Naunyn-Schmiedeherg's Arch. Pharmacol. 291,359--370 (1975) 9 by Springer-Verlag 1975

Pre- and Postjunctional Neuromuscular Blockade by Carbachol R o b e r t L. Volle a n d E d w a r d G. H e n d e r s o n I)epartment of Pharmacology University of Connecticut Health Center, Farmington, Connecticut Received June 10 / Accepted September 9, 1975

Summary. Carbachol, when applied to the bathing t~inger solution of frog sartorius muscles, caused depolarization of the endplate and a blockade of endplate potentials (EPP's), miniature EPP's (mepp's) and the iontophoretic acetyleholine potential. In muscles treated with an analog of hemicholinium-3, cr ammonium acetaldehyde diethylacetal)-p-pqdiaeetylbiphenyl dibromide (I)MAE), depolarization of the endplate by carbachol was blocked and the blockade by earbaehol of the iontophoretie acetylcholine potential was prevented. These responses to earbaehol were attributed to a postjunetional action that was antagonized by I)MAE. In contrast, the blockade by carbachol of EPP's and mepp's was enhanced in I)MAE-treated muscles at a time when carbachol-induced depolarization was blocked. This response to carbaehol was attributed to a prejunctional action. Carbaehol either blocked transmitter release by a mechanism that was insensitive to DMAE or enhanced the prejunctional blocking actions of I)MAE. Succinylcholine had actions similar to earbachol. DMAE prevented depolarization by succinylcholine but enhanced neuromuscular blockade by succinylcholine. SKF 525-A (fl-diethylaminoethyl diphenylpropylacetate hydrochloride), like I)MAE, prevented depolarization but not transmission blockade caused by carbachol. Key words: Acetylcholine receptors -- Carbaehol -- Neuromuscular blockade -Neuromuscular transmission -- Nicotinic drugs. Nicotinic drugs are r e g a r d e d g e n e r a l l y as causing n e u r o m u s c u l a r b l o c k a d e in two phases. The first p h a s e of n e u r o m u s c u l a r b l o c k a d e b y nicotinic drugs is a s s o c i a t e d w i t h d e p o l a r i z a t i o n of the e n d p l a t e region a n d t h e second p h a s e w i t h a b l o c k a d e of t h e a c e t y l c h o l i n e r e c e p t o r s a t a t i m e a f t e r t h e d e p o l a r i z a t i o n has subsided (Zaimis, 1953; Thesleff, 1955). D e p e n d i n g u p o n t h e muscle t e s t e d a n d t h e drugs studied, differenees in t h e e x t e n t o f d e p o l a r i z a t i o n a n d b l o c k a d e can be observed. I n frog muscle t h e r e l a t i o n s h i p b e t w e e n d e p o l a r i z a t i o n of the endp l a t e a n d r e c e p t o r b l o c k a d e is unclear. F o r e x a m p l e , succinylcholine

Send o]/print reque~.ts to: R. L. Volle, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A.

360

I~. L. Volle and E. G. Henderson

caused greater depolarization and less blockade than did decamethonium (Gissen and Nastuk, 1970). Of particular interest, was the finding that during depolarization by decamethonium, blockade occurred even though the membrane potential was far below --57 mV, the critical Em for depolarization blockade (Jenerick and Gerard, 1953). Further, it has been shown that nicotine, in doses too small to depolarize the endplate, blocked neuromuscular transmission and that lobeline, a drug with ganglionic stimulating and other nicotinic properties, blocked transmission without causing depolarization (Wang and Narahashi, 1972 ; Steinberg and Volle, 1972; Hancock and Henderson, 1972). Transmission blockade by nicotinic drugs may also include a prejunctional component. I t has been noted that a decrease in transmitter output occurred during the period of endplate depolarization by nicotinic drugs (Edwards and Ikeda, 1962; Steinberg and Volle, 1972). The mechanism responsible for the depression of transmitter release is not known. Depolarization of the motor nerve terminals could result in decreased transmitter release secondary to an alteration in the amplitude and duration of the nerve terminal action potential (Takeuchi and Takeuchi, 1962; Dudel, 1971). However, some evidence has been obtained to show that nerve terminal depolarization was not necessarily required to obtain a decrease in transmitter output by acetyleholine (Hubbard et al., 1965). A novel way to study the relationship between endplate depolarization and neuromuscular blockade by nicotinic drugs was provided by a curious pharmacological property of an analog of hemieholinium-3. cr acetaldehyde diethylacetal)-p-p'-diacetylbiphenyl dibromide (DMAE) in concentrations that had no effect on transmission at the neuromuscular junction and at sympathetic ganglia selectively depressed nicotinic excitatory responses (Wong and Long, 1968; Jaramillo, 1968; Miyamoto and Volle, 1974). Although there is no satisfactory explanation of the selective blocking activity of DMAE, this property of DMAE provides a useful way to study the relationship between transmission blockade and endplate depolarization by nicotinic drugs. If depolarization of the endplate and blockade of transmission are due to the same mechanisms, then DMAE should antagonize both responses to the nicotinic drugs. However, if the depolarization of the endplate is unrelated to transmission blockade, then DMAE should have no effect on nicotine-induced blockade of transmission at a time when depolarization is suppressed. The actions of DMAE on depolarization and neuromuscular blockade by earbaehol were compared with those of fl-diethylaminoethyl diphenylpropylaeetate hydrochloride (SKF 525-A). SKF 525-A has been shown to enhance the neuromuscular blocking actions of deeamethonium

Neuromuscular Depolarization and Blockade

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a n d succinylcholine (Della Bella et al., 1962) a n d to p r o m o t e receptor desensitization (VyskoSil a n d Magazanik, 1972; Terrar, 1974). A p r e l i m i n a r y a c c o u n t of the findings has been made Volle, 1975).

Methods The sciatic nerve-sartorius muscle was removed from Rana p~pien~ and allowed to equilibrate in Ringer solution containing: NaC1, 97 mM; KCI, 2.5 mM; CaCl2, 1.8 mM; NaH2P04, 0.85 mM and Na,~HPQ, 2.5 mM. MgCI 2 (6 to I0 mM) was added to the Ringer solution to unmask the endplate potentials (EPP's) but was omitted from the bathing solution when only miniature EPP's (mepp's) were studied. Enbanccment of the amplitude of mepp's was produced in all experiments by neostigmine (10 -6 M). The solutions were used at room temperature (20--22 ~ C). Osmolalities of solutions were checked by measurement of the depression of freezing point of water. Solutions were perfused at a rate of 6 ml/min. The muscles were fixed by miniature pins to the bed of a small bath (2 ml capacity) with the inner surface facing upward. Only the fibers in the superficial layer were used. Rectangular wave pulses of supramaximal voltage and 0.5 msec duration were delivered to the sciatic nerve through a suction electrode and

stimulus isolation unit. Electrical activity was monitored using glass microelectrodes filled with 3 M KCI (5-- 15 megohms). Potentials were displayed on an oscilloscope and permanent records made by photographic means. The appearance of EPP's with rise times of 1.5--2.0 msec and the appearance of mepp's were used to determine the location of endplates. Responses from the same endplate were measured before, during and after the administration of drugs. With selected cells, it was possible to obtain stable impalements for more than 1 or 2 hrs. EPP's were evoked by continuous nerve stimulation at a rate of 0.5 Hz. More rapid stimulation in the presence of DMAE resulted in the progressive fade of the EPP's (Voile, 1973). The mean amplitude of 30 consecutive EPP's was used to estimate neuromuscular function at various times during the course of the experiment. The mean amplitude of mepp's was estimated from measurements of 60 consecutive mepp's. The basal frequency of mcpp's was of the order of 0.5 to 2/sec. Endplate responses to the iontophoretic application of acetylcholine were evoked at a rate of 0.5/see. The drug pipettes contained 3 M acetylcholine which was ejected by applying current pulses of 5 to 10 msee duration. The iontophoretie electrodes had resistances of 10--20 megohms and were placed within 50 ~z of the intracellular voltage recording electrode. The amplitude of depolarization produced by the bath application of nicotinic drugs was estimated from the difference between resting Em and the maximum depolarization produced by the drug, usually occurring 2 to 4 rain after application to the bath. In most instances, the nicotinic drugs were applied for 5 to 10 rain. With prolonged washing, Em usually returned to control values upon removal of the nicotinic drug from the bath.

Results The effects of D M A E (1 • 10 -6 M) o n the d e p o l a r i z a t i o n of the sartorius muscle caused b y carbachol were studied i n the first series of e x p e r i m e n t s (Fig. 1). The muscles were soaked i n n o r m a l R i n g e r s o l u t i o n c o n t a i n i n g n e o s t i g m i n e a n d endplates were identified b y the presence of mepp's. As shown graphically i n Fig. 1, the blockade b y D M A E of the carbachol-induced d e p o l a r i z a t i o n was n o t a n t a g o n i z e d b y large

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Fig. 3. Blockade of EPP's and depolarization by carbachol (CARB) before and during exposure of the muscle to DMAE. The nerve was stimulated at a rate of 0.5 Hz. Each point on the upper graph represents the mean value for 15 consecutive EPP's. The same endplate was used throughout

doses of carbachol. I n four additional muscles, it was found that the depolarization (21.4 ~ 1.0 mV; mean ~ S.E.) caused by succinylcholine (1 • 10-5 M) was almost completely prevented (2.0 ~ 0.4 mV) by DMAE (1 • 10-6 M). A more systematic study of the blockade by DMAE of sueeinylcholine-induced depolarization was not made. I n the next series of experiments, the inhibition by carbaehol of the amplitudes of E P P ' s evoked at a rate of 0.5 Hz in Mg2+-Ringer solution was studied before and in the presence of DMAE (1 • 10-6 M). The results obtained are presented as a partial dose-response curve in Fig. 2. The blockade of transmission produced by carbachol was enhanced by DMAE. The sequence of drug application was randomized so that in some instances the effects of carbachol on transmission were studied first in the absence, then in the presence of DMAE (Fig. 3). The reverse sequence was used in other experiments. The effects of DMAE were easily reversed by removal of the drug from the bath. The results obtained in individual experiments are given in Figs. 3 and 4. Concentrations of DMAE (1 to 3 • 10-s M) having no effect on the amplitudes of E P P ' s (Volle, 1973) or mepp's (Volle, 1973; Miyamoto and Volle, 1974) prevented completely the depolarization produced by carbachol (2 • 10-5 M; Figs. 3 and 4). However, the blockade of E P P ' s

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and mepp's produced by carbactiol was intensified by DMAE (Figs. 3 and 4). This differential blocking action of DMAE was observed also when succinylcholine (1 • 10-6 M) was used to block transmission (3 additional experiments). DMAE prevented the depolarization, but enhanced the blockade of E P P ' s or mepp's produced by suecinylcholine. The acetylcholine potential evoked by iontophoretie application to endplates, like the E P P ' s and the mepp's, was blocked by bath applied carbachol (2 x 10 -6 M; Table 1). At DMAE-treated endplates, however, bath applied carbachol failed to depolarize the endplate and had no effect on the acetylcholine potential at a time when EPP's were blocked (Table 1 ; Fig. 5). I t should be noted also that DMAE (4 X l0 -6 M) depressed the E P P ' s but had no effect on the amplitudes of the acetylcholine potential. Concentrations of DMAE (2 • 10 -8 M) that prevented endplate depolarization by carbachol had no effect on either E P P ' s or the acetylcholine potential (Table 1). Like DMAE, SKF 525-A (2 x 10 -6 M) blocked the depolarization but not the blockade of mepp's caused by carbachol (Fig. 6). The depolarization produced by carbachol (2 • 10-5 M) was 19.0 ~ 1.4 mV before and 2.7 :~ 1.0 mV (6 experiments) in the presence of SKF 525-A (2 x 10-6 M). Whereas SKF 525-A (2• 10 -6 M) had no effect on the amplitude of the mepp's, the mean amplitude of mepp's was depressed by approximately 50~ when the concentration of SKF 525-A was raised to 6 • 10-6 M. The effects of SKF 525-A on E P P ' s of Mg~+-treated junctions were not studied. Unlike those of DMAE, the effects of SKF 525-A were difficult to reverse and required prolonged washing (Fig. 6). Discussion That DNAE prevented the slowly occurring depolarization of the endplate but not the transmission blockade produced by carbachol is the main finding reported here. Thus, at junctions treated with DMAE the blockade of transmission by carbachol was unrelated to endplate depolarization. The dissociation by DMAE of the two actions of carbachol can be interpreted in several ways. There is the possibility that carbachol caused transmission block by actions at more than one site. As noted before, nicotinic drugs have been shown to depress transmitter release at the frog neuromuscular junction (Edwards and Ikeda, 1962; Steinberg and Volle, 1972). An action of carbachol on the motor nerve terminals to depress transmitter release would explain the failure of DMAE to antagonize the earbacholinduced depression of the EPP's at a time when the carbachol-induced depolarization had been prevented. A prejunctional site of action for carbachol wo~]d account also for the blockade by carbachol of E P P ' s

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Fig. 5A--C. EPP's (left) and acetylcholine potentials (right) of frog sartorius endplates in Mg2+-Ringer solution. Top row of records (A) show responses to nerve stimulation (0.5 Hz) and iontophoretie ACh-potential (0.5 Hz) of untreated endplates. Middle row (B) shows responses after DMAE (4 • 10-6 M). Bottom row (C) shows responses to bath applied carbachoI (2 • 10 5 M) of DMAE-treated endplate. The same endplate was used throughout. The cell had an Em of 88 inV. The response to ACh was 20 mV

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Fig. 6. Effects of SKF 525-A on the blockade of mepp's and depolarization produced by carbachol (CAleB). Experimental conditions are the same as those of the experiment of Fig. 4 site of action. Nicotinic drugs depress evoked transmitter output presumably as a consequence of nerve terminal depolarization and reduction in the amplitude of the incoming nerve terminal action potential. Although depolarization of the nerve terminals by carbaehol has been shown to increase the frequency of mepp's (Miyamoto and Volle, 1974), there is no evidence to show that a reduction in the size of the mepp's is associated with nerve terminal depolarization. In the absence of DMAE the decreased amplitude of mepp's produced by nicotinic drugs is related to postjunctional depolarization. I t is worth noting that DMAE has important prejunctional actions. At some endplates, DMAE (4• 10 -6 M) depressed E P P ' s but not the iontophoretie acetyleholine potential. In addition to the differential blocking action of DMAE on E P P ' s and the iontophoretie acetyleholine potential, DMAE depressed transmitter release by a frequency-dependent process (Volle, ][973 ; Branisteanu et al., 1975). Concentrations of DMAE t h a t have no effect on the ~mplitude of EI~P's evoked by low frequency stimulation (0.5 Itz), caused a marked blockade when the frequency of stimulation was increased to 5 or 10 Itz. The blockade occurring under these conditions w~s due to a decrease in the number of quanta of transmitter release by the nerve impulse. The possibility exists, therefore, that carbachol enhanced the prejunctional blocking actions of 25 iNaunyn-Schmiedeberg'sArch. 1)harmacol., Vol. 291

368

R. L. Volle and E, G. Henderson

DMAE and, in this regard, was similar to repetitive nerve stimulation. This meehanism would account for the depression by earbaehol of both the EPP's and mepp's. Clearly, DMAE has blocking actions on the endplate receptor complex. DMAE, in concentrations having no effect on the amplitudes of EPP's, mepp's or the aeetyleholine potential, depressed the slowly occurring depolarization produced by bath applied earbaehol. In addition, DMAE prevented the blockade of the acetyleholine potential produced by earbachol. Obviously, both of these actions of earbaehol and their prevention by DMAE were due to the effects of the drugs on the endplate. The ability of DMAE to discriminate between the slowly occurring depolarization of bath applied nicotinic drugs and that of iontophoretically applied nicotinic drugs or the transmitter is not understood. This action of DMAE is found not only at the neuromuscular junction of the frog but also at sympathetic ganglia. Wong and Long (1968) found that ganglionic responses to preganglionie stimulation required about 1000 times more DMAE for blockade than did the response to nicotine. Jaramillo (1968) reported a 10-fold difference in the sensitivity to DMAE between depolarization of ganglia by nicotinic drugs and by preganglionic stimulation. In frog muscle, EPP's evoked at a rate of 0.5 tIz were about 10-times less sensitive to DMAE than was depolarization by nicotine or earbaehol. The differential blocking action of DMAE and the dissociation by DMAE of the several actions of earbaehol could reflect differences in the activation of the aeetyleholine receptor complex by the transmitter, the iontophoretic application of acetyleholine and bath applied earbachol. The blockade by DMAE of the slowly occurring responses to nicotinic drugs may have been due to the development of receptor desensitization and to enhancement of the desensitization process by DMAE. Desensitization would not be expected to occur when the onset of drug action was rapid and the duration of drug application was brief. There is ample precedent for postulating such a mechanism for DMAE. SKF 525-A has been shown to enhance receptor desensitization by an inhibitory effect on the ion conducting system rather than by an action on the formation of the transmitter receptor complex (VyskoSil and Magazanik, 1972). There is also some evidence that cr acts on the conductance system (Magazanik and VyskoSil, 1972; Albuquerque et al., 1972). It should be noted that SKF 525-A blocked ion exchange in skeletal muscle (Henderson and Volle, 1974) and that DMAE modified calcium exchange in peripheral nerve (Greenberg e~ al., 1972). Thus, both compounds have a demonstrated capacity to block ion conductance systems.

Neuromuscular Depolarization and Blockade

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I t is also possible t h a t the ability of carbachol in the presence of DMAE to block the E P P ' s and mepp's but not the acetylcholine potential was due to differences in the activation of endplate ionic pathways b y iontophoretically applied acetyleholine and b y the transmitter. I t is known, for example t h a t the parameters of activating ionic channels differ widely for various nicotinic substances (Katz and Miledi, 1971; Colquhoun et al., 1975) and similar differences m a y exist between the transmitter and nicotinic drugs. A more detailed description of the several parameters of receptor activation and inactivation b y nicotinic drugs and the transmitter is required before this possibility can be tested. Although a complete comparison between bath applied carbaehol and suecinyleholine was not made, it appears t h a t both substances had the same actions on the neuromuscular junction. I f this is so, then succinylcholine, like earbachol, probably acted on the motor nerve terminals to suppress transmitter release (see also, Edwards and Ikeda, 1962). Similarly, S K F 525-A and DMAE had some common pharmacological properties. S K F 525-A blocked the slow depolarization produced b y b a t h applied carbaehol but not the depression of mepp's b y carbaehol. I n 3 to 4-fold higher concentrations, S K F 525-A blocked mepp's. However, unlike DMAE, there is no evidence t h a t S K F 525-A has a depressant effect on transmitter release. Blockade of neuromuscular transmission b y S K F 525-A was not frequency dependent and did not cause fade of E P P ' s as did DMAE (unpublished experiments).

Acknowledgement. Supported by Grant NS-07540-8, Institute of Neurological Diseases and Stroke, NItt, U.S.P.H.S. I)MAE was a gift from Dr. J. P. Long, University of Iowa and SKF 525-A was a gift from Dr. S. S. Walkenstein, Smith, Kline and French Laboratories, Philadelphia, Penna. References Albuquerque, E. X., Barnard, E. C., Chiu, T. H., Lapa, A. J., Dolly, J. 0., Jansson, S.-T., I)aly, J., Witkop, B. : Acetylcholine receptor and ion conductance modulator sites at the marine neuromuscular junction: evidence from specific toxin reactions. Proc. nat. Acad. Sci. (Wash.) 70, 949--953 (1973) Branisteanu, D. I)., Miyamoto, ~. D., Volle, R.L.: Quantal release parameters during fade of endplatc potentials. Naunyn-Schmiedeberg's Arch. Pharmaeol. 288, 323--327 (1975) Colqifimun, I)., I)ionne, V.W., Steinbach, J. H., Stevens, C.F.: Conductance of channels opened by acetylcholine-like drugs in muscle end-plate. Nature (Lond.) 258, 204--206 (1975) Della Bella, I)., Rognoni, F., Teotino, Z.: Effect of fl-diethylaminoethyl-3,3-diphenylpropylacetate on the action of suxamethonium and other neuromuscular blocking drugs. Brit. J. Pharmaeol. 18, 563--571 (1962) I)udel, J. : The effect of polarizing current on action potential and transmitter release in crayfish motor nerve terminals. Pfliigers Arch. 824, 227--248 (1971) 25*

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