European Journal of Pharmacology, 32 ( 1975) 267--272 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
D A N T R O L E N E EFFECTS ON N E U R O M U S C U L A R FUNCTION IN CAT SOLEUS MUSCLE H ERBE R T E. LOWNDES
Department of Pharmacology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen Street, Newark, New Jersey 07103, U.S.A. Received 16 January 1975, revised MS received 3 March 197 5, accepted 27 March 1975 H.E. LOWNDES, Dantrolene effects on neuromuscular function in cat soleus muscle, European J. Pharmacol. 32 (1975) 267--272. Dantrolene sodium (DS) was investigated for its effects on cat soleus muscle contractile properties and motor nerve terminal activity in particular. DS, 0.1--1.5 mg/kg i.v., caused a dose-dependent depression of indirectly elicited contractile strength which was more pronounced at lower frequencies of stimulation. Maximum tetanic strength at frequencies of 10--400 Hz was depressed to a lesser degree than contractile responses evoked by lower frequencies of stimulation~ the twitch/tetanus contraction ratios were reduced with increasing dose, primarily because of diminished twitch. DS was without effect on motor nerve terminals as evidenced by normal post-tetanic repetition in the nerves following DS administration. Post-tetanic potentiation became relatively larger in amplitude as contractile strength was diminished. These data suggest that DS depresses neuromuscular function at a site other than the neural apparatus at the neuromuscular junction. Motor nerve terminals
Dantrolene sodium
1. Introduction Dantrolene sodium (DS) is a new muscle relaxant which has been evaluated for clinical efficacy in the therapy of spasticity (Snyder et al., 1967; Chyatte et al., 1971; Chipman et al., 1974). In contrast to other 'muscle relaxants', the mechanism of action of DS has been ascribed to an uncoupling action on excitation--contraction coupling in the muscle {Ellis and Carpenter, 1972; Putney and Bianchi, 1974). While Putney and Bianchi {1974) ascribed a significant portion of the muscle relaxant actions of DS to such an uncoupling, they did not rule out other possible mechanisms. Even though muscle action potentials are n o t altered by DS ( H o n k o m p et al., 1970; Ellis and Bryant, 1972; Nott and Bowman, 1973; 1974; Odette and Atwood, 1974), the possibility of a motor nerve terminal depressant action of DS has not been explored. A direct effect of DS on motor nerve terminals could be contributory to the pharmacological actions of DS.
Muscle relaxant
Contractile strength
The m o t o r nerve terminals in cat soleus are extremely sensitive to a wide variety of pharmacological agents, both depressant and facilitatory (Riker and Okamoto, 1969). Monitoring of the responsiveness of these nerve terminals provides a sensitive indicator of any depressant effect of the agent on the nerve endings. This communication reports the responsiveness of m o t o r nerve terminals in the presence of DS. Also, the actions of DS on contractile responses of this nerve--muscle preparation have been assessed, and the dose--response relationship of DS-induced muscle paralysis is reported.
2. Materials and methods
2.1. Surgical preparation Adult cats of either sex were anesthetized with a-chloralose, 80 mg/kg i.v., and surgically prepared for either contractile or motor
268
nerve terminal responses as previously described (Riker et al., 1957). Briefly, in experiments on contractile responses the sciatic nerve was sectioned at the sciatic notch, the gastrocnemius muscles and their innervation excised and the leg was held rigidly in a modified Brown--Schuster myograph by means of steel drill rods driven through the bone. The preparation was covered with liquid paraffin oil and maintained at 37 ° C by radiant heat. Platinum wire stimulating electrodes were placed on the soleus nerve and isometric contractile tension responses recorded by connecting the strain gauge o u t p u t to a pen-writer. The preparations were stimulated supramaximally at 0.4 Hz with square pulses of 0.1 msec duration. Tetani were delivered as trains of 10 sec duration at frequencies described in the text. Motor nerve terminal responses in the presence of dantrolene sodium were recorded in preparations which had been surgically prepared as described for contractile responses, except that the sciatic nerves were not sectioned. In addition, a dorsal laminectomy was performed to expose the ventral roots innervating the soleus muscles (L7 and $1). The dura was opened under paraffin oil and the ventral roots split to isolate functionally single filaments supplying the soleus muscle. Electrical activity in the filaments in response to a 400 Hz, 10 sec conditioning tetanus applied peripherally to the soleus nerve was monitored using platinum wire electrodes. It was then amplified and stored on magnetic tape for later analysis and photography.
2.2. Administration o f dantrolene sodium Dantrolene sodium is a drug which is poorly soluble in saline. This difficulty was circumvented by heating, but not boiling, the salt in 0.1 N NaOH. Boiling DS in NaOH to produce a concentrated solution may cause inactivation of the c o m p o u n d by opening the h y d a n t o i n ring (Dr. A.W. Castellion, personal communication). The solution was then diluted with warm saline and administered while still warm (< 37°C) in a final volume of approximately
H.E. LOWNDES
3 ml. The volume of NaOH used in any injection did not exceed 1 ml. The same volume of 0.1 N NaOH was without effect on any of the parameters measured. All concentrations of dantrolene sodium are expressed in terms of the salt. Blood pressure was monitored by means of a cannula inserted into the femoral artery of the contralateral leg.
3. Results Dantrolene sodium (DS), administered i.v. in concentrations of 0.1--1.5 mg/kg caused a dose-dependent reduction in contractile strength of indirectly stimulated soleus muscle (fig. 1, also fig. 4, middle trace). Twitch depression occurred within 10--20 sec of DS administration, remained maximally depressed for approximately 2 0 - 2 5 min and recovered to predrug levels over the subsequent 90 min. Fig. 1 illustrates that increasing DS concentration from 1.0 to 1.5 mg/kg resulted only in a disproportionately small increase in twitch depression, suggesting that a ceiling effect was being approached. Even at the largest dose employed, DS did not cause any appreciable degree of respiratory paralysis, hence the cats did not require mechanical ventilation.
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Fig. 1. Percent depression (± S.E.M.) of indirectly elicited contractile strength of cat soleus muscle following various dosages of dantrolene, n, number of determinations at each dose.
NEUROMUSCULAR PHARMACOLOGY OF DANTROLENE 100
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Fig. 2. Dantrolene depression of contractile strength of cat soleus muscle in response to varying frequencies of indirect stimulation, measured as percent (+S.E.M.) of pre-drug control. Each p o i n t represents 5 determinations.
Contractile tension of the soleus muscle elicited by various frequencies of indirect stimulation was measured before and after administration of DS. Contractile tension measured 10 sec after initiation of the test stimulus frequencies was also reduced in a dose-dependent fashion (fig. 2). On the average, the depression of contractile strength was less marked in preparations stimulated at 10 Hz than at frequencies of less than 10 Hz. The suggestion that contractile strength was less susceptible to depression at higher frequencies o f stimulation was investigated further by determining soleus muscle responses to tetanic frequencies of 10--400 Hz (fig. 3). Soleus muscle normally develops twitch contractile strength that is 15--25% of the maximum tetanic strength, depending on tetanic frequency {fig. 3, control). The twitch/tetanus (t/T) ratios were decreased by DS as a function of dose at all tetanic frequencies. At 1.5 mg/kg, DS caused the t/T ratio to fall at a b o u t 20% of that of untreated preparations. This dose-effect relationship did not strictly hold for the 10 Hz responses (fig. 3). Fig. 4 illustral~es the typical results obtained in a single experiment. Following a control 400 Hz tetanus, DS was administered; a test tetanus was given when contractile tension was maximally depressed (middle trace).
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Fig. 3. Effect o f d a n t r o l e n e on t w i t c h / T e t a n u s (t/T) ratios in cat soleus muscles. Control data were determined in u n t r e a t e d animals. Each p o i n t represents a m i n i m u m of 4 determinations. The x and z axes are neither linear nor logarithmic, but rather equally spaced for graphic purposes.
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Fig. 4. Typical results f r o m a single e x p e r i m e n t with simultaneous recording o f post-tetanic r e p e t i t i o n (top trace), contractile responses (middle trace) and blood pressure ( b o t t o m trace). T: 400 Hz, 10 sec tetanus.
270
H.E. LOWNDES
Repetitive electrical activity evoked by the tetani was monitored before and after DS administration (top traces) and blood pressure was simultaneously recorded ( b o t t o m trace). Dantrolene was without measurable effect on blood pressure as is illustrated in the bott o m trace, fig. 4. The transient variation in blood pressure observed was ascribed to an artefact of the injection of up to 3 ml of solution. Of particular interest were the tetanic responses elicited by 10 sec, 400 Hz trains of stimuli, since Standaert (1963) has demonstrated that this is the o p t i m u m conditioning frequency for evoking motor nerve terminal post-tetanic repetition (PTR) and also the resultant post-tetanic potentiation (PTP) in cat soleus muscle (Standaert, 1964a). Fig. 4, middle trace, demonstrates that a 400 Hz tetanus still evoked vigorous PTP following a dose of DS that caused a 50% reduction in twitch height. PTP in response to 400 Hz tetanus over a wide range of doses of DS (fig. 5) was quantitated in the following manner. PTP was measured before administration of DS as the increase in contractile tension immediately post-tetanically over pre-tetanus contractile strength. This provided control PTP measurements. Following DS administration post-
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Fig. 5. Post-tetanic potentiation (PTP) (+- S.E.M.) elicited by 400 Hz tetani following dantrolene administration, expressed as percent of control (see text for explanation of measurement of PTP). Each point represents 5 determinations.
tetanic contractile strength was again measured as increase over pre-tetanic contractile strength, which was decreased at this time as a consequence of the drug. This post-drug PTP increase was then expressed as a percentage of pre-drug PTP (fig. 5). This figure illustrates that as contractile strength was diminished with increasing dose, PTP became progressively larger with respect to pre-tetanic contractile strength. The repetitive electrical activity which underlies PTP was not altered by DS. For example, fig. 4, top trace, indicates that PTR was not altered by a dose of DS that reduced twitch height by approximately 50%. PTR was not altered in either amplitude or duration by any concentration of DS investigated.
4. Discussion Standaert (1964b) demonstrated that posttetanic repetitive electrical events originating in cat soleus m o t o r nerve terminals are extremely sensitive to depressant drugs. For example, he showed that doses of d-tubocurarine, too small to affect contractile responses, were capable of decreasing and then abolishing this post-tetanic repetition. As a consequence, muscle PTP was diminished or blocked. The demonstration that PTR was not altered in amplitude or duration by dantrolene sodium provides evidence that this c o m p o u n d is without effect on the motor nerve terminals in cat soleus muscle. Any depressant action would have been signalled by a quantitative attenuation in PTR. Conversely, any facilitating action would have been manifested by an increase in the a m o u n t or duration of PTR. Dantrolene did not abolish post-tetanic potentiation of contractile strength in the present study. Similarly, Nott and Bowman (1974) reported DS to have little effect on this posttetanic p h e n o m e n o n in cat soleus muscle. However, in their study, Nott and Bowman employed tetanic trains of 60 Hz for 3 sec; in the present investigation tetanic stimulating fre-
NEUROMUSCULAR PHARMACOLOGY OF DANTROLENE quencies of up to 400 Hz for 10 sec were used. Standaert (1964a) showed that maximum PTP of contractile tension in cat soleus is elicited by 400 Hz, 10 sec tetani. Therefore, the tetanic frequencies used by Nott and Bowman (1974) were not sufficient to evoke maximum PTP and the effects of DS on PTP could not be ascertained. Nott and Bowman (1974} also investigated the actions of DS on cat anterior tibialis muscle. They found no relative depression of posttetanic augmentation of contractile strength in anterior tibialis muscles exposed to DS. However, these results are not directly comparable with those from soleus muscles in the present study. PTP in cat soleus muscle is largely of neural origin, being consequent upon generation o f PTR in the nerve terminals (Standaert, 1963; 1964a}. In a fast-contracting muscle, such as the anterior tibialis, post-tetanic facilitation is of post-junctional origin, distinct from the pre-junctional origin of this event in slowcontracting soleus muscle (cf. Standaert, 1964a). The results are comparable to the extent that they demonstrate that DS does not impair PTP whether it is of neurogenic (soleus muscle) or myogenic (anterior tibialis) origin. Therefore, DS does not impair either the pre- or post-junctional influences of high-frequency conditioning volleys. If DS acted via an action on neuromuscular transmission, an impaired transmission process would be further embarrassed b y the additional stress of tetanic stimulation, whereas increasing the stimulus frequency tended to increase the DS-reduced contractile tensions (figs. 2, 3 and 5). Additionally, indirectly elicited post-synaptic muscle action potentials are not altered by DS, regardless of the concentration (Honkomp et al., 1970; Ellis and Bryant, 1972; Nott and Bowman, 1974; Odette and Atwood, 1974). Therefore, DS must be exerting its effects at a site distal to the neuro muscular junction. Fig. 5 suggests that PTP is proportionately greater in DS-treated preparations than in normal muscles. While higher frequencies of stimulation proved effective in overcoming DS-induced twitch reduction (vide supra), the maxi-
271
m u m tetanic strength of the soleus muscles was n o t altered by any of the concentrations of DS studied. PTP is not, in fact, increased by dantrolene; the exaggerated PTP/twitch ratios following DS result from the depressed twitch and n o t an increase in PTP. The proportional increase in post-tetanic amplitude is therefore n o t significant. Putney and Bianchi (1974) similarly reported DS to have a lesser effect on development of tetanus tension than on twitch tension. Respiratory depression was not a feature of the actions of dantrolene in the present study. The cats did not require mechanical ventilation even following administration of the largest dose of DS investigated. Nott and Bowman (1974) employed a wider range of doses of DS (up to 32 mg/kg cumulative dose) and reported a similar observation, even when indirectly elicited twitch tension of skeletal muscle was reduced b y as much as 90%. Changes in heart rate and blood pressure in the present study and that of Nott and Bowman (1974) were neither remarkable nor attributable to DS. These findings suggest that DS may have a reasonable safety factor in clinical use. Putney and Bianchi (1974) suggested that DS owes at least part of its effect to an inhibition of a triggering step in excitation-contraction coupling (ECC). Their evidence indicates that DS may interfere with the movement of calcium, in its role as the 'trigger'. The present investigation indirectly supports this hypothesis with t w o facts. First, the action of DS is n o t mediated via a depressant action on neuromuscular transmission. This, combined with the observation of normal muscle action potentials suggests an action on an event subsequent to neuromuscular transmission, or ECC. Secondly, the observation that DS-induced reduction of twitch contractions can be partly reversed by higher frequencies of stimulation supports the idea of an action of DS on calcium movement in the muscle. Bianchi and Shanes (1959) showed that calcium release is enhanced by stimulation of a muscle. This additional calcium, elicited by higher frequency stimulation, could conceivably provide the
272
basis of the partial reversal of twitch reduction caused by DS.
Acknowledgements This investigation was supported by a grant from New Jersey Medical School. Dantrolene sodium was generously supplied by Dr. A.W. Castellion of the Norwich Pharmacol. Co., Norwich, N.Y.
References Bianchi, C.P. and A.M. Shanes, 1959, Ca-influx in skeletal muscle at rest, during activity and during K-contracture, J. Gen. Physiol. 42, 803. Chipman, M., S. Kaul and M. Lambi, 1974, Efficacy of dantrolene sodium in the treatment of spasticity, Dis. Nerv. Syst. 35, 427. Chyatte, S.B., J.H. Birdsong and B.A. Bergman, 1971, The effects of dantrolene sodium on spasticity and motor performance in hemiplegia, South. Med. J. 64, 180. Ellis, K.O. and S.H. Bryant, 1972, Excitation--contraction uncoupling in skeletal muscle by dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 107. Ellis, K.O. and J.F. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium. A skeletal muscle relaxant, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Honkomp, L.J., R.P. Halliday and F.L. Wessels, 1970, Dantrolene, 1-(5-(p-nitrophenyl)-furfurylidene) amino hydantoin, a unique skeletal muscle relaxant, The Pharmacologist 12, 301.
H.E. LOWNDES Nott, M.W. and W.C. Bowman, 1973, Effects of dan ° trolene sodium on contractions of cat skeletal muscles, Naunyn-Schmiedeb. Arch. Pharmacol. 279 {Suppl.), R30. Nott, M.W. and W.C. Bowman, 1974, Actions of dantrolene sodium on contractions of the tibialis anterior and soleus muscles of cats under chloralose anaesthesia, Clin. Exptl. Pharmacol. Physiol. 1,113. Odette, L.L. and H.L. Atwood, 1974, Dantrolene sodium: Effects on crustacean muscle, Can. J. Physiol. Pharmacol. 52, 887. Putney, J.W. and C.P. Bianchi, 1974, Site of action of dantrolene in frog sartorius muscle, J. Pharmacol. Exptl. Therap. 189, 202. Riker, W.F., Jr. and M. Okamoto, 1969, Pharmacology of motor nerve terminals, Ann. Rev. Pharmacol. 9, 173. Riker, W.F., Jr., J. Roberts, F.G. Standaert and H. Fujimori, 1957, The motor nerve terminal as the primary focus for drug-induced facilitation of neuromuscular transmission, J. Pharmacol. Exptl. Therap. 121, 286. Snyder, H.R., Jr., C.S. Davis, R.K. Bickerton and R. Halliday, 1967, 1-fi-(5-arylfurfurylidene)-amino)hydantoins. A new class of muscle relaxants, J. Med. Chem. 10, 807. Standaert, F.G., 1963, Post-tetanic repetitive activity in the cat soleus nerve. Its origin, course and mechanism of generation, J. Gen. Physiol. 47, 53. Standaert, F.G., 1964a, The mechanisms of posttetanic potentiation in cat soleus and gastrocnemius muscles, J. Gen. Physiol. 47,987. Standaert, F.G., 1964b, The action of d-tubocurarine on the motor nerve terminal, J. Pharmacol. Exptl. Therap. 143, 181.
D A N T R O L E N E EFFECTS ON N E U R O M U S C U L A R FUNCTION IN CAT SOLEUS MUSCLE H ERBE R T E. LOWNDES
Department of Pharmacology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, 100 Bergen Street, Newark, New Jersey 07103, U.S.A. Received 16 January 1975, revised MS received 3 March 197 5, accepted 27 March 1975 H.E. LOWNDES, Dantrolene effects on neuromuscular function in cat soleus muscle, European J. Pharmacol. 32 (1975) 267--272. Dantrolene sodium (DS) was investigated for its effects on cat soleus muscle contractile properties and motor nerve terminal activity in particular. DS, 0.1--1.5 mg/kg i.v., caused a dose-dependent depression of indirectly elicited contractile strength which was more pronounced at lower frequencies of stimulation. Maximum tetanic strength at frequencies of 10--400 Hz was depressed to a lesser degree than contractile responses evoked by lower frequencies of stimulation~ the twitch/tetanus contraction ratios were reduced with increasing dose, primarily because of diminished twitch. DS was without effect on motor nerve terminals as evidenced by normal post-tetanic repetition in the nerves following DS administration. Post-tetanic potentiation became relatively larger in amplitude as contractile strength was diminished. These data suggest that DS depresses neuromuscular function at a site other than the neural apparatus at the neuromuscular junction. Motor nerve terminals
Dantrolene sodium
1. Introduction Dantrolene sodium (DS) is a new muscle relaxant which has been evaluated for clinical efficacy in the therapy of spasticity (Snyder et al., 1967; Chyatte et al., 1971; Chipman et al., 1974). In contrast to other 'muscle relaxants', the mechanism of action of DS has been ascribed to an uncoupling action on excitation--contraction coupling in the muscle {Ellis and Carpenter, 1972; Putney and Bianchi, 1974). While Putney and Bianchi {1974) ascribed a significant portion of the muscle relaxant actions of DS to such an uncoupling, they did not rule out other possible mechanisms. Even though muscle action potentials are n o t altered by DS ( H o n k o m p et al., 1970; Ellis and Bryant, 1972; Nott and Bowman, 1973; 1974; Odette and Atwood, 1974), the possibility of a motor nerve terminal depressant action of DS has not been explored. A direct effect of DS on motor nerve terminals could be contributory to the pharmacological actions of DS.
Muscle relaxant
Contractile strength
The m o t o r nerve terminals in cat soleus are extremely sensitive to a wide variety of pharmacological agents, both depressant and facilitatory (Riker and Okamoto, 1969). Monitoring of the responsiveness of these nerve terminals provides a sensitive indicator of any depressant effect of the agent on the nerve endings. This communication reports the responsiveness of m o t o r nerve terminals in the presence of DS. Also, the actions of DS on contractile responses of this nerve--muscle preparation have been assessed, and the dose--response relationship of DS-induced muscle paralysis is reported.
2. Materials and methods
2.1. Surgical preparation Adult cats of either sex were anesthetized with a-chloralose, 80 mg/kg i.v., and surgically prepared for either contractile or motor
268
nerve terminal responses as previously described (Riker et al., 1957). Briefly, in experiments on contractile responses the sciatic nerve was sectioned at the sciatic notch, the gastrocnemius muscles and their innervation excised and the leg was held rigidly in a modified Brown--Schuster myograph by means of steel drill rods driven through the bone. The preparation was covered with liquid paraffin oil and maintained at 37 ° C by radiant heat. Platinum wire stimulating electrodes were placed on the soleus nerve and isometric contractile tension responses recorded by connecting the strain gauge o u t p u t to a pen-writer. The preparations were stimulated supramaximally at 0.4 Hz with square pulses of 0.1 msec duration. Tetani were delivered as trains of 10 sec duration at frequencies described in the text. Motor nerve terminal responses in the presence of dantrolene sodium were recorded in preparations which had been surgically prepared as described for contractile responses, except that the sciatic nerves were not sectioned. In addition, a dorsal laminectomy was performed to expose the ventral roots innervating the soleus muscles (L7 and $1). The dura was opened under paraffin oil and the ventral roots split to isolate functionally single filaments supplying the soleus muscle. Electrical activity in the filaments in response to a 400 Hz, 10 sec conditioning tetanus applied peripherally to the soleus nerve was monitored using platinum wire electrodes. It was then amplified and stored on magnetic tape for later analysis and photography.
2.2. Administration o f dantrolene sodium Dantrolene sodium is a drug which is poorly soluble in saline. This difficulty was circumvented by heating, but not boiling, the salt in 0.1 N NaOH. Boiling DS in NaOH to produce a concentrated solution may cause inactivation of the c o m p o u n d by opening the h y d a n t o i n ring (Dr. A.W. Castellion, personal communication). The solution was then diluted with warm saline and administered while still warm (< 37°C) in a final volume of approximately
H.E. LOWNDES
3 ml. The volume of NaOH used in any injection did not exceed 1 ml. The same volume of 0.1 N NaOH was without effect on any of the parameters measured. All concentrations of dantrolene sodium are expressed in terms of the salt. Blood pressure was monitored by means of a cannula inserted into the femoral artery of the contralateral leg.
3. Results Dantrolene sodium (DS), administered i.v. in concentrations of 0.1--1.5 mg/kg caused a dose-dependent reduction in contractile strength of indirectly stimulated soleus muscle (fig. 1, also fig. 4, middle trace). Twitch depression occurred within 10--20 sec of DS administration, remained maximally depressed for approximately 2 0 - 2 5 min and recovered to predrug levels over the subsequent 90 min. Fig. 1 illustrates that increasing DS concentration from 1.0 to 1.5 mg/kg resulted only in a disproportionately small increase in twitch depression, suggesting that a ceiling effect was being approached. Even at the largest dose employed, DS did not cause any appreciable degree of respiratory paralysis, hence the cats did not require mechanical ventilation.
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80
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Fig. 1. Percent depression (± S.E.M.) of indirectly elicited contractile strength of cat soleus muscle following various dosages of dantrolene, n, number of determinations at each dose.
NEUROMUSCULAR PHARMACOLOGY OF DANTROLENE 100
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Contractile tension of the soleus muscle elicited by various frequencies of indirect stimulation was measured before and after administration of DS. Contractile tension measured 10 sec after initiation of the test stimulus frequencies was also reduced in a dose-dependent fashion (fig. 2). On the average, the depression of contractile strength was less marked in preparations stimulated at 10 Hz than at frequencies of less than 10 Hz. The suggestion that contractile strength was less susceptible to depression at higher frequencies o f stimulation was investigated further by determining soleus muscle responses to tetanic frequencies of 10--400 Hz (fig. 3). Soleus muscle normally develops twitch contractile strength that is 15--25% of the maximum tetanic strength, depending on tetanic frequency {fig. 3, control). The twitch/tetanus (t/T) ratios were decreased by DS as a function of dose at all tetanic frequencies. At 1.5 mg/kg, DS caused the t/T ratio to fall at a b o u t 20% of that of untreated preparations. This dose-effect relationship did not strictly hold for the 10 Hz responses (fig. 3). Fig. 4 illustral~es the typical results obtained in a single experiment. Following a control 400 Hz tetanus, DS was administered; a test tetanus was given when contractile tension was maximally depressed (middle trace).
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Fig. 3. Effect o f d a n t r o l e n e on t w i t c h / T e t a n u s (t/T) ratios in cat soleus muscles. Control data were determined in u n t r e a t e d animals. Each p o i n t represents a m i n i m u m of 4 determinations. The x and z axes are neither linear nor logarithmic, but rather equally spaced for graphic purposes.
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Fig. 4. Typical results f r o m a single e x p e r i m e n t with simultaneous recording o f post-tetanic r e p e t i t i o n (top trace), contractile responses (middle trace) and blood pressure ( b o t t o m trace). T: 400 Hz, 10 sec tetanus.
270
H.E. LOWNDES
Repetitive electrical activity evoked by the tetani was monitored before and after DS administration (top traces) and blood pressure was simultaneously recorded ( b o t t o m trace). Dantrolene was without measurable effect on blood pressure as is illustrated in the bott o m trace, fig. 4. The transient variation in blood pressure observed was ascribed to an artefact of the injection of up to 3 ml of solution. Of particular interest were the tetanic responses elicited by 10 sec, 400 Hz trains of stimuli, since Standaert (1963) has demonstrated that this is the o p t i m u m conditioning frequency for evoking motor nerve terminal post-tetanic repetition (PTR) and also the resultant post-tetanic potentiation (PTP) in cat soleus muscle (Standaert, 1964a). Fig. 4, middle trace, demonstrates that a 400 Hz tetanus still evoked vigorous PTP following a dose of DS that caused a 50% reduction in twitch height. PTP in response to 400 Hz tetanus over a wide range of doses of DS (fig. 5) was quantitated in the following manner. PTP was measured before administration of DS as the increase in contractile tension immediately post-tetanically over pre-tetanus contractile strength. This provided control PTP measurements. Following DS administration post-
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Fig. 5. Post-tetanic potentiation (PTP) (+- S.E.M.) elicited by 400 Hz tetani following dantrolene administration, expressed as percent of control (see text for explanation of measurement of PTP). Each point represents 5 determinations.
tetanic contractile strength was again measured as increase over pre-tetanic contractile strength, which was decreased at this time as a consequence of the drug. This post-drug PTP increase was then expressed as a percentage of pre-drug PTP (fig. 5). This figure illustrates that as contractile strength was diminished with increasing dose, PTP became progressively larger with respect to pre-tetanic contractile strength. The repetitive electrical activity which underlies PTP was not altered by DS. For example, fig. 4, top trace, indicates that PTR was not altered by a dose of DS that reduced twitch height by approximately 50%. PTR was not altered in either amplitude or duration by any concentration of DS investigated.
4. Discussion Standaert (1964b) demonstrated that posttetanic repetitive electrical events originating in cat soleus m o t o r nerve terminals are extremely sensitive to depressant drugs. For example, he showed that doses of d-tubocurarine, too small to affect contractile responses, were capable of decreasing and then abolishing this post-tetanic repetition. As a consequence, muscle PTP was diminished or blocked. The demonstration that PTR was not altered in amplitude or duration by dantrolene sodium provides evidence that this c o m p o u n d is without effect on the motor nerve terminals in cat soleus muscle. Any depressant action would have been signalled by a quantitative attenuation in PTR. Conversely, any facilitating action would have been manifested by an increase in the a m o u n t or duration of PTR. Dantrolene did not abolish post-tetanic potentiation of contractile strength in the present study. Similarly, Nott and Bowman (1974) reported DS to have little effect on this posttetanic p h e n o m e n o n in cat soleus muscle. However, in their study, Nott and Bowman employed tetanic trains of 60 Hz for 3 sec; in the present investigation tetanic stimulating fre-
NEUROMUSCULAR PHARMACOLOGY OF DANTROLENE quencies of up to 400 Hz for 10 sec were used. Standaert (1964a) showed that maximum PTP of contractile tension in cat soleus is elicited by 400 Hz, 10 sec tetani. Therefore, the tetanic frequencies used by Nott and Bowman (1974) were not sufficient to evoke maximum PTP and the effects of DS on PTP could not be ascertained. Nott and Bowman (1974} also investigated the actions of DS on cat anterior tibialis muscle. They found no relative depression of posttetanic augmentation of contractile strength in anterior tibialis muscles exposed to DS. However, these results are not directly comparable with those from soleus muscles in the present study. PTP in cat soleus muscle is largely of neural origin, being consequent upon generation o f PTR in the nerve terminals (Standaert, 1963; 1964a}. In a fast-contracting muscle, such as the anterior tibialis, post-tetanic facilitation is of post-junctional origin, distinct from the pre-junctional origin of this event in slowcontracting soleus muscle (cf. Standaert, 1964a). The results are comparable to the extent that they demonstrate that DS does not impair PTP whether it is of neurogenic (soleus muscle) or myogenic (anterior tibialis) origin. Therefore, DS does not impair either the pre- or post-junctional influences of high-frequency conditioning volleys. If DS acted via an action on neuromuscular transmission, an impaired transmission process would be further embarrassed b y the additional stress of tetanic stimulation, whereas increasing the stimulus frequency tended to increase the DS-reduced contractile tensions (figs. 2, 3 and 5). Additionally, indirectly elicited post-synaptic muscle action potentials are not altered by DS, regardless of the concentration (Honkomp et al., 1970; Ellis and Bryant, 1972; Nott and Bowman, 1974; Odette and Atwood, 1974). Therefore, DS must be exerting its effects at a site distal to the neuro muscular junction. Fig. 5 suggests that PTP is proportionately greater in DS-treated preparations than in normal muscles. While higher frequencies of stimulation proved effective in overcoming DS-induced twitch reduction (vide supra), the maxi-
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m u m tetanic strength of the soleus muscles was n o t altered by any of the concentrations of DS studied. PTP is not, in fact, increased by dantrolene; the exaggerated PTP/twitch ratios following DS result from the depressed twitch and n o t an increase in PTP. The proportional increase in post-tetanic amplitude is therefore n o t significant. Putney and Bianchi (1974) similarly reported DS to have a lesser effect on development of tetanus tension than on twitch tension. Respiratory depression was not a feature of the actions of dantrolene in the present study. The cats did not require mechanical ventilation even following administration of the largest dose of DS investigated. Nott and Bowman (1974) employed a wider range of doses of DS (up to 32 mg/kg cumulative dose) and reported a similar observation, even when indirectly elicited twitch tension of skeletal muscle was reduced b y as much as 90%. Changes in heart rate and blood pressure in the present study and that of Nott and Bowman (1974) were neither remarkable nor attributable to DS. These findings suggest that DS may have a reasonable safety factor in clinical use. Putney and Bianchi (1974) suggested that DS owes at least part of its effect to an inhibition of a triggering step in excitation-contraction coupling (ECC). Their evidence indicates that DS may interfere with the movement of calcium, in its role as the 'trigger'. The present investigation indirectly supports this hypothesis with t w o facts. First, the action of DS is n o t mediated via a depressant action on neuromuscular transmission. This, combined with the observation of normal muscle action potentials suggests an action on an event subsequent to neuromuscular transmission, or ECC. Secondly, the observation that DS-induced reduction of twitch contractions can be partly reversed by higher frequencies of stimulation supports the idea of an action of DS on calcium movement in the muscle. Bianchi and Shanes (1959) showed that calcium release is enhanced by stimulation of a muscle. This additional calcium, elicited by higher frequency stimulation, could conceivably provide the
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basis of the partial reversal of twitch reduction caused by DS.
Acknowledgements This investigation was supported by a grant from New Jersey Medical School. Dantrolene sodium was generously supplied by Dr. A.W. Castellion of the Norwich Pharmacol. Co., Norwich, N.Y.
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