974). The entry of labelled calcium into the innervated region of the mouse diaphragm muscle. J. Phy8iol. 240, 511-533. FORD, L. E. & PODOLSKY, R. J. (1970). Regenerative calcium release with muscle cells. Science, N.Y. 167, 58-59. FORD, L. E. & PODOL5KY, R. J. (1972). Intracellular calcium movements in skinned muscle cells. J. Phy8iol. 223, 21-33. FRNKxAEusER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Phy8iol. 137, 218-244. GORDON, TESSA & JONES, ROSEMARY (1971). The influence of CaO+ on the sensitivity of denervated muscle to acetylcholine. J. Phyeiol. 218, 34P. HODGKIN, A. L. & HOROwIcz, P. (1960). The effect of nitrate and other anions on the mechanical response of single muscle fibres. J. Phy8iol. 153, 404-412. JENDEN, J. J. & REGER, J. F. (1963). The role of resting potential changes in the contractile failure of frog sartorius muscles during calcium deprivation. J. Physiol. 169, 889-901. JENKNsoN, D. H. (1960). The antagonism between tubocurarine and substances which depolarize the motor end-plate. J. Physiol. 152, 309-324. JENKiNsON, D. H. & NicHoias, J. G. (1961). Contractures and permeability changes produced by acetylcholine in depolarized denervated muscle. J. Phy-iol. 159, 1 11127. KERRICK, W. G. L. & DONALDSON, S. K. B. (1972). The effects of Mg on subminimum CaO+-activated tension in skinned fibres of frog skeletal muscle. Biochim. biophy8. Acta 275, 117-122. Koxsmu, K. (1969). Calcium and the excitable cell membrane. Neuro8ci. Re8. Progr. Bull. 2, 1-39. LopmovI6, K. (1970). Acetylcholine contractures of denervated muscles in sodium free solutions. Am. J. Phyaiol. 219, 1496-1504. MANERY, J. P. (1966). Effects of Ca ions on membranes. Fedn Proc. 25, 1804-1810. MANTmY, A. A. (1974). Changes in Ca permeability of muscle fibers during desensitization to carbamylcholine. Am. J. Phy-iol. 226, 481-489. MARco, L. A., MIKITEN, T. M. & NAsTUK, W. L. (1969). Histochemical detection of calcium released at neuromuscular junctions of oscillating frog muscle fibres. Fedn Proc. 28, 457. NAsTuK, W. L. & Lru, J. H. (1966). Muscle post-junctional membrane: changes in chemosensitivity produced by calcium. Science, N. Y. 154, 266-267. PORTZEHL, H., ZAORELEK, P. & GAUDIN, J. (1969). The activation by Ca of the ATPase of extracted muscle fibre with variation of ionic strength, pH and concentration of MaATP. Biochim. biophy8. Acta 189, 440-448. SHANEs, A. M. (1958). Electrochemical aspects of physiological and pharmacological action in excitable cells. Part I. The resting cell and its alterations by extrinsic factors. Phar-mac. Rev. 10, 59-164. TAEuCHI, N. (1963). Effects of calcium on the conductance change of the end-plate membrane during the action of transmitter. J. Phyiol. 167, 141-155. T~xEucm, A. & TAEUCHI, N. (1960). On the permeability of end-plate membrane during the action of transmitter. J. Phyeiol. 154, 52-67. TAYLOR, D. B. (1973). The role of inorganic ions in ion exchange processes at the cholinergic receptor of voluntary muscle. J. Pharmacy. exp. Ther. 186, 537-551.
575
THE EFFECT OF EXTERNAL CALCIUM AND MAGNESIUM IONS ON THE RESPONSE OF DENERVATED MUSCLE TO ACETYLCHOLINE
BY TESSA GORDON From the Department of Phy8iology, Medical School, Birmingham B15 2TJ
(Received 6 November 1974) SUIMMARY
1. The effect of external Ca and Mg on the membrane depolarization and contracture of rat denervated muscle in response to acetylcholine, ACh, was studied. 2. Raising external Ca concentration reduced the rate of rise and the amplitude of the ACh contracture, and prolonged its time course. 3. Increasing external Ca reduced the membrane depolarization in response to ACh. The dose increment required to elicit depolarization in high Ca concentration increased with external Ca, and was greater for depolarization than for contracture. 4. External Mg was less effective than Ca in reducing ACh depolarization but was more effective in reducing contracture. In contrast to Ca, Mg did not alter the time course of relaxation. 5. It is concluded that external Ca has two opposing effects on the ACh contracture: one of stabilizing the membrane and the other of increasing intracellular Ca concentration. External Mg may interfere with Ca influx and hence reduce contractility. INTRODUCTION
The external concentration of Ca is known to influence the ability of muscle to contract, but it is still unclear whether the influence is exerted on the contractile process directly or indirectly as a consequence of an action on the surface membrane. In skeletal muscle, extracellular Ca ions can influence contractility by affecting the electrical properties of the membrane. Thus, the failure of the K-contracture in Ca-free solutions can be accounted for, not by loss of Ca required for activation of the contractile proteins, but rather by the combined effect of the lowered membrane potential (Edman & Grieve, 1961; Jenden & Reger, 1963), and decreased contractile threshold (Costantin, 1968). On the other hand, there is evidence to suggest that the influx of 19-2
TESSA GORDON extracellular Ca ions across the chemically excited membrane of skeletal muscle may be more directly involved in activation ofthe contractile proteins: in denervated rat muscle which has been completely depolarized by K+, acetylcholine (ACh) causes an increased Ca influx and a contracture which varies in amplitude with the extracellular Ca concentration (Jenkinson & Nicholls, 1961). Under these experimental conditions, ACh does not change the membrane potential and presumably depolarization-induced Ca release does not occur. The effects of high Ca on the muscle membrane may, however, influence the contracture since there is evidence from studies on normally innervated frog muscle that high concentrations of Ca reduce carbachol depolarizations (Nastuk & Liu, 1966; see also Taylor, 1973). If high concentrations of Ca similarly reduce the ACh depolarization of the chemosensitive membrane of denervated muscle, then the enhancing action of Ca on the contracture may well be opposed by the effect on the membrane. It therefore seemed of interest to study the effects of changes in external Ca and Mg on depolarization and contracture responses of mammalian denervated muscle to ACh. 576
METHODS All experiments were performed in vitro on rat soleus muscles at room temperature. Adult albino rats of between 150 and 200 g body weight were used. The sciatic nerve was sectioned unilaterally or bilaterally in the thigh under ether anaesthesia, using aseptic precautions; 3-13 days later, the denervated soleus muscles were excised and secured in an organ bath for membrane potential or isometric tension recording. Membrane potential was recorded intracellularly with conventional 3 m-KCl-filled glass micropipettes (10-20 Ml resistance) connected by a short Ag-AgCl wire to a negative capacitance amplifier ELS-ELSA-2 and displayed on an oscilloscope (Tektronix 502) and with a pen recorder (Devices Ltd M2). Isometric tension was measured with Statham strain gauges (4 oz.) and recorded on a Devices M2 pen recorder. The muscles set up for potential recording were perfused with oxygenated Krebs-Henseleit solution at a rate of 4-8 ml./min. Fibres were impaled at a discrete site outside the former end-plate region but not in the myotendinous junctions, and doses of ACh or carbachol were given at regular intervals. Although the depolarization was maximal within 9-15 sec in all the solutions used, the muscles were exposed to the drug for 30-60 see; the exposure time was constant throughout each experiment. In the experiments in which ACh contractures were studied, the drug was in contact with the muscle until the maximum tension was attained. It was then washed out. Solutions. The normal Krebs-Henseleit solution used contained (mM): KCl, 4-7; NaCl, 118-4; CaCl2, 2-5; MgSO4, 1-2; KH2PO4, 1-11; NaHCO3, 25; glucose, 5-53. The solutions were aerated continuously with 95 % 02 and 5 % CO2 to attain a pH of 7-3. In the solutions which contained high concentrations of Cat+ or Mg2+, NaCl was replaced isomotically by CaCl2 or MgSO4, respectively. In the solution containing high concentrations of Ca, the Mg concentration remained at 1-2 mM; similarly in solutions containing high Mg concentrations 2-5 mm-Ca was present. The concentration of Ca could not be raised further than 12-5 mm without precipitating the Ca since the solubility coefficient of Ca2(PO4)3 is low. In the experiments in which Ca-
Ca-ACh INTERACTION IN SKELETAL MUSCLE
577
free solutions were used, 1-2 mM chelating agent, ethyleneglycol bis (aminoethylether)-N,N tetra-acetic acid (EGTA), was included in the bathing solution. The pH of the solution was adjusted to 7-3 by adding NaOH. The drugs used in these experiments were acetylcholine chloride (Laboratoire Lematte et Boinot Ltd), and carbachol (B.D.H. Ltd). All dilutions were made up in the same solution as that bathing the muscle. The dose of the drug was expressed in FuM for ACh and mm for carbachol. RESULTS
Parallel experiments were done in which ACh depolarization or contractures were measured. In solutions containing 2-5 mM-Ca, first a measurable contracture was recorded in response to doses of ACh between 0-55 and 1-0 /SM. The maximum tension and maximum rate of rise of tension increased with the dose until maximal responses were obtained at doses greater than 100 /2M (Fig. 2). The dose to elicit a measurable depolarization in single fibres was the same as for threshold ACh contractures and the amplitude of the response increased with ACh over the same dose range in the lower 50 % of the dose-response curve (Fig. 4). It was impossible to record depolarizations larger than 35 mV because of movement of the muscle.
Effect of Ca on ACh contracture The ACh contracture was reduced by raising the external concentration of Ca. Fig. 1 shows that the maximum tension developed by a muscle in response to ACh was less in the solution containing 12-5 mM-Ca. In 2-5 mM-Ca solution, muscles develop maximal tension in 3-7 sec, and then relax spontaneously. In solutions containing high concentrations of Ca, the time taken to reach peak tension is greatly prolonged and the muscles then relax very slowly, if at all. ACh would be expected to have diffused beyond the surface fibres after 7 sec but its effect on deeper fibres would normally be masked by the spontaneous relaxation of those at the surface. It is thus likely that, because the fibres do not relax, more became activated by ACh applied in solutions containing high concentrations of Ca. It is interesting in this respect that lowering the temperature of the muscle from 22 to 120 C mimics the effect of a fivefold increase in external Ca in reducing the rate of spontaneous relaxation and thereby prolonging the time course of contracture. Dose-response curves were plotted for each muscle in solutions containing the normal and raised Ca concentrations. The maximal tension and the maximum rate of rise of tension were measured. The tension developed by the muscle in solutions containing raised Ca concentrations was also measured at the time when that muscle reached peak tension in the normal solution. This latter tension has been used to plot the dose-response curves
TESSA GORDON 578 in Fig. 2, which shows that the curve is shifted to the right by raising the external Ca concentration while the slope of the curve and the maximum response are unaltered. The effect of raised Ca concentration is rapid and reversible. The ACh contractures in the experiment shown in Fig. 2 were reduced within 2-3 min of raising the Ca concentration, and this effect was reversible in the same time. 2*5mMCa
12-5 mmCa
r
-A
_ -1.1 ,uM
JN~~~~~~~~~~~~~W ~~2.7 PM
+
+
12z it
~~~5-5 FUM ACh 10 sec
Fig. 1. The effect of a fivefold increase in external Ca concentration on ACh contractures of a 4-day-denervated muscle.
As the Ca concentration was raised in steps from 2-5 to 12-5 mm, the dose-response curves were shifted progressively further to the right. Since the curves were parallel, the dose increment to elicit the same response in the high Ca solution was the same for different response amplitudes. The dose increment was expressed as the ratio of the doses of ACh which produced the same response in solutions containing high and normal concentrations of Ca. The ratio for three different responses was averaged for each experiment (Jenkinson, 1960) and plotted against the rise in Ca concentration which was expressed as the ratio of the high tQ the normal concentration. The plot shown in Fig. 3B summarizes the results from
Ca-ACI INTERACTION IN SKELETAL MUSCLE 579 twenty-four experiments. If the response was measured as the maximal rate of rise of tension, the dose increments were identical with those shown in the Figure. When the maximum tension was used as the parameter of response irrespective of the time at which it occurred, the dose increment was less. 5
4A
/
C
0
E
21 A
I
I
I
I
I
I
I
I
I
100 1000 Am ACh dose Fig. 2. Dose-response curves relating tension to ACh dose are plotted on 10
semilogarithmic scale for 13-day-denervated muscle in solutions containing 2*5 (@) and 12*5 mm-Ca (A). The tension recorded in 2-5 mm-Ca after being in high Ca solution is shown by open circles (0).
Effect of Ca on membrane potential changes produced by ACh Depolarization of superficial fibres was recorded in response to a range of ACh doses in solutions containing the normal and raised Ca concentrations. As the external Ca was raised two to five times above the normal concentration, ACh depolarization was reduced. This is shown by the shift of the dose-response curve for depolarization farther to the right with increasing Ca concentration (Fig. 4). The effects of raised Ca concentrations were rapid and reversible. The mean resting potential was 66 mV in solutions containing 2-5 mmCa and increased to 69 mV in 12-5 mM-Ca solutions (Table 1). The relationship between ACh sensitivity and external Ca is summarized in Fig. 3A. The plot shows that the increase in ACh dose required to depolarize the membrane is proportional to the rise in external Ca
580 TESSA GORDON concentration. The dose increment was the same whether ACh or carbachol was applied so that it is unlikely that Ca reduces sensitivity by altering cholinesterase activity. The slope of the curve obtained for the contractile response was 0 5 (Fig. 3B) as compared with 1P0 (Fig. 3A) for the depolarization response. If the contractile response to ACh was dependent only on voltage dependent release of internal Ca, the two plots might be expected
4E 63
1
2
3
4
5
6
1
[Caln /[Ca], 12 10
:2
4
2
3
4
5
6
10
12
[Ca]n /[Cal,
:I~~a:-
" /S
46 6
8
1
1
2
.8
10
12
2
6
-
2 2
4
6
[Mg] n /[Mg],
4
6
8
[Mg]n /[MgJ,
Fig. 3. The ACh dose increment, [ACh]/[ACh], to elicit response in high, and normal concentrations of Ca or Mg is plotted against the increase in Ca or Mg concentration, [Ca],, and [Mg],,, above normal, [Ca]1 and [Mg],. The dose increments in A and C are for depolarization response. In B and D the dose increments are to elicit contracture. The interrupted lines in each graph show the relationship between ACh dose and Ca or Mg concentration for contracture A and C, and depolarization, B and D.
to be similar. However, the depolarization was more reduced than the contracture by an increase in Ca, presumably because the enhancing effect of external Ca on the contracture is countered by the reduced depolarization of the surface membrane. The depolarization response of the fibres to ACh was unaltered in Ca-free solutions. It is probable that the diffusion of Ca from the membranes of fibres in the muscle
Ca-ACh INTERACTION IN SKELETAL MUSCLE
581
centre could replace Ca released from the superficial fibres in which potential was recorded. When the chelating agent was included in the bathing solution to help remove Ca from the membrane, the muscle fibres became depolarized. A small and variable drop of potential was measured in solutions containing 1 mM-EGTA; in these fibres there was a small increase in ACh depolarization. In solutions containing 2 mm-EGTA, resting membrane potential dropped significantly and ACh depolarization was depressed.
A
B
0
A
mV
30/ /
20 10
1|
2
C
|10120 mVL 110 10 sec
~
mV
p
20 20
10
1
D / 40 * |-
0//_
-
v
so1:0
10
1
PM
1
10 JAM
Fig. 4. Dose-response curves are plotted for depolarization in response to ACh. Responses in 2-5 mm-Ca are shown by filled circles. Responses recorded in solutions with raised Ca concentration are shown by filled triangles. A, 5-0 mM-Ca; B, 7-5 mM-Ca; C, 10 mi-Ca; D, 12-5 mM-Ca. Responses in solutions containing 2-5 mm-Ca, after muscles had been in high Ca solutions, are shown by open circles. Sample records of depolarizations recorded in presence of maintained concentrations of A, 1-67; B, 5-0; C and D, 2-7 j/M-ACh are shown in normal and high Ca solutions. The drug was administered at the beginning of each trace.
The effect of external Mg on ACh depolarization Raising the external Mg concentration also reduced the depolarization response of denervated muscle but Mg was less effective than Ca. Increasing the Mg concentration tenfold had the same effect on ACh depolarization as raising the Ca concentration twofold. The resting potential was not significantly altered by raising the Mg concentration (Table 1).
TESSA GORDON
582
TABLE 1. Resting potentials in normal and high external Ca and Mg bathing solutions Divalent ion concentration (mM)
Ca
Mg
5*0
1*2
7*5
1P2
10.0
1P2
12-5
1*2
2-5
6-0
2-5
12*0
Membrane potential (mY)
66*6+3-3, n = 20 (normal 66-6 ± 3 5, = 20 (normal 66-7 ± 3 9, 67-8+4-3, n = 20 (normal 66-7 ± 4*7, 69*0 ± 4*2, n = 30 (normal 66-2 ± 3-6, 66-5±3-7, n = 20 (normal 66*4 ± 3-2, 66-8±3-3, n = 20
n = 30)
67'2±2-8 n
(normal 66-9±441,
n = 40) n = 20) n =
50)
n =
20)
n =
20)
Values represent means and S.D. of observation; the normal solution contained 2*5 mm-Ca and 12 mm-Mg.
Mg and the ACh contracture If Mg ions in the extracellular medium influence only the muscle membrane during the action of ACh, it might be expected that the effect of raising the concentration on the contractile response to ACh would be as small as that on depolarization. However, Mg had a rather marked depressant effect on the contractile response. The peak tension developed in response to ACh was immediately reduced by raising the external concentration of Mg. The effect of Mg was concentration dependent, as shown in Fig. 5. It can be seen that raised Mg concentrations reduced the tension developed and the rate of rise of tension without altering the time course of the response. In all Mg solutions, the time taken for the muscle to reach peak tension in response to a dose of ACh varied between 6 and 9 see which contrast with 15-40 sec taken by muscles in solutions containing 12-5 mM-Ca. Moreover, the tension fell spontaneously after the peak tension: the fall of tension followed an exponential time course characteristic of the normal ACh contracture. The effect of the raising external Mg concentrations on the depolarization and contractile response was evaluated in the same way as in the experiments with increased external Ca. The shift of the dose-response curve to the right by raising Mg concentration was again measured by averaging the dose increment for three responses. The effects of Mg on ACh depolarizations and contractures are compared with the effects of Ca on these responses in Fig. 3. For both responses, the dose increment
583 Ca-ACh INTERACTION IN SKELETAL MUSCLE increased with the rise in the Ca or Mg concentration. With increasing Ca concentration, the dose increment for depolarization was twice that for
contracture (Fig. 3A, B). With Mg, on the other hand, the dose increment for contracture was five times that for depolarization.
1.2 mmMg
12 mmMgp
5.5 isM
]]
u
,M 55~
10 sec ACh Fig. 5. The effect of Mg on tension developed by denervated muscle in response to doses of ACh. High concentrations of Mg reduce the rate of rise and amplitude of ACh contractures without affecting the relaxation of the muscle.
DISCUSSION
It is known that ACh depolarizes chemosensitive membranes by increasing conductance to Na and K ions (Takeuchi & Takeuchi, 1960). Ca conductance is also increased so that Ca ions flow into the sarcoplasm (Jenkinson & Nicholls, 1961). At the end-plate region of innervated muscle, this Ca influx is small and it is only when the Ca concentration is raised to 80 mm that a localized contractile response can be observed on application of ACh (Takeuchi, 1963). Manthey (1974) has recently measured these localized contractions in response to carbachol in frog muscles which have been depolarized by high K solutions. He showed that the degree of contraction at the end-plate was closely related to the extracellular Ca concentration and excluded the possibility that carbachol could directly mobilize sequestered intracellular stores of Ca as suggested by Marco, Mikiten & Nastuk ( 1969). When Ca influx occurs along the entire length of the chemo-sensitive membrane of denervated muscle, ACh contractures can be elicited even when the membrane is completely depolarized by K ions (Jenkinson & Nicholls, 1961; Lorkovic, 1970). The results of the
TESSA GORDON present study show that the ACh contracture is diminished when external Ca concentration is raised, although it is known that the Ca influx into the muscle increases with the external concentration. It appears that in a muscle which is not depolarized but has a resting membrane potential, external Ca contributes only a small part of the Ca for activation of contraction and internal sources of Ca must also be involved. Since depolarization of the membrane is known to control the release of Ca ions from the sarcoplasmic reticulum (Hodgkin & Horowicz, 1960; Ashley & Ridgeway, 1970) the effect of external Ca in reducing ACh contractures could be accounted for by the stabilizing effect of Ca on the membrane. Ca has a well established effect on the electrical excitability of membranes: Na and K conductance of resting and active nerve and muscle membranes depends on the external Ca concentration (Brink, 1954; Shanes, 1958; Manery, 1966; Koketsu, 1969). To account for the finding that changes in external Ca concentration have similar effects to changes in membrane potential on the systems that control Na and K movements through nerve membranes, Frankenhaeuser & Hodgkin (1957) suggested that Ca is adsorbed on to the outer surface of the membrane and, by neutralizing negative fixed charges, sets up a local positive potential which would increase the electrical field within the membrane. A change in potential across the membrane would therefore alter the adsorption of the ions and thereby allow monovalent ions to pass through the membrane more readily. The effect of raising the external Ca concentration on the chemically excitable membrane is to reduce the depolarization in response to ACh. It could be that following interaction of ACh with the receptor, a conformational change would alter the binding of Ca to a site on or near the receptor, and allow Na and K to pass through the membrane readily: on this basis, it could be imagined that Ca ions would be less readily displaced in solutions containing high Ca concentrations, so that the ACh depolarization is reduced. The experiments of Taylor (1973) provide evidence that the cholinergic receptor at the mammalian end-plate is normally occupied by Ca and Mg ions and that these are displaced by quaternary ammonium agonists in an ion-exchange process. Since two monovalent carbachol ions exchange for one Mg or Ca ion, Taylor suggests that receptors are located in pairs that are bridged by a divalent cation and that the displacement of the cation leads to structural rearrangement which precedes and leads to 584
depolarization. The effect of external Ca in reducing ACh contracture was consistent with the reduced ACh depolarization, but the finding that external Ca reduced the contracture less than membrane depolarization indicated that Ca influenced the contractile response to ACh in opposing ways. By
585 Ca-ACh INTERACTION IN SKELETAL MUSCLE reducing the membrane depolarization, Ca depresses the voltage dependent release of Ca from internal stores and so reduces the contractile ability of the muscle. At the same time, external Ca enters the fibre to enhance the contracture and this Ca influx increases with the external concentration. The balance between the two opposing effects remains constant over a range of Ca concentrations. The finding of Jenkinson & Nicholls (1969) that the tension developed by denervated muscles to ACh was very much less when the membrane was completely depolarized also suggests that Ca influx normally contributes only a part of the Ca required for contraction. The Ca that enters from the extracellular medium may enhance contracture by increasing the release of Ca from internal stores (Endo, Tanaka & Ogawa, 1970; Ford & Podolsky, 1970, 1972). Ca influx into the sarcoplasm could also disturb the balance betwen release and uptake by the reticular system and it is in this way that high concentrations of Ca may interfere with the spontaneous relaxation following ACh depolarization. In contrast to Ca, high external Mg markedly inhibited the ACh contracture at concentrations which have little effect on ACh depolarization. There is evidence that the Ca influx at the end-plate is reduced by raising extracellular Mg concentration (Evans, 1974). Extracellular Mg could also act at sites other than the surface membrane. Since ACh increases membrane permeability to all cations, Mg ions may enter the cell and inhibit the contractile proteins directly. Experiments using isolated myofibrillar preparations (Portzehl, Zaoralek & Gaudin, 1969) and skinned muscle fibres (Kerrick-& Donaldson, 1972) have shown that more Ca is required for contraction when the intracellular Mg concentration is raised. I would like to thank Dr Gertra Vrbova for her continuous support and helpful advice during this investigation and I am grateful to the Wellcome Trust for their support. Thanks are due also to Dr Rosemary Jones with whom I collaborated in some of the experiments. A preliminary report of some of this work has been published with Dr Jones in this Journal (Gordon & Jones, 1971). This work forms part of a thesis accepted by Birmingham University for the degree of Ph.D. REFERENCES Asui Y, C. P. C. & RIDGEWAY, E. B. (1970). On the relationships between potential calcium transient and tension in single barnacle muscle fibres. J. Phy8iol. 209,
105-130. BRINK, F. (1954). The role of calcium in neural processes. Pharmac. Rev. 6, 233-298. COSTANTIN, L. L. (1968). The effect of calcium on contraction and conductance thresholds in frog skeletal muscle. J. Physiol. 195, 119-132. EDXAN, K. A. P. & GRIEVE, D. W. (1961). The role of calcium and zinc in the electrical and mechanical responses of frog sartorius muscle. Experientia 17, 557558.
ENDO, M., TANSxA, M. & OGAWA, Y. (1970). Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature, Lond. 228, 34-36.
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TESSA GORDON
EvANs, R. H. (1974). The entry of labelled calcium into the innervated region of the mouse diaphragm muscle. J. Phy8iol. 240, 511-533. FORD, L. E. & PODOLSKY, R. J. (1970). Regenerative calcium release with muscle cells. Science, N.Y. 167, 58-59. FORD, L. E. & PODOL5KY, R. J. (1972). Intracellular calcium movements in skinned muscle cells. J. Phy8iol. 223, 21-33. FRNKxAEusER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Phy8iol. 137, 218-244. GORDON, TESSA & JONES, ROSEMARY (1971). The influence of CaO+ on the sensitivity of denervated muscle to acetylcholine. J. Phyeiol. 218, 34P. HODGKIN, A. L. & HOROwIcz, P. (1960). The effect of nitrate and other anions on the mechanical response of single muscle fibres. J. Phy8iol. 153, 404-412. JENDEN, J. J. & REGER, J. F. (1963). The role of resting potential changes in the contractile failure of frog sartorius muscles during calcium deprivation. J. Physiol. 169, 889-901. JENKNsoN, D. H. (1960). The antagonism between tubocurarine and substances which depolarize the motor end-plate. J. Physiol. 152, 309-324. JENKiNsON, D. H. & NicHoias, J. G. (1961). Contractures and permeability changes produced by acetylcholine in depolarized denervated muscle. J. Phy-iol. 159, 1 11127. KERRICK, W. G. L. & DONALDSON, S. K. B. (1972). The effects of Mg on subminimum CaO+-activated tension in skinned fibres of frog skeletal muscle. Biochim. biophy8. Acta 275, 117-122. Koxsmu, K. (1969). Calcium and the excitable cell membrane. Neuro8ci. Re8. Progr. Bull. 2, 1-39. LopmovI6, K. (1970). Acetylcholine contractures of denervated muscles in sodium free solutions. Am. J. Phyaiol. 219, 1496-1504. MANERY, J. P. (1966). Effects of Ca ions on membranes. Fedn Proc. 25, 1804-1810. MANTmY, A. A. (1974). Changes in Ca permeability of muscle fibers during desensitization to carbamylcholine. Am. J. Phy-iol. 226, 481-489. MARco, L. A., MIKITEN, T. M. & NAsTUK, W. L. (1969). Histochemical detection of calcium released at neuromuscular junctions of oscillating frog muscle fibres. Fedn Proc. 28, 457. NAsTuK, W. L. & Lru, J. H. (1966). Muscle post-junctional membrane: changes in chemosensitivity produced by calcium. Science, N. Y. 154, 266-267. PORTZEHL, H., ZAORELEK, P. & GAUDIN, J. (1969). The activation by Ca of the ATPase of extracted muscle fibre with variation of ionic strength, pH and concentration of MaATP. Biochim. biophy8. Acta 189, 440-448. SHANEs, A. M. (1958). Electrochemical aspects of physiological and pharmacological action in excitable cells. Part I. The resting cell and its alterations by extrinsic factors. Phar-mac. Rev. 10, 59-164. TAEuCHI, N. (1963). Effects of calcium on the conductance change of the end-plate membrane during the action of transmitter. J. Phyiol. 167, 141-155. T~xEucm, A. & TAEUCHI, N. (1960). On the permeability of end-plate membrane during the action of transmitter. J. Phyeiol. 154, 52-67. TAYLOR, D. B. (1973). The role of inorganic ions in ion exchange processes at the cholinergic receptor of voluntary muscle. J. Pharmacy. exp. Ther. 186, 537-551.
