Acta physiol. scand. 1978. 103. 160-164 From the Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden
Pharmacological properties of curare-sensitive receptors mediating post-tetanic potentiation in rat masseter muscle BY
LENNART WALDENLIND Received 29 November 1977
Abstract WALDENLIND, L. Pharmuco~ogicu~ properties of curare-sensitive receptors mediating posttetutiic potetitiation it1 rut masseter mrtscle. Acta physiol. scand. 1978. 103. 160-164. Post-tetanic potentiation (PTP) of single muscle twitches after “tetanic” stimulation at 1 to 20 Hz in rat masseter muscle has been studied. PTP was inhibited by d-tubocurarine (2-6 ,ug/kg), hexamethonium (1.75-2.25 mg/kg), succinylcholine (5-7pg/kg), prostigmin (l.O-I.Spg/kgj, lidocain ( 3 4 mg/kg) and ouahain (215-240 pgjkg). The doses did not affect the control twitches. Prostigmin could not reverse the blocking effect on the PTP caused by these drugs. Atropin was without any effect on the PTP. The nicotinic receptors mediating PTP differ from both end-plate and ganglionic receptors since drugs blocking both types of receptors are effective. The sensitivity to the blocking agents is higher or the same (in the case of hexamethonium) as for the two other types of nicotinic receptors. Thus the PTP is dependent on activity of a “nicotinic” receptor active during physiological conditions, suggesting the presence of an additional mechanism for neuromuscular transmission in rat twitch fibres.
Tetanic stimulation of fast skeletal muscle causes a transitory increase in the peak tension of the isometric twitch response (post-tetanic potentiation, PTP), whereas a decrease is noted in slow mammalian muscles (Brown and Euler 1938, Bowman 1962, Standaert 1964 and Close and Hoh 1968). PTP is absent in muscles perfused with saline solutions (Brown and Euler 1938) suggesting the importance of a humoral factor. It has been shown that PTP is abolished in thiamine-deficiency induced by pyrithiamine, or by d-tubocurarine in doses lower than those affecting the normal single twitches (Waldenlind. In Press). Since thiamine has been shown to bind to nicotinic receptors (Waldenlind el al. I n Press) and since the role of thiamine for the development of PTP was found to be unrelated to the cocarbovylase functions of thiamine-diphosphate, it was thought that thiamine is important for the function of curare-sensitive receptors necessary for the occurrence of PTP. In the present article the pharmacological properties of curare-sensitive receptors mediating PTP are studied. 160
RECEPTORS MEDIATING POST-TETANIC POTENTIATION
161
Fig. 1. Effect of hexamethonium on PTP. The first arrow indicates the infusion of hexamethonium (2.0 mg/ kg). Part of the PTP is still left. N o effect on the single twitches is seen. The next PTP is, however, significantly smaller. The second bar indicates the infusion of an additional dose of hexamethonium (0.5 mg/kg). The PTP is now totally abolished and the single twitches are still the same as in the beginning. The pretetanic contraction force is equivalent to 55 g.
Methods Male Sprague-Dawley rats (250-500 g) were anesthetized with meburnal sodium (30 mg/kg). The trachea was intubated with a plastic tube just below the larynx. The external jugular vein was cannulated to allow i.v. injections. The normal blood circulation of the masseter muscle was intact. The masseteric nerve was prepared and cut off proximally. Supramaximal stimulation (1.5 times that required for maximal stimulation) of the masseteric nerve was then carried out both directly and by field stimulation (0.2 Hz, 0.2 ms) by a Grass S D 9 stimulator using platinum electrodes. The isometric twitches of the masseter muscle were measured by a thread through the mandibular bone. The thread was connected to a Grass force-displacement transducer (FT 10) and a Grass polygraph (model 7 B). The contraction force registered is therefore proportional to the tension in the masseter muscle. The initial tension was equivalent to 150 g. The contraction force is given in the individual figures. The PTP was provoked by a brief tetanic stimulation at 2 H z for 5 s. Since the untreated muscle responded with individual twitches at frequences at 20 Hz (typical for fast muscle) the term PTP is somewhat incorrect. However, it is an accepted way of describing the phenomenon studied. All substances were dissolved in 150 mM NaCl (0.3 ml) and injected i.v.
Materials d-tubocurarine chloride and hexamethonium bromide were obtained from Sigma Chemical Co; ouabain and sodium pentobarbital from ACO, Sweden; prostigmin and diazepam from Hoffman LaRoche, succinylcholine from Vitrum, Sweden and haloperidol from LEO, Sweden.
Results Hexamethonium inhibited PTP dose-dependently. The ordinary twitches were unaffected (Fig. 1). Prostigmin (1-5 pg/kg) did not reverse the effect of hexamethonium, on the contrary, a potentiation of the hexamethonium effect was noted. PTP was inhibited by doses of hexamethonium (ED,, = 1.75-2.0 mg/kg, n =4) slightly higher than those inhibiting the action of nicotine on the isolated guinea-pig ileum (Waldenlind 1977). Succinylcholine blocked PTP dose-dependently in doses (ED,, = 5-7 ,ug/kg, n =4; ED,,, = 9-13 pg/kg, n = 4 ) which did not affect the ordinary muscle twitches (Fig. 2). At higher =40-50 ,ug/kg, n =4). The effect of doses the ordinary muscle twitches were reduced (EDSO succinylcholine on the PTP could not be reversed by prostigmin. The ordinary muscle twitches increased after prostigmin administration at a dose when the PTP was totally inhibited by succinylcholine showing that the end plate receptors were unaffected (Fig. 2). 11 - 785876
162
LENNART WALDENLIND
Fig. 2 . Effect of succinylcholine and prostigniin on the PTP. The first arrow indicates the infusion of succinylcholinc (10 lfg'kg). The PTP is almost completely abolished. The second bar shows the infusion of proqtigmin (6 pg!kg). The single twitches at low frequency stimulation increases but PTP is still inhibited. The pretetanic contractim force is equivalent to 44 g.
3-4 mg/kg, n = 5 ) as did ouabain (ED,, 215-240 pglkg. n - 5 ) (Fig. 3). Both lidocain and ouabain completely inhibited the PTP without any effect on the ordinary twitches. Pretreatment with a dose of d-tubocurarine which was about half of that having a minimal effect on the PTP, gave a dose-dependent inhibition of the P T P by lidocain (50-100 mg/kg) in doses which otherwise were without any effect. The inhibition of the PTP by lidocain or ouabain was not reversed by prostigmin. The single twitches could always be increased by prostigmin even if the PTP was totally inhibited. Prostigmin inhibited P T P dose-dependently (ED,, -= 1.0-1.5 pg/kg, n "4; ED,,, -2.02.5 Icgllig. n 4). The ordinary muscle twitches began to increase at a dose of 3-4 pg/kg. Atropin was without any effect on the PTP in doses up to 50 mg/kg (Fig. 4). Haloperidol (10 mg/kg), mebumal (50 nig/kg) and diazepam ( 2 mg/kg) were without any effect o n the PTP. Lidocain also inhibited the PTP dose-dependently (ED,,
=
a
b
Fig. 3. Effect of ouabain on PTP. a ) PTP after stimulation at 20 Hz for 5 s of untreated preparation. b) Trtanic stimulations at 30 Hz for 5 s and at 50 Hz for 20 s after infusion of 120 pg/kg ouabain. No PTP develops after stirnulation at 20 Hr or at 50 Hz (for 20 s). The pretetanic contraction force is equivalent to 41 g. The 50 Hz stimulation for 70 s reached the maximum level of the Grass. Therefore the maximum contraction force can not be calculated.
RECEPTORS MEDIATING POST-TETANIC POTENTIATION
163
Fig. 4. The effect of atropin on PTP. Atropin (75 mg/kg) has been infused after the first tetanus and no effect o n either the single twitches o r the PTP can be seen. The pretetanic contraction force is equivalent to 63 g.
Discussion The results show that PTP is inhibited by nicotinic receptor blocking agents in doses lower than those affecting the ordinary muscle twitches. Inhibition of PTP occurred both with d-tubocurarine and hexamethonium indicating that the “nicotinic” receptors mediating PTP are undifferentiated and thus constitute a third class of nicotinic receptors active during physiological conditions. In Table I it is clearly seen that nicotinic receptors mediating PTP in fast mammalian twitch muscles are pharmacologically different from end plate receptors and nicotinic receptors mediating post-tetanic repetition in slow muscles, such as the cat soleus muscle. This difference is also seen in Fig. 2 where PTP is totally inhibited, but still prostigmin could increase the ordinary twitches. The end plate receptors must therefore be unaffected since succinylcholine blocks ordinary muscle twitches irreversibly. In the case of ouabain and lidocain, the single twitches could always be increased even if PTP was fully inhibited. Since the increase in single twitches after prostigmin treatment is due to post-tetanic repetition, PTP and post-tetanic repetition seem to have different pharmacological characteristics. It is probable that the action of lidocain is on nicotinic receptors since a small dose of d-tubocurarine which has no effect itself on the PTP potentiates the action of lidocain or ouabain. Presynaptic nicotinic receptors have been isolated which bind both local anesthetics and ouabain in low concentrations (Denburg and O’Brien 1973). TABLE I. The table summarizes the effects of drugs on end plate receptors, nicotinic receptors mediating PTP in fast mammalian muscles and nicotinic receptors mediating post-tetanic repetition. The effects of drugs o n post-tetanic repetition are from Riker (1969).
d-tubocurarine hexamethonium succinylcholine prostigmin atropin lidocain mebumal ouabain
end plate receptors
PTP
post-tetanic repetition
reversible inhibition 0
nonreversible inhibition nonreversible inhibition nonreversible inhibition nonreversible inhibition 0 inhibition 0 nonreversible inhibition
reversible inhibition 0
depolarizing nonreversible stimulation (by AChE-effect) 0 inhibition 0
0
stimulation stimulation 0 inhibition depression ?
164
LENNART WALDENLIND
It should be noted that during physiological conditions, only end-plate receptors and receptors mediating PTP in fast mammalian muscles are active. PTP in fast muscles begins to develop at 1 Hz and is maximal after about 10 Hz, post-tetanic repetition in slow muscles, such as the soleus muscle, is only seen after stimulation at 400 Hz which is an unphysiological stimulation frequency. The present results show that PTP in the rat masseter muscle is mediated via the activation of nicotinic receptors which have different properties in comparison with the end plate receptors, they are “nondifferentiated”, i.e. they are inhibited by both d-tubocurarine and hexamethonium. Since these nicotinic receptors are activated at physiological stimulation frequences (1 Hz) there evidently exists an additional synaptic mechanism in neuromuscular transmission in fast mammalian muscles. The possibility exists that activation of presynaptic nicotinic receptors are of importance for stimulation above 1 Hz in rat masseter muscle.
References B O W M ~ NW. . C., A. A. J. GOLDBEKG and C. RAPER,A comparison between the effects of a tetanus and the effects of sympathomimetic amines on fast- and slow-contracting mammalian muscles. Brit. J . Pharmacol. 1961. 19. 464-484. BKOWV.G . L. and U. S . v. EULER,The after effects of a tetanus on mammalian muscle. J. Phvsiol. (Lond.) 1938. Y3. 39-60. CLOSE.R. and J. F. Y.HOH.The after-effects of repetitive stimulation on the isometric twitch contraction of rat fast skeletal muscle. J . P/i.rsiol. (Lond.) 1968. 197. 461477. DFNRURC, J. L. and R . D. O ’ B R l f N , Axonal cholinergic binding macromolecule. Response to neuroactive drugs. J. t i i d Chcrii. 1973. 16. 57-60. KIKEK.W. F. and M. OKAMOTO. Pharmacology of motor nerve terminals. Ann. R E C .Pharinacol. 1969. 173208.
STAN~AER F.TG., . The mechanisms of post-tetanic potentiation in cat soleus and gastrocnemius muscle. J . 8c.n. Physiol. 1964. 47. 987-1001. WALDENLINO, L., Possible role of thiamine for neuromuscular transmission. To be published.
Pharmacological properties of curare-sensitive receptors mediating post-tetanic potentiation in rat masseter muscle BY
LENNART WALDENLIND Received 29 November 1977
Abstract WALDENLIND, L. Pharmuco~ogicu~ properties of curare-sensitive receptors mediating posttetutiic potetitiation it1 rut masseter mrtscle. Acta physiol. scand. 1978. 103. 160-164. Post-tetanic potentiation (PTP) of single muscle twitches after “tetanic” stimulation at 1 to 20 Hz in rat masseter muscle has been studied. PTP was inhibited by d-tubocurarine (2-6 ,ug/kg), hexamethonium (1.75-2.25 mg/kg), succinylcholine (5-7pg/kg), prostigmin (l.O-I.Spg/kgj, lidocain ( 3 4 mg/kg) and ouahain (215-240 pgjkg). The doses did not affect the control twitches. Prostigmin could not reverse the blocking effect on the PTP caused by these drugs. Atropin was without any effect on the PTP. The nicotinic receptors mediating PTP differ from both end-plate and ganglionic receptors since drugs blocking both types of receptors are effective. The sensitivity to the blocking agents is higher or the same (in the case of hexamethonium) as for the two other types of nicotinic receptors. Thus the PTP is dependent on activity of a “nicotinic” receptor active during physiological conditions, suggesting the presence of an additional mechanism for neuromuscular transmission in rat twitch fibres.
Tetanic stimulation of fast skeletal muscle causes a transitory increase in the peak tension of the isometric twitch response (post-tetanic potentiation, PTP), whereas a decrease is noted in slow mammalian muscles (Brown and Euler 1938, Bowman 1962, Standaert 1964 and Close and Hoh 1968). PTP is absent in muscles perfused with saline solutions (Brown and Euler 1938) suggesting the importance of a humoral factor. It has been shown that PTP is abolished in thiamine-deficiency induced by pyrithiamine, or by d-tubocurarine in doses lower than those affecting the normal single twitches (Waldenlind. In Press). Since thiamine has been shown to bind to nicotinic receptors (Waldenlind el al. I n Press) and since the role of thiamine for the development of PTP was found to be unrelated to the cocarbovylase functions of thiamine-diphosphate, it was thought that thiamine is important for the function of curare-sensitive receptors necessary for the occurrence of PTP. In the present article the pharmacological properties of curare-sensitive receptors mediating PTP are studied. 160
RECEPTORS MEDIATING POST-TETANIC POTENTIATION
161
Fig. 1. Effect of hexamethonium on PTP. The first arrow indicates the infusion of hexamethonium (2.0 mg/ kg). Part of the PTP is still left. N o effect on the single twitches is seen. The next PTP is, however, significantly smaller. The second bar indicates the infusion of an additional dose of hexamethonium (0.5 mg/kg). The PTP is now totally abolished and the single twitches are still the same as in the beginning. The pretetanic contraction force is equivalent to 55 g.
Methods Male Sprague-Dawley rats (250-500 g) were anesthetized with meburnal sodium (30 mg/kg). The trachea was intubated with a plastic tube just below the larynx. The external jugular vein was cannulated to allow i.v. injections. The normal blood circulation of the masseter muscle was intact. The masseteric nerve was prepared and cut off proximally. Supramaximal stimulation (1.5 times that required for maximal stimulation) of the masseteric nerve was then carried out both directly and by field stimulation (0.2 Hz, 0.2 ms) by a Grass S D 9 stimulator using platinum electrodes. The isometric twitches of the masseter muscle were measured by a thread through the mandibular bone. The thread was connected to a Grass force-displacement transducer (FT 10) and a Grass polygraph (model 7 B). The contraction force registered is therefore proportional to the tension in the masseter muscle. The initial tension was equivalent to 150 g. The contraction force is given in the individual figures. The PTP was provoked by a brief tetanic stimulation at 2 H z for 5 s. Since the untreated muscle responded with individual twitches at frequences at 20 Hz (typical for fast muscle) the term PTP is somewhat incorrect. However, it is an accepted way of describing the phenomenon studied. All substances were dissolved in 150 mM NaCl (0.3 ml) and injected i.v.
Materials d-tubocurarine chloride and hexamethonium bromide were obtained from Sigma Chemical Co; ouabain and sodium pentobarbital from ACO, Sweden; prostigmin and diazepam from Hoffman LaRoche, succinylcholine from Vitrum, Sweden and haloperidol from LEO, Sweden.
Results Hexamethonium inhibited PTP dose-dependently. The ordinary twitches were unaffected (Fig. 1). Prostigmin (1-5 pg/kg) did not reverse the effect of hexamethonium, on the contrary, a potentiation of the hexamethonium effect was noted. PTP was inhibited by doses of hexamethonium (ED,, = 1.75-2.0 mg/kg, n =4) slightly higher than those inhibiting the action of nicotine on the isolated guinea-pig ileum (Waldenlind 1977). Succinylcholine blocked PTP dose-dependently in doses (ED,, = 5-7 ,ug/kg, n =4; ED,,, = 9-13 pg/kg, n = 4 ) which did not affect the ordinary muscle twitches (Fig. 2). At higher =40-50 ,ug/kg, n =4). The effect of doses the ordinary muscle twitches were reduced (EDSO succinylcholine on the PTP could not be reversed by prostigmin. The ordinary muscle twitches increased after prostigmin administration at a dose when the PTP was totally inhibited by succinylcholine showing that the end plate receptors were unaffected (Fig. 2). 11 - 785876
162
LENNART WALDENLIND
Fig. 2 . Effect of succinylcholine and prostigniin on the PTP. The first arrow indicates the infusion of succinylcholinc (10 lfg'kg). The PTP is almost completely abolished. The second bar shows the infusion of proqtigmin (6 pg!kg). The single twitches at low frequency stimulation increases but PTP is still inhibited. The pretetanic contractim force is equivalent to 44 g.
3-4 mg/kg, n = 5 ) as did ouabain (ED,, 215-240 pglkg. n - 5 ) (Fig. 3). Both lidocain and ouabain completely inhibited the PTP without any effect on the ordinary twitches. Pretreatment with a dose of d-tubocurarine which was about half of that having a minimal effect on the PTP, gave a dose-dependent inhibition of the P T P by lidocain (50-100 mg/kg) in doses which otherwise were without any effect. The inhibition of the PTP by lidocain or ouabain was not reversed by prostigmin. The single twitches could always be increased by prostigmin even if the PTP was totally inhibited. Prostigmin inhibited P T P dose-dependently (ED,, -= 1.0-1.5 pg/kg, n "4; ED,,, -2.02.5 Icgllig. n 4). The ordinary muscle twitches began to increase at a dose of 3-4 pg/kg. Atropin was without any effect on the PTP in doses up to 50 mg/kg (Fig. 4). Haloperidol (10 mg/kg), mebumal (50 nig/kg) and diazepam ( 2 mg/kg) were without any effect o n the PTP. Lidocain also inhibited the PTP dose-dependently (ED,,
=
a
b
Fig. 3. Effect of ouabain on PTP. a ) PTP after stimulation at 20 Hz for 5 s of untreated preparation. b) Trtanic stimulations at 30 Hz for 5 s and at 50 Hz for 20 s after infusion of 120 pg/kg ouabain. No PTP develops after stirnulation at 20 Hr or at 50 Hz (for 20 s). The pretetanic contraction force is equivalent to 41 g. The 50 Hz stimulation for 70 s reached the maximum level of the Grass. Therefore the maximum contraction force can not be calculated.
RECEPTORS MEDIATING POST-TETANIC POTENTIATION
163
Fig. 4. The effect of atropin on PTP. Atropin (75 mg/kg) has been infused after the first tetanus and no effect o n either the single twitches o r the PTP can be seen. The pretetanic contraction force is equivalent to 63 g.
Discussion The results show that PTP is inhibited by nicotinic receptor blocking agents in doses lower than those affecting the ordinary muscle twitches. Inhibition of PTP occurred both with d-tubocurarine and hexamethonium indicating that the “nicotinic” receptors mediating PTP are undifferentiated and thus constitute a third class of nicotinic receptors active during physiological conditions. In Table I it is clearly seen that nicotinic receptors mediating PTP in fast mammalian twitch muscles are pharmacologically different from end plate receptors and nicotinic receptors mediating post-tetanic repetition in slow muscles, such as the cat soleus muscle. This difference is also seen in Fig. 2 where PTP is totally inhibited, but still prostigmin could increase the ordinary twitches. The end plate receptors must therefore be unaffected since succinylcholine blocks ordinary muscle twitches irreversibly. In the case of ouabain and lidocain, the single twitches could always be increased even if PTP was fully inhibited. Since the increase in single twitches after prostigmin treatment is due to post-tetanic repetition, PTP and post-tetanic repetition seem to have different pharmacological characteristics. It is probable that the action of lidocain is on nicotinic receptors since a small dose of d-tubocurarine which has no effect itself on the PTP potentiates the action of lidocain or ouabain. Presynaptic nicotinic receptors have been isolated which bind both local anesthetics and ouabain in low concentrations (Denburg and O’Brien 1973). TABLE I. The table summarizes the effects of drugs on end plate receptors, nicotinic receptors mediating PTP in fast mammalian muscles and nicotinic receptors mediating post-tetanic repetition. The effects of drugs o n post-tetanic repetition are from Riker (1969).
d-tubocurarine hexamethonium succinylcholine prostigmin atropin lidocain mebumal ouabain
end plate receptors
PTP
post-tetanic repetition
reversible inhibition 0
nonreversible inhibition nonreversible inhibition nonreversible inhibition nonreversible inhibition 0 inhibition 0 nonreversible inhibition
reversible inhibition 0
depolarizing nonreversible stimulation (by AChE-effect) 0 inhibition 0
0
stimulation stimulation 0 inhibition depression ?
164
LENNART WALDENLIND
It should be noted that during physiological conditions, only end-plate receptors and receptors mediating PTP in fast mammalian muscles are active. PTP in fast muscles begins to develop at 1 Hz and is maximal after about 10 Hz, post-tetanic repetition in slow muscles, such as the soleus muscle, is only seen after stimulation at 400 Hz which is an unphysiological stimulation frequency. The present results show that PTP in the rat masseter muscle is mediated via the activation of nicotinic receptors which have different properties in comparison with the end plate receptors, they are “nondifferentiated”, i.e. they are inhibited by both d-tubocurarine and hexamethonium. Since these nicotinic receptors are activated at physiological stimulation frequences (1 Hz) there evidently exists an additional synaptic mechanism in neuromuscular transmission in fast mammalian muscles. The possibility exists that activation of presynaptic nicotinic receptors are of importance for stimulation above 1 Hz in rat masseter muscle.
References B O W M ~ NW. . C., A. A. J. GOLDBEKG and C. RAPER,A comparison between the effects of a tetanus and the effects of sympathomimetic amines on fast- and slow-contracting mammalian muscles. Brit. J . Pharmacol. 1961. 19. 464-484. BKOWV.G . L. and U. S . v. EULER,The after effects of a tetanus on mammalian muscle. J. Phvsiol. (Lond.) 1938. Y3. 39-60. CLOSE.R. and J. F. Y.HOH.The after-effects of repetitive stimulation on the isometric twitch contraction of rat fast skeletal muscle. J . P/i.rsiol. (Lond.) 1968. 197. 461477. DFNRURC, J. L. and R . D. O ’ B R l f N , Axonal cholinergic binding macromolecule. Response to neuroactive drugs. J. t i i d Chcrii. 1973. 16. 57-60. KIKEK.W. F. and M. OKAMOTO. Pharmacology of motor nerve terminals. Ann. R E C .Pharinacol. 1969. 173208.
STAN~AER F.TG., . The mechanisms of post-tetanic potentiation in cat soleus and gastrocnemius muscle. J . 8c.n. Physiol. 1964. 47. 987-1001. WALDENLINO, L., Possible role of thiamine for neuromuscular transmission. To be published.
