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On-line version ISSN 1806-907X
Rev. Bras. Anestesiol. vol.57 no.1 Campinas Jan./Feb. 2007
Influence of procainamide on the neuromuscular blockade caused by rocuronium and investigation on the mechanism of action of procainamide on the neuromuscular junction*
Influencia de la procainamida sobre el bloqueo neuromuscular producido por el rocuronio e investigación sobre el mecanismo de acción de la procainamida en la junción neuromuscular
Thalita Duque MartinsI; Yolanda Christina S. LoyolaII; Angélica de Fátima de Assunção Braga, TSAIII
em Farmacologia pelo Departamento de Farmacologia da FCM da UNICAMP
IIDoutora em Farmacologia pelo Departamento de Farmacologia da FCM da UNICAMP
IIIProfessora Associada do Departamento de Anestesiologia da FCM da UNICAMP
OBJECTIVES: It has already been proved that procainamide potentiates the
neuromuscular blockade of d-tubocurarine; however, the mechanism of this potentiation
is controversial. The aim of this study was to assess the influence of procainamide
on the neuromuscular blockade produced by rocuronium and investigate the mechanisms
of this interaction.
METHODS: Fifteen rats (250 to 300 g) were used in the preparation described by Bülbring. They were divided in three groups (n = 5 each): procainamide 20 µg.mL-1 (Group I); rocuronium 4 µg.mL-1 (Group II); and rocuronium 4 µg.mL-1 and procainamide 20 µg.mL-1 (Group III). The following parameters were evaluated: 1) amplitude of muscle contractions under indirect stimulation, before and after the administration of the drugs; 2) miniature end plate potentials (MEPPs); and 3) the efficacy of 4-aminopyridine in reverting the muscular blockade. The mechanism of the interaction was studied in Biventer cervicis (n = 5) and in the denervated rat diaphragm (n = 5), observing the influence of procainamide in the response to acetylcholine.
RESULTS: Procainamide alone did not change the neuromuscular responses. Group III presented a 68.6% ± 7.1% blockade, which represented a statistically significant difference (p = 0.0067) when compared with Group II (10.4% ± 4.5%), which was reverted by 4-aminopiridine. Procainamide increased the frequency of the MEPP, followed by a blockade that was reverted by 4-aminopiridine. In Biventer cervicis, procainamide increased the contraction in response to acetylcholine, which was not observed in the denervated diaphragm.
CONCLUSIONS: Procainamide potentiated the blockade caused by rocuronium. The changes observed with MEPP and Biventer cervicis identified pre-synaptic action. The antagonism of 4-aminopiridine on the blockade of the MEPP suggested receptor desensitization by procainamide.
Key Words: ANIMAL: rat; ANTI-ARRHYTHMICS: procainamide; DRUGS: interaction; NEUROMUSCULAR BLOCKERS, Nondepolarizing: rocuronium.
Y OBJETIVOS: La potenciación de la procainamida sobre el bloqueo
neuromuscular producido por la d-tubocurarina ya está comprobada, pero
sin embargo el mecanismo es controvertido. El objetivo del estudio fue el de
evaluar la influencia de la procainamida en el bloqueo neuromuscular producido
por el rocuronio e investigar los mecanismos de esa interacción.
MÉTODO: Se utilizaron 15 ratones (250 a 300 g) en preparación descrita por Bülbring. Se formaron los siguientes grupos (n = 5 cada): procainamida 20 µg.mL-1 (Grupo I); rocuronio 4 µg.mL-1 (Grupo II) y rocuronio - 4µg.mL-1 y procainamida - 20µg.mL-1 (Grupo III). Se evaluó: 1) la amplitud de las contracciones musculares bajo la estimulación indirecta en cada grupo, antes y después de la adición de los fármacos; 2) los potenciales de placa terminal en miniatura (PPTM); 3) la eficacia de la 4-aminopiridina en la reversión del bloqueo neuromuscular. El mecanismo de la interacción se estudió en Biventer cervicis (n = 5) y diafragma de ratón desnervado (n = 5), observándose la influencia de la procainamida en la respuesta a la acetilcolina antes y después de la adición de la procainamida.
RESULTADOS: De forma aislada, la procainamida no alteró las respuestas neuromusculares. El bloqueo producido con el Grupo III fue de 68,6% ± 7,1%, con una diferencia significativa (p = 0,0067) con relación al Grupo II (10,4% ± 4,5%), revertido por la 4-aminopiridina. La procainamida ocasionó un aumento en la frecuencia de los PPTM, seguido de bloqueo revertido por la 4-aminopiridina. En Biventer cervicis, la procainamida aumentó la respuesta a la acción de contracción de la acetilcolina, resultado no observado con el diafragma desnervado.
CONCLUSIONES: La procainamida potenció el bloqueo producido por el rocuronio. Las alteraciones observadas con PPTM y Biventer cervicis identificaron una acción presináptica. El antagonismo de la 4-aminopiridina sobre el bloqueo de los PPTMs sugirió la desensibilización de los receptores por la procainamida.
Procainamide, a synthetic analogue of the local anesthetic procaine, developed for the treatment of ventricular and supraventricular arrhythmias, is longer acting than procaine because its amidic action is resistant to esterases 1.
Its chronic use may promote the development of antinuclear antibodies with biochemical evidence of a lupus syndrome, which hinders its oral use for a prolonged time. However, intravenously it has been useful in the treatment of cardiac arrhytmias 1,2.
Some clinical reports demonstrated that high plasma concentrations of procainamide associated with factors that compromise neuromuscular transmission, such as neuromuscular diseases (latent myasthenia gravis without clinical manifestations), drugs, such as amiodarone, diabetes mellitus, and renal failure, can cause myasthenia-like symptoms 3-5. These effects have been observed in the absence of anti-acetylcholine receptor antibodies (Ach) 2.
Although there are studies trying to explain such effects, the mechanism by which procainamide interferes with neuromuscular transmission is still unknown 6-10.
It is important to discover the interaction between procainamide and non-depolarizing neuromuscular blockers, which would allow for the safe use of these drugs.
The potentiation of the effects of d-tubocurarine by procainamide has already been shown 11; however, there is a lack of comparative studies to demonstrate the interactions of procainamide with other neuromuscular blockers used nowadays.
Rocuronium is a non-depolarizing, aminosteroid, neuromuscular blocker with an intermediate duration but fast onset of action, which differentiates it from the other non-depolarizing blockers and makes it an alternative agent to succinylcholine in situations of rapid-sequence induction 12-14.
The aim of this study was to evaluate, in an experimental model, the effects of procainamide in the neuromuscular transmission and its influence on the neuromuscular blockade produced by rocuronium and to investigate the probable mechanism of the interaction between procainamide and rocuronium.
This study complied with the ethical principles of the Colégio Brasileiro de Experimentação Animal (COBEA) approved by the Ethics Commission on Animal Research of the Instituto de Biologia da Universidade Estadual de Campinas (UNICAMP).
To assess the effects of procainamide on neuromuscular transmission and its influence on the neuromuscular blockade caused by rocuronium, the rat phrenic nerve diaphragm preparation proposed by Bulbring 15 was used. Fifteen Wistar rats, weighing 250 to 300 g, were sacrificed under general anesthesia with the intraperitoneal administration of 10% chloral hydrate (250 mg.kg-1) by bleeding them through the sectioned neck vessels. The hemidiaphragms with the corresponding phrenic nerves were removed and fixed in a basin containing 40 mL of a nutrient solution of Tyrode with the following composition (in mM): NaCl 137; KCl 2.7; CaCl2 1.8; NaHCO3 11.9; MgCl2 0.25; NaH2PO4 0.3; and glucose 11. The solution was aerated constantly with carbogen (95% O2 and 5% CO2). The nerve was placed over platinum electrodes connected to a Grass S48 stimulator. The diaphragm was kept under constant tension (5.0 g) through its tendinous portion by a wire connected to the Load Cell BG59 GMS isometric transducer and submitted to indirect stimulation of 1.0 Hz and 0.2 msec, and tension variations produced by diaphragmatic contractions were recorded by a Gould RS 3400 physiograph.
The rats were divided in three groups (n = 5 in each one) according to the drug added to the preparation: Group I procainamide (20 µg.mL-1); Group II rocuronium (4 µg.mL-1); Group III rocuronium (4 µg.mL-1) in a preparation exposed previously to procainamide (20 µg.mL-1). In Group III (procainamide-rocuronium), the rocuronium was added to the preparation 30 minutes after the procainamide. The neuromuscular responses to indirect stimulation were recorded for 90 minutes after the drugs were added. To revert the neuromuscular blockade produced by the association procainamide-rocuronium, 4-aminopyridine (4-AP) - 20 µg.mL-1, was added to the preparation 60 minutes after the neuromuscular blockade. The phrenic nerve diaphragm preparation was also used to study the effects of procainamide on miniature end plate potentials (MEPP).
The chick Biventer cervicis preparations (n = 5) were assembled according to the method described by Ginsborg16 and kept in 5 mL of a nutrient Krebs solution composed of (in mM): NaCl 136; KCl 5.0; CaCl2 2.5; KH2PO4 1.2; NaHPO3 11.9; and glucose 11.2, under a tension of 5.0 g, temperature of 37°C, and aerated with carbogen. The muscles received indirect stimulation with supramaximal pulses (intensity of 20-80 V, 0.2 msec duration, and frequency of 0.1 Hz). The responses to acetylcholine (10 µg.mL-1) were recorded for 30 minutes in a Gould RS 3400 physiograph before and 30 minutes after the addition of procainamide (20 µg.mL-1) to the preparation.
For the chronically denervated rat diaphragm preparation, the diaphragms were denervated 17 and 15 to 30 days later the left hemidiaphragms were removed and placed in a 40 mL basin containing a nutrient solution of Tyrode, aerated, and kept at 37°C, as described for the normal preparations. The responses to acetylcholine (4 µg.mL-1) were recorded by a Gould SR 3400 physiograph before and 30 minutes after the addition of procainamide (20 µg.mL-1). Results were expressed as mean and standard deviation. The Student t test was used for the statistical analysis. A level of 5% (a = 5%) was considered significant. The test power was calculated and we obtained a b > 20% (power > 80%).
Procainamide at the concentration used in this study and applied to the rat phrenic nerve diaphragm preparation did not reduce the amplitude of the muscular responses to indirect electrical stimulation (Figure 1).
In the preparations exposed previously to procainamide, rocuronium produced a 68.5% ± 7.1% blockade, with a statistically significant difference (p = 0.0067) when compared to the blockade produced by rocuronium alone (10.4% ± 4.5%) (Figure 2). 4-aminopyrydine effectively reversed the blockade produced by rocuronium in the preparations exposed to procainamide (Figure 3).
In the chick Biventer cervicis preparation, procainamide (20 µg.mL-1) increased the contraction provoked by acetylcholine, which was not observed in the chronically denervated rat hemidiaphragm (Figures 4 and 5).
The effects on the miniature end plate potentials were characterized, initially, by an increase in frequency followed by a blockade 20 minutes after the addition of the drug. This effect was reversed by 4-aminopyridine (Figure 6).
Figures 1 and 3 show that procainamide alone does not have a direct effect on neuromuscular transmission, but it is capable of potentiating the blockade caused by rocuronium. These results confirm the results of prior studies demonstrating that procainamide can potentiate the effects of neuromuscular blockers, such as d-tubocurarine 11. Prolonged exposure to procainamide can reduce the number of receptors in the neuromuscular junction, reducing the safety margin of receptors in this region. This can probably explain the potentiation of the neuromuscular blockade caused by rocuronium 11.
4-aminopyridine, a plasma membrane K+ channel blocker that, in the presence of an action potential, increases the concentration of acetylcholine in the synaptic cleft, antagonized this blockade. The blockade of the K+ channels maintains the membrane depolarized for longer and, consequently, the voltage-dependent Ca++ channels are opened, increasing the amount of Ca++ inside the pre-synaptic terminal, which is directly proportional to the release of neurotransmitter. Initially, the increase in the amount of acetylcholine released induced by 4-aminopyridine, reversing the neuromuscular blockade caused by the interaction rocuronium-procainamide, allows us to infer that this is a competitive blockade.
These results are similar to those obtained by Lee et al. 6 Using a preparation of rat Digitorum longus muscle, they studied the effects of different concentrations of procainamide on miniature end plate potentials (MEPP), terminal plate potentials (TPP), and release of quanta of acetylcholine in the neuromuscular junction, elaborated three hypothesis regarding the actions of procainamide on the neuromuscular junction: 1) inhibits the synthesis or release of acetylcholine in the terminal neuron and reduced transmission of nerve impulses; 2) stabilizes membranes; 3) competes with acetylcholine for the post-synaptic nicotinic receptor.
The last hypothesis was confirmed by other authors 7-10, but goes against the results of this study, since procainamide did not reduce the amplitude of the muscular responses in the chronically denervated rat hemidiaphragm or in the chick Biventer cervicis preparation, demonstrating the lack of competition between procainamide and acetylcholine.
The chick Biventer cervicis muscle contracts in response to acetylcholine because the density of nicotinic receptors along the muscle fibers is much greater than that of mammalian muscles. The same can be observed in the denervated diaphragm, since the denervation process promotes the neoformation of nicotinic receptors along the muscle fiber, increasing considerably the sensitivity of the receptors to the neurotransmitter17. The difference between both preparations is that the denervated muscle lacks the pre-synaptic terminal of the neuromuscular junction and the events observed in this preparation reflect post-synaptic effects.
In this study, procainamide increased the contraction in response to acetylcholine in the chick Biventer cervicis preparation, which was not seen in the denervated diaphragm. This led to the assumption that procainamide facilitates the actions of acetylcholine on post-synaptic nicotinic receptors, most likely through a pre-synaptic mechanism, since the facilitation was observed only in preparations in which the innervation was preserved.
This facilitating action of acetylcholine on post-synaptic nicotinic receptors could also be evidenced by the influence of the drug on MEPP (Figure 6). Miniature end plate potentials presented a biphasic response to procainamide. In the first phase, there was an increase in MEPP frequency, followed by a blockade of those potentials (second phase) around 20 minutes after the addition of procainamide. This can be attributed to a peculiar characteristic of ionic type receptors, such as those present in the neuromuscular junction, that remain in a state of desensitization, i.e., refractory to new stimuli when they are depolarized for a prolonged time by their agonists 18,19.
The hypothesis of nicotinic receptor desensitization is reinforced by observing that 4-aminopyridine was effective in reversing the blockade of MEPP caused by procainamide 18-21. Although the reversal of the blockade with 4-aminopyridine can be explained by the increased amount of acetylcholine in the synaptic cleft when the action potential is initiated, at rest the amount of acetylcholine is not increased. In the absence of nerve stimulation, the blockade of K+ channels does not prolong membrane depolarization and, consequently, there is no Ca++ flow into the pre-synaptic terminal 18-20. Studies to investigate the mechanism by which 4-aminopyridine reverts the response of the receptors in the neuromuscular junction indicate that this drug antagonizes the desensitization of those receptors 18-21.
This study demonstrated that the interference of procainamide on the neuromuscular junction is based on the desensitization of nicotinic receptors and indicates that this desensitization is secondary to a pre-synaptic action of that drug, which increased the release of acetylcholine into the synaptic cleft. Besides, the concomitant use of procainamide and non-depolarizing neuromuscular blockers, such as rocuronium, or in situations that compromise the safety margin of the neuromuscular junction, can have clinical implications that demand monitoring of the neuromuscular transmission.
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Dra. Thalita Duque Martins
Rua Dr. Shigeo Mori, 589
13083-760 Campinas, SP
Submitted em 17
de março de 2006
Accepted para publicação em 8 de setembro de 2006
* Received from Departamento de Farmacologia da Faculdade de Ciências Médicas da Universidade Estadual de Campinas (FCM-UNICAMP), Campinas, SP