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Acetylcholine-induced currents in denervated mouse soleus muscle: Effects of antagonists
Neuropharmacologl Vol. 29, No. 6, PP. K-577, Printed in Great Britain. All rights reserved
1990
0028-3908/90 53.00 + 0.00 Copyright Q 1990 Pergamon Press plc
ACETYLCHOLINE-INDUCED CURRENTS IN DENERVATED MOUSE SOLEUS MUSCLE: EFFECTS ANTAGONISTS
OF
H LORKOVI~ Abteilung
fiir Allgemeine
Physiologie Federal
der Universitat Ulm, Einsteinallee Republic of Germany
11, D-7900
Ulm,
(Accepfed 22 January 1990) Summary--Acetylcholine-induced currents were measured in partially depolarized mouse soleus muscles, denervated for 336 days by using a point voltage clamp. When 0.25 PM d-tubocurarine (d-Tc) was used, the weak currents provoked by 0. I PM ACh. at a holding potential of -20 mV, were barely affected, while the large currents provoked by 2-5 nM ACh were decreased by more than 50%. By contrast, weak and strong ACh-induced currents were proportionally diminished when, under similar conditions, 20-100 p M ipratropium was used. Currents were proportionally diminished by d-Tc when the holding potential was set at + 15 mV, a level corresponding to the reversal potential of the current provoked by small concentrations of ACh. In non-denervated flexor digitorum brevis muscles, d-Tc had the same re!ative effect at small and at large concentrations of ACh, independent of the holding potential. The reversal potential for the ACh-induced currents was about + 14 mV for small concentrations of ACh and decreased to about + 3 mV with 4 PM ACh in denervated soleus muscles. It was concluded that denervated soleus muscles, in contrast to the endplate regions of non-denervated mouse muscles, contain a small proportion of highly ACh-sensitive, weakly d-Tc-sensitive. predominantly Na+-permeable ACh receptors. These receptors are presumably responsible for the non-fading ACh-induced currents, described before, for the denervated mouse soleus muscle. K~J, tt,ord.r-ACh
The
membrane
current
receptor,
provoked
denervated
by
muscle,
endplate,
d-tubocurarine,
ipratropium.
those used in the work of Beranek and VyskoEil (1967). The desensitizing receptors of denervated muscles are not to be confounded with the ACh receptors present at the endplates. The contribution of the latter receptors to the current signals recorded in this work was shown to be negligibly small.
acetylcholine
in denervated mouse soleus muscles and measured by the point voltage-clamp technique has two components, the major one (comprising more than 90% of the current provoked by S-10 ,uM ACh, at a holding potential of - 30 mV), decaying with a half-time of about 15 set, the other showing no decay during exposure times to ACh not exceeding 2min. This non-fading current may be elicited by concentrations of ACh smaller than those required for the fading current. Other evidence, e.g. a distinctly more positive reversal potential for the non fading current, pointed to the presence, in denervated mouse soleus muscles, of two types of acetylcholine receptor (Lorkovic, 198 1). Pharmacological differences between the AChreceptor occurring in normal endplates of nondenervated mammalian muscles and those found in non-junctional areas of denervated muscles (Beranek and VyskoEil, 1967) are known to be related to types of ACh receptor. differing in at least one subunit (c vs ;‘; for a brief review see Schuetze, 1986). Pharmacological differentiation was recently extended to ACh receptors present in juvenile muscles, by using dichohnes (Lorkovic, 1989). In this work, it was desirable to see whether the putative desensitizing and non-desensitizing receptors of denervated muscles could be distinguished by means similar to (ACh)
METHODS Female
adult
mice (NMRI,
Interfauna,
Tuttlingen,
F.R.G.) were anaesthetised with ether and a portion of the ischiadicus nerve was excised. Three to 6 days after denervation the soleus muscle was dissected, split longitudinally and trimmed to a thickness of less than 0.2 mm. The preparations were placed in a narrow flow-through chamber and superfused (at about 1 ml/min) by a solution containing (in mM) Na+ 121, K+ 30, Mg *+ 1.0, Ca*+ 0.1, 3(N-morpholino)propane sulphonic acid (MOPS) buffer (Sigma M 1254) 1.O, methanesulphonate 150 and Cl2.2. The large concentration of K+ and the small concentration of Ca2+ were used to avoid contractions caused by ACh. The pH of the solutions was 7.2 and the temperature 20-22’C. The membrane current, caused by ACh, was measured by a conventional two-microelectrode voltage clamp apparatus. With a muscle fibre length exceeding 7 mm and an electric length constant less 573
574
H.
bRKOVk
than 2 mm, a uniform clamping of the membrane voltage was certainly not achieved at rest and it must have been even less complete during the action of ACh. To check for possible artefacts arising from this and from the fact that under the conditions described one part of the muscle was affected by the agonist before those parts lying more distally in the superfusion channel, measurements of current were made by using another chamber in which the muscle was placed, such that its long axis was perpendicular to the flow of solution. Less than 1 mm of the muscle fibre length was exposed to the solution (and thus to the agonist) under these conditions, the rest of the muscle being mechanically clamped by a tightly fitting plastic frame covered with Vaseline. Except for the smaller size of the current signals measured under the latter conditions, there was no difference between the results obtained with the two methods. The microelectrodes, used to measure the membrane potential, had resistances of 30-50 MR, while current-passing microelectrodes (made of twocompartment glass tubing) had resistances of 4-7 MR when filled with 2 M citrate solution. The resting potentials of denervated soleus muscle fibres, measured in the specified solutions, were -30 to - 38 mV. Non-denervated flexor digitorum superficialis muscles were used to measure the membrane current provoked by succinylcholine in normal endplates. The reason was that amplitudes of membrane currents. recorded from these muscles, were much less variable than those measured in non-denervated soleus muscles. Succinylcholine was used to avoid agonist hydrolysis by choline esterase. After a successful impalement of a muscle fibre by the two microelectrodes, recognized by the current of about 1 nA being required for a 10 mV change in holding potential. ACh, in concentrations ranging from IO-’ to 1.6 x 10m5 M. was applied for 4-20 set by injecting the appropriate solution into the chamber while the agonist-free superfusion was stopped. Currents provoked by ACh, ranging from lo-‘” to 3 x IO-' A, could be measured reliably. Depending on the concentration of ACh used, the application of the agonist was followed by a lo-200 set pause, in which the agonist was washed out. The peak (or the plateau, at small concentrations of ACh) amplitude of the ACh current was read directly from the screen of a Tektronix D 11 oscilloscope. Antagonistic drugs were applied in the (AChfree) superfusing and in the injection solutions. Tubocurarine (d-Tc, ASTA, Bielefeld, F.R.G.) and ipratropium bromide (Boehringer, Ingelheim, F.R.G.) were mainly used. Neither of these drugs had any influence on the rate of desensitization, when applied in concentrations 10 times as large as those used in the work reported here. A superfusion period of at least 1 min. in the presence of an antagonist, was allowed to pass before the measurement of the (diminished) ACh current was made. In successful
runs 12-14 applications of ACh were made in one single fibre, the microelectrodes remaining impaled for up to 25 min. Only those results were evaluated in which the control current responses (usually that to 2 x 10e6 ACh at the beginning of the series and following the washout of an antagonist) differed by less than 5%. The variability of the amplitudes of the currents was such that the SEM, calculated from 3-7 fibres, was less than 10% of the average value. Variability was less when the data were normalized with respect to the ACh current, measured in the presence of the largest ACh concentrations used. The SEM, calculated in this way, was less than the size of the symbols used in the illustrations, except for the smallest concentrations of ACh used. The data were plotted as log-log graphs, permitting an adequate representation of the smallest current responses. RESULTS
Typical changes in membrane currents caused by a large concentration of ACh at holding potentials ranging from -25 to + 15 mV are shown in Fig. 1. A reversal potential of about --2mV was obtained from the muscle fibre used. Inspection revealed that the time course of the current changes, e.g. for the holding potential of - 5 and + 5 mV, were not the same, the current returning to the resting level at a faster rate at + 5 than at - 5 mV and undershooting that level after washout of ACh with +5 mV. The behaviour of another fibre is shown at high current gain in Fig. 2. The holding potential was +8 mV. The time course of the change in current 15
-----
-I
5 h.p. (mV)
-5
----.
-15 -25
-
-. b
t
8 XICFM EAChI
Fig. I. Holding potentials (h.p., upper lines) and membrane current changes (lower lines) caused by 8 PM ACh in a mouse soleus muscle fibre denervated for 4 days. Arrow downward: application of ACh, arrow upward: washout of ACh, in this and in the rest of the figures.
Antagonists on ACh currents +amv -J
t tACh1
x~O-~M
Fig. 2. Changes in membrane current caused in a mouse soleus muscle fibre denervated for 5 days at two different concentrations of ACh. The holding potential was 8 mV for both records. (upper tracing, large concentrations of ACh) is shown to be complicated: following the initial negative (inward) deflection, the current trace turned positive only to return to the negative after the washout of ACh. Such a result suggests that the direction of current depends on the concentration of ACh prevailing at the point of insertion of the microelectrode, smaller concentrations of ACh causing inward, larger concentrations outward membrane current at the same holding potential. This explanation is supported by the lower tracing, obtained in the same muscle fibre and at the same holding potential, with a concentration of ACh equal to one tenth of that used for the upper tracing. A monophasic, inward current was now provoked. The reversal potential, as a function of the concentration of ACh, was studied in two muscles. With records like the one presented in Fig. 2, the reversal potential was determined from the larger current component. The results obtained are shown in Fig. 3. The reversal potential was close to + 15 mV at small concentrations of ACh and it decreased to + 3 mV as the concentration was raised, without reaching a constant value in the range of the concentrations of
IL -
515
ACh used. The relative permeability ratio (PNa/PK at small concentrations of ACh, divided by P,,/P,at large concentrations) was calculated from the Goldman equation assuming the concentrations of Ki, K,, Na, and Na, to be 150, 30, 7 and 121 mM, respectively. The value obtained was 1.71, suggesting that the ACh receptor channel was relatively more permeable to Na+ at small rather than at large concentrations of ACh. An alternative (and more likely) explanation is that two types of acetylcholine receptors are involved in the current response, the type responsible for the current at small concentrations of ACh having a greater P,,/P,ratio than the one activated by large concentrations. It may be mentioned that the same relative permeability ratio was obtained independent of whether ionic concentrations or activities were considered. The relation between the reversal potential and the concentration of ACh was shifted to larger concentrations of ACh when d-tubocurarine (d-Tc) was present (Fig. 3) as expected. The shift appeared to be greater at large, rather than small concentrations but, because of the small slope of the curves at small concentrations, the shift could not be measured with adequate precision. A more convenient method was to measure the effect of d-Tc on the amplitude of the ACh current. The results are shown in Figs 4 and 5. The upper, high-gain current records were obtained by using 0.3 PM ACh and d-Tc was applied 1 min before ACh was applied, to obtain the middle record. Barely any effect of d-Tc can be seen. When a larger concentration of ACh was used (Fig. 4, lower, high-gain records), the current deflection measured at the same holding potential as before (- 20 mV) was nearly 50% less in the presence of the same concentration of d-Tc as used before (0.25 PM) than in its absence. The relative blocking effect of d-Tc increased continuously as the concentration of ACh was increased from 0.1 to 4 p M. Most of the change in the effectiveness of d-Tc occurred between 0.2 and 1.OPM ACh (Fig. 5). These results suggest that the putative, highly ACh-sensitive and Na-permeable ACh receptor is less sensitive to the
12. lomV
66-
01
d -Tc
0.1 0.2 0.4
1
2 L 8
2.5 x lO+M
[ACh] x10-‘M Fig. 3. Reversal potential (ordinate, mV) as a function of the concentration of ACh ([ACh]); average values i SEM. Mouse soleus muscle denervated for 4 days, four fibres per point.
Fig. 4. Changes in membrane current provoked by ACh (0.3 and 8.0 uM) in a mouse soleus muscle fibre, denervated for 3 days. ‘Both records were obtained at a holding potential (h.p.) of -20 mV; d-Tc (0.25 PM) was applied for I min before and during application of ACh (middle records).
Fig. 5. Maximum amplitude of the current changes proby ACh. Mouse soleus muscle, denervated for 5 days, 5 fibres per point. Full circles: control, open circles: 0.25 PM d-Tc.
concentrations of ACh. As with d-Tc. the curve for ipratropium was shifted to larger concentrations of ACh as the concentration of blocker was increased. These results suggest that the putative, highly Napermeable ACh receptors are about equally sensitive to ipratropium as the less ACh-sensitive bulk of ACh receptors of the denervated mouse soleus muscle. The effect of atropine was similar to that of ipratropium, except that the current tended to saturate at a level lower than maximum at larger concentrations of the drug. The time course of the current responses suggested that, in addition to its wellknown antagonistic effect, atropine may enhance desensitization. Two local anaesthetics, tetracaine and lidocaine, were also used. The effects appeared to be similar to those of d-Tc, but desensitization problems prevented definite conclusions to be made.
voked
blocking action of d-Tc than the less ACh-sensitive bulk of the receptors.
When larger concentrations of d-Tc (0.5, 1.0, 2.5, 10.0 p M) were used, the curve representing the maximum current in the presence of d-Tc was shifted to the right but its slope at any current amplitude was not changed much. In Lineweaver-Burk plots of the data (with l/fi on the ordinate, where I is the measured current amplitude, cf. e.g. Adams, 1975) the slopes of the lines connecting the data points were always smaller for small rather than for large concentrations of ACh. The difference in the slopes was increased in the presence of d-Tc (not shown). Blocking effects, different from those obtained with d-Tc, were found with the muscarinic ACh receptorblocker ipratropium. The effect of a small concentration of the drug is shown in Fig. 6. The relative diminution of the current responses to ACh was, if anything, somewhat greater at small than at large den. SOL 30 mM K+
1
@I/
/
Ipr. 2x16%
/
,6L!-_ 0.3
.ld’
1.0 M ACh
CONTROL
EXPERIMENTS
The existence of two reversal potentials for the currents provoked by ACh makes it possible to check the idea of differential pharmacological sensitivity of the two putative types of receptor by using holding potentials corresponding to either reversal potential. In one series of such experiments, the holding potential was + 15 mV, the reversal potential for the more sensitive of ACh type receptor. The control curve and the curve for 0.5 PM d-Tc were running parallel to each other, as expected for conditions under which the current through the highly AChsensitive ACh receptor was negligible. The results obtained with 100pM ipratropium were similar to those obtained with d-Tc under these conditions. In another check, Na-free solutions were used. The current through the highly-sensitive ACh receptor should be weak under these conditions. With all of the Na replaced by K, the currents recorded at a holding potential of - 30 mV were again similarly affected by d-Tc and by ipratropium. as expected. In a third check, currents through the membrane of non-denervated muscle fibres of the flexor digitorum superficialis were investigated. The highly AChsensitive receptor, if present at all. makes little contribution to the ACh current in this preparation (Lorkovic, 1981). As in the other control experiments, the effects of d-Tc were similar to those of ipratropium, no difference in blocking effectiveness having been seen at small and at large concentrations of ACh. DISCUSSION
3.0
Fig. 6. Maximum amplitude of the current changes provoked by ACh. Mouse soleus muscle, denervated for 4 days. Full circles: control, open circles: 20 PM ipratropium. Four fibres per point.
Two types of ACh receptor, in muscles of different animals, are known to exist and a great deal of attention has been given to them recently in connection with the attempts at elucidating the role of individual receptor subunits (Schuetze, 1986). Thus, the question arises, whether the types of receptor dealt with in this work, are identical with the two
577
Antagonists on ACh currents types described in the literature. One variant of this possibility is that all fading currents depend on the activation of ACh receptors, located at the endplate. If this were so, the contribution of the fading currents in denervated muscles should decrease with increasing distance from the endplate. This was not so; fading currents were about the same amplitude, wherever in the muscle the measurements were made. This means that the bulk of ACh receptors in denervated mouse muscles belong to the densitizing type. Another variant of the possibility mentioned is that ACh receptors, identical in nature with those located at the endplate, were also distributed in extrajunctional areas of denervated muscles in sufficient numbers to account for densensitization. The evidence from the work of others (see Schuetze and Role, 1987; Brehm and Henderson, 1988) is against this possibility: the proportion of ACh receptors belonging to the high-conductance, brief open-time receptor type, normally found at the endplates of adult muscles is small (+ 10%) at the periphery of denervated muscles, too small to account for the strongly fading currents observed in the latter muscles in the presence of large concentrations of ACh. The non-fading current, provoked by ACh in denervated mouse soleus muscle (and in other denervated muscles of mice and rats. but not of frogs-unpublished data), proved to be less sensitive to d-Tc than the fading current. Beranek and VyskoEil(l967) found the denervated muscles of rats to be less sensitive to d-Tc than non-denervated muscles. The question may be asked, whether this result was due to the activation of the putative non-desensitizing receptors. The answer is likely to be negative because the electrophoretic technique of application of ACh was used by these authors. In this technique, a large concentration of ACh is applied for brief time intervals. It is not probable that the non-densensitizing receptors alone would be affected under these conditions. In fact, desensitization was first demonstrated with this technique (Katz and Thesleff, 1957). Endplates of juvenile mammals contain the ACh receptor, displaying low conductance and long opentimes, also characteristic of the ACh receptor of
denervated muscles (Schuetze and Role, 1987). It is not known whether part of the ACh receptor, present in juvenile endplates, belong to the non-desensitizing type. No variation of the reversal potential with the concentration of the ACh has been reported for those endplates. This work suggests that two naturally-occurring types of ACh receptor differ in their pharmacology and ion selectivity. Previous work, suggesting differential ionic selectivity of ACh receptors in muscle, proved to be methodically inadequate (Feltz and Mallart, 1971, Mallart, Dreyer and Peper. 1976). In this work, the error of undue extrapolation has been avoided. It remains to be seen whether differential ionic selectivity and pharmacological sensitivity, may be confirmed by single channel recording.
REFERENCES
Adams P. R. (1975) An analysis of the dose-response curve at voltage-clamped frog endplates. Pftigers Arch. 360: 145.--153.
B&trek R. and Vyskofil F. (1967) The action of tubocarine and atropine on the normal and denervated rat diaphragm. J. Physiol., Land. 188: 53-66. Brehm P. and Henderson L. (1988) Regulation of acetylcholine receptor channel function during development of skeletal muscle. Dec. Biol. 129: I-11. Feltz A. and Mallart A. (197 1) An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. J. Physiol., Lond. 218: 85-100. Katz B. and Thesleff S. (1957) A study of the “densensitization” produced by acetylcholine at the motor end-plate. J. Phy.Gol., Lond. 138: 63-80.
Lorkovic H. (1981) Desensitization in denervated mouse muscles. Pfriigers Arch. 391: 171-177. LorkoviC H. (1989) Sensitivity of rodent skeletal muscles to dicholines: dependence on innervation and age. Neuropharmacology 28: 373-377.
Mallart A., Dreyer F. and Peper K. (1976) Current-voltage relation and reversal potential at junctional and extrajunctional ACh-receptors of the frog neuromuscular junction. PJltgers Arch. 362: 43-47. Schuetze S. M. (1986) Embryonic and adult acetylcholine receptors: molecular basis of developmental changes in ion channel properties. Trends Neurosci. 9: 386-388. Schuetze S. M. and Role L. W. (1987) Developmental regulation of nicotinic acetylcholine receptors. Ann. Rec. Neurosci. 10: 403-457.
1990
0028-3908/90 53.00 + 0.00 Copyright Q 1990 Pergamon Press plc
ACETYLCHOLINE-INDUCED CURRENTS IN DENERVATED MOUSE SOLEUS MUSCLE: EFFECTS ANTAGONISTS
OF
H LORKOVI~ Abteilung
fiir Allgemeine
Physiologie Federal
der Universitat Ulm, Einsteinallee Republic of Germany
11, D-7900
Ulm,
(Accepfed 22 January 1990) Summary--Acetylcholine-induced currents were measured in partially depolarized mouse soleus muscles, denervated for 336 days by using a point voltage clamp. When 0.25 PM d-tubocurarine (d-Tc) was used, the weak currents provoked by 0. I PM ACh. at a holding potential of -20 mV, were barely affected, while the large currents provoked by 2-5 nM ACh were decreased by more than 50%. By contrast, weak and strong ACh-induced currents were proportionally diminished when, under similar conditions, 20-100 p M ipratropium was used. Currents were proportionally diminished by d-Tc when the holding potential was set at + 15 mV, a level corresponding to the reversal potential of the current provoked by small concentrations of ACh. In non-denervated flexor digitorum brevis muscles, d-Tc had the same re!ative effect at small and at large concentrations of ACh, independent of the holding potential. The reversal potential for the ACh-induced currents was about + 14 mV for small concentrations of ACh and decreased to about + 3 mV with 4 PM ACh in denervated soleus muscles. It was concluded that denervated soleus muscles, in contrast to the endplate regions of non-denervated mouse muscles, contain a small proportion of highly ACh-sensitive, weakly d-Tc-sensitive. predominantly Na+-permeable ACh receptors. These receptors are presumably responsible for the non-fading ACh-induced currents, described before, for the denervated mouse soleus muscle. K~J, tt,ord.r-ACh
The
membrane
current
receptor,
provoked
denervated
by
muscle,
endplate,
d-tubocurarine,
ipratropium.
those used in the work of Beranek and VyskoEil (1967). The desensitizing receptors of denervated muscles are not to be confounded with the ACh receptors present at the endplates. The contribution of the latter receptors to the current signals recorded in this work was shown to be negligibly small.
acetylcholine
in denervated mouse soleus muscles and measured by the point voltage-clamp technique has two components, the major one (comprising more than 90% of the current provoked by S-10 ,uM ACh, at a holding potential of - 30 mV), decaying with a half-time of about 15 set, the other showing no decay during exposure times to ACh not exceeding 2min. This non-fading current may be elicited by concentrations of ACh smaller than those required for the fading current. Other evidence, e.g. a distinctly more positive reversal potential for the non fading current, pointed to the presence, in denervated mouse soleus muscles, of two types of acetylcholine receptor (Lorkovic, 198 1). Pharmacological differences between the AChreceptor occurring in normal endplates of nondenervated mammalian muscles and those found in non-junctional areas of denervated muscles (Beranek and VyskoEil, 1967) are known to be related to types of ACh receptor. differing in at least one subunit (c vs ;‘; for a brief review see Schuetze, 1986). Pharmacological differentiation was recently extended to ACh receptors present in juvenile muscles, by using dichohnes (Lorkovic, 1989). In this work, it was desirable to see whether the putative desensitizing and non-desensitizing receptors of denervated muscles could be distinguished by means similar to (ACh)
METHODS Female
adult
mice (NMRI,
Interfauna,
Tuttlingen,
F.R.G.) were anaesthetised with ether and a portion of the ischiadicus nerve was excised. Three to 6 days after denervation the soleus muscle was dissected, split longitudinally and trimmed to a thickness of less than 0.2 mm. The preparations were placed in a narrow flow-through chamber and superfused (at about 1 ml/min) by a solution containing (in mM) Na+ 121, K+ 30, Mg *+ 1.0, Ca*+ 0.1, 3(N-morpholino)propane sulphonic acid (MOPS) buffer (Sigma M 1254) 1.O, methanesulphonate 150 and Cl2.2. The large concentration of K+ and the small concentration of Ca2+ were used to avoid contractions caused by ACh. The pH of the solutions was 7.2 and the temperature 20-22’C. The membrane current, caused by ACh, was measured by a conventional two-microelectrode voltage clamp apparatus. With a muscle fibre length exceeding 7 mm and an electric length constant less 573
574
H.
bRKOVk
than 2 mm, a uniform clamping of the membrane voltage was certainly not achieved at rest and it must have been even less complete during the action of ACh. To check for possible artefacts arising from this and from the fact that under the conditions described one part of the muscle was affected by the agonist before those parts lying more distally in the superfusion channel, measurements of current were made by using another chamber in which the muscle was placed, such that its long axis was perpendicular to the flow of solution. Less than 1 mm of the muscle fibre length was exposed to the solution (and thus to the agonist) under these conditions, the rest of the muscle being mechanically clamped by a tightly fitting plastic frame covered with Vaseline. Except for the smaller size of the current signals measured under the latter conditions, there was no difference between the results obtained with the two methods. The microelectrodes, used to measure the membrane potential, had resistances of 30-50 MR, while current-passing microelectrodes (made of twocompartment glass tubing) had resistances of 4-7 MR when filled with 2 M citrate solution. The resting potentials of denervated soleus muscle fibres, measured in the specified solutions, were -30 to - 38 mV. Non-denervated flexor digitorum superficialis muscles were used to measure the membrane current provoked by succinylcholine in normal endplates. The reason was that amplitudes of membrane currents. recorded from these muscles, were much less variable than those measured in non-denervated soleus muscles. Succinylcholine was used to avoid agonist hydrolysis by choline esterase. After a successful impalement of a muscle fibre by the two microelectrodes, recognized by the current of about 1 nA being required for a 10 mV change in holding potential. ACh, in concentrations ranging from IO-’ to 1.6 x 10m5 M. was applied for 4-20 set by injecting the appropriate solution into the chamber while the agonist-free superfusion was stopped. Currents provoked by ACh, ranging from lo-‘” to 3 x IO-' A, could be measured reliably. Depending on the concentration of ACh used, the application of the agonist was followed by a lo-200 set pause, in which the agonist was washed out. The peak (or the plateau, at small concentrations of ACh) amplitude of the ACh current was read directly from the screen of a Tektronix D 11 oscilloscope. Antagonistic drugs were applied in the (AChfree) superfusing and in the injection solutions. Tubocurarine (d-Tc, ASTA, Bielefeld, F.R.G.) and ipratropium bromide (Boehringer, Ingelheim, F.R.G.) were mainly used. Neither of these drugs had any influence on the rate of desensitization, when applied in concentrations 10 times as large as those used in the work reported here. A superfusion period of at least 1 min. in the presence of an antagonist, was allowed to pass before the measurement of the (diminished) ACh current was made. In successful
runs 12-14 applications of ACh were made in one single fibre, the microelectrodes remaining impaled for up to 25 min. Only those results were evaluated in which the control current responses (usually that to 2 x 10e6 ACh at the beginning of the series and following the washout of an antagonist) differed by less than 5%. The variability of the amplitudes of the currents was such that the SEM, calculated from 3-7 fibres, was less than 10% of the average value. Variability was less when the data were normalized with respect to the ACh current, measured in the presence of the largest ACh concentrations used. The SEM, calculated in this way, was less than the size of the symbols used in the illustrations, except for the smallest concentrations of ACh used. The data were plotted as log-log graphs, permitting an adequate representation of the smallest current responses. RESULTS
Typical changes in membrane currents caused by a large concentration of ACh at holding potentials ranging from -25 to + 15 mV are shown in Fig. 1. A reversal potential of about --2mV was obtained from the muscle fibre used. Inspection revealed that the time course of the current changes, e.g. for the holding potential of - 5 and + 5 mV, were not the same, the current returning to the resting level at a faster rate at + 5 than at - 5 mV and undershooting that level after washout of ACh with +5 mV. The behaviour of another fibre is shown at high current gain in Fig. 2. The holding potential was +8 mV. The time course of the change in current 15
-----
-I
5 h.p. (mV)
-5
----.
-15 -25
-
-. b
t
8 XICFM EAChI
Fig. I. Holding potentials (h.p., upper lines) and membrane current changes (lower lines) caused by 8 PM ACh in a mouse soleus muscle fibre denervated for 4 days. Arrow downward: application of ACh, arrow upward: washout of ACh, in this and in the rest of the figures.
Antagonists on ACh currents +amv -J
t tACh1
x~O-~M
Fig. 2. Changes in membrane current caused in a mouse soleus muscle fibre denervated for 5 days at two different concentrations of ACh. The holding potential was 8 mV for both records. (upper tracing, large concentrations of ACh) is shown to be complicated: following the initial negative (inward) deflection, the current trace turned positive only to return to the negative after the washout of ACh. Such a result suggests that the direction of current depends on the concentration of ACh prevailing at the point of insertion of the microelectrode, smaller concentrations of ACh causing inward, larger concentrations outward membrane current at the same holding potential. This explanation is supported by the lower tracing, obtained in the same muscle fibre and at the same holding potential, with a concentration of ACh equal to one tenth of that used for the upper tracing. A monophasic, inward current was now provoked. The reversal potential, as a function of the concentration of ACh, was studied in two muscles. With records like the one presented in Fig. 2, the reversal potential was determined from the larger current component. The results obtained are shown in Fig. 3. The reversal potential was close to + 15 mV at small concentrations of ACh and it decreased to + 3 mV as the concentration was raised, without reaching a constant value in the range of the concentrations of
IL -
515
ACh used. The relative permeability ratio (PNa/PK at small concentrations of ACh, divided by P,,/P,at large concentrations) was calculated from the Goldman equation assuming the concentrations of Ki, K,, Na, and Na, to be 150, 30, 7 and 121 mM, respectively. The value obtained was 1.71, suggesting that the ACh receptor channel was relatively more permeable to Na+ at small rather than at large concentrations of ACh. An alternative (and more likely) explanation is that two types of acetylcholine receptors are involved in the current response, the type responsible for the current at small concentrations of ACh having a greater P,,/P,ratio than the one activated by large concentrations. It may be mentioned that the same relative permeability ratio was obtained independent of whether ionic concentrations or activities were considered. The relation between the reversal potential and the concentration of ACh was shifted to larger concentrations of ACh when d-tubocurarine (d-Tc) was present (Fig. 3) as expected. The shift appeared to be greater at large, rather than small concentrations but, because of the small slope of the curves at small concentrations, the shift could not be measured with adequate precision. A more convenient method was to measure the effect of d-Tc on the amplitude of the ACh current. The results are shown in Figs 4 and 5. The upper, high-gain current records were obtained by using 0.3 PM ACh and d-Tc was applied 1 min before ACh was applied, to obtain the middle record. Barely any effect of d-Tc can be seen. When a larger concentration of ACh was used (Fig. 4, lower, high-gain records), the current deflection measured at the same holding potential as before (- 20 mV) was nearly 50% less in the presence of the same concentration of d-Tc as used before (0.25 PM) than in its absence. The relative blocking effect of d-Tc increased continuously as the concentration of ACh was increased from 0.1 to 4 p M. Most of the change in the effectiveness of d-Tc occurred between 0.2 and 1.OPM ACh (Fig. 5). These results suggest that the putative, highly ACh-sensitive and Na-permeable ACh receptor is less sensitive to the
12. lomV
66-
01
d -Tc
0.1 0.2 0.4
1
2 L 8
2.5 x lO+M
[ACh] x10-‘M Fig. 3. Reversal potential (ordinate, mV) as a function of the concentration of ACh ([ACh]); average values i SEM. Mouse soleus muscle denervated for 4 days, four fibres per point.
Fig. 4. Changes in membrane current provoked by ACh (0.3 and 8.0 uM) in a mouse soleus muscle fibre, denervated for 3 days. ‘Both records were obtained at a holding potential (h.p.) of -20 mV; d-Tc (0.25 PM) was applied for I min before and during application of ACh (middle records).
Fig. 5. Maximum amplitude of the current changes proby ACh. Mouse soleus muscle, denervated for 5 days, 5 fibres per point. Full circles: control, open circles: 0.25 PM d-Tc.
concentrations of ACh. As with d-Tc. the curve for ipratropium was shifted to larger concentrations of ACh as the concentration of blocker was increased. These results suggest that the putative, highly Napermeable ACh receptors are about equally sensitive to ipratropium as the less ACh-sensitive bulk of ACh receptors of the denervated mouse soleus muscle. The effect of atropine was similar to that of ipratropium, except that the current tended to saturate at a level lower than maximum at larger concentrations of the drug. The time course of the current responses suggested that, in addition to its wellknown antagonistic effect, atropine may enhance desensitization. Two local anaesthetics, tetracaine and lidocaine, were also used. The effects appeared to be similar to those of d-Tc, but desensitization problems prevented definite conclusions to be made.
voked
blocking action of d-Tc than the less ACh-sensitive bulk of the receptors.
When larger concentrations of d-Tc (0.5, 1.0, 2.5, 10.0 p M) were used, the curve representing the maximum current in the presence of d-Tc was shifted to the right but its slope at any current amplitude was not changed much. In Lineweaver-Burk plots of the data (with l/fi on the ordinate, where I is the measured current amplitude, cf. e.g. Adams, 1975) the slopes of the lines connecting the data points were always smaller for small rather than for large concentrations of ACh. The difference in the slopes was increased in the presence of d-Tc (not shown). Blocking effects, different from those obtained with d-Tc, were found with the muscarinic ACh receptorblocker ipratropium. The effect of a small concentration of the drug is shown in Fig. 6. The relative diminution of the current responses to ACh was, if anything, somewhat greater at small than at large den. SOL 30 mM K+
1
@I/
/
Ipr. 2x16%
/
,6L!-_ 0.3
.ld’
1.0 M ACh
CONTROL
EXPERIMENTS
The existence of two reversal potentials for the currents provoked by ACh makes it possible to check the idea of differential pharmacological sensitivity of the two putative types of receptor by using holding potentials corresponding to either reversal potential. In one series of such experiments, the holding potential was + 15 mV, the reversal potential for the more sensitive of ACh type receptor. The control curve and the curve for 0.5 PM d-Tc were running parallel to each other, as expected for conditions under which the current through the highly AChsensitive ACh receptor was negligible. The results obtained with 100pM ipratropium were similar to those obtained with d-Tc under these conditions. In another check, Na-free solutions were used. The current through the highly-sensitive ACh receptor should be weak under these conditions. With all of the Na replaced by K, the currents recorded at a holding potential of - 30 mV were again similarly affected by d-Tc and by ipratropium. as expected. In a third check, currents through the membrane of non-denervated muscle fibres of the flexor digitorum superficialis were investigated. The highly AChsensitive receptor, if present at all. makes little contribution to the ACh current in this preparation (Lorkovic, 1981). As in the other control experiments, the effects of d-Tc were similar to those of ipratropium, no difference in blocking effectiveness having been seen at small and at large concentrations of ACh. DISCUSSION
3.0
Fig. 6. Maximum amplitude of the current changes provoked by ACh. Mouse soleus muscle, denervated for 4 days. Full circles: control, open circles: 20 PM ipratropium. Four fibres per point.
Two types of ACh receptor, in muscles of different animals, are known to exist and a great deal of attention has been given to them recently in connection with the attempts at elucidating the role of individual receptor subunits (Schuetze, 1986). Thus, the question arises, whether the types of receptor dealt with in this work, are identical with the two
577
Antagonists on ACh currents types described in the literature. One variant of this possibility is that all fading currents depend on the activation of ACh receptors, located at the endplate. If this were so, the contribution of the fading currents in denervated muscles should decrease with increasing distance from the endplate. This was not so; fading currents were about the same amplitude, wherever in the muscle the measurements were made. This means that the bulk of ACh receptors in denervated mouse muscles belong to the densitizing type. Another variant of the possibility mentioned is that ACh receptors, identical in nature with those located at the endplate, were also distributed in extrajunctional areas of denervated muscles in sufficient numbers to account for densensitization. The evidence from the work of others (see Schuetze and Role, 1987; Brehm and Henderson, 1988) is against this possibility: the proportion of ACh receptors belonging to the high-conductance, brief open-time receptor type, normally found at the endplates of adult muscles is small (+ 10%) at the periphery of denervated muscles, too small to account for the strongly fading currents observed in the latter muscles in the presence of large concentrations of ACh. The non-fading current, provoked by ACh in denervated mouse soleus muscle (and in other denervated muscles of mice and rats. but not of frogs-unpublished data), proved to be less sensitive to d-Tc than the fading current. Beranek and VyskoEil(l967) found the denervated muscles of rats to be less sensitive to d-Tc than non-denervated muscles. The question may be asked, whether this result was due to the activation of the putative non-desensitizing receptors. The answer is likely to be negative because the electrophoretic technique of application of ACh was used by these authors. In this technique, a large concentration of ACh is applied for brief time intervals. It is not probable that the non-densensitizing receptors alone would be affected under these conditions. In fact, desensitization was first demonstrated with this technique (Katz and Thesleff, 1957). Endplates of juvenile mammals contain the ACh receptor, displaying low conductance and long opentimes, also characteristic of the ACh receptor of
denervated muscles (Schuetze and Role, 1987). It is not known whether part of the ACh receptor, present in juvenile endplates, belong to the non-desensitizing type. No variation of the reversal potential with the concentration of the ACh has been reported for those endplates. This work suggests that two naturally-occurring types of ACh receptor differ in their pharmacology and ion selectivity. Previous work, suggesting differential ionic selectivity of ACh receptors in muscle, proved to be methodically inadequate (Feltz and Mallart, 1971, Mallart, Dreyer and Peper. 1976). In this work, the error of undue extrapolation has been avoided. It remains to be seen whether differential ionic selectivity and pharmacological sensitivity, may be confirmed by single channel recording.
REFERENCES
Adams P. R. (1975) An analysis of the dose-response curve at voltage-clamped frog endplates. Pftigers Arch. 360: 145.--153.
B&trek R. and Vyskofil F. (1967) The action of tubocarine and atropine on the normal and denervated rat diaphragm. J. Physiol., Land. 188: 53-66. Brehm P. and Henderson L. (1988) Regulation of acetylcholine receptor channel function during development of skeletal muscle. Dec. Biol. 129: I-11. Feltz A. and Mallart A. (197 1) An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. J. Physiol., Lond. 218: 85-100. Katz B. and Thesleff S. (1957) A study of the “densensitization” produced by acetylcholine at the motor end-plate. J. Phy.Gol., Lond. 138: 63-80.
Lorkovic H. (1981) Desensitization in denervated mouse muscles. Pfriigers Arch. 391: 171-177. LorkoviC H. (1989) Sensitivity of rodent skeletal muscles to dicholines: dependence on innervation and age. Neuropharmacology 28: 373-377.
Mallart A., Dreyer F. and Peper K. (1976) Current-voltage relation and reversal potential at junctional and extrajunctional ACh-receptors of the frog neuromuscular junction. PJltgers Arch. 362: 43-47. Schuetze S. M. (1986) Embryonic and adult acetylcholine receptors: molecular basis of developmental changes in ion channel properties. Trends Neurosci. 9: 386-388. Schuetze S. M. and Role L. W. (1987) Developmental regulation of nicotinic acetylcholine receptors. Ann. Rec. Neurosci. 10: 403-457.