British Journal of Anaesthesia, 2003, Vol. 90, No. 1 66-71
© 2003 The Board of Management and Trustees of the British Journal of Anaesthesia
Laboratory Investigations |
Effects of epinephrine and phosphodiesterase III inhibitors on bupivacaine-induced myocardial depression in guinea-pig papillary muscle
Department of Anaesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan E-mail: mazuma@med.hokudai.ac.jp
Accepted for publication: August 30, 2002
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Abstract |
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Background. This study was designed to investigate the effects of epinephrine and the phosphodiesterase III inhibitors amrinone and milrinone on bupivacaine-induced myocardial depression in guinea-pig papillary muscles using an electrophysiological method.
Methods. Electrophysiological studies of the effects of bupivacaine, epinephrine, amrinone and milrinone in normal and high K+ Tyrode’s solution were measured with guinea-pig papillary muscles. Specifically, epinephrine, amrinone and milrinone reversal of bupivacaine-induced depression was measured.
Results. Bupivacaine reduced the action potential duration (APD), the maximum rate of rise of the AP (V·max) and contractile force. Although epinephrine increased the contractile force similarly to amrinone and milrinone, it shortened the APD at 50% repolarization (APD50) and 90% repolarization (APD90). A high concentration of amrinone shortened APD, while milrinone did not affect APD except for a prolongation of APD20. In high K+ Tyrode’s solution (25 mM), epinephrine, amrinone and milrinone increased the APD and the contractile force. Epinephrine reversed bupivacaine depression of APD and contractile force to control levels. Amrinone and milrinone restored not only the contractile force but also APD. There was an incomplete recovery of APD50 for amrinone and the prolongation of APD20 for milrinone.
Conclusions. Our results suggest that bupivacaine decreases the Ca+ current (ICa) and Na+ current (INa). Epinephrine and amrinone may increase ICa and the delayed outward current (Ik), whereas milrinone may increase ICa. The profound cardiovascular depression caused by bupivacaine was effectively reversed by amrinone and milrinone in a manner similar to epinephrine.
Br J Anaesth 2003; 90: 66–71
Keywords: anaesthetics local, bupivacaine; enzymes, phosphodiesterase, inhibition; nerve, transmission; sympathetic nervous system, epinephrine
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Introduction |
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Bupivacaine is known not only for its potent and long-lasting effects, but also for its cardiodepressive properties. Cardiovascular collapse caused by bupivacaine is difficult to treat. Bupivacaine blocks the fast inward current, using a single sucrose gap voltage clamp technique,1 and the slow inward current using an electrophysiological method, in guinea-pig ventricular muscle.2 Bupivacaine also inhibits the transient outward current (Ito).3 Epinephrine is commonly used to treat bupivacaine-induced cardiovascular depression. However, this use remains controversial.4
Inhibition of phosphodiesterase (PDE) III leads to increased intracellular concentrations of cyclic adenosine monophosphate (cAMP)5 and activation of protein kinases,6 which in turn explain the positive inotropic effects of the drug. It has been shown that amrinone (PDE III inhibitor) increases cardiac output (CO) because of an increase in myocardial contractility7 and effectively reverses profound bupivacaine-induced cardiovascular depression in pigs through intracellular Ca2+-release mechanisms.8 Milrinone (another PDE III inhibitor), in a manner similar to amrinone, enhances cardiac contractility, increases CO,4 acts on Ca2+ channels in the sarcolemma, and increases cAMP, which activates Ca2+ influx and Ca2+ release/uptake from the sarcoplasmic reticulum.9 10 In this study, we evaluated whether amrinone and milrinone would reverse bupivacaine-induced cardiodepression in a manner similar to epinephrine.
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Methods |
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This study was approved by Hokkaido University Graduate School of Medicine Animal Care and Use Committee. Electrophysiological experiments were performed as previously described.11 Hearts were rapidly removed from guinea-pigs weighing 250–460 g that had been anaesthetized with diethyl ether. Right ventricular papillary muscles were carefully excised in oxygenated Tyrode’s solution at room temperature and transferred to a 5 ml tissue chamber and superfused at a rate of 10 ml min–1 with Tyrode’s solution of the following composition (mM): NaCl 125; KCl 4; NaH2PO4 1.8; MgCl2 0.5; CaCl2 2.7; NaHCO3 25; and glucose 5.5. The solution was gassed with oxygen 95% and carbon dioxide 5%. The temperature was kept constant at
33.0°C.
One end of the papillary muscle was hooked to an extension of the lever arm of a force transducer (Nihon Kohden, JZ-802J, Tokyo, Japan), and the other end was pinned to the bottom of the tissue chamber. The transducer was mounted on a micromanipulator and resting tension was progressively increased to 200 mg. These preparations were stimulated at 0.5 Hz through platinum field electrodes with rectangular pulses of 1 ms duration at twice the threshold voltage, delivered by an electronic stimulator (Nihon Kohden, SEN 3301) through an isolation unit (Nihon Kohden, SS-102J). Transmembrane potentials were recorded using glass microelectrodes filled with KCl 3 M (resistance of 10–30 M
). The microelectrodes were coupled through an Ag/AgCl junction to a high-impedance capacitance-neutralizing amplifier (Nihon Kohden, MZE2801). An agar bridge containing KCl 3 M was used as a reference electrode. An electronic differentiator with linear output 50–1000 V s–1 was used for measurement of the maximum rate of rise of the fast action potential (AP). This rate is denoted by V·max. These amplified signals were displayed on a digital oscilloscope (Hewlett Packard 54501A, Palo Alto, CA, USA) and recorded on a chart recorder (Hewlett Packard, 2225AJ). The preparations were stabilized for 
60 min before commencement of the experiments. After a 30 min stabilization period for the AP, control recordings were made and the preparations were exposed to solutions containing various concentrations of bupivacaine, epinephrine, amrinone and milrinone. The concentration was increased in a stepwise fashion at intervals of 30 min and recordings were made when the changes in the AP and the developed tension reached a steady state. In the experiments to test the recovery of bupivacaine-induced depression, the preparations were exposed for 30 min to bupivacaine and then to the solutions in the absence of bupivacaine, containing various concentrations of epinephrine, amrinone and milrinone.
To induce slow AP, contributing to an indirect estimate of the Ca2+-mediated slow inward current, a 25 mM K+ Tyrode’s solution for depolarizing the papillary muscles was made by isotonic substitution of NaCl with KCl. Resting membrane potential (RMP) was usually depolarized to approximately –50 mV in this solution, which is sufficient to inactivate fast Na+ channels. Slow AP was induced electrically by delivering rectangular pulses of 10–11 ms duration to the field electrodes at a frequency of 0.5 Hz. After a 15 min stabilization period of the slow AP, the experimental procedure commenced. A concentration of 5x10–5 M bupivacaine was selected for the slow AP study because this concentration caused a 75% decrease in contractile force with normal Tyrode’s solution. Epinephrine 5x10–7 M, amrinone 10–3 M and milrinone 5x10–5 M were used because these concentrations produced recovery of the contractile force to control values after bupivacaine-induced depression.
The following drugs were used: bupivacaine (Sigma Chemical, St Louis, MO, USA); epinephrine (Daiichi Pharmaceuticals, Tokyo, Japan); amrinone (Meiji Pharmaceuticals, Tokyo, Japan); and milrinone (Yamanouchi Pharmaceuticals, Tokyo, Japan).
All data are mean (SEM). Student’s t-test was used for analysis of paired and unpaired observations. P<0.05 was considered statistically significant.
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Results |
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The effects of bupivacaine on AP variables and contractile force in guinea-pig papillary muscles with normal Tyrode’s solution are shown in Table 1. Bupivacaine shortened the APD and decreased contractile force in a concentration-dependent manner. At a high concentration, bupivacaine decreased V·max and AP amplitude (APA). It did not influence RMP. A bupivacaine concentration of 5x10–5 M decreased contractile force to
75% of the control value.
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The effects of bupivacaine on slow AP were examined in muscles depolarized to approximately –49 mV by 25 mM K+ Tyrode’s solution. Bupivacaine (5x10–5 M) significantly shortened the APD50 and decreased contractile force (Table 2).
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Although epinephrine markedly increased contractile force in a concentration-dependent manner, it shortened the APD in normal Tyrode’s solution as shown in Table 3. Epinephrine significantly shortened the APD50 and the APD90 while it increased APA in a concentration-dependent manner. Epinephrine did not influence RMP or V·max. Epinephrine (5x10–7 M) increased contractile force to 307.9 (27.4)%. In high K+ Tyrode’s solution, epinephrine (5x10–7 M) prolonged APD50 and increased contractile force (Table 2).
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Amrinone also increased contractile force and APA in a concentration-dependent manner. It also significantly shortened APD at 5x10–4 M and 10–3 M (Table 3). However, 10–3 M amrinone, which increased the contractile force to 253.0 (38.0)% in normal Tyrode’s solution, prolonged APD50 and increased contractile force in high K+ Tyrode’s solution (Table 2).
Milrinone increased contractile force and APA, but did not influence APD except for a prolongation of APD20 (Table 3). Milrinone at 5x10–5 M, which, in normal Tyrode’s solution, increased contractile force to 237.6 (26.2)%, also prolonged APD50 and increased contractile force in high K+ Tyrode’s solution (Table 2).
As shown in Table 4, epinephrine 5x10–7 M administration restored APD and contractile force to control levels (Fig. 1A). Amrinone at 5x10–4 M and 10–3 M restored APD20 and APD90, but not APD50, to control levels. Contractile force was restored to control at amrinone concentration of 10–3 M (Fig. 1B). For milrinone, 10–5 M and 5x10–5 M caused recovery of APD50 and APD90 to control and prolonged APD20. Contractile force was also restored to control at 10–5 M, and significantly increased at 5x10–5 M. V·max was restored at both concentrations (Fig. 1C).
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In order to clarify whether bupivacaine-induced depression was irreversible, the time course of bupivacaine washout was examined (n=6). The decreases in APD, APA and V·max were not restored after 60 min washout with drug-free Tyrode’s solution. Contractile force was 61.6 (5.2)% of control at 60 min after bupivacaine removal.
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Discussion |
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Bupivacaine caused a concentration-dependent decrease in contractile force and a shortening of APD in isolated guinea-pig papillary muscles. For slow AP, induced by strong electrical stimuli in high K+ Tyrode’s solution, bupivacaine significantly decreased contractile force and shortened APD50. Such behaviour could be explained if bupivacaine depressed the channels responsible for slow inward current (ICa), which contributes to the AP plateau. Bupivacaine also decreased V·max, which is used as an indicator of sodium current activity, in a concentration-dependent manner. This finding suggests that bupivacaine might inhibit the sodium channel (INa).1
Epinephrine caused AP shortening in guinea-pig papillary muscles. However, it also increased contractile force in normal Tyrode’s solution. For slow AP in high K+ Tyrode’s solution, epinephrine prolonged APD50 and increased contractile force. This finding suggests that epinephrine increases Ca2+ influx through the myocardial Ca2+ channel. It has been reported that beta adrenoceptor agonists increase ICa and the delayed outward current (IK) in ventricular cells. Bennett and Begenisich12 suggested that an increase in IK produced by catecholamines may serve to limit the degree of calcium-current-induced AP prolongation during increased sympathetic tone and rapid heart rates. Our observations may indicate a possible mechanism to explain the AP shortening caused by epinephrine which may result from an increase in IK in the guinea-pig ventricular muscle. Epinephrine may not only enhance ICa, but also IK, both of which contribute to the APD.
Amrinone shortened APD at 5x10–4 M and 10–3 M and increased contractile force. For the slow AP, amrinone prolonged APD50 and increased contractile force. These results suggest that amrinone increases Ca2+ influx through myocardial Ca2+ channels. However, the shortening of APD at 5x10–4 M and 10–3 M in normal Tyrode’s solution may be explained by an increase in IK. It is well documented that an increase in intracellular cAMP leads to an increase in the K+ current in isolated ventricular myocytes.13 We suggest that the underlying mechanism of amrinone may be to increase ICa and Ik via phosphorylation of the channel secondary to an increase in cAMP.
Conversely, milrinone did not affect APD50 or APD90, but increased contractile force and prolonged APD20. Under slow AP conditions, milrinone prolonged APD50 and increased contractile force. The positive inotropic effect of milrinone may be attributable to a greater influx of calcium during the AP and more efficient intracellular calcium handling.10 Indeed, milrinone increased ICa measured using conventional microelectrode and patch clamp techniques.14
In the ‘reversal study’, the washout of bupivacaine with drug-free Tyrode’s solution did not restore APD nor contractile force to control level. Epinephrine (5x10–7 M) restored contractile force, APD and V·max, while amrinone restored these only at 10–3 M. Milrinone also produced recovery of contractile force at 10–5 M, and an increase at 5x10–5 M.
Comparisons between epinephrine, amrinone and milrinone of the concentration required to produce >200% increase in the contractile force, indicated that epinephrine was the most potent and amrinone the least potent. Epinephrine induced severe tachycardia, hypertension and cardiac arrhythmia during resuscitation from ropivacaine-induced cardiovascular toxicity in pigs, whereas milrinone did not.4 Amrinone was also superior to epinephrine for the treatment of bupivacaine-induced cardiovascular depression in sevoflurane-anaesthetized dogs according to a study in which tachycardia occurred in response to epinephrine, compared with those receiving amrinone.15
In conclusion, for reversal of the cardiodepressive effects of bupivacaine in guinea-pig papillary muscles, amrinone and milrinone were similar to epinephrine. Recovery of cardiodepression may be because of the positive inotropic effect of epinephrine, amrinone and milrinone accompanied by an increase in ICa.
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Acknowledgement |
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We would like to thank A. Sakai for secretarial assistance.
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References |
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