Inhibition by local anaesthetic drugs at low and high stimulation frequencies

Neuropharmacology Vol. 23, No. 1, pp. X3-88, 1984 Printed in Great Britain. All rights reserved

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0028-3908/84 $3.00 + 0.00 1984 Pergamon Press Ltd

INHIBITION BY LOCAL ANAESTHETIC DRUGS AT LOW AND HIGH STIMULATION FREQUENCIES A COMPARISON BETWEEN THE ISOLATED PHRENIC NERVE OF THE RAT AND THE PHRENIC NERVE-DIAPHRAGM PREPARATION P. BRODIN and A. RGED Department of Physiology and Biochemistry, Dental Faculty, University of Oslo, Box 1052, Blindern, Oslo 3, Norway (Accepted 17 June 1983)

effects of lidocaine (0.25 mM), prilocaine (0.3 mM), diphenylhydantoin (0.24 mM), tetracaine (0.005mM) and dibucaine (0.002mM) on the isolated rat phrenic nerve, neuromuscular transmission and the directly stimulated rat diaphragm, were observed at low (twitch) and high (tetanic) stimulation frequency, The phrenic nerve compound action potential and muscle tension during indirect Summary-The

and direct stimulation were compared. At high frequency stimulation all drugs caused high frequency inhibition of both nerve and muscle. The high frequency inhibition was usually biphasic with an initial decrease in the amplitude of compound action potential and tetanic tension and a subsequent stabilization at a reduced plateau level. The duration of the initial phase increased with the potency of the drugs and was similar for nerve and muscle. A specific high frequency inhibition during indirect stimulation was found with diphenylhydantoin and prilocaine, indicating pre- and post-synaptic inhibition of neuromuscular transmission. At low frequency stimulation, the drugs induced a basal inhibition of the nerve compound action potential. In spite of that, the twitches were not depressed during indirect stimulation of the muscle, illustrating the margin of safety with neuromuscular transmission. These results indicate that the action of the drug was similar at the excitable nerve and muscle membranes when stimulated at high frequency, but different at low frequency stimulation. Key words: local anaesthetics, frequency-dependent action potential, tetanic tension.

inhibition, neuromuscular transmission, compound

brane stabilizing drugs which caused high frequency inhibition also induced a basal inhibition of the compound action potential at low frequency stimulation (Courtney, 1975). However, in mammalian muscle high frequency inhibition could be observed without a concomitant basal inhibition; the twitch contractions were unchanged or even increased (Harvey, 1939; Lilleheil and Reed, 1971). These similarities and differences in effects of membrane stabilizing drugs on amphibian nerve axons and mammalian muscle cells have not been compared in nerve and muscle preparations from the same species. In the present experiments, therefore, the effects of five membrane stabilizing drugs on the compound action potential of the isolated rat phrenic nerve were compared with the effects of the drugs on the indirectly and directly stimulated rat phrenic nerve-diaphragm preparation during low and high frequency stimulation. In this way, the actions of the drugs on the excitable membrane of a nerve and its muscle could be evaluated under identical experimental conditions. Furthermore, by indirect stimulation of the nerve-muscle preparation, the vulnerability of the process of neuromuscular transmission

Membrane stabilizing drugs, like local anaesthetics, block the action potential in nerves by inhibiting sodium conductance (Ritchie, 1979; Taylor, 1959). The inhibitory effect of most drugs is enhanced when the stimulation frequency is increased as shown in voltage clamp experiments (Courtney, 1975; Hille, 1977; Strichartz, 1973). High frequency inhibition has also been observed for the compound action potential in isolated frog nerves (Courtney, Kendig and Cohen, 1978) and for the tetanic tension in the mammalian muscle both during indirect and direct tetanic stimulation (Harvey, 1939; Lilleheil and Rered, 1971). In most cases the high frequency inhibition consisted of two phases: an initial phase with a transient decrease of electrical and/or mechanical activity (initial phase), followed by a phase where the activity was stabilized at a reduced level (plateau phase). Recordings from frog nerve and rat muscle have shown that the number of stimulation pulses needed to complete the initial phase varied for different drugs (Courtney et al., 1978; Reed, unpublished observations). In frog nerve libres, the concentration of mem83

P. BRDDIN and A. Rn~v

84

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Fig I. lnhihition of the compound actiun putzntiA uf the isolated rat phrenic ncrw and of kl;rnic ~ensinn of the indirectly stinmlaled rat diaphragm after exposure IO five mcmhriinc-;t;i~~li~i~~~ dtvgs. The basal inhibition of the nerve wan recur&d ~1 1 Hz. and the high frcqucncy InhIbItion 01 the ncrvc and I~USCIO was measured at the end of 5%~ stimulation periods al IOOHr ~meaa v:dIuc tSEM).

Frequency-dependent

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effects of local anaesthetics

stimulation of the muscle were performed after complete curarization (0.005 mM d-tubocurarine Cl; Sigma). A Grass Model S48 Stimulator was used for stimulation. When stimulating the nerve, supramaximum pulses of 0.05 msec duration were used. For indirect or direct stimulation of the muscle, supramaximum pulses of 0.05 or 0.5 msec duration, respectively, were used. A Grass Model CCUI Constant Current Unit was incorporated for direct stimulation. Low frequency stimulation was performed at 1 Hz for the nerve and at 0.1 Hz for the neuromuscular preparation. High frequency stimulation was obtained with a Ssec stimulation period at 100 Hz in all preparations. For recording, the compound action potential of the nerve was amplified by a Grass Model P 16 Amplifier, displayed on a Tektronix S I 13 Dual Beam Storage Oscilloscope and photographed. A Grass Model FT 03 or FT IO Force Displacement Transducer was used for tension recordings. Electromyogram tetanic and twitch tension of the muscle were monitored on a Grass Model 7 Polygraph. The effects during low and high frequency stimulation of five membrane stabilizing drugs were tested on the isolated phrenic nerve and on the indirectly-

as well as the mode of action of drug on the neuromuscular junction could be compared with the effects on excitable membranes. METHODS

Wistar rats of either sex, weighing 15&200 g, were anaesthetized with ether and decapitated. The left phrenic nerve or the left and right phrenic nervediaphragm preparation was dissected free and placed in a bath containing Tyrode solution (150 mM Na+, 2.7mM K+, l.8mM Ca”-, O.lmM Mg’+, l40mM Cl-, l2mM HCO;, 0.4mM H,PO<, I I mM dextrose). The bath was supplied with 95% Or and 5% CO*, the pH was 7.2, and the temperature was 37 C. The isolated phrenic nerve was mounted between two suction electrodes. One was used for stimulation and the other for recording of the compound action 1970). The phrenic nervepotential (Lilleheil, diaphragm preparation was mounted by a Bulbring technique (1946). The perspex holder anchoring the muscle contained two silver electrodes. These were used for recording of the electromyogram (EMG) during indirect stimulation or for direct stimulation of the muscle surface. Experiments involving direct

MUSCLE NERVE

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DIRECT

CONTROL

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Fig. 2. Pattern of phrenic nerve compound action potential and tension or the diaphragm (indirectly or directly evoked) during 5 set periods of 100 Hz stimulation. before (control) and 30 min after exposure to various drugs.

86

P. BRODINand A.

or directly-stimulated phrenic nerve-diaphragm preparation. The response to high frequency stimulation was tested before and at 10 min intervals after addition of drug. The following drugs were tested: lidocaine HCl (Astra), prilocaine HCl (Astra), tetracaine HCl (Sigma), diphenylhydantoin Na (Sigma) and dibucaine HCl (ICN). The concentrations of the drugs were selected as those causing about 50% basal inhibition in preliminary dose-response experiments. The experiments were performed in triplicate. The results were presented as mean +SEM. To test the reversibility of the effects, the recordings were repeated after washing the preparations with Tyrode solution 40min after addition of the drugs. RESULTS

RCED

pleted at the second stimulation pulse, and for diphenylhydantoin tetracaine and dibucaine after about 15, 25 and 300 pulses, respectively. With prilocaine the tetanic tension did not stabilize at a reduced level, but showed a continuous fall towards the baseline (Fig. 2). Muscle tension during direct stimulation The effects on the twitches were the same as during indirect stimulation. The pattern of the high frequency inhibition was also the same, except for diphenylhydantoin and prilocaine (Fig. 2). Diphenylhydantoin caused a continuous fall of the tetanic tension during the 5 set test periods, while with prilocaine a stable plateau was obtained. Lidocaine and prilocaine apparently caused a more extensive high frequency inhibition during direct than during indirect stimulation (Fig. 2).

Compound action potentials of the phrenic nerve

Reversibility of drug effects

All drugs caused a basal inhibition of the compound action potential at low frequency stimulation. However, a steady state was not obtained within the experimental period (Fig. 1). Ten minutes after addition, all drugs induced high frequency inhibition (Fig. 1). The high frequency inhibition of the nerve consisted of an initial decrease of the amplitude of the compound action potential (initial phase) followed by a stable reduced level (plateau phase) (Fig. 2). The duration of the initial phase was approximately the same after exposure to the drug for 10, 20, 30 and 40 min, but varied between the drugs. For prilocaine (0.3 mM) and lidocaine (0.25 mM) the initial phase was completed at the second stimulation pulse, for diphenylhydantoin (0.24 mM) after 10-15 pulses, for tetracaine (0.005 mM) after 25-30 pulses and for dibucaine (0.002 mM) after approx. 250 pulses (Fig. 2). The basal inhibition and the high frequency inhibition of the compound action potential of the nerve showed a parallel development during the 40 min experimental period (Fig. 1).

All effects of the drugs described above were reversed after washing with Tyrode solution. The effects of lidocaine and prilocaine were completely reversed 10min after washing, but with tetracaine, diphenylhydantoin and dibucaine the recovery phase was of longer duration.

Muscle tension and EMG during indirect stimulation

At low frequency stimulation no basal inhibition of the twitch contractions of the muscle was observed. Diphenylhydantoin, prilocaine and lidocaine caused potentiation of the twitch, whereas tetracaine and dibucaine did not affect the twitches. However, at high frequency stimulation all drugs caused high frequency inhibition of the tetanic tension (Fig. 2) and electromyogram of the muscle. The duration of the initial phase of the electromyogram and tetanic tension was essentially the same at high frequency test periods 10, 20, 30 and 40 min after addition of the drugs. The muscle was relatively more depressed than the nerve during the plateau phase of high frequency inhibition (Fig. 1). Recordings of the electromyogram showed that for lidocaine and prilocaine the initial phase was com-

DISCUSSION As shown in the results, both the nerve, neuromuscular transmission and the muscle cells were susceptible to drug action. The slow development of the inhibitory effects (Fig. 1) suggested that the basal inhibition, as well as the high frequency inhibition, may be exerted from the inside of the membranes. This site of action is partly in accordance with the theory of local anaesthesia presented by Narahashi, Frazier and Yamada (1970). Based on the knowledge of a pHdependent dissociation between a neutral and a positively-charged form of the drug, they suggested that the neutral form penetrated the membrane and that the positive form inhibited the ion conductance from the inside after renewed dissociation. However, this theory is partly contradicted by the present results where the negatively charged diphenylhydantoin also caused a slow development of both a basal inhibition and a high frequency inhibition. The order of potency of the drugs in causing high frequency inhibition was similar in the isolated nerve and in the muscle. Moreover, the pattern of the high frequency inhibition was the same in the isolated nerve and in the directly-stimulated muscle (except for the antiepileptic drug diphenylhydantoin, which caused a continuous reduction of tension during tetanic stimulation). For all the local anaesthetic drugs, the same number of pulses was needed to complete the initial depression phase in the nerve and in the muscle, suggesting a similar effect of each drug

Frequency-dependent effects of local anaesthetics on the excitable membranes of nerve axons and muscle cells to high frequency stimulation. However, the muscle was relatively more depressed than the isolated nerve during the plateau phase of the high frequency inhibition. This was in accordance with previous observations with propranolol (Lilleheil and Rned, 1971). Comparisons of the high frequency inhibition of muscle tension during indirect and direct stimulation showed that the local anaesthetics with high potency had similar effects, whereas the local anaesthetics with low potency, lidocaine and prilocaine, caused a more marked inhibition during direct stimulation. This was also observed for propranolol (Lilleheil and Rsed, 1971) and indicated that the high frequency inhibition was dependent on the stimulus pulse which excited the sarcolemma. Thus, the end-plate potential was more effective than the shorter, directly-applied pulse to antagonize the high frequency inhibition of the drugs. Accordingly, the high frequency inhibition may be explained by an interference by the drugs with the gating mechanisms regulating the excitability of the sarcolemma. Prilocaine caused a continuous decrease of tension during indirect tetanic stimulation. This pattern of high frequency stimulation differed from the responses which could be localized to the excitable membranes, and may therefore indicate the occurrence of a post-synaptic frequency-dependent inhibition of ion transfer, secondary to activation of receptors. Interaction with these processes was suggested by Akerman and Sokoll (1969) for other local anaesthetic agents. Diphenylhydantoin was the only drug which caused a more marked high frequency inhibition at the plateau phase during indirect than direct stimulation. The pattern of the high frequency inhibition was similar to that found in the isolated phrenic nerve. The effect may therefore be localized to the nerve terminal, which is known to be excitable (Katz and Miledi, 1965). All drugs caused a basal inhibition of the nerve at concentrations giving a high frequency inhibition of the isolated nerve as well as of the indirectly- and directly-stimulated muscle. However, a basal inhibition at these concentrations was not observed in the muscle, where instead the three drugs with lowest potency, diphenylhydantoin, lidocaine and prilocaine, caused a twitch potentiation. This potentiation was similar during indirect and direct stimulation and should therefore result from an effect on the muscle cell. This different susceptibility of the nerve axons and muscle cells to the effect of membrane-stabilizing drugs at low stimulation frequencies might be related to known differences between the ion conductances in nerve and muscle cells. The sarcolemma contains a late potassium channel which carries an outward potassium current during the repolarization of the action potential (Adrian, Chandler and Hodgkin,

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1970). A similar channel in the squid axon membrane was susceptible to block by the membrane-stabilizing drug, quinine (Yeh, Oxford and Narahashi, 1976). The inhibition of this potassium current by a drug may prolong the muscle action potential. An increase of the area of the action potential will ensue, and thus, more calcium is released from the sarcoplasmic reticulum through the excitation
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twitch. Accordingly, this result supported the suggestion of a considerable margin of safety of neuromuscular

transmission

Salpeter and

(Paton

Eldefrawi,

and

Waud,

196’7;

1973).

Acknou,leLl’gemenl-The work was supported by the Norwegian Research Council Science and the Humanities.

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