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Rev. Bras. Anestesiol. vol.56 no.2 Campinas Mar/Apr. 2006
Influence of nifedipine on the neuromuscular block produced by atracurium and cistracurium. Study in rat phrenic-diaphragmatic nerve preparation*
Influencia de la nifedipina en el bloqueo neuromuscular producido por atracurio y cisatracurio. Estudio en preparación nervio frénico diafragma de ratón
Silmara Rodrigues de Sousa, M.D.I; Angélica de Fátima de Assunção Braga, TSA, M.D.II; Glória Maria Braga Potério, TSA, M.D. II; Franklin Sarmento da Silva Braga, M.D.III; Yolanda Christina S Loyola, M.D.I; Samanta Cristina Antoniassi Fernandes, M.D.I
do Curso de Pós-Graduação do Departamento de Farmacologia
da FCM da UNICAMP
IIProfessora Associada do Departamento de Anestesiologia da FCM da UNICAMP
IIIProfessor Doutor do Departamento de Anestesiologia da FCM da UNICAMP
AND OBJECTIVES: Calcium channel blockers may interact with neuromuscular blockers,
increasing its effects. Research studies about this interaction display controversial
results. In some studies these drugs produced neuromuscular blockage, or contracture,
or no effect at all was proved over skeletal neuromuscular response. This study
assessed the nifedipine effects over muscular responses and its possible interaction
with neuromuscular blockers in rat diaphragm.
METHODS: A number of 25 rats were used, weighing between 250 and 300 g and sacrificed under anesthesia with intraperitoneal pentobarbital (40 mg.kg-1). Preparation was mounted according to the technique described by Bulbring. Diaphragm was kept under tension, connected to an isometric transducer and subjected to an indirect stimulation of 0.1 Hz frequency. Diaphragm contractions were registered on a physiograph. In order to evaluate the effect of these drugs on neuromuscular transmission, they were added separately or associated to the preparation, on the following concentrations: nifedipine (4 µg.mL-1); atracurium (20 µg.mL-1); cistracurium (3 µg.mL-1). On phrenic-nerve preparation, the assessed items were: 1) the extent of diaphragm muscle response to indirect stimulation, before and 45 minutes after adding nifedipine and neuromuscular blockers separately and after the association of both drugs; 2) nifedipine effects on membrane potentials (MP) and miniature end-plate potentials (MEPP).
RESULTS: Employed separately, nifedipine did not alter the extent of muscular responses, but it did significantly increase the neuromuscular blocking activity of atracurium and cistracurium. Nifedipine did not alter the membrane potential and caused an initial increase on MEPP frequencies, followed by a blockage.
CONCLUSIONS: Nifedipine, on the employed concentration, increased the neuromuscular blockage produced by atracurium and cistracurium. Electrophysiological studies demonstrate the existence of presynaptic action and absence of depolarizing action over the muscle fiber.
Key words: ANIMALS: rats; CALCIUM CHANNEL BLOCKERS: nifedipine; NEUROMUSCULAR BLOCKERS, Nondepolarizing: atracurium, cistracurium.
Y OBJETIVOS: Los bloqueadores de canales de calcio pueden reaccionar con los
bloqueadores neuromusculares potenciando sus efectos. Los estudios sobre esta
interacción presentan resultados controvertidos. En algunos estudios estas
drogas produjeron el bloqueo neuromuscular, o contractura, o no se observó
ningún efecto sobre las respuestas musculares esqueléticas. El estudio
evaluó los efectos de la nifedipina sobre la respuesta muscular y su posible
relación con los bloqueadores neuromusculares en el diafragma del ratón.
MÉTODO: Fueron utilizados 25 ratones, con peso entre 250 y 300 g sacrificadas con anestesia con pentobarbital (40 mg.kg-1) por vía intraperitoneal. La preparación fue montada de acuerdo con la técnica descripta por Bulbring. El diafragma fue mantenido bajo tensión, conectado con un transductor isométrico y sometido a estímulo indirecto de 0,1 Hz de frecuencia. Las contracciones del diafragma fueron registradas en un fisiógrafo. Para la evaluación de los efectos de las drogas en la transmisión neuromuscular, las mismas fueron añadidas aisladamente o asociadas a la preparación en las siguientes concentraciones: nifedipina (4 µg.mL-1); atracurio (20 µg.mL-1); cisatracurio (3 µg.mL-1 ). En las preparaciones nervio frénico-diafragma se evaluaron: 1) la amplitud de las respuestas del músculo diafragma al estímulo indirecto, antes y 45 minutos después de la adición de nifedipina y de los bloqueadores neuromusculares aisladamente y después de la asociación de las drogas; 2) los efectos de la nifedipina en los potenciales de la membrana (PM) y potenciales de la placa terminal en miniatura (PPTM).
RESULTADOS: La nifedipina, cuando empleada aisladamente, no cambió la amplitud de las respuestas musculares, pero aumentó significativamente la actividad bloqueadora neuromuscular del atracurio y del cisatracurio, no cambió el potencial de membrana y produjo el aumento inicial en la frecuencia de los PPTM, seguida de bloqueo.
CONCLUSIONES: La nifedipina, en la concentración empleada, potenció el bloqueo neuromuscular que el atracurio e cisatracurio produjeron. Estudios electrofisiológicos demostraron una acción presináptica y la ausencia de acción despolarizante sobre la fibra muscular.
Calcium channel blockers are widely used to treat cardiovascular diseases such as angina pectoris, cardiomyopathy, arterial hypertension, and supraventricular dysrhythmia. Although several studies have been performed in order to investigate the effects of calcium channel blockers on muscular respon-ses and their interaction with neuromuscular blockers (NMB), the results are still conflicting. Some studies showed that calcium channel blockers produce neuromuscular blockage 1-5, while others reported muscular contracture 6,7 or even no effect on skeletal musculature was found 8-10. It is less probable that, when administered on therapeutic doses, the Ca++ blockers may determine neuromuscular blockage. But when the safety margin of neuromuscular transmission is compromised by NMB use or by neuromuscular diseases, there may be an exacerbation of muscular paralysis 11. Therefore, research works point to the interaction between calcium channel blockers and neuromuscular blockers with the increase of neuromuscular blockers effects 2,4,8-10,12-14. Nifedipine is a calcium channel blocker which derives from dihydropyridine and it is available for oral use only. It also has a powerful vasodilative and coronary peripheral activity with a minimal effect on the capacitance vessels 15.
This study aimed at assessing the nifedipine effect on neuromuscular transmission and its influence on neuromuscular blockage produced by atracurium and cistracurium.
Both, experimental study and used procedures comply with the ethical principles about tests on animals adopted by the COBEA (Brazilian Association for Laboratory Animal Science) and were also approved by the Ethical Commission of Animal Experimentation from Biology Institute - UNICAMP (Universidade Estadual de Campinas).
The study used 25 male rats from Wistar lineage, weighing between 250 and 300 g, sacrificed under anesthesia with intraperitoneal pentobarbital (40 mg.kg-1), and bleeding by means of neck vessels section. Preparation was mounted according to the technique described by Bulbring 16. The hemidiaphragms with corresponding phrenic nerves were removed and placed on a vat containing 40 mL of Tyrode nourishing solution, composed of the following elements in mM: NaCl 137; KCl 2.7; CaCl2 1.8; NaHCO3 11.9; MgCl2 0.25; NaH2PO4 0.3, and glycose 11. This solution was constantly aerated with carbogen (95% O2 + 5% CO2) and kept at 37ºC (99ºF). The nerve was placed onto platinum electrodes connected to a S48 Grass stimulator. Diaphragm was maintained by its tendinous portion under constant tension (5.0 g), through a wire connected to a Load Cell BG50 GMS isometric transducer, and subjected to an indirect stimulation of 0.1 Hz frequency and lasting 0.2 msec. Tension variations produced by diaphragm contractions were registered on a Gould RS 3400 physiograph. Three groups were formed (n = 5) in order to evaluate the effects of the drugs on neuromuscular transmission when employed separately: nifedipine (4 µg.mL-1); atracurium (20 µg.mL-1); cistracurium (3 µg.mL-1). Two other groups (n = 5) study the effects of associating nifedipine with both neuromuscular blockers (atracurium and cistracurium) over the extent of muscular responses. Muscular responses to indirect stimulation were registered during 45 minutes after adding the drugs. Rat phrenic-nerve preparation (FND) was also used to study the effects of nifedipine on miniature end-plate potentials and on membrane potentials. The following items were assessed: 1) the extent of diaphragm muscle response to indirect stimulation, both before and 45 minutes after adding the nifedipine and neuromuscular blockers separately; 2) the extent of diaphragm muscle response to indirect stimulation, both before and 45 minutes after adding the association between nifedipine and the neuromuscular blocker; 3) nifedipine effects over membrane potentials (MP) and miniature end-plate potentials (MEPP). The results were expressed on mean and standard deviations. For a statistical analysis, the Student t and Student t for paired samples tests were used. A significant level of 5% (a = 5%) was assumed. The power of test was calculated and a value of b > 20% was found (power > 80%).
Nifedipine, on the studied concentration and separately employed on FND specimens, did not cause extent reduction on the muscular responses to indirect electric stimulation (Figure 1).
On preparation treated with nifedipine, the neuromuscular blockage produced by atracurium was 64.19% ± 9.37%, being significantly higher (p = 0.0053) then when atracurium was separately employed (45.02% ± 6.33%) (Figure 2). Blockage produced by the association between nifedipine and cistracurium was 74.04% ± 10.12%, significantly higher (p = 0.0038) than the one observed when cistracurium was employed separately (48.01 ± 10.33%) (Figure 3).
It was not noted any significant effect of nifedipine over membrane potentials. The effects over miniature end-plate potentials initially feature a frequency increase, noted 30 minutes after adding the drug, followed by a blockage at 60 minutes (Figure 4).
There are evidences that calcium channel blockers may interact and increase the effects of neuromuscular blockers commonly used on general anesthesia 4,9,12,17-20. These effects were experimentally proved in isolated preparation, on intact animal and they were also confirmed at the clinical practice, on a patient with Duchenne muscular dystrophy who developed acute respiratory failure after the administration of verapamil to treat atrial flutter 2-4,8-14,17,18,21, as well as a higher difficulty to antagonize neuromuscular blockage on a patient chronically treated with verapamil. Those intercurrences are attributed to the neuromuscular blockage property of verapamil 22. The use of a separated nerve muscle preparation (in vitro), properly aerated and kept at a constant temperature make it possible to evaluate the nifedipine and neuromuscular effects over muscular responses to indirect stimulation, excluding any other factors that may interfere on neuromuscular transmission. Besides, experiments performed on rats are considered more appropriate to investigate postsynaptic events when compared to the tests performed on dogs 9.
Although the effects of calcium channel blockers have been extensively investigated on cardiac, vascular smooth, respiratory, and intestinal muscles, there are only a few studies about the action of these drugs on neuromuscular activity. The skeleton muscle contains calcium channels of the slow type, similar to the ones observed on cardiac and vascular smooth muscles, with special connection sites for calcium channel blockers. On vascular smooth and cardiac muscles, those channels are located all over the muscular membrane and display a high sensitivity to calcium channel blockers, while on skeleton muscle they present lesser sensitivity and are located on the transverse tubular system 23. Due to these particularities, some physiologic processes on neuromuscular junction or at the muscle may be altered by these drugs.
The choice of neuromuscular blockers dose was established on a pilot project and then adjusted until obtaining a neuromuscular blockage which progressively installs itself during 45 minutes. Nifedipine concentration used on this study was 4 µg.mL-1, determined from the data presented on studies by BikhazI et al 9. The separate use of nifedipine did not produce any compromising of neuromuscular transmission, this result being similar to the ones observed on other research works 1,9. These authors reported on experiments performed in vitro and in vivo that nifedipine and verapamil produced minimal or no change on the muscular responses to isolated stimuli. However, these results are opposed to the ones obtained by Del Pozo and Baeyens 5, who studied on rat's phrenic-nerve diaphragm preparation the effects of several nifedipine, verapamil and diltiazem concentrations and observed a concentration-dependant decrease of the diaphragm muscle answer to indirect stimulation. Similar results were also proved on other experimental studies using other calcium channel blockers 2-4,17.
On the likeness of prior studies, nifedipine increase the neuromuscular blockage produced by atracurium and by cistracurium. Verapamil or nifedipine concentrations (in vitro) or doses (in vivo) that separately presented little or no effect on muscular response increase the power of atracurium, vecuronium and pancuronium, fact proved by a DE50 decrease of these drugs. This is considered a more than additive effect 1,9.
Although the reasons for this interaction and increase of neuromuscular blockage are not fully elucidated, several mechanisms have been proposed in order to explain it. Some research works performed in vivo and in vitro suggest that calcium channel blockers prevent the calcium inflow through the membranes of calcium slow channels, altering calcium and presynaptic cyclic adenosine monophosphate (cAMP) concentrations. This harms the neurotransmitter mobilization and liberation, consequently inhibiting neuromuscular transmission and muscular contraction 3,4,14,17-19,24-27. These drugs also display a local anesthetic activity that may contribute to generate a depressor effect on muscular contraction, probable due to its action on the sodium fast channel 15,17,21. It has been reported on literature that verapamil reduces the acetylcholine release on motor nerve ending, suggesting a presynaptic action 4,21,25. In other works, several explanations have been proposed for the direct effects of verapamil on skeletal muscle. Acting at calcium channels on the skeleton muscle, this drug prevents calcium entrance on muscular cells, impairing the activation mechanism and consequently the exciting-contraction pro-cess 13,28,29. Alternatively, Chiarandini and Bentley 30 reported that verapamil, on toad preparation, blocks the muscular response induced by acetylcholine and suggests that, on a similar way to nondepolarizing neuromuscular blockers, it may have an effect on the ionic channels activated by acetylcholine on the end plate, reflecting a postjunctional effect 1,30. On the other hand, results about the inhibitory effects of verapamil on muscular response to indirect stimulation are higher than the ones obtained with direct stimuli. It may mean that the calcium channel blocker also act directly at the nerve 3,19.
When assessing bioelectrical potentials, it was noted that nifedipine, on the employed concentration, does not alter the membrane potential of muscular fibers; therefore demonstrating that it has no depolarizing action over the skeleton muscular fiber. However, it was noted an influence on the miniature end-plate potentials (mepps), which may be attributed to a possible presynaptic action.
At some moment, anesthesiologists may come across this interaction when performing anesthesia on patients with chronic use of calcium channel blockers or even when there is a need to use these drugs on the treatment of cardiocirculatory complications during surgery. Results of in vitro studies with the association between calcium channel blockers and neuromuscular blockers suggest that the interaction between these two groups of drugs may differ according to the usage form of the calcium channel blocker. On patients with chronic use of calcium channel antagonist, these drugs may accumulate on the muscles and a great part of the blockage may be attributed to the calcium antagonist. Its administration during anesthesia may temporarily increase the degree of neuromuscular blockage, however with no increase in the action duration 9. Results show that nifedipine, on the employed concentration, increases the neuromuscular blockage produced by atracurium and cistracurium. Alterations of the miniature end-plate potentials exteriorize a presynaptic action and the absence of effect on membrane potential proves that it has no depolarizing action on the muscular fiber.
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Dra. Angélica de Fátima de Assunção Braga
Rua Luciano Venere Decourt, 245 - Cidade Universitária
13084-040 Campinas, SP
for publication 26 de setembro de 2005
Accepted for publication 09 de dezembro de 2005
* Received from Departamento de Farmacologia da Faculdade de Ciências Médicas da Universidade de Campinas (FCM UNICAMP), Campinas, SP