Ca antagonistic and non-specific effects of diltiazem on the rat phrenic nerve diaphragm preparation

European Journal of Pharmacology, 159 (1989) 61-71

61

Elsevier EJP 50578

Ca antagonistic and non-specific effects of diitiazem on the rat phrenic nerve diaphragm preparation Asbjorn Roed Department of Physiology and Biochemistry, Dental Faculty, University of Oslo, P.O. Box 1052, Blindern, 0316 Oslo 3, Norway

Received 21 April 1988, revised MS received 19 September 1988, accepted 4 October 1988

The calcium antagonist diltiazem (2.8 x 10 -4 M) blocked the twitches of a rat phrenic nerve diaphragm preparation after a period of twitch potentiation. Its ability to block twitches was greater during indirect than direct stimulation. Experiments on the isolated phrenic nerve indicated that the excitability of the nerve was blocked. Diltiazem (2.3-9.0 X 10 -5 M) caused a similar inhibition of indirectly and directly elicited tetanic contractions and EMG. Experiments with d-tubocurarine and lowered temperature disclosed a separate inhibition at the neuromuscular junction. High Ca 2+ did not reverse the diltiazem-affected twitch or tetanic contractions, which suggests that they are non-specific effects. KCI (100 mM)-induced contractures were antagonized at low (2.3-4.5 x 10 -5 M) but not at high (1 raM) concentrations of diltiazem. Diltiazem depressed the initial phase of the two-phasic caffeine (10 raM) contracture and increased and accelerated the slow phase. Diltiazem greatly reduced the amplitude and duration of the caffeine-potentiated KCI contracture, and reduced and delayed the slow phase of the KCl-potentiated caffeine contracture. The effects on the combined contractures (caffeine-induced, KCl-potentiated) were partly antagonized by a high Ca 2+ (2.2 x 10 -5 M) solution, which suggests that diltiazem has calcium antagonistic effects. Diltiazem; Ca 2+ antagonists; KC1 contracture; Caffeine contracture; Diaphragm (rat); (Use-dependent inhibition)

1. Introduction Skeletal m u s c l e cells have a very high d e n s i t y of C a channels in their transverse (T) t u b u l a r m e m b r a n e s ( F o s s e t et al., 1983; Curtis a n d Catterall, 1984; J a i m o w i c h et al., 1986). However, a clear-cut p h y s i o l o g i c a l f u n c t i o n for these c h a n n e l s has b e e n difficult to establish (Miller a n d F r e e d m a n , 1984). T h e r e a s o n for this m a y b e that the channels are ' s i l e n t ' , i.e. they have n o significant f u n c t i o n in o r d i n a r y muscle p e r f o r m a n c e . Nevertheless, a f u n c t i o n m i g h t b e disclosed u n d e r e x t r e m e situations, such as muscle fatigue o r contractures. T h e slow Ca channels, which are k n o w n to be affected b y o r g a n i c C a a n t a g o n i s t s ( C o g n a r d et al., 1986), m a y be especially m o r e affected d u r i n g such cond i t i o n s t h a n d u r i n g o r d i n a r y twitch a n d tetanic c o n t r a c t i o n s . A recent finding in our l a b o r a t o r y has c o n f i r m e d this h y p o t h e s i s : the d i h y d r o p y r i -

d i n e C a a n t a g o n i s t n i f e d i p i n e p o t e n t i a t e s subtetanic c o n t r a c t i o n s a n d delays the d e v e l o p m e n t of fatigue ( R o e d , in p r e p a r a t i o n ) . The p r e s e n t e x p e r i m e n t s were p l a n n e d to investigate the effects of the b e n z o t h i a z e p i n e Ca a n t a g o n i s t d i l t i a z e m on e x t r e m e m u s c u l a r performance, such as fatiguing a n d m y o t o n i c activity a n d contractures. T h e initial e x p e r i m e n t d i s c l o s e d a u s e - d e p e n d e n t i n h i b i t i o n of tetanic c o n t r a c t i o n s , which was n o t f o u n d with nifedipine. This difference b e t w e e n d i l t i a z e m a n d n i f e d i p i n e has also been r e p o r t e d for the h e a r t b y Lee a n d Tsien (1983). T h e y f o u n d a m o r e m a r k e d u s e - d e p e n d e n c e with v e r a p a m i l t h a n w i t h n i f e d i p i n e . V e r a p a m i l causes a u s e - d e p e n d e n t i n h i b i t i o n of N a channels, giving the d r u g m e m b r a n e stabilizing or local a n a e s t h e t i c (Strichartz, 1976). Verap a m i l also causes a local a n a e s t h e t i c u s e - d e p e n d ent i n h i b i t i o n of skeletal muscle ( G a l l a n t a n d

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

62 Goettl, 1985; Kotsias and Muchnik, 1985), and this effect, rather than a concomitant block of Ca channels, might inhibit muscle contraction (Van der Kloot and Kita, 1975). In the present experiments we first described and localized the effects of diltiazem on twitch and tetanic contractions and on the E M G of the isolated rat phrenic nerve diaphragm preparation. The effects on the compound action potential of the isolated phrenic nerve were also recorded. Secondly, the effect on myotonic preparations were investigated. Thirdly, the effects of diltiazem on contractures induced by caffeine and KCI and by combinations of these agents were described. The observed effects were characterized as being Ca antagonistic or unspecific, dependent upon whether they could be antagonized by a high Ca 2÷ solution or not.

2. Materials and methods

2.1. Materials Wistar albino rats of either sex, weighing 100200 g, were anaesthetized with diethylether and decapitated. Each hemidiaphragm with its phrenic nerve was dissected as described by Biilbring (1946). The preparations were suspended in a Tyrode solution of the following composition (in mM): Na + 150; K ÷ 2.7; Ca 2+ 1.8; Mg 2÷ 0.1; C1140; HCO 3 12; H2PO 4- 0.4; dextrose 11. In some of the experiments the K ÷ concentration was increased to 100 mM by adding KC1 to induce contractures or the concentration of Ca 2+ was increased by adding 18 mM CaC12 to antagonize the effects of diltiazem. Experiments in which MgC12 was added instead of CaC12 were performed to differentiate between specific Ca antagonistic effects and effects due to an altered membrane surface potential. The solutions were kept at 3 7 ° C and were continuously aerated with 95% 02 + 5% CO2, which gave a pH 7.4. The following drugs were used: diltiazem hydrochloride, parahydroxymercuribenzoate sodium salt (pOHMB), d-tubocurarine hydrochloride (dTC) and caffeine. All drugs were from Sigma. The drugs were added to the organ bath.

2.2. Contraction and EMG recording The preparations were suspended with the rib end of the fibres pushed into a perspex holder with Ag/AgC1 wires serving as electrodes for direct stimulation. Alternatively, these electrodes could be used for recording the E M G during indirect stimulation. The electrode used for indirect (nerve) stimulation was of the type described by Biilbring (1946). The stimulating square wave pulses were delivered by a Grass Model S 48 stimulator. The voltage for indirect stimulation was about 3 times that needed for maximum muscle contraction, and the pulse duration was 50 its. The voltage for direct stimulation was 150 V and the pulse duration was 0.5 ms. The frequency was 0.1 Hz during twitch stimulation. Tetanic stimulation was performed with a pulse train of 50 Hz in 10 s. During direct stimulation, the pulses were delivered through a Grass Model CCU 1 constant current unit. The tendinous end of the muscle was tied to a Grass force-displacement transducer (Model FT.03C or FT10C) which was connected to a Grass Model 7 polygraph for recording the muscle contractions. The E M G could be recorded simultaneously.

2.3. Compound action potential recording The phrenic nerve was dissected, transferred to the bath and connected to stimulating and recording electrodes of the Ag/AgC1 suction type as described by Lilleheil (1970). The stimulating pulse was delivered through a Grass Model SIU 5 stimulus isolation unit. The voltage was about 3 times that needed for maximum amplitude of the compound action potential, and the duration was 50 #S. The compound action potential was amphfied by a Grass Model P 16 A C / D C microelectrode amplifier, displayed on a Tektronix Model 5030 dual-beam oscilloscope and photographed with a Grass Model C4N kymograph camera.

2.4. Statistics The results are presented as the means + S.E. (n = No. of observations). The statistical significance of the results was analysed with Student's

63

t-test for un-paired observations. A probability of P < 0.05 was regarded as being statistically significant.

TABLE 1 Twitch-potentiating effect of diltiazem. The values are the means-+ S.E. (No. of observations). Diltiazem cOne.

3. Results

x l 0 -5 M

3.1. Effects of diltiazem on muscle contractions

1.1 2.3 4.5 9.0 CaCI2, 18 mM 2.3 CaCI 2, 9 mM 4.5

3.1.1. Effects on twitch contractions Diltiazem caused three obvious effects on the phrenic nerve diaphragm preparations: a twitch potentiation, followed by a twitch block (fig. la) and a specific inhibitory effect on the tetanic contractions (fig. 2). A significant twitch potentiation could be observed over the concentration range 2.3 × 10 -5 to 10 - 3 M. Table 1 shows data from the lower concentration range. At the lowest concentration there was no significant difference between the twitch potentiation during indirect and direct

A

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Time(rain) Fig. 1. The effect of diltiazem (2.8x10 -4 M) added at ~ on the diaphragnl twitch contraction and on the phrenic nerve compound action potential. Stimulation frequency: 0.1 Hz. (A) Twitches of the indirectly (via the phrenic nerve) stimulated diaphragm. After complete block, twitches could be elicited by direct stimulation (arrow) on the muscle surface until a complete block ensued. (B) Amplitude of the compound action potential (cAP) of the isolated phrenic nerve after addition of dihiazem. The values are the m e a n s + S.D. of 8 experiments in % of control.

Twitch amplitudes after 10 mJn drug exposure as a % of predrug twitch values Dir. stim.

Ind. stim.

115.3+4.5 (7) 139.8+3.7 (6) 152.4+6.4(5)

101.5+0.9 114.3+2.2 123.2+1.5 163.4--+9.7

(8) (12) (44) a (5)

117.8+4.0

(5) b

119.0+__7.4 (4) b

a Significant difference between the responses elicited by direct and indirect stimulation, b Not significantly different from responses without pretreatment with CaC12.

stimulation. When the concentration was increased to 4.5 x 10 -5 M, a larger potentiation during direct stimulation was observed and was probably due to a simultaneously developing inhibition of the indirectly elicited twitches. The indirectly stimulated preparation was completely blocked by 2.8 x 10 - 4 M diltiazem in 25.2 + 2.7 min ( n - 5). The preparations were then stimulated directly, and 3 of the preparations were blocked within 60 min. The irregular profile of the twitch decay curves, which was most evident during direct stimulation (fig. la), could indicate that inhibition was due to an increase in the threshold for excitation. Two series of experiments were performed in the high Ca solution. The twitch potentiating effect was not significantly altered by the high Ca 2÷ concentrations (table 1), but the blocking effects of diltiazem (2.8 × 10 - 4 M) were potentiated during indirect (complete block in 18.3 + 1.1 rain; n = 4) and during direct (3 of 4 blocked in 60 rnin) stimulation. The effect of diltiazem (2.8 x 10 - 4 M) on the compound action potential of the isolated phrenic nerve was investigated and compared with its effect on the block induced by indirect stimulation. As seen in fig. lb, the compound action potential was not completely obliterated at the time of complete neuromuscular block. However, the ac-

64

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t Fig. 2. The effect of diltiazem, added at ~ , on the twitch and tetanic activity during direct and indirect stimulation. The twitch stimulation frequency was 0.1 Hz. One 10-s period of tetanic 50 Hz stimulation before and similar periods every 10 min after addition of diltiazem showed the development of the tetanic inhibition. Note that the paper speed was increased and the amplifier sensitivity (vertical bars) reduced during the tetanic periods. (A) The effect of diltiazem (9.0 × 10-5 M) on contractions during direct stimulation of the completely curarized preparation at 37 o C. A control tetanus was recorded before the addition of diltiazem. (B) The effect of diltiazem (9.0 x 10 s M) on contractions during indirect stimulation at 37 o C. Note that the effects of diltiazem on the twitch and tetanic contractions were similar during indirect and direct stimulation at 37 o C. (C) EMG recorded during indirect stimulation, demonstrating that inhibition of the tetanic tension was accompanied by inhibition of the tetanic EMG. (D) The effect of diltiazem (4.5 x 10 -5 M) on tetanic contractions eficited indirectly (at 10, 20 and 50 n-fin) and directly (from arrow; at 55 min) at 24 o C. Comparison of the two latter tetani shows that the tetanic inhibition was more marked during indirect stimulation.

tion potentials of the individual fibres might well be below the threshold for neuromuscular transmission when the whole preparation were blocked during indirect stimulation. 3.1.2. Effects on tetanic contractions

The inhibitory effect of diltiazem (2.3-9.0 × M) on the tetanic contractions was tested by eliciting 50 Hz tetanic contractions of 10 s duration every 10 min after addition of the drug. The 10 -6

tests were performed during direct stimulation of the completely curarized preparation to disclose effects located beyond the neuromuscular junction, and during indirect stimulation to include possible effects on the nerve or neuromuscular junction. Figure 2 shows the development of the tetanic inhibition during a 30-min exposure to the drug. The tetanic contraction became biphasic, with an initial peak followed by a plateau phase, and the inhibition by diltiazem was dose-depen-

65

A

15QI

B

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Fig. 3. Test for synergism between diltiazem and drugs that act at or distal to the neuromuscular junction. The pattern of stimulation and variation in paper speed and amplifier sensitivity as in fig. 2. Diltiazem added at ~ . (A) Pretreatment with a low concentration of dTC (3.6 x 10-7 M added at O ) caused a moderate frequency-dependent inhibition at the neuromuscular junction. Addition of a low concentration of diltiazem (1.1 × 10 5 M) caused a marked enhancement of the tetanic inhibition. (B) Pretreatment with a low concentration of lidocaine (9.3 × 10 5 M added at 0 ) caused a moderate frequency-dependent inhibition which might be localized to the excitable sarcolemma. Addition of a low concentration of diltiazem (1.1 × 10-5 M) did not potentiate the development of tetanic inhibition.

dent (table 2). There was no significant difference between the responses during indirect and direct stimulation. When a similar experiment was performed in the high Ca solution, there was a significant increase in the inhibition during both indirect and direct stimulation (table 2). In some of the experiments with indirect stimulation, the E M G was recorded in parallel with the muscle tension (fig. 2c). A qualitatively similar type of inhibition of the E M G and tension was

observed. When the same stimulation pattern was continued for 1 h after washing, a nearly complete recovery of the tetanic tension and the E M G amplitude and appearance was observed. Since the use-dependent inhibition by diltiazem was similar during indirect and direct stimulation, the sarcolemma might be the c o m m o n site of

A

TABLE 2 Inhibitory effect of diltiazem on tetanic contractions. The values are the means_+ S.E. (No. of observations). Diltiazem conc. ×10 5 M

Endpoint tension of a 50 Hz tetanic contraction of 10 s duration after 30 rain of drug exposure. Values are % of predrug twitch tension Dir. stim.

0 (control) 2.3 4.5 9.0 CaC12, 9 mM 4.5

492.2+_24.0 180.0+_64.3 90.4+18.6 25.8_+ 7.8

B

A

5 mi'~n lSg

Ind. stim. (13) (3) (7) (5)

12.0_+ 2.2 (4) a

436.6+22.3 303.3_+43.3 131.7_+13.7 13.8_+ 2.2

(13) (3) (9) (5)

23.7_+ 8.1 (3) a

a Significant change from the response determined in the normal solution; P < 0.05.

Fig. 4. The inhibitory effect of diltiazem on a myotonic response elicited at the excitable sarcolemma. Myotonia was induced by pOHMB (10 -4 M) added at A. The myotonia can be observed as a blackening of the trace due to delayed relaxation of the contractions. Inserts of single recordings at a higher paper speed show the myotonia before and after addition of diltiazem. (A) Diltiazem (2.3x10 -5 M) and (B) Diltiazem (4.5×10 -5 M) added at ~ . Note the biphasic blocking effect.

66

action. Tests for parallel effects on the isolated phrenic nerve showed no evidence of a use-dependent inhibition of the compound action potential, not even when the concentration of diltiazem was increased beyond that which causes a complete tetanic inhibition of the whole preparation. The responses obtained after the bath temperature had been lowered indicated that diltiazem had different inhibitory effects during indirect and direct stimulation. The tetanic inhibitory effect of diltiazem (4.5 x 10 -3 M) at 2 4 ° C is shown in fig. 2d. The inhibition was considerably more marked

A

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during indirect than during direct stimulation. The twitch potentiation was not observed at 24 ° C. The possibility that diltiazem has an inhibitory effect at the neuromuscular junction was also tested in experiments where the neuromuscularblocking drug dTC was used in combination with diltiazem (fig. 3a). Both drugs were added to the bath in concentrations that cause minimal use-dependent inhibition when applied separately. The combination of the drugs caused a marked use-dependent inhibition of the preparation. This suggests a synergistic effect of diltiazem and dTC on

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Fig. 5. Effects of diltiazem on the KC1 contracture and on the KC1 plus caffeine contracture. Open arrow: Addition of KCI (100 mM). Filled arrow: Addition of caffeine (10 mM). ( ~ ) Addition of diltiazem. ( ~ ) Addition of CaCI 2. The increase of the paper speed during the initial transient phase of the KC1 contracture is indicated by . . . . . . ; 10 s between the twitches. (A) The control KC1 contracture and the effect of the KCI contracture on the subsequent caffeine contracture. (B) The effect of diltiazem (4.5 x 10- 5 M) on the KCI and the KCI plus caffeine contracture. (C) The effect of diltiazem (1 mM) on the KCI contracture. (D) The antagonism by nigh Ca 2+ (18 mM CaC12 added) of the diltiazern (4.5 x 10 -5 M) effect on the KCI plus caffeine contracture. (E) The effect of high Ca 2÷ on the KC1 contracture. The initial rapid phase was depressed, whereas a second slow phase appeared. The second phase was depressed by diltiazem (not shown).

67

the neuromuscular junction. A synergistic effect was also observed for the inhibition of twitch contractions during indirect stimulation with higher concentrations of the same drugs (dTC: 7.2 x 10 - 7 M ; diltiazem: 4.5 × 10 -5 M). Since the sarcolemma is a probable site of action for diltiazem, we also investigated the possible interaction between diltiazem and a membrane-stabilizing drug, lidocaine. M e m b r a n e stabilizing drugs are known to cause a use-dependent inhibition of excitable membranes, especially at the sarcolemma (Raed, 1983). However, the two drugs were no more effective when applied together than when applied separately at the same concentrations (fig. 3b). 3.1.3. Effects on fatiguing subtetanic contractions A low concentration of diltiazem (1.1 × 10 -5 M; a concentration below that which affects the twitch and tetanic contractions) was tested for an effect on subtetanic tension during continuous stimulation (20 Hz). The m a x i m u m tension was 137.2 + 4.1% (n = 45) and 120.7 + 8.9 (n = 6) of the pretetanic twitch in control and diltiazem-exposed preparations, respectively. As a measure of fatigue, the tension decayed to 50% of the pretetanic twitch in 75.6 + 2.0 (n = 45) and 50.8 + 2.5 s (n = 6) in control and diltiazem-exposed preparations, respectively. The difference in amplitude was not significant, whereas the development of fatigue was significantly accelerated by diltiazem. 3.1.4. Effects on myotonic contractions Testing the effect of diltiazem on myotonia might also indicate whether the sarcolemma is a site of action. Myotonia is a high-frequency discharge of action potentials initiated by each lowfrequency (0.1 Hz) stimulus to the sarcolemma, and leads to a tetanic contraction in response to each twitch. Myotonia was produced by exposing the diaphragm preparation to p O H M B ( 1 0 - 4 M ; Roed, 1981). When the m a x i m u m level of myotonic contraction was obtained after about 15 rain, the addition of diltiazem (2.3 × 10 -5 M) caused a shortening of the myotonic contraction (fig. 4, upper trace). A higher concentration of diltiazem

TABLE 3 Effect of diltiazem on the KCI (100 raM) contracture. The values are the m e a n s ± S.E. (No. of observations). Diltiazem cone. x 1 0 -5 M

Amplitude of maximum contracture in % of initial twitch contraction

0 (control) 2.3 4.5 100.0 CaCI 2, 18 mM 0 (control) 4.5 100.0

73.6± 2.8 19.3_+ 5.2 32.7± 10.8 73.7_+10.1

1. phase

2. phase (39) (8)a (7) a (7) 64.1 ± 7.3 (7) 31.5 +_3.0 (8) b

85.3_+ 5.9 (4)

a Significant decreases when compared with the control; P < 0.05. b Significant decrease when compared with the control in high CaC12 solution; P < 0.05.

(4.5 x 10 -5 M) caused a complete and immediate disappearance of the myotonia (fig. 4, lower trace). 3.2. Effects of diltiazem on contractures 3.2.1. Effects on the KCI contracture Diltiazem had a complex interaction with the depolarization contracture induced by KC1 (100 mM). The control KC1 contracture is shown in fig. 5a. A low concentration of diltiazem (2.3 x 10 -5 M) markedly inhibited the contracture (fig. 5b and table 3). However, the amplitude of the KC1 contractures varied considerably after diltiazem, but an accelerated decay of the tension of the contractures with the highest amplitudes could be observed (fig. 5b). A high concentration of diltiazem

TABLE 4 Effect of diltiazem on the caffeine (10 raM) contracture. The values are the means + S.E. (No. of observations). Diltiazem cone. xlO -s M

Amplitude; % of predrug twitch tension Initial phase

Max. phase

0(control) 2.3 4.5 1~.0

12.3±0.6 8.0±1.4 5.2±1.4 a

63.3±2.4 88.6±5.8 a 64.6±5.7 103.3±5.5 a

Time to max. phase (rain)

12.3±0.9 (108) 6.0±0.4 a (5) 6.2±0.4 a (5) 4.7±0.3 a (6)

a Significantly different from control values; P < 0.05.

68

(1 mM) did not reduce the amplitude of the KC! contracture, but did shorten the contracture duration (fig. 5c). When the KCI contracture was elicited in the high Ca 2+ solution (18 mM CaCI 2 added), the rapid depolarization phase was strongly inhibited, whereas a second, slowly developing phase appeared (fig. 5e). This phase did not appear in the high Mg 2+ solution. Diltiazem caused a significant inhibition of this Ca-dependent phase (table 3). The initial rapid contracture caused by KCi in the presence of diltiazem (1 mM) was not inhibited by high Ca 2÷ (table 3).

TABLE 5 Effect of dihiazem on the KCl-affected caffeine contracture. The values the means_+ S.E. (No. of observations). Diltiazem conc.

× 1 0 -5 M

0(c ont rol ) 2.3 4.5 CaCI z, 18 mM 4.5 MgCI 2, + 1 8 mM 4.5

Amplitude; % of predrug twitch tension Initial phase

Time to max. phase (min)

Max. phase

60.6_+ 5.5 ~ 117.9_+ 3.7 " 4.7_+0.7 a (8) 46.2+- 7.9 52.5_+ 5 . 4 b l l . 7 + 2 . 7 h ( 4 ) 52.3 ! 6.5 63.3 -+ 6.9 ~' 20.8-+ 2.8 b (4) 75.2+ 5.4 73.5+_12.7

118.0-+ 5.0

3.6+_0.4

(6)

75.3-+12.4 b 13.0-+2.2 b (6)

a Significantly different from the control caffeine contracture in table 4; P < 0.05. b Significantly different from control values; P < 0.05.

3.2.2. Effects on the caffeine contracture Pretreatment with diltiazem reduced the rapid initial phase and increased the second maximum phase of the biphasic contracture elicited by caffeine (10 mM) (fig. 6a,b; table 4). The most marked effect was a reduction of the time taken to obtain the maximum phase of the caffeine contracture the two phases could not be separated at the highest concentration of diltiazem (1 mM).

4~

3.2.3. Effects on contractures caused by a combination of KCI and caffeine As shown in figs. 5 and 6, and tables 5 and 6, KC1 and caffeine potentiated the contracture caused by the other agent. When a caffeine con-

TA B LE 6 Effect of diltiazern on the caffeine-affected KC1 contracture. The values are the means_+ S.E. (No. of observations). Fig. 6. Effects of diltiazem ( 4 . 5 × 1 0 -5 M) on the caffeine contracture and on the caffeine plus KCI contracture. Filled arrow: addition of caffeine (10 raM). Open arrow: addition of KC1 (100 mM). ( ~ ) Addition of diltiazem. ( 0 ) Addition of CaC12. (A) The control caffeine contracture. At the maximu m of this contracture, KCI was added and caused a large, longlasting additional contracture. (B) Diltiazem pretreatment changed the caffeine contracture and speeded it up. Moreover, the additional contracture caused by KC1 was markedly inhibited. (C) C a C I 2 (18 mM added) reversed the effect of diltiazem on the caffeine contracture and on the caffeine plus KCI contracture,

Diltiazem conc. x 10 5 M

Max. ampl., % of predrug twitch

Time to 50% decay (min)

0 (control) 2.3 4.5 CaC12, 18 mM 4.5 MgCI 2, 18 mM 4.5

138.0 + 6.3 70.8+2.1 a 54.2+6.9 a

10.5 + 1.6 (9) 1.3_+0.5 a (4) 0 . 9 + 0 . 2 " (5)

59.0__+7.0 ~

6.6_+1.6

64.3_+7.4 a

1.2_+0.2 a (6)

a Significantly different from control values; P < 0.05.

(4)

69

tracture was induced after a rapid and transient KC1 contracture, both phases were potentiated, and the time taken to reach the maximum phase was reduced (fig. 5a and table 5; compare with fig. 6a and table 4). Diltiazem reduced the amplitude of the maximum phase and markedly increased the time taken to obtain this phase (fig. 5b); these changes were significant (table 5). These effects of diltiazem were completely obliterated when the experiments were performed in the high Ca 2+ solution, but not when the experiments were performed in the high Mg 2+ solution (fig. 5d; table 5). When KC1 was added at the maximum of the caffeine contracture, a rapid and long-lasting additional increase in tension developed (fig. 6a, table 6). This tension could be partly inhibited by diltiazem; the amplitude as well as the duration of the KCl-induced tension were significantly reduced (fig. 6b; table 6). The effect on the duration was antagonized when the experiments were performed in the high Ca 2÷ solution, but not when the experiments were performed in the high Mg 2÷ solution; the depressive effect on the amplitude was not antagonized in these solutions (fig. 6c, table 6).

4. Discussion

Of the several effects of diltiazem on the phrenic nerve diaphragm preparation, some were nonspecific, while others could probably be related to the Ca antagonistic action of the drug, since they could be antagonized by an increased concentration of Ca 2+.

4.1. Non-specific effects The marked use-dependent inhibition by diltiazem could not be antagonized by high C a 2+ concentrations. This inhibition was the same during indirect and direct stimulation at 37 ° C. Contrary to this observation, Sato and Ono (1981) observed tetanic fade only during indirect stimulation of the tibialis anterior of the dog in situ, whereas Gallant and Goettl (1985) report a mod-

crate tetanic fade in directly stimulated extensor digitorum longus of the mouse. We determined that diltiazem had two sites of action for its inhibitory effects: one at the neuromuscular junction and the other at a site distal to the neuromuscular junction. Its action at the neuromuscular junction was indicated, by the synergism between diltiazem and d T C during both twitch and tetanic stimulation. The inhibitory effect of diltiazem during indirect stimulation had a different temperature sensitivity than the use-dependent inhibition observed during direct stimulation. Previous experiments have shown that the effects of neuromuscular-blocking drugs are augmented by the Ca antagonist, verapamil (Bikhazi et al., 1982; Durant et al., 1984). Ilias and Steinbereithner (1985) showed that Ca 2+ antagonists have synergistic effects with the non-depolarizing neuromuscular blocker, pancuronium bromide. The enhancement of this effect in the high Ca 2+ solution is also in accordance with a curare-like action of diltiazem, since high Ca 2+ concentrations potentiate the effect of dTC due to an increased desensitization (Manthey, 1966). The curare-like property of diltiazem may be related to the similar property of several local anesthetic drugs (Harrah et al., 1970). The nerve terminal and post-synaptic mechanisms have been suggested to be the site of action for these effects (Gibb and Marshall, 1984). The use-dependent inhibition during direct stimulation and the inhibitory action of diltiazem on the myotonia were probably due to an effect of the drug on the excitability of the sarcolemma. The Ca antagonist verapamil has been shown previously to inhibit the sodium current in a use-dependent way (a local anaesthetic effect) in skeletal muscle cells (Van der Kloot and Kita, 1975). However, we found no synergism between the use-dependent inhibitory effects of diltiazem and the local anaesthetic drug lidocaine. The gating mechanism of the activation or inactivation of this current represents several possible sites of action. The lack of synergism may suggest that there is an interaction with separate parts of these complex and multistage mechanisms. The lack of use-dependent inhibition by diltiazem of the compound action potential of the isolated phrenic nerve also

70 suggests a mechanism separate from that of lidocaine, which inhibits this nerve in a use-dependent way (Brodin and R~ed, 1984). The lack of Ca antagonism suggests that the diltiazem effect was not due to an inhibition of a Ca component of the action potential. Unlike nifedipine, diltiazem did not have a potentiating and fatigue-antagonistic effect on subtetanic and tetanic contractions at low concentrations. Instead, increased fatigue was observed during 20 Hz subtetanic stimulation. This was probably an expression of the use-dependent inhibition.

4.2. Effects dependent on Ca antagonism Some of the effects of diltiazem on the contractures induced by KC1 and caffeine might be an expression of a Ca antagonistic action. Especially, the depressive effect of diltiazem on the second slow phase of the KC1 contracture in the high Ca 2+ solution was probably due to an inhibitory effect on the C a 2+ flux. The effect was not mimicked by local anaesthetics in concentrations that showed equipotence regarding use-dependent inhibition, but it was observed with verapamil (observations in our laboratory). Diltiazem had complex effects on the initial KC1 contracture phase, with a depressive effect on the amplitude and an accelerating effect on the speed of decay. We could not determine whether Ca antagonized these effects, since high Ca by itself inhibits this phase of the contracture. These two effects of the low concentration of diltiazem could have a common mechanism of action, but the lack of effect of the high diltiazem concentration on the amplitude might indicate separate lowand high-concentration effects of diltiazem on the KCI contracture. The failure of high Ca 2÷ to depress the KC1 contracture in the presence of diltiazem (10 3 M) might support this conclusion. We could not test whether Ca 2+ antagonized the effects of diltiazem on the caffeine contracture (a reduction of the initial phase, a potentiation of the second maximum phase, and a marked speeding up of the second phase) because of the great variability of the caffeine contracture in the high Ca 2+ solution (own unpublished observations).

However, the effects of diltiazem on the combined contractures showed clear evidence of Ca antagonism. The marked inhibitory effect on the caffeine + KCI contracture could be partly antagonized by high Ca 2+, and the diltiazem-induced delay of the KC1 + caffeine contracture could be completely antagonized by high Ca 2+. Since these antagonistic effects were not observed in the high Mg 2+ solution, they were not due to a change of the membrane surface potential caused by the divalent cations. It may be difficult to ascribe all these effects of diltiazem to a single mechanism of action. However, since the effects could be affected by the Ca added to the organ bath, the T tubules, which communicate with the external solution (Franzini-Armstrong and Porter, 1964) might be the site of action. The T tubules have a very high density of Ca channels (Curtis and Catterall, 1984; Schwartz and Triggle, 1984; Glossmann et al., 1985). Ca currents have been demonstrated in mammalian skeletal muscle (Donaldson and Beam, 1983), and they can be blocked by diltiazem (Walsh et al., 1986).

4.3. Conclusion The effects of diltiazem on the stimulus-induced contractions were probably not related to the Ca 2+ antagonistic effect of the drug. In contrast, diltiazem in the same concentration range showed Ca antagonistic effects on several types of contractures. This could be in accordance with the observation that skeletal muscle cells exhibit two types of Ca channels: one 'transient' type, which may be related to action potential activity and is insensitive to organic Ca channel modulators, and one 'long-lasting' type, which may be active during slowly developing contractures and is sensitive to the Ca antagonists (Cognard et al., 1986; Schwartz, 1987). Under normal physiological conditions in vivo, skeletal muscle contraction is always induced by stimulation from the motor nerves via action potentials in the sarcolemma. The contractures, which are independent of action potential activity, may, however, serve as models for some pathological defects of the Ca transport mechanisms of the muscle, such as malignant hy-

71

perthermia (Britt, 1979). Diltiazem could be beneficial to patients with such conditions.

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