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The effects of verapamil on tetanic contractions of frog's ckeletal muscle
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Comp. Biochem. Physiol. Vol. 107C, No. 3, pp. 321-329, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/94 $6.00 + 0.00
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The effects of verapamil on tetanic contractions of frog's skeletal muscle M. Oz and G. B. Frank Department of Pharmacology, 9-70 Medical Sciences Building, Edmonton, Alberta, Canada, T6G 2H7 The effects of the organic calcium channel antagonist, verapamil, were tested on twitches and tetanic contractions (100 Hz, 2 sec) in frog toe muscles. At low concentrations (3 x 10 -6 M), verapamil had no effect on the maximum amplitudes of twitches, but significantly reduced the size of the tetanic responses. This depression was observed as an inability to maintain the maximum tetanic tension for more than 0.5 sec. With increasing concentrations up to 10 -4 M of verapamil, its depressant effect on tetanic responses gradually increased, and at very high concentrations (10 -4 M ) of verapamil, twitches were also blocked. Intraceilular microelectrode recordings showed that there was no block of the action potentials during the stimulus train at the concentration of 3 x 10 -6 M of verapamii. These results support the concept that during tetanic responses, the voltage sensitive Ca 2+ channels in the t-tubules open and the Ca 2÷ ions entering via these channels are required to maintain the full strength of the contraction. At higher concentrations, verapamil blocked Na + action potentials during the stimulus trains in a concentration and use-dependent manner. Key words: Calcium channel; Calcium channel blockers; Muscle contraction.
Comp. Biochem. Physiol. 107C, 321-329, 1994.
Introduction Blockade of voltage sensitive, slow calcium channels (VSSCCs) by verapamil and D-600, has been demonstrated in several different studies using amphibian and mammalian skeletal muscle (Beaty and Stefani, 1976; Frank, 1984; Walsh et al., 1986). In a previous study from this laboratory, it was reported that verapamil and D-600 block high K ÷ induced contractures, but not twitches, in frog's skeletal muscle at concentrations that block voltage sensitive slow Ca z÷ channels (Frank, 1984). Blockade of high K ÷ induced contractures by voltage dependent Ca 2÷ channel
Correspondence to: M. Oz, Laboratory of Molecular & Cellular Neurobiology, National Institute on Alcohol Abuse & Alcoholism, 12501 Washington Ave., Rockville, Maryland, 20852, U.S.A. Received 25 October 1993; accepted 17 November 1993. 321
blockers is also consistent with previous studies indicating that high K ÷ induced contractures require the influx of extracellular Ca 2+ ions for excitation-contraction (E-C) coupling (Frank, 1958, 1960, 1982; Liittgau and Spiecker, 1979). In addition to high K ÷ induced contractures, another type of maintained response in skeletal muscle is tetanic contractions. We have reported recently (Oz and Frank, 1991) that during removal of extracellular Ca z+ ions, tetanic responses were depressed in area before twitches were reduced in amplitude. Furthermore, nitrendipine, an organic Ca 2+ channel blocker, also greatly depressed the area under the tetanic tension curve without blocking the development of action potentials during repetitive stimulations. In the present study, the effects of verapamil on skeletal muscle membrane excitability and tetanic contractions were investigated.
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Preliminary results of some of the experiments described here were presented previously (Frank and Oz, 1991).
Materials and Methods Materials All experiments were carried out at room temperature (18-21°C) using a skeletal muscle isolated from the frog, Rana pipiens. The extensor longus digiti IV (toe) muscle of the frog was used in all experiments. The muscles were allowed to equilibrate for a period of 30-45 min in oxygenated Ringer's solution before an experiment was started. Drug solutions were prepared by dissolving verapamil hydrochloride (Knoll A. G., Ludwig Shafen) in the appropriate Ringer's solution. To accelerate the dissolving of verapamil, beakers with this drug plus solution were placed in a hot water bath for 5 to 10 min. The composition of the frog Ringer's solution was as follows: 111.87 mM NaC1, 2.47 mM KCI, 1.08mM CaC12, 0.087mM NaH2PO 4, 2.38mM NaHCO3 and 11.1 m M dextrose, D-Tubocurarine (0.1 mg/ml) was added to the Ringer's solution. Throughout the mechanical recording experiments, the bathing solution was bubbled with a gas mixture of 99.5% O z + 0.5% C02. Tension studies Toe muscles were dissected from the frog in a way similar to that described by Frank (1960) and were mounted vertically in an 8-ml bath containing Ringer's solution. The lower end of the muscle was fixed near the bottom of the bath and the upper end was attached to the arm of the strain gauge by means of a silk thread. Solutions were changed by draining the bath at the bottom and adding new solution at the top with a syringe. Tension was recorded by means of a strain gauge whose active elements consisted of two pixie transducers (Endevco model 81214) in a wheatstone bridge configuration. The resting tension on the muscle was adjusted so that the muscle just failed to remain in a vertical position when the bath solution was drained and returned to a vertical position when the bath was refilled with solution. This length is presumably near or at the resting length because the largest twitches were produced at this muscle length. For twitch recording, supramaximal rectangular pulses, 1.5msec in duration, were applied to the muscle by means of two platinum electrodes; one situated at the bottom and the other at the top part of the bath.
For tetanic responses, the muscles were stimulated with 1.5msec supramaximal rectangular pulses at 100, 50 or 25 Hz for 2 sec once every 10-15min in experiments lasting up to 3 hr. Twitches were recorded 15 sec to 1 min before each tetanus. Effects on twitches were evaluated by measuring the twitch tension and the effects on tetani by measuring the tension x time area of the response. The transducer outputs were digitized and recorded using a Tekmar Lab Master A/D converter board connected to a Compaq portable computer. Records were analyzed using a Compaq Deskpro 286 and the responses were drawn on a Roland D G P R - 1212A printer. Responses were recorded with 1000 points/sweep at all sweep speeds. Programs for collecting, transmitting, storing and analyzing data were written in Turbo Pascal.
Electrophysiological studies For measurements of the late negative afterpotential (LAP), the muscles were mounted horizontally in a bath and viewed from above with a dissecting microscope. Illumination was provided by light passing up through the transparent bottom of the bath. In order to reduce muscle movement and consequent membrane damage during the recordings of the action potentials and the after-potentials, the toe muscles were stretched and wrapped around a glass rod of 2ram diameter which was suspended across the bath in the Ringer's solution which was oxygenated l0 min prior to and during the recordings (Stefani and Schmidt, 1972; Oz and Frank, 1991). Resting membrane and action potentials of the frog's toe muscle fibers were recorded intracellularly using conventional glass capillary microelectrodes (10~40Mfl resistance) filled with 3 M KCI. A small proportion of the muscle fibers was electrically stimulated by an extracellular, bipolar platinum-filled pore electrode placed on the surface of the muscle 5-10 mm from the recording electrode. The bipolar stimulating electrode was constructed with 0.4 mm platinum wires placed in polyethylene tubing and cemented together so that only the ends of the wires were exposed to solution. The wire ends rested on the surface of the muscle 1.5 mm apart. The muscle fibers were stimulated with supramaximal pulses delivered through a stimulus isolater unit (WP Instruments, model 305). The stimulating pulse consisted of either a single supramaximal stimulus pulse or a train of square waves of 1.5 msec duration. The glass micro-electrode was held in a micro-electrode holder which was connected through a probe to the main amplifier (WP Instruments, model MA4). The output from the
Verapamil on tetanic responses
amplifier was displayed on either a Model 4094 or 3091 Nicolet digital oscilloscope. The recordings were made with either 16,000 (Model 4094) or 4000 (Model 3091) points/sweep at all sweep speeds. These records were stored on floppy disks and pictures of action potentials were drawn using a digital plotter.
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Results The effect of verapamil twitches and tetani In our preliminary observations, depression of single action potentials was observed only at concentrations higher than 10 -4 M and therefore these higher concentrations were not used in the remainder of this study. Experiments were conducted with 23 muscles using verapamil in the concentration range of 10-6-10-4M. The measurements made in these studies are plotted in Fig. 1. The twitch amplitudes were slightly increased by exposing the muscles to 5 x 10 -6 M and 5 x 10 -6 M verapamil (Fig. 1, top section). The potentiation of the twitch by organic Ca 2+ channel blockers at concentrations which block Ca 2+ channels in skeletal muscle
,
0
-4
Log [verapamil] Fig. 2. Concentration-response curve for 10 min exposures to verapamil on tetanic areas. The numbers of experiments (n) at each concentration are given in the legend for Fig. 1.
has been reported in a number of earlier studies (Marwaha and Treffers, 1980; Frank, 1984; Singh and Dryden, 1988). Depression of action potentials and thus twitches was only observed at very high concentrations (0.3 mM or higher) of verapamil or D-600 (Van Der Kloot, 1975; Bondi, 1978; Dorrscheidt-Kafer, 1977; Frank, 1984). Verapamil decreased the area under the 2 set: i50 Twitch Tension tetanic force curve at 100Hz stimulation frequency over the concentration range of 3 x 10 - 6 to 10-4M verapamil (Fig. 1 bottom section). The effect of verapamil over this range was large and the area under the tetanic tension curve was depressed by up to 96% of control values by iO0 10-4M verapamil. With all the concentrations used, the effect of verapamil reached its equilibrium value by 5-10 min. The dose-response curve obtained using the data taken at 10 min is 4-J g shown in Fig. 2. ¢J 5O In a previous study with nitrendipine, the Tetanic Area amplitude of the tetanus always declined gradually during the 2 sec stimulus train (Oz and 100 Frank, 1991). In contrast, verapamil produced a variable depression pattern on the tetanic responses (Fig. 3). Verapamil, at the concentration of 3 x 10-6M, like nitrendipine produced only a gradual tension decline in tetanic tension beginning 0.5-1.0 sec after the start of the stimulus train (Fig. 3A). At concentrations of 5 x 10 - 6 and 10-SM verapamil, there was 0 often a rapid decline in tension to zero followed 0 I0 20 30 40 50 50 70 by another rise in tension during the stimulus Time {min) train (Fig. 3B). This rapid depressant effect of verapamil on tetanic responses at a concenFig. I. Effects over time of different verapamil concentration of 5 × 10 -6 M did not occur when using trations on maximum twitch tension (at the top) and tetanus lower stimulation frequencies such as 50Hz areas (at the bottom) in frog toe muscles. Means and standard errors. Standard error bars were omitted when (Fig. 4). Increasing the concentration of verathey overlapped or when they were smaller than the size of pamil to 5 x 10 -5 or to 10-4M caused a rapid the symbol. The verapamil concentrations (molar) and the block of the tetanic contraction during the number of experiments (n) were as follows: A , A : 10 -6, (4); stimulus train at 100Hz (Fig. 3C). At these 0 , 0 : 3 x 10 -6 (5); I - I , l : 5 x 10 -6, (4); O , O : 10 -5, (4); x,x: high concentrations of verapamil, the maximum 5 x 10 -5, (4); and + , + : 10 -4, (5).
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M. Oz and G. B. Frank
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10m Fig. 3. Effects of different verapamil concentrations on the tetanic contractions of frog skeletal muscle. The concentrations of verapamil used and stimulation frequencies during 2 sec tetanic contractions were indicated on the left side of the figure.
tension developed during a tetanus was also decreased to a great extent.
The effect of different concentrations of verapamil on intracellularly recorded action potentials At concentrations of 5 x l0 -6 M and above, using 100Hz stimulation, verapamil always blocked some of the action potentials during the stimulation train. Depending on the concentration used, the time to block the development of repetitive action potentials was about 1.5 sec to tens of milliseconds (Fig. 5). Occasionally, we were able to record the repetitive action potentials occurring in a biphasic manner (Fig. 6). This biphasic development of action potentials seen in the intracellular recording was consistent with the biphasic force traces often seen in tension measurements (Fig. 3B). Although there was a significant depression of tetanic contractions at the concentration of 3 x 10-6M (Fig. 3A), at this concentration there was no blockade of action potentials
during 2 sec trains at 100 Hz of tetanic stimuli (Fig. 5). The use-dependent block of Na + action potentials varied with the stimulus frequency and the concentration of verapamil used. The use-dependent blockade of Na + action potentials by verapamil was reversed by using lower stimulation frequencies, such as 50 and 25 Hz. These effects are shown in Fig. 7. Only the loss of a few responses near the end of the stimulus train was produced by 5 × 1 0 - 6 M verapamil and this was prevented by lowering the stimulus frequency to 5 0 H z (Fig. 7A). A larger depression was produced by 10-SM verapamil and was only eliminated by reducing the stimulus frequency to 25 Hz (Fig. 7B). Discussion
It is known that verapamil blocks voltage dependent, show Ca :+ channels and
Verapamil on tetanic responses
325
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Fig. 4. Effects of lower stimulation frequency on the blockade of tetanic responses. Blockade of tetanic responses elicited by 50 Hz stimulation frequency for 2 sec by 5 x 10-6 M verapamil.
inhibits Ca 2+ dependent processes such as the Solandt effect, acetylcholine contractures and Ca 2+ dependent activation of phosphorylase kinase, at the relatively low concentrations of 10-5-10-6M in skeletal muscle (Chirandini and Bentley, 1973; Carlsen et aL, 1985). At such low concentrations, although both verapamil and D-600 have been shown to block high K ÷ induced contractures (Kaumann and Uchitel, 1976; Bondi, 1978; Frank, 1984), caffeine contractures were not depressed (Bondi, 1978; Marwaha and Treffers, 1980). These findings support the suggestion that verapamil blocks K+-induced contractures which require the influx of extracellular Ca 2+ ions for E-C coupling (Frank, 1958, 1982).
In addition to K÷-induced contractures, another type of maintained contraction is tetanic contractions in which repetitively developed action potentials produce a maintained tension. The function of slow Ca 2+ channels during tetanic contractions has been the subject of a few studies (Carlsen et al., 1985; Kostsias et al., 1986; Oz and Frank, 1991) and in other studies the importance of extracellular Ca ~÷ ions in tetanic responses has been mentioned (Spiecker et al., 1979; Blinks et al., 1978; Kostsias et al., 1986). In our studies (Oz and Frank, 1990, 1991), the areas under the tetanic force versus time curve during tetani were decreased when toe muscles were kept in Ca2+-free solutions. The fact that
A
B
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Fig. 5. Depression of intracellularly recorded action potentials by different concentrations of verapamil, during 2 sec stimulus trains at 100 Hz, A, B, C different toe muscles. On the left control recordings. On the fight responses to the stimulus train recorded in different fibers after 10rain in solutions with verapamil; in A, 3 x 10-6M (n:3); in B, 5 x 10-6M (n:3); in C, 10-4M (n:5). Some small stimulus artifacts can be seen in between the repetitive action potentials.
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I
50mV
I
I
2 sec
Fig. 6. Blockade of action potentials and rebursting during a 2 sec stimulus train at 100 Hz recorded in a toe muscle fiber exposed to 10-5 M verapamil for 10 min. Some stimulus artifacts can be seen between these action potentials during repetitive activities. Between the bursts, these artifacts have been reduced in size by whiting them out. Due to the limited time resolution of our digital oscilloscope used in this recording, the peaks of the spikes are usually missed and a liaising effect is seen (see also Oz and Frank, 1991).
nitrendipine, a n organic Ca2+-channel blocking drug, also reduced the (time x tension) area showed that extracellular Ca 2÷ ions need to enter the skeletal muscle via slow Ca 2÷ c h a n n e l s d u r i n g a tetanus in order for the muscle to m a i n t a i n the full tetanic t e n s i o n (Oz a n d F r a n k , 1991). It was d e m o n s t r a t e d some time ago that d u r i n g a series o f rapidly repeated action poten-
A1
A=
tials in skeletal muscle, K ÷ ions, which leak out with each action potential, a c c u m u l a t e in the t-tubules a n d only slowly achieve diffusion e q u i l i b r i u m with the s u r r o u n d i n g extracellular fluid ( F r e y g a n g et al., 1964; Kirsch et al., 1977). This K + a c c u m u l a t i o n causes a small b u t significant depolarization o f a b o u t 20-25 m V d u r i n g a train of tetanic action potentials a n d when the stimuli stop, the muscle fiber m e m b r a n e remains
BI
B2
A3 B_
Ai
B4
150rnV I
2 sec
Fig. 7. Intracellularly recorded action potentials for tetanic stimulation trains in frog toe muscle fibers. Recordings from different single fibers are presented in column A I through A4 and Bt through B4. Control recordings are A t and Bj. Verapamil at the concentration of 5 x 10 -~ M and 10-5 M used in recordings A2-A4 and B2-B4 respectively. Frequencies of stimulations used during recordings are 100; 100; 50 and 25 for At, B~; A2, B2; A3, B3; and A4, B4, respectively.
Verapamil on tetanic responses depolarized and only very slowly repolarizes (Fig. 5, control recordings); this slow repolarization was called the late negative after-potential (LAP). In our previous report, we suggested that the t-tubular depolarization at the time of the repetitive action potentials was sufficient to open the voltage sensitive, slow calcium channels (VSSCC) and allow some influx of Ca 2+ ions. Although this influx is not required to begin the tetanus, we suggested that it was required to maintain the full tension during the tetanic contraction. Our observation that nitrendipine reduced the tetanic contraction size without reducing the t-tubule depolarization supports this hypothesis. In a more recent study, it was shown that verapamil produced a block of the Ca 2+ fluxes in an isolated t-tubule vesicle preparation that were otherwise produced by depolarizing these vesicles by an amount equivalent to the size of the LAP (Oz et al., 1992, 1993). This finding supports our suggestions that the small depolarization during the tetanus is sufficient to open the VSSCCs and allow the influx of Ca 2÷ ions and that this influx could be blocked by Ca2+-channel blocking drugs. A more detailed discussion of this hypothesis with earlier supporting evidence from the literature can be found elsewhere (Oz and Frank, 1991; Frank and Oz, 1992). In addition, other studies, in agreement with our hypothesis, have demonstrated that VSSCCs have functional roles during long term skeletal muscle activities such as tetanic contractions and the treppe effect (Garcia et al., 1990; Williams, 1990; Williams and Ward, 1991). The results obtained in the present study were more complicated than our earlier findings with nitrendipine. Although we were careful to use only lower verapamil concentrations which did not block single twitches, still most of the concentrations we used did result in a usedependent block of some of the action-potentials during the stimulus trains at 100 Hz for 2 sec (Frank and Oz, 1991). Even so, we were able to demonstrate a clear reduction of the tetanus area due to the block of VSSCCs by verapamil under two experimental conditions. The first was a significant reduction of the area by 3 x 10-6M verapamil (Fig. 2) which was not a high enough concentration to produce a use-dependent action potential block during 100 Hz stimulation for 2 sec (Fig. 5). The second was the reduction of the tetanus area produced by 5 x 10-6M verapamil (Fig. 4) when the stimulus frequency was reduced to 50 Hz at which frequency this verapamil concentration did not produce any use-dependent action potential block (Fig. 7). In frog skeletal muscle, in addition to a block of the slow Ca 2÷ channels, verapamil at slightly
327
higher concentrations also blocks Na ÷ channels. Our results indicate that the repetitive firing of action potentials faciitates the blockade of Na ÷ action potentials in frog skeletal muscle fibers. At the concentration range of 5 x 1 0 - 6 to 10 -5 M, although no blockade of single action potential was observed, repetitive production of action potentials at the stimulation frequency of 100 Hz, elicited the use-dependent blockade of Na ÷ channels (see Figs 6 and 7). This usedependent blockade of Na÷-channels was reversed with the use of lower stimulation frequencies such as 50 or 25 Hz. At the concentration of 5 x 10 -6 M verapamil, using a 50 Hz stimulation frequency was enough to remove the blockade of Na ÷ channels. On the other hand, with 10 -5 M verapamil, a lower stimulation frequency of 25 Hz, which could produce only an unfused tetanus, was required to eliminate the blockade of Na ÷ channels. At the concentrations range of 5 x 1 0 - 6 to 10-SM verapamil, we sometimes observed that the blockade of Na ÷ action potentials followed a biphasic time course (see Fig. 6). In about 0.5-1 sec, there was a complete blockade of action potentials and a decrease in the cell membrane depolarization. This was followed by a second burst of full size action potentials which lasted a few hundred milliseconds. This second activation of action potentials during 2 sec pulse stimulations, was also consistent with the tension recordings in which a second rise in the tension curve was observed (see Fig. 3B). These findings suggested that the inactivation of the Na ÷ channels by verapamil declined during the time between these two activation periods (see Fig. 5). In addition to removal of the use-dependent blockade of Na ÷ channels by the absence of action potentials, the gradual reduction of the cell membrane depolarization might also contribute to the removal of the Na ÷ channel inactivation, and thus lead to the production of a second burst of action potentials. The time required for recovery from the use-dependent blockade of action potentials by verapamil was a few hundred milliseconds. This approximate recovery time is also quite similar to that seen with local anesthetics such as lidocaine that cause a use-dependent blockade of Na ÷ channels (Hille, 1988). Similar effects of verapamil have also been observed on the slow soleus muscle fibres of the rat (Kostsias and Muchnik, 1986). In conclusion, in the present study, the effects of verapamil have been observed on frog's skeletal muscle. At relatively low concentrations, verapamil causes an inhibition of tetanic contractions by the blockade of voltage sensitive slow Ca 2÷ channels. At slightly higher concentrations, verapamil, in addition to
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its known use-dependent blockade of calcium channels (Frank, 1986), also produces a use-dependent blockade effect on Na + channels of frog's skeletal muscle. Summary Verapamil exerts differential effects on skeletal muscle contractions that are dependent on the drug concentration and on the pattern of stimulation. It appears that at low concentrations, verapamil depresses the tetanic contractions without blocking action potentials. This depression was suggested to be related to the blockade of VSSCCs. At higher concentrations of verapamil, there was a blockade of sodium channels, and this blockade had usedependent properties. Acknowledgements--We would like to thank Joan McGinnis for her skilful technical assistance. This study was supported by grants from the Medical Research Council of Canada.
References Beaty G. N. and Stefani E. (1976) Calcium dependent electrical activity in twitch muscle fibres of the frog. Proc. R. Soc. London Set. B. 194, 141-150. Blinks J. R., Rudel R. and Taylor S. R. (1978) Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. 277, 291-323. Bondi A. Y. (1978) Effects of verapamil on excitationcontraction coupling in frog sartorius muscle. J. Pharmac. exp. Ther. 205, 49-57. Carlsen R. C., Larson D. B. and Walsh D. A. (1985) A fast-twitch oxidative glycolytic muscle with a robust inward calcium current. Can. J. Physiol. Pharmac. 63, 958. Chirandini D. J. and Bentley P. J. (1973) The effects of verapamil on metabolism and contractility of the toad skeletal muscle. J. Pharmac. exp. Ther. 186, 52-59. Frank G. B. (1958) Inward movement of calcium as a link between electrical and mechanical events in contraction. Nature 182, 1800-1801. Frank G. B. (1960) Effects of changes in extracellular calcium concentration on the potassium-induced contracture of frog's skeletal muscle. J. Physiol. (London) 151, 518-538. Frank G. B. (1982) The effects of reducing the extracellular calcium concentration on the twitch in isolated frog's skeletal muscle fibres. Jpn, J. Physiol. 32, 589~i08. Frank G. B. (1984) Blockade of Ca 2+ channels inhibits K* contractures but not twitches in skeletal muscle fibres. Can. J. Physiol. Pharmac. 62, 374~378. Frank G. B. (1986) A pharmacological explanation of the use-dependency of the verapamil (and D-600) block of slow calcium channels. J. Pharmac. exp. Ther. 236, 505-511. Frank G. B. and Oz M. (1991) Use-dependent block of sodium channels by verapamil in skeletal muscle during repetitive stimulation. Proc. West. Pharmac. Soc. 34, 409. Frank G. B. and Oz M. (1992) The functional role of T-tubular Ca 2+ channels in skeletal muscle contractions. In Excitation-Contraction Coupling in Skeletal, Cardiac and Smooth Muscle (Edited by Frank, G. B., Bianchi,
C. P. and ter Keurs, H. E. D. J.), pp. 123-136. Plenum Press, New York. Freygang W. H. Jr., Golstein D. A. and Hellam D. C. (1964) The after potential that follows trains of impulses in frog muscle fibres. J. gen. Physiol. 47, 929--952. Garcia J., Avila-Sakar A. J. and Stefani E. (1990) Repetitive stimulation increases the activation rate of skeletal muscle Ca 2+ current. Pfliigers Arch. 416, 210-212. Hille B. (1988) Ionic channels: Molecular pores of excitable membranes. In The Harvey Lectures 82, 1 23. Alan R. Liss, New York. Kaumann A. J. and Uchitel O. D. (1976) Reversible inhibition of potassium contractures by optical isomers of verapamil and D-600 on slow muscle fibres of the frog. Naunyn-Schmiedeberg's Arch. Pharmac. 292, 21-27. Kirsch G. E., Nichols R. A. and Nakajima S. (1977) Delayed rectification in the transverse tubules. J. gen. Physiol. 70, 1-21. Kostsias B. A., Muchnik S. and Obejero Paz C. A. (1986) Co z+, low Ca 2+ and verapamil reduce mechanical activity in rat skeletal muscles. Am. J. Physiol. 250, C40~16. Lee K. S. and Tsien R. W. (1983) Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature 3112, 790 794. Liittgau H. C. and Spiecker W. (1979) The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibers of the frog. J. Physiol. (London). 296, 411~129. Marwaha J. and Treffers R. C. (1980) Actions of a calcium antagonist, the D-600, on electrical and mechanical properties of frog skeletal muscle. Prog. NeuroPsychopharmac. 4, 145-152. Oz M. A., Dunn S. M. J. and Frank G. B. (1992) 45Ca2+ etllux studies in rabbit t-tubule membrane preparations in the range of late after potentials during contractions. In Excitation-Contraction Coupling in Skeletal, Cardiac and Smooth Muscle, pp. 419421. Plenum Press, New York. Oz M. A. and Frank G. B. (1990) Organic calcium channel blocking drugs and calcium free solution effects on tetanic responses in frog's skeletal muscle. Proe. Can. Fed. Biol. Soc. 33, 017. Oz M. A. and Frank G. B. (1991) Decrease in the size of tetanic responses by nitrendipine or by extracellular calcium ion removal without blocking twitches or action potentials in skeletal muscle. J. Pharmac. exp. Ther. 257, 575-581. Oz M. A., Frank G. B. and Dunn S. M. J. (1993) Voltage dependent calcium influx across rabbit skeletal muscle transverse tubule membranes in the range of late after potentials. Can. J. Pharmac. Physiol. 71, 518-521. Singh Y. N. and Dryden W. F. (1988) Sites of action of dihydropyridine drugs in the mouse hemidiaphragm muscle. Eur. J. Pharmac. 148, 247-255. Spiecker W., Melzer W. and L/ittgau H. C. (1979) Extracellular Ca 2+ and excitation-contraction coupling. Nature 280, 158-160. Stefani E. and Schmidt H. (1972) A convenient method for repeated intracellular recording of action potentials from the same muscle fibre without membrane damage. Pfliigers Arch. 334, 276-278. Tanabe T., Takeshima H., Mikamo A., Flockerzi V., Takahashi H., Kangoawa K., Kojima M., Matsuo H., Hirose T. and Numa S. (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313-318. Walsh K. B., Bryant S. H. and Schwartz A. (1986) Effect of Calcium Antagonist Drugs on Calcium Currents in Mammalian Skeletal Muscle Fibers. J. Pharmac. exp. Ther. 236, 403-417.
Verapamil on tetanic responses Williams J. H. (1990) Effects of low calcium and calcium antagonists on skeletal muscle staircase and fatigue. Muscle and Nerve 13, 1118-1124.
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Williams J. H. and Ward C. W. (1991) Dihydropyridine effects on skeletal muscle fatigue. J. Physiol. (Paris) 85, 235-238.
Comp. Biochem. Physiol. Vol. 107C, No. 3, pp. 321-329, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/94 $6.00 + 0.00
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0742-8413(93)E0043-U
The effects of verapamil on tetanic contractions of frog's skeletal muscle M. Oz and G. B. Frank Department of Pharmacology, 9-70 Medical Sciences Building, Edmonton, Alberta, Canada, T6G 2H7 The effects of the organic calcium channel antagonist, verapamil, were tested on twitches and tetanic contractions (100 Hz, 2 sec) in frog toe muscles. At low concentrations (3 x 10 -6 M), verapamil had no effect on the maximum amplitudes of twitches, but significantly reduced the size of the tetanic responses. This depression was observed as an inability to maintain the maximum tetanic tension for more than 0.5 sec. With increasing concentrations up to 10 -4 M of verapamil, its depressant effect on tetanic responses gradually increased, and at very high concentrations (10 -4 M ) of verapamil, twitches were also blocked. Intraceilular microelectrode recordings showed that there was no block of the action potentials during the stimulus train at the concentration of 3 x 10 -6 M of verapamii. These results support the concept that during tetanic responses, the voltage sensitive Ca 2+ channels in the t-tubules open and the Ca 2÷ ions entering via these channels are required to maintain the full strength of the contraction. At higher concentrations, verapamil blocked Na + action potentials during the stimulus trains in a concentration and use-dependent manner. Key words: Calcium channel; Calcium channel blockers; Muscle contraction.
Comp. Biochem. Physiol. 107C, 321-329, 1994.
Introduction Blockade of voltage sensitive, slow calcium channels (VSSCCs) by verapamil and D-600, has been demonstrated in several different studies using amphibian and mammalian skeletal muscle (Beaty and Stefani, 1976; Frank, 1984; Walsh et al., 1986). In a previous study from this laboratory, it was reported that verapamil and D-600 block high K ÷ induced contractures, but not twitches, in frog's skeletal muscle at concentrations that block voltage sensitive slow Ca z÷ channels (Frank, 1984). Blockade of high K ÷ induced contractures by voltage dependent Ca 2÷ channel
Correspondence to: M. Oz, Laboratory of Molecular & Cellular Neurobiology, National Institute on Alcohol Abuse & Alcoholism, 12501 Washington Ave., Rockville, Maryland, 20852, U.S.A. Received 25 October 1993; accepted 17 November 1993. 321
blockers is also consistent with previous studies indicating that high K ÷ induced contractures require the influx of extracellular Ca 2+ ions for excitation-contraction (E-C) coupling (Frank, 1958, 1960, 1982; Liittgau and Spiecker, 1979). In addition to high K ÷ induced contractures, another type of maintained response in skeletal muscle is tetanic contractions. We have reported recently (Oz and Frank, 1991) that during removal of extracellular Ca z+ ions, tetanic responses were depressed in area before twitches were reduced in amplitude. Furthermore, nitrendipine, an organic Ca 2+ channel blocker, also greatly depressed the area under the tetanic tension curve without blocking the development of action potentials during repetitive stimulations. In the present study, the effects of verapamil on skeletal muscle membrane excitability and tetanic contractions were investigated.
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Preliminary results of some of the experiments described here were presented previously (Frank and Oz, 1991).
Materials and Methods Materials All experiments were carried out at room temperature (18-21°C) using a skeletal muscle isolated from the frog, Rana pipiens. The extensor longus digiti IV (toe) muscle of the frog was used in all experiments. The muscles were allowed to equilibrate for a period of 30-45 min in oxygenated Ringer's solution before an experiment was started. Drug solutions were prepared by dissolving verapamil hydrochloride (Knoll A. G., Ludwig Shafen) in the appropriate Ringer's solution. To accelerate the dissolving of verapamil, beakers with this drug plus solution were placed in a hot water bath for 5 to 10 min. The composition of the frog Ringer's solution was as follows: 111.87 mM NaC1, 2.47 mM KCI, 1.08mM CaC12, 0.087mM NaH2PO 4, 2.38mM NaHCO3 and 11.1 m M dextrose, D-Tubocurarine (0.1 mg/ml) was added to the Ringer's solution. Throughout the mechanical recording experiments, the bathing solution was bubbled with a gas mixture of 99.5% O z + 0.5% C02. Tension studies Toe muscles were dissected from the frog in a way similar to that described by Frank (1960) and were mounted vertically in an 8-ml bath containing Ringer's solution. The lower end of the muscle was fixed near the bottom of the bath and the upper end was attached to the arm of the strain gauge by means of a silk thread. Solutions were changed by draining the bath at the bottom and adding new solution at the top with a syringe. Tension was recorded by means of a strain gauge whose active elements consisted of two pixie transducers (Endevco model 81214) in a wheatstone bridge configuration. The resting tension on the muscle was adjusted so that the muscle just failed to remain in a vertical position when the bath solution was drained and returned to a vertical position when the bath was refilled with solution. This length is presumably near or at the resting length because the largest twitches were produced at this muscle length. For twitch recording, supramaximal rectangular pulses, 1.5msec in duration, were applied to the muscle by means of two platinum electrodes; one situated at the bottom and the other at the top part of the bath.
For tetanic responses, the muscles were stimulated with 1.5msec supramaximal rectangular pulses at 100, 50 or 25 Hz for 2 sec once every 10-15min in experiments lasting up to 3 hr. Twitches were recorded 15 sec to 1 min before each tetanus. Effects on twitches were evaluated by measuring the twitch tension and the effects on tetani by measuring the tension x time area of the response. The transducer outputs were digitized and recorded using a Tekmar Lab Master A/D converter board connected to a Compaq portable computer. Records were analyzed using a Compaq Deskpro 286 and the responses were drawn on a Roland D G P R - 1212A printer. Responses were recorded with 1000 points/sweep at all sweep speeds. Programs for collecting, transmitting, storing and analyzing data were written in Turbo Pascal.
Electrophysiological studies For measurements of the late negative afterpotential (LAP), the muscles were mounted horizontally in a bath and viewed from above with a dissecting microscope. Illumination was provided by light passing up through the transparent bottom of the bath. In order to reduce muscle movement and consequent membrane damage during the recordings of the action potentials and the after-potentials, the toe muscles were stretched and wrapped around a glass rod of 2ram diameter which was suspended across the bath in the Ringer's solution which was oxygenated l0 min prior to and during the recordings (Stefani and Schmidt, 1972; Oz and Frank, 1991). Resting membrane and action potentials of the frog's toe muscle fibers were recorded intracellularly using conventional glass capillary microelectrodes (10~40Mfl resistance) filled with 3 M KCI. A small proportion of the muscle fibers was electrically stimulated by an extracellular, bipolar platinum-filled pore electrode placed on the surface of the muscle 5-10 mm from the recording electrode. The bipolar stimulating electrode was constructed with 0.4 mm platinum wires placed in polyethylene tubing and cemented together so that only the ends of the wires were exposed to solution. The wire ends rested on the surface of the muscle 1.5 mm apart. The muscle fibers were stimulated with supramaximal pulses delivered through a stimulus isolater unit (WP Instruments, model 305). The stimulating pulse consisted of either a single supramaximal stimulus pulse or a train of square waves of 1.5 msec duration. The glass micro-electrode was held in a micro-electrode holder which was connected through a probe to the main amplifier (WP Instruments, model MA4). The output from the
Verapamil on tetanic responses
amplifier was displayed on either a Model 4094 or 3091 Nicolet digital oscilloscope. The recordings were made with either 16,000 (Model 4094) or 4000 (Model 3091) points/sweep at all sweep speeds. These records were stored on floppy disks and pictures of action potentials were drawn using a digital plotter.
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Results The effect of verapamil twitches and tetani In our preliminary observations, depression of single action potentials was observed only at concentrations higher than 10 -4 M and therefore these higher concentrations were not used in the remainder of this study. Experiments were conducted with 23 muscles using verapamil in the concentration range of 10-6-10-4M. The measurements made in these studies are plotted in Fig. 1. The twitch amplitudes were slightly increased by exposing the muscles to 5 x 10 -6 M and 5 x 10 -6 M verapamil (Fig. 1, top section). The potentiation of the twitch by organic Ca 2+ channel blockers at concentrations which block Ca 2+ channels in skeletal muscle
,
0
-4
Log [verapamil] Fig. 2. Concentration-response curve for 10 min exposures to verapamil on tetanic areas. The numbers of experiments (n) at each concentration are given in the legend for Fig. 1.
has been reported in a number of earlier studies (Marwaha and Treffers, 1980; Frank, 1984; Singh and Dryden, 1988). Depression of action potentials and thus twitches was only observed at very high concentrations (0.3 mM or higher) of verapamil or D-600 (Van Der Kloot, 1975; Bondi, 1978; Dorrscheidt-Kafer, 1977; Frank, 1984). Verapamil decreased the area under the 2 set: i50 Twitch Tension tetanic force curve at 100Hz stimulation frequency over the concentration range of 3 x 10 - 6 to 10-4M verapamil (Fig. 1 bottom section). The effect of verapamil over this range was large and the area under the tetanic tension curve was depressed by up to 96% of control values by iO0 10-4M verapamil. With all the concentrations used, the effect of verapamil reached its equilibrium value by 5-10 min. The dose-response curve obtained using the data taken at 10 min is 4-J g shown in Fig. 2. ¢J 5O In a previous study with nitrendipine, the Tetanic Area amplitude of the tetanus always declined gradually during the 2 sec stimulus train (Oz and 100 Frank, 1991). In contrast, verapamil produced a variable depression pattern on the tetanic responses (Fig. 3). Verapamil, at the concentration of 3 x 10-6M, like nitrendipine produced only a gradual tension decline in tetanic tension beginning 0.5-1.0 sec after the start of the stimulus train (Fig. 3A). At concentrations of 5 x 10 - 6 and 10-SM verapamil, there was 0 often a rapid decline in tension to zero followed 0 I0 20 30 40 50 50 70 by another rise in tension during the stimulus Time {min) train (Fig. 3B). This rapid depressant effect of verapamil on tetanic responses at a concenFig. I. Effects over time of different verapamil concentration of 5 × 10 -6 M did not occur when using trations on maximum twitch tension (at the top) and tetanus lower stimulation frequencies such as 50Hz areas (at the bottom) in frog toe muscles. Means and standard errors. Standard error bars were omitted when (Fig. 4). Increasing the concentration of verathey overlapped or when they were smaller than the size of pamil to 5 x 10 -5 or to 10-4M caused a rapid the symbol. The verapamil concentrations (molar) and the block of the tetanic contraction during the number of experiments (n) were as follows: A , A : 10 -6, (4); stimulus train at 100Hz (Fig. 3C). At these 0 , 0 : 3 x 10 -6 (5); I - I , l : 5 x 10 -6, (4); O , O : 10 -5, (4); x,x: high concentrations of verapamil, the maximum 5 x 10 -5, (4); and + , + : 10 -4, (5).
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M. Oz and G. B. Frank
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tension developed during a tetanus was also decreased to a great extent.
The effect of different concentrations of verapamil on intracellularly recorded action potentials At concentrations of 5 x l0 -6 M and above, using 100Hz stimulation, verapamil always blocked some of the action potentials during the stimulation train. Depending on the concentration used, the time to block the development of repetitive action potentials was about 1.5 sec to tens of milliseconds (Fig. 5). Occasionally, we were able to record the repetitive action potentials occurring in a biphasic manner (Fig. 6). This biphasic development of action potentials seen in the intracellular recording was consistent with the biphasic force traces often seen in tension measurements (Fig. 3B). Although there was a significant depression of tetanic contractions at the concentration of 3 x 10-6M (Fig. 3A), at this concentration there was no blockade of action potentials
during 2 sec trains at 100 Hz of tetanic stimuli (Fig. 5). The use-dependent block of Na + action potentials varied with the stimulus frequency and the concentration of verapamil used. The use-dependent blockade of Na + action potentials by verapamil was reversed by using lower stimulation frequencies, such as 50 and 25 Hz. These effects are shown in Fig. 7. Only the loss of a few responses near the end of the stimulus train was produced by 5 × 1 0 - 6 M verapamil and this was prevented by lowering the stimulus frequency to 5 0 H z (Fig. 7A). A larger depression was produced by 10-SM verapamil and was only eliminated by reducing the stimulus frequency to 25 Hz (Fig. 7B). Discussion
It is known that verapamil blocks voltage dependent, show Ca :+ channels and
Verapamil on tetanic responses
325
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Fig. 4. Effects of lower stimulation frequency on the blockade of tetanic responses. Blockade of tetanic responses elicited by 50 Hz stimulation frequency for 2 sec by 5 x 10-6 M verapamil.
inhibits Ca 2+ dependent processes such as the Solandt effect, acetylcholine contractures and Ca 2+ dependent activation of phosphorylase kinase, at the relatively low concentrations of 10-5-10-6M in skeletal muscle (Chirandini and Bentley, 1973; Carlsen et aL, 1985). At such low concentrations, although both verapamil and D-600 have been shown to block high K ÷ induced contractures (Kaumann and Uchitel, 1976; Bondi, 1978; Frank, 1984), caffeine contractures were not depressed (Bondi, 1978; Marwaha and Treffers, 1980). These findings support the suggestion that verapamil blocks K+-induced contractures which require the influx of extracellular Ca 2+ ions for E-C coupling (Frank, 1958, 1982).
In addition to K÷-induced contractures, another type of maintained contraction is tetanic contractions in which repetitively developed action potentials produce a maintained tension. The function of slow Ca 2+ channels during tetanic contractions has been the subject of a few studies (Carlsen et al., 1985; Kostsias et al., 1986; Oz and Frank, 1991) and in other studies the importance of extracellular Ca ~÷ ions in tetanic responses has been mentioned (Spiecker et al., 1979; Blinks et al., 1978; Kostsias et al., 1986). In our studies (Oz and Frank, 1990, 1991), the areas under the tetanic force versus time curve during tetani were decreased when toe muscles were kept in Ca2+-free solutions. The fact that
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Fig. 5. Depression of intracellularly recorded action potentials by different concentrations of verapamil, during 2 sec stimulus trains at 100 Hz, A, B, C different toe muscles. On the left control recordings. On the fight responses to the stimulus train recorded in different fibers after 10rain in solutions with verapamil; in A, 3 x 10-6M (n:3); in B, 5 x 10-6M (n:3); in C, 10-4M (n:5). Some small stimulus artifacts can be seen in between the repetitive action potentials.
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I
50mV
I
I
2 sec
Fig. 6. Blockade of action potentials and rebursting during a 2 sec stimulus train at 100 Hz recorded in a toe muscle fiber exposed to 10-5 M verapamil for 10 min. Some stimulus artifacts can be seen between these action potentials during repetitive activities. Between the bursts, these artifacts have been reduced in size by whiting them out. Due to the limited time resolution of our digital oscilloscope used in this recording, the peaks of the spikes are usually missed and a liaising effect is seen (see also Oz and Frank, 1991).
nitrendipine, a n organic Ca2+-channel blocking drug, also reduced the (time x tension) area showed that extracellular Ca 2÷ ions need to enter the skeletal muscle via slow Ca 2÷ c h a n n e l s d u r i n g a tetanus in order for the muscle to m a i n t a i n the full tetanic t e n s i o n (Oz a n d F r a n k , 1991). It was d e m o n s t r a t e d some time ago that d u r i n g a series o f rapidly repeated action poten-
A1
A=
tials in skeletal muscle, K ÷ ions, which leak out with each action potential, a c c u m u l a t e in the t-tubules a n d only slowly achieve diffusion e q u i l i b r i u m with the s u r r o u n d i n g extracellular fluid ( F r e y g a n g et al., 1964; Kirsch et al., 1977). This K + a c c u m u l a t i o n causes a small b u t significant depolarization o f a b o u t 20-25 m V d u r i n g a train of tetanic action potentials a n d when the stimuli stop, the muscle fiber m e m b r a n e remains
BI
B2
A3 B_
Ai
B4
150rnV I
2 sec
Fig. 7. Intracellularly recorded action potentials for tetanic stimulation trains in frog toe muscle fibers. Recordings from different single fibers are presented in column A I through A4 and Bt through B4. Control recordings are A t and Bj. Verapamil at the concentration of 5 x 10 -~ M and 10-5 M used in recordings A2-A4 and B2-B4 respectively. Frequencies of stimulations used during recordings are 100; 100; 50 and 25 for At, B~; A2, B2; A3, B3; and A4, B4, respectively.
Verapamil on tetanic responses depolarized and only very slowly repolarizes (Fig. 5, control recordings); this slow repolarization was called the late negative after-potential (LAP). In our previous report, we suggested that the t-tubular depolarization at the time of the repetitive action potentials was sufficient to open the voltage sensitive, slow calcium channels (VSSCC) and allow some influx of Ca 2+ ions. Although this influx is not required to begin the tetanus, we suggested that it was required to maintain the full tension during the tetanic contraction. Our observation that nitrendipine reduced the tetanic contraction size without reducing the t-tubule depolarization supports this hypothesis. In a more recent study, it was shown that verapamil produced a block of the Ca 2+ fluxes in an isolated t-tubule vesicle preparation that were otherwise produced by depolarizing these vesicles by an amount equivalent to the size of the LAP (Oz et al., 1992, 1993). This finding supports our suggestions that the small depolarization during the tetanus is sufficient to open the VSSCCs and allow the influx of Ca 2÷ ions and that this influx could be blocked by Ca2+-channel blocking drugs. A more detailed discussion of this hypothesis with earlier supporting evidence from the literature can be found elsewhere (Oz and Frank, 1991; Frank and Oz, 1992). In addition, other studies, in agreement with our hypothesis, have demonstrated that VSSCCs have functional roles during long term skeletal muscle activities such as tetanic contractions and the treppe effect (Garcia et al., 1990; Williams, 1990; Williams and Ward, 1991). The results obtained in the present study were more complicated than our earlier findings with nitrendipine. Although we were careful to use only lower verapamil concentrations which did not block single twitches, still most of the concentrations we used did result in a usedependent block of some of the action-potentials during the stimulus trains at 100 Hz for 2 sec (Frank and Oz, 1991). Even so, we were able to demonstrate a clear reduction of the tetanus area due to the block of VSSCCs by verapamil under two experimental conditions. The first was a significant reduction of the area by 3 x 10-6M verapamil (Fig. 2) which was not a high enough concentration to produce a use-dependent action potential block during 100 Hz stimulation for 2 sec (Fig. 5). The second was the reduction of the tetanus area produced by 5 x 10-6M verapamil (Fig. 4) when the stimulus frequency was reduced to 50 Hz at which frequency this verapamil concentration did not produce any use-dependent action potential block (Fig. 7). In frog skeletal muscle, in addition to a block of the slow Ca 2÷ channels, verapamil at slightly
327
higher concentrations also blocks Na ÷ channels. Our results indicate that the repetitive firing of action potentials faciitates the blockade of Na ÷ action potentials in frog skeletal muscle fibers. At the concentration range of 5 x 1 0 - 6 to 10 -5 M, although no blockade of single action potential was observed, repetitive production of action potentials at the stimulation frequency of 100 Hz, elicited the use-dependent blockade of Na ÷ channels (see Figs 6 and 7). This usedependent blockade of Na÷-channels was reversed with the use of lower stimulation frequencies such as 50 or 25 Hz. At the concentration of 5 x 10 -6 M verapamil, using a 50 Hz stimulation frequency was enough to remove the blockade of Na ÷ channels. On the other hand, with 10 -5 M verapamil, a lower stimulation frequency of 25 Hz, which could produce only an unfused tetanus, was required to eliminate the blockade of Na ÷ channels. At the concentrations range of 5 x 1 0 - 6 to 10-SM verapamil, we sometimes observed that the blockade of Na ÷ action potentials followed a biphasic time course (see Fig. 6). In about 0.5-1 sec, there was a complete blockade of action potentials and a decrease in the cell membrane depolarization. This was followed by a second burst of full size action potentials which lasted a few hundred milliseconds. This second activation of action potentials during 2 sec pulse stimulations, was also consistent with the tension recordings in which a second rise in the tension curve was observed (see Fig. 3B). These findings suggested that the inactivation of the Na ÷ channels by verapamil declined during the time between these two activation periods (see Fig. 5). In addition to removal of the use-dependent blockade of Na ÷ channels by the absence of action potentials, the gradual reduction of the cell membrane depolarization might also contribute to the removal of the Na ÷ channel inactivation, and thus lead to the production of a second burst of action potentials. The time required for recovery from the use-dependent blockade of action potentials by verapamil was a few hundred milliseconds. This approximate recovery time is also quite similar to that seen with local anesthetics such as lidocaine that cause a use-dependent blockade of Na ÷ channels (Hille, 1988). Similar effects of verapamil have also been observed on the slow soleus muscle fibres of the rat (Kostsias and Muchnik, 1986). In conclusion, in the present study, the effects of verapamil have been observed on frog's skeletal muscle. At relatively low concentrations, verapamil causes an inhibition of tetanic contractions by the blockade of voltage sensitive slow Ca 2÷ channels. At slightly higher concentrations, verapamil, in addition to
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its known use-dependent blockade of calcium channels (Frank, 1986), also produces a use-dependent blockade effect on Na + channels of frog's skeletal muscle. Summary Verapamil exerts differential effects on skeletal muscle contractions that are dependent on the drug concentration and on the pattern of stimulation. It appears that at low concentrations, verapamil depresses the tetanic contractions without blocking action potentials. This depression was suggested to be related to the blockade of VSSCCs. At higher concentrations of verapamil, there was a blockade of sodium channels, and this blockade had usedependent properties. Acknowledgements--We would like to thank Joan McGinnis for her skilful technical assistance. This study was supported by grants from the Medical Research Council of Canada.
References Beaty G. N. and Stefani E. (1976) Calcium dependent electrical activity in twitch muscle fibres of the frog. Proc. R. Soc. London Set. B. 194, 141-150. Blinks J. R., Rudel R. and Taylor S. R. (1978) Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J. Physiol. 277, 291-323. Bondi A. Y. (1978) Effects of verapamil on excitationcontraction coupling in frog sartorius muscle. J. Pharmac. exp. Ther. 205, 49-57. Carlsen R. C., Larson D. B. and Walsh D. A. (1985) A fast-twitch oxidative glycolytic muscle with a robust inward calcium current. Can. J. Physiol. Pharmac. 63, 958. Chirandini D. J. and Bentley P. J. (1973) The effects of verapamil on metabolism and contractility of the toad skeletal muscle. J. Pharmac. exp. Ther. 186, 52-59. Frank G. B. (1958) Inward movement of calcium as a link between electrical and mechanical events in contraction. Nature 182, 1800-1801. Frank G. B. (1960) Effects of changes in extracellular calcium concentration on the potassium-induced contracture of frog's skeletal muscle. J. Physiol. (London) 151, 518-538. Frank G. B. (1982) The effects of reducing the extracellular calcium concentration on the twitch in isolated frog's skeletal muscle fibres. Jpn, J. Physiol. 32, 589~i08. Frank G. B. (1984) Blockade of Ca 2+ channels inhibits K* contractures but not twitches in skeletal muscle fibres. Can. J. Physiol. Pharmac. 62, 374~378. Frank G. B. (1986) A pharmacological explanation of the use-dependency of the verapamil (and D-600) block of slow calcium channels. J. Pharmac. exp. Ther. 236, 505-511. Frank G. B. and Oz M. (1991) Use-dependent block of sodium channels by verapamil in skeletal muscle during repetitive stimulation. Proc. West. Pharmac. Soc. 34, 409. Frank G. B. and Oz M. (1992) The functional role of T-tubular Ca 2+ channels in skeletal muscle contractions. In Excitation-Contraction Coupling in Skeletal, Cardiac and Smooth Muscle (Edited by Frank, G. B., Bianchi,
C. P. and ter Keurs, H. E. D. J.), pp. 123-136. Plenum Press, New York. Freygang W. H. Jr., Golstein D. A. and Hellam D. C. (1964) The after potential that follows trains of impulses in frog muscle fibres. J. gen. Physiol. 47, 929--952. Garcia J., Avila-Sakar A. J. and Stefani E. (1990) Repetitive stimulation increases the activation rate of skeletal muscle Ca 2+ current. Pfliigers Arch. 416, 210-212. Hille B. (1988) Ionic channels: Molecular pores of excitable membranes. In The Harvey Lectures 82, 1 23. Alan R. Liss, New York. Kaumann A. J. and Uchitel O. D. (1976) Reversible inhibition of potassium contractures by optical isomers of verapamil and D-600 on slow muscle fibres of the frog. Naunyn-Schmiedeberg's Arch. Pharmac. 292, 21-27. Kirsch G. E., Nichols R. A. and Nakajima S. (1977) Delayed rectification in the transverse tubules. J. gen. Physiol. 70, 1-21. Kostsias B. A., Muchnik S. and Obejero Paz C. A. (1986) Co z+, low Ca 2+ and verapamil reduce mechanical activity in rat skeletal muscles. Am. J. Physiol. 250, C40~16. Lee K. S. and Tsien R. W. (1983) Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells. Nature 3112, 790 794. Liittgau H. C. and Spiecker W. (1979) The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibers of the frog. J. Physiol. (London). 296, 411~129. Marwaha J. and Treffers R. C. (1980) Actions of a calcium antagonist, the D-600, on electrical and mechanical properties of frog skeletal muscle. Prog. NeuroPsychopharmac. 4, 145-152. Oz M. A., Dunn S. M. J. and Frank G. B. (1992) 45Ca2+ etllux studies in rabbit t-tubule membrane preparations in the range of late after potentials during contractions. In Excitation-Contraction Coupling in Skeletal, Cardiac and Smooth Muscle, pp. 419421. Plenum Press, New York. Oz M. A. and Frank G. B. (1990) Organic calcium channel blocking drugs and calcium free solution effects on tetanic responses in frog's skeletal muscle. Proe. Can. Fed. Biol. Soc. 33, 017. Oz M. A. and Frank G. B. (1991) Decrease in the size of tetanic responses by nitrendipine or by extracellular calcium ion removal without blocking twitches or action potentials in skeletal muscle. J. Pharmac. exp. Ther. 257, 575-581. Oz M. A., Frank G. B. and Dunn S. M. J. (1993) Voltage dependent calcium influx across rabbit skeletal muscle transverse tubule membranes in the range of late after potentials. Can. J. Pharmac. Physiol. 71, 518-521. Singh Y. N. and Dryden W. F. (1988) Sites of action of dihydropyridine drugs in the mouse hemidiaphragm muscle. Eur. J. Pharmac. 148, 247-255. Spiecker W., Melzer W. and L/ittgau H. C. (1979) Extracellular Ca 2+ and excitation-contraction coupling. Nature 280, 158-160. Stefani E. and Schmidt H. (1972) A convenient method for repeated intracellular recording of action potentials from the same muscle fibre without membrane damage. Pfliigers Arch. 334, 276-278. Tanabe T., Takeshima H., Mikamo A., Flockerzi V., Takahashi H., Kangoawa K., Kojima M., Matsuo H., Hirose T. and Numa S. (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313-318. Walsh K. B., Bryant S. H. and Schwartz A. (1986) Effect of Calcium Antagonist Drugs on Calcium Currents in Mammalian Skeletal Muscle Fibers. J. Pharmac. exp. Ther. 236, 403-417.
Verapamil on tetanic responses Williams J. H. (1990) Effects of low calcium and calcium antagonists on skeletal muscle staircase and fatigue. Muscle and Nerve 13, 1118-1124.
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Williams J. H. and Ward C. W. (1991) Dihydropyridine effects on skeletal muscle fatigue. J. Physiol. (Paris) 85, 235-238.