Neurochemistry International 40 (2002) 655–659
Presynaptic inhibitory effects of rocuronium and SZ1677 on [3H]acetylcholine release from the mouse hemidiaphragm preparation Shunichi Takagi a , Yushi U. Adachi a , Albert J. Saubermann b , E. Sylvester Vizi a,∗ b
a Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450 Budapest, Hungary Department of Anesthesiology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY 10461, USA
Received 22 June 2001; accepted 15 September 2001
Abstract It has been shown that nondepolarizing muscle relaxants may have effects on nicotinic acetylcholine receptors (nAChRs) other than those located on the skeletal muscle: some of them possess inhibitory effects on neuronal nAChRs [Anesth. Analg. 59 (1980) 935; Trends Pharmacol. Sci. 9 (1988) 16; Pharmacol. Ther. 73 (1997) 75]. It was shown that, e.g. (+)-tubocurarine and pancuronium are able to inhibit ACh release from the axon terminals of hemidiaphragm preparations and produce tetanic fade indicating their presynaptic effect. In this study rocuronium, a nondepolarizing steroidal muscle relaxant with shorter onset of action, and SZ1677 [1-(3␣-hydroxy-17-acetyloxy)-2-(1.4-dioxa-8-azaspiro-[4,5]-dec-8-yl)-(5␣-androstane-16-yl)-1-(2-propenyl) pyrrolidinium bromide], a short-acting muscle relaxant [Ann. New York Acad. Sci. 757 (1995b) 84] inhibited the release of ACh in response to axonal stimulation, while ␣-bungarotoxin failed to reduce the stimulation evoked release of ACh and did not produce tetanic fade. These results indicate that in addition to their postsynaptic effect, rocuronium and SZ1677 have presynaptic inhibitory effects on neuronal nAChRs at the neuromuscular junction. The finding that ␣-bungarotoxin does not inhibit the release and does not produce tetanic fade indicates that it possesses affinity only for the postsynaptic muscle nAChRs. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Rocuronium; SZ1677; Presynaptic effect; Neuromuscular junction; ACh release
1. Introduction Nondepolarizing neuromuscular blocking agents are widely used in clinical settings of anesthesia. A series of reviews had dealt with these drugs, however, these mainly focused on their clinical effects and paid little attention to their mode of action (Foldes, 1960; Bowman, 1982, 1990; Lien, 1994; Miller and Ehrenburg, 1994; Bartkowski, 1999; Atherton and Hunter, 1999). Nondepolarizing neuromuscular blocking agents are considered competitive antagonists of nicotinic acetylcholine receptors (nAChRs) located on the skeletal muscle (Chang et al., 1967; Fletcher and Forrester, 1975). In addition, they have effects on neuronal nAChRs (Vizi and Lendvai, 1997; Marchi et al., 1999) and on muscarinic (M2 and M3 ) receptors as well (Narita et al., 1992; Okanlami et al., 1996; Vizi and Lendvai, 1997; Cembala et al., 1998). Using sensitive radioactive method (Foldes et al., 1984), the effect of neuromuscular blocking agents on the release of ACh from the presynaptic axon terminals was investigated (Vizi et al., 1985, 1987, 1995a; ∗
Corresponding author. Tel.: +36-1-210-9421; fax: +36-1-210-9423. E-mail address:
[email protected] (E.S. Vizi).
Wessler et al., 1986, 1987; Somogyi and Vizi, 1987; Vizi and Somogyi, 1989): (+)-tubocurarine (Vizi et al., 1985, 1987; Wessler et al., 1986) and pancuronium (Vizi et al., 1987), drugs widely used in the clinical practice, reduced the release of ACh in response to axonal stimulation. It was concluded that this presynaptic inhibition of ACh release may be partly responsible for tetanic fade (Bowman et al., 1988; Vizi et al., 1987; England et al., 1997). Several new nondepolarizing muscle relaxants showing almost ideal property has been developed. However, the data for these newly developed neuromuscular blocking agents concerning the effect on ACh release is limited (Vizi and Lendvai, 1997). Therefore in the present investigation, the effect of rocuronium, a nondepolarizing steroidal muscle relaxant with shorter onset of action, and SZ1677 a short-acting, nondepolarizing muscle relaxant was studied on the release of ACh in in vitro phrenic nerve-hemidiaphragm preparation; their effect on the release and on tetanic fade were compared with the effect of ␣-bungarotoxin. Both compounds reduced the release and produced tetanic fade, but as compared to their potencies to inhibit twitch tension, SZ1677 was less potent. ␣-Bungarotoxin failed to affect the release, and did not produce tetanic fade.
0197-0186/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 1 ) 0 0 1 1 1 - 5
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S. Takagi et al. / Neurochemistry International 40 (2002) 655–659
2. Materials and methods Male Swiss-Webster mice, weighing 25–30 g were lightly anesthetized with ether and decapitated. Both hemidiaphragms (about 2–3 mm to the right and left of the insertions of the phrenic nerves) were prepared and the phrenic nerves were cut 1 mm from the muscle. 2.1. Stimulation The preparations were stimulated with a Grass S-88 stimulator using platinum electrodes placed above the proximal and below the distal parts of the preparation (field stimulation). Every 10 s trains of supramaximal (50 V) square wave impulses were delivered (pulse duration: 1.0 ms, frequency: 50 Hz, train duration: 0.8 s). 2.2. Incubation The preparations were incubated for 60 min in Krebs solution containing 3 H-choline (methyl-3 H-choline, 185 kBq ml−1 , 5 Ci ml−1 , Amersham Corp. (Arlington Heights, IL)). Krebs solution was gassed with a mixture of 95% O2 and 5% CO2 at 37 ◦ C and the composition was (mM): NaCl 113, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2 PO4 1.2, glucose 11.5. In order to facilitate the incorporation of 3 H-choline into the acetylcholine pool of the motor nerve terminal, the preparations were stimulated during the first 45 min as described above. For the next 15 min, the preparations were allowed to rest. After being washed three times to remove excess 3 H-choline, each preparation was transferred into a fourchannel microvolume perfusion system. Two pieces of diaphragms (right and left side) were placed into each chamber, the preparation was superfused with Krebs solution for 60 min. From then on, hemicholinium-3 (10 M) containing solution was superfused at a rate of 0.5 ml min−1 . Hemicholinium-3 was added to prevent reuptake of 3 H-choline. After the 60 min preincubation period, 3 min aliquots of the effluents were collected by an automated fraction collector. The preparations were stimulated as described above for 3 min during the 3rd (S0 ), 13th (S1 ) and 22nd (S2 ) fraction, delivering a total of 720 shocks in each case (Fig. 1). Neuromuscular blocking agents were added from the 19th fraction, 9 min before S2 , until the end of the experiment. The 2 × ED95 and higher concentrations (rocuronium 6 and 80 M, SZ1677 1 and 16 M) of neuromuscular blocking agents were used for the experiment. At the end of the experiment, tissue pieces were removed from the chamber and homogenized in 0.5 ml of 10% trichloroacetic acid. A 0.5 ml aliquot of the superfusate or 0.1 ml of the tissue supernatant were added to 2 ml of scintillation cocktail (Ultima GOLD Packard). Tritium content was measured with a Packard 1900 TR liquid scintillation counter using an internal standard.
Fig. 1. Release of 3 H-choline from hemidiaphragm in response to field stimulation. The tissue was superfused (0.5 ml min−1 ) and 3 min fractions were collected. The tissue was stimulated three times, at the 3rd (S0 ) 13th (S1 ) and 22nd (S2 ) fractions (50 Hz, 1 ms, 720 shocks).
2.3. Calculation of resting and evoked release of radioactivity Data were expressed as fractional release (FR), that is the percentage of the total radioactivity in the tissue at the moment of the collection of the sample. It is calculated from the absolute amount of radioactivity (measured in Bq g−1 ) of the tissue after experiment plus the fractions collected subsequently. Thus the loss of radioactivity during experiment is corrected for. Field stimulation evoked FR was calculated by subtracting the values of the basal release from the total release during the stimulation period. Basal release was defined as the average FR of the two fractions before stimulation. Stimulation evoked FR values were calculated from the three fractions during and after each stimulation (S1 and S2 ). The effect of the drugs on electrical stimulation-induced outflow were expressed as the ratio of FR during the second (S2 ) compared with the first (S1 ) stimulation (FRS2 /FRS1 ): drugs were present in the superfusion fluid from the 19th collection period and maintained throughout the experiment. 2.4. Materials The following materials were used: 3 H-methylcholine chloride (78 Ci ml−1 ), obtained from Amersham Corp., rocuronium obtained from Organon Inc. (West Orange, NJ, USA), ␣-bungarotoxin (Sigma Chemical, St. Louis, MO) and SZ1677 [1-(3␣-hydroxy-17-acetyloxy)-2-(1.4-dioxa8-azaspiro-[4,5]-dec-8-yl)-(5␣-androstane-16-yl)-1-(2-pro penyl) pyrrolidinium bromide], obtained from Gedeon Richter (Budapest, Hungary) and Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan). All other chemicals were obtained from Sigma Chemical. 2.5. Statistical analysis All data are expressed as mean (±S.E.M.). Statistical significance was determined using analysis of variance
S. Takagi et al. / Neurochemistry International 40 (2002) 655–659
(ANOVA) followed by Dunn’s test. P < 0.05 was accepted as significant.
Table 2 Effects of rocuronium, SZ1677 and ␣-bungarotoxin on twitch tension and tetanic maintenancea Compounds
Inhibition of twitch tension (%)
Tetanic fade (%)
Rocuronium SZ1677 ␣-Bungarotoxin
19.5 (±2.6) 20.7 (±1.4) 18.9 (±1.6)
81.2 (±2.9) 29.6 (±1.4) 2.2 (±0.3)
3. Results 3.1. Content of tritiated acetylcholine in the hemidiaphragm The content of radioactivity after 60 min incubation with 5 Ci ml−1 of 3 H-choline was 10.50 (±1.38) × 104 Bq g−1 (n = 4). The radioactivity released by the electrical stimulations (S0 , S1 , S2 ) and between them was 28.97 (±5.73) × 103 Bq g−1 amounting to 27.2% (±2.7) of the total radioactivity present in the tissue after loading. At rest, during the 3 min collection periods 1.01% (±0.11) of the total radioactivity content was released. 3.2. Effect of nondepolarizing neuromuscular blockers and α-bugarotoxin on stimulation-evoked release The FR during the first stimulation was 3.7% (±0.3), calculated as the sum of three fractions during and after stimulation (the absolute amount of released radioactivity was 3.95 (±0.81) × 103 Bq g−1 ). When the stimulation was repeated (S2 ) the release was fairly constant: the ratio between the amount of radioactivity released by consecutive stimulation (FRS2 /FRS1 ) was 0.789 (±0.034). In the experiment, rocuronium and SZ1677 were added to the perfusion solution 9 min before the second (S2 ) stimulation and kept in perfusion fluid throughout the experiments. As shown in Table 1, rocuronium and SZ1677 significantly reduced the stimulation evoked release of ACh. When concentrations of rocuronium and SZ1677 were equipotent (they were set to cause 20% inhibition of twitch tension), SZ1677 was a Table 1 Effect of nondepolarizing neuromuscular blocking agents and ␣-bungarotoxin on stimulation-evoked release of 3H-acetylcholine from mouse hemidiaphragma Drugs
Concentration (M)
Control
n
FRS2 /FRS1
9
0.789 (±0.034)
Rocuronium
6 20 80
6 6 4
0.496 (±0.025)b 0.310 (±0.018)c 0.094 (±0.024)c
SZ1677
1 3 16
6 6 4
0.686 (±0.030)b 0.445 (±0.031)c 0.296 (±0.010)c
6 6
0.837 (±0.035) 0.823 (±0.060)
␣-Bungarotoxin
0.01 0.1
a The effect of the drugs on electrical stimulation-induced outflow is expressed as the ratio of FR in the second (S2 ) compared with the first (S1 ) stimulation (FRS2 /FRS1 ). Data are expressed as mean (±S.E.M.). b P < 0.05. c P < 0.001; compared with control.
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a The drugs were administered at concentrations able to produce an about 20% steady state neuromuscular block and subsequently a 50 Hz tetanus was applied for 2 s. Twitch tension was produced by an indirect stimulation of phrenic nerve with 0.1 Hz stimulation.
significantly (P < 0.001) less potent then rocuronium in exerting presynaptic effects. ␣-Bungarotoxin caused a small non-significant increase in the basal efflux, but it had no effect on the stimulation evoked release of ACh. 3.3. Effect on twitch tension and maintenance of tetanic contraction Table 2 shows the effect of muscle relaxants and ␣-bungarotoxin on twitch tension and on tetanic fade.
4. Discussion The aim of the present investigation was to evaluate the presynaptic effects of rocuronium and SZ1677 on the stimulation-evoked release of ACh from the mouse hemidiaphragm preparation and to compare their effects with ␣-bungarotoxin, an antagonist known to have high affinity for skeletal muscle nAChRs (Arias, 2000; Testai et al., 2000). Our results clearly demonstrated, in agreement with earlier observations with (+)-tubocurarine and pancuronium, that rocuronium and SZ1677 decreased the release of ACh and produced tetanic fade. In producing tetanic fade, rocuronium was more potent than SZ1677. Although ␣-bungarotoxin has previously been shown to modulate basal ACh release (Bukharaeva et al., 2000), no effect was found on the stimulus evoked release of [3 H]ACh (Apel et al., 1995). In addition, no significant tetanic fade was induced by ␣-bungarotoxin (Bradley et al., 1987). In agreement with these results, in our preparation the FRS2 /FRS1 value was unchanged by ␣-bungarotoxin, and tetanic fade was not induced. The nondepolarizing neuromuscular blocking agents might be able to reduce the margin of safety in two ways: (1) by inhibiting the effect of ACh on the nicotinic receptors located on the skeletal muscle and (2) by reducing the amount of ACh released via inhibition of positive-feedback modulation of ACh release (Bowman, 1980; Bowman et al., 1988; Vizi and Lendvai, 1997; Vizi et al., 1987). Any reduction in ACh release may produce a devastating ‘fade’ in muscular strength, when a proportion of the nAChRs located on the muscle are occupied (Vizi et al., 1987).
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Acetylcholinesterase inhibitors are used to reverse neuromuscular block in clinical setting. However, the effect of the acetylcholinesterase inhibitors to reverse neuromuscular block is affected by the amount of ACh released from axon terminals. SZ1677 significantly reduced the release of ACh; the concentration needed for a presynaptic inhibitory effect (1 M) is comparable to concentration required for the inhibition of postsynaptic nAChRs. This indicates that SZ1677 at concentration expected to be present in the plasma has a presynaptic effect. The clinical experiments will be critical in answering the question whether it is an advantage that a compound has both pre- and postsynaptic effect at the neuromuscular junction. The nAChRs located at both sides of the junction are different in subunit composition (Vizi and Lendvai, 1997). On the other hand, ␣-bungarotoxin, a selective inhibitor of skeletal muscle nAChRs ((␣1)2 1␦⑀) failed to reduce the release of ACh and did not produce tetanic fade. These findings support that tetanic and train-of-four fade produced by nondepolarizing muscle relaxants is due to their presynaptic effects. The margin of safety (Paton and Waud, 1967) for SZ1677 might be wider than that for rocuronium. SZ1677 does not inhibit vagal-induced depression of heart rate in cats (Vizi et al., 1995b), and does not have antimuscarinic effects in vitro experiments (Vizi and Lendvai, 1997). Sato et al. (1999) reported that SZ1677 does not potentiate norepinephrine release. These results support that SZ1677 is less potent for presynaptic receptors, which may be preferable for the clinical use. Since all voluntary muscle movements in man and other mammals are elicited by short trains of tetani (Zierler, 1974), the presynaptic inhibition of the evoked release of ACh by muscle relaxants at high frequency stimulation may have considerable clinical consequences. Tetanic and train-of-four fade are more sensitive to nondepolarizing muscle relaxants than the single twitch, therefore they are important signs for evaluating the residual effect of neuromuscular blocking agents for recovery phase (Foldes et al., 1981). The recovery of the tetanic fade is always delayed from that of the twitch tension elicited by single impulse. The difference between the recovery of tetanic fade and the responses to the twitch tension seems to indicate presynaptic involvement in relaxation. References Apel, C., Ricny, J., Wagner, G., Wessler, I., 1995. Alpha-bungarotoxin, kappa-bungarotoxin, alpha-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm. Naunyn Schmiedebergs Arch. Pharmacol. 352, 646–652. Arias, H.R., 2000. Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. Neurochem. Int. 36, 595–645. Atherton, D.P., Hunter, J.M., 1999. Clinical pharmacokinetics of the newer neuromuscular blocking drugs. Clin. Pharmcokinet. 36, 169–189. Bartkowski, R.R., 1999. Recent advances in neuromuscular blocking agents. Am. J. Health Syst. Pharm. 56, S14–17.
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