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Print version ISSN 0034-7094
Rev. Bras. Anestesiol. vol.55 no.5 Campinas Sept./Oct. 2005
Effect of ropivacaine on neuronal norepinephrine reuptake in smooth muscle*
Efecto de la ropivacaína en la recaptación neuronal de noradrenalina en un músculo liso
Carlos Alberto de Souza Martins, TSA, M.D.I; Pedro Wanderley de Aragão, M.D.II; Sonia Maria de Farias Freire, M.D.III; Mahiba Mattar Rahbani de Souza Martins, M.D.IV; Marilene Oliveira da Rocha Borges, M.D.V; Antonio Carlos Romão Borges, M.D.VI
IResponsável pelo Serviço
de Anestesiologia da Clínica São Marcos; Professor Adjunto da Disciplina
de Farmacologia da UFMA. Mestre em Ciências da Saúde pela UFMA
IIProfessor Adjunto da Disciplina de Farmacologia da UFMA; Coordenador do Centro de Estudos da Clínica São Marcos; Mestre em Ciências da Saúde pela UFMA
IIIProfessora Adjunta da Disciplina de Farmacologia e Diretora do Biotério Central da UFMA; Mestre em Farmacologia pela Universidade Federal de São Paulo, UNIFESP
IVProfessora Adjunta da Disciplina de Farmacologia da UFMA; Mestre em Ciências da Saúde pela UFMA
VProfessora Adjunta da Disciplina de Farmacologia e Coordenadora do Curso de Mestrado em Ciências da Saúde da UFMA; Doutora em Farmacologia pela UNIFESP
VIProfessor Adjunto da Disciplina de Farmacologia da UFMA; Coordenador do Programa de Monitoração de Propaganda e Publicidade de Produtos sob Vigilância Sanitária no Maranhão
BACKGROUND AND OBJECTIVES: In addition
to local anesthetic action, ropivacaine has clinically significant vasoconstrictor
effects, which may be observed at infiltrative anesthesia, making it an important
anesthetic for field blockade. This study aimed at characterizing the constrictor
mechanism of ropivacaine on smooth muscles.
METHODS: Norepinephrine concentration-effect curves in the absence or presence of ropivacaine were plotted on isolated preparations of vas deferens of rats. In another series of experiments rats were treated with reserpine (10 mg.kg-1, i.p.) to evaluate vas deferens reactivity to tyramine or norepinephrine, in the absence or presence of ropivacaine.
RESULTS: Ropivacaine 5 or 10 µg.mL-1 potentiated maximum norepinephrine effect (Emax) in 47% and 35%, respectively, while higher concentrations (50 or 100 µg.mL-1) inhibited its maximum effect. In isolated vas deferens of rats treated with reserpine, ropivacaine (10 or 20 µg.mL-1) potentiated (150% and 25%, respectively) norepinephrine-induced contractions, while higher concentrations (50 or 100 µg.mL-1) have not changed responses to norepinephrine.
CONCLUSIONS: Ropivacaine blocks neuronal norepinephrine reuptake by sympathetic nerve terminals.
Key Words: ANESTHESIA, Experimental; ANESTHETICS, Local: ropivacaine; AUTONOMOUS NERVOUS SYSTEM, Simpatico
JUSTIFICATIVA Y OBJETIVOS: Además
de la acción anestésica local, la ropivacaína presenta un efecto
vasoconstrictor, clínicamente significativo y puede ser observado cuando
de la anestesia infiltrativa, con esto lo hace un anestésico importante
en el bloqueo del campo. Este trabajo tuvo por objetivo caracterizar el mecanismo
de acción constrictor de la ropivacaína en músculo liso.
MÉTODO: En preparaciones separadas del conducto deferente de ratones fueron construidas curvas concentración-efecto de noradrenalina en la ausencia o en la presencia de la ropivacaína. En otra serie de experimentos los ratones fueron tratados con reserpina (10 mg.kg-1, por la via intraperitoneal) para evaluar la reactividad de los conductos deferentes a la tiramina o noradrenalina, en la ausencia o presencia de la ropivacaína.
RESULTADOS: La ropivacaína en las concentraciones de 5 ó 10 µg.mL-1 potenció el efecto máximo (Emax) de la noradrenalina en un 47% y 35%, respectivamente, mientras que en las concentraciones de 50 ó 100 µg.mL-1 inhibió el efecto máximo producido por este agonista. En conductos deferentes separados de ratones reserpinizados, la ropivacaína (10 ó 20 µg.mL-1) potenció (150% y 25%, respectivamente) las contracciones inducidas por la noradrenalina, mientras que las concentraciones de 50 ó 100 µg.mL-1 no alteraron las respuestas a la noradrenalina.
CONCLUSIONES: Los resultados logrados permiten concluir que la ropivacaína bloquea la recaptación neuronal de noradrenalina por los terminales nerviosos simpáticos.
Ropivacaine (1-propyl-2'-6 pipecoloxylidide hydrochloride) is a long-lasting local anesthetic chemically homologous to mepivacaine and bupivacaine. Pre-clinical trials have shown that ropivacaine is less toxic for central nervous and cardiovascular systems as compared to bupivacaine 1, which is the most widely used drug for anesthetic blocks as a function of its prolonged action.
In clinical trials, healthy volunteers submitted to intravenous ropivacaine have shown less intense reactions on these two systems 2,3, which is extraordinarily interesting due to the always present possibility of high concentrations reaching the blood as a consequence of inadvertent intravascular injection. Although ropivacaine exhibits sensory nerve blocking properties similar to those observed with bupivacaine 4, the same is not true with regard to motor block because there is a well shown sensory-motor separation with ropivacaine 5,6. Another property of ropivacaine is its clinically significant vasoconstrictor action 7, observed during infiltrative anesthesia, which makes it attractive for field blockade 8.
The association of epinephrine to ropivacaine is not a major factor for decreasing absorption from injection sites or for its prolonged effect 9, as occurs to other local anesthetics, except cocaine. Pre-clinical trials have shown that high ropivacaine concentrations promote spinal cord vasoconstriction in rats, which is reversible within 20 minutes 10. This vasoconstrictor action of ropivacaine and cocaine is not shared by other local anesthetics, such as bupivacaine, in spite of their chemical similarities. Conversely, bupivacaine induces vascular relaxation after contraction by chemical or electrical stimulation. This muscle relaxing effect is due to depression of adrenergic neurotransmission as well as to inhibition of vascular smooth muscle activity 11.
Considering that local anesthetic effect is a consequence of action on sodium channels and that these have little influence in vascular smooth muscle contraction, it is feasible to assume that ropivacaine-induced vasoconstriction is promoted by interference with sympathetic autonomous nervous system, similarly to cocaine, which is an alkaloid obtained from eritroxylon coca and is characterized by local anesthetic action and ability to block neuronal norepinephrine reuptake 12. This study aimed at characterizing mechanisms of the constrictor action of ropivacaine on smooth muscles.
This study was performed with Wistar rats of the species Rattus norvegicus and variety albino (200-220 g, 60 days of age), supplied by the Lab Animals Facility, Universidade Federal, Maranhão. Animals were adapted to the pharmacology laboratory for 15 days before starting the experiment and were kept under controlled conditions. Animals received free water and food during the study period. All procedures and protocols of this trial were submitted to and approved by the Research Ethics Committee, Hospital Universitário Presidente Dutra, UFMA.
Rats were anesthetized with sulfuric ether and sacrificed by cervical vessels section. Vasa deferentia were dissected and their lumen washed with vesicle nutritional fluid (VNF) with the following composition in mM: NaCl = 138; KCl = 5.7; NaH2PO4 = 0.4; NaHCO3 = 15; Glucose = 5.5; CaCl2 = 1.8. Preparations were assembled in isolated organ contraction chamber with aerated VNF at 30 ºC and adapted to a recording system preloaded with 1 g tension. After system stabilization (30 min), norepinephrine concentration-effect curves were plotted in the absence or presence of ropivacaine or cocaine. Concentration-effect curves were also plotted from rats treated with intraperitoneal reserpine (10 mg.kg-1) and sacrificed 24 hours later to evaluate vas reactivity to tyramine and norepinephrine in the absence or presence of ropivacaine.
Percentage of contraction as a function of maximum norepinephrine induced contraction in control curves was related to negative logarithms of agonist molar concentrations. EC50 (effective 50% concentration), pD2 (EC50 negative logarithm) and Emax (maximum effect) produced by norepinephrine in the absence or presence of ropivacaine or cocaine were determined from obtained concentration-effect curves. Values of pD2 and Emax were expressed as mean ± standard error (S.E.M.) and differences between norepinephrine effects in the absence or presence of ropivacaine or cocaine were determined by Student's t test for a significance level of 5% (p < 0.05) 15.
Successive norepinephrine addition has produced concentration-dependent contractions, reaching Emax with 0.1 mM and generating pD2 = 5.2 ± 0.03. Incubation in ropivacaine (5, 25 or 100 µg.mL-1) caused concentration-independent low amplitude contraction. In addition, it did not significantly change a1-adrenoreceptor system sensitivity (pD2) to norepinephrine in vas deferens of rats (Figure 1 and Table I).
On the other hand, 5 or 25 µg.mL-1 ropivacaine concentrations potentiated norepinephrine Emax, while 50 or 100 µg.mL-1 inhibited Emax induced by this agonist. Cocaine (1 µM) also potentiated norepinephrine Emax without changing pD2 value (Figure 1).
In vasa of rats treated with reserpine, 5 and 25 µg.mL-1 ropivacaine potentiated norepinephrine Emax (submaximum concentration, 3 µM) in 116% and 75%, respectively, while cocaine potentiated it in 57.6%. In concentrations of 50 and 100 µg.mL-1, ropivacaine did not change norepinephrine-induced Emax (Figure 2). The addition of tyramine (30 µM) after norepinephrine did not change basal tone of isolated vasa of rats treated with reserpine (Figure 2).
Our results have shown that ropivacaine modulates rat vas deferens reactivity to norepinephrine in a two-phase manner. Ropivacaine in 5 and 25 µg.mL-1 concentrations promoted potentiation, while in 50 to 100 g.mL-1 concentrations decreased maximum effect of concentration curves induced by norepinephrine in vas deferens of rats.
It is currently well-established that norepinephrine-induced contraction in vas deferens of rats involves the activation of a1-adrenoreceptors bound to phospholipase C activating G protein, which promotes hydrolysis of phosphatidinositol originating the second-messengers inositol triphosphate (IP3) and diacylglycerol (DAG), which mobilize intracellular calcium for muscle contraction 16,17.
Similarly to low bupivacaine doses, 1 µM cocaine has also potentiated maximum effect of norepinephrine-induced contraction curves, which agrees with the literature and shows that cocaine inhibits neuronal norepinephrine reuptake system in peripheral tissue 12,18,19.
Our results suggest that low ropivacaine concentrations have potentiated vas deferens reactivity to norepinephrine, characterized by increased Emax of this agonist, probably due to neuronal amine reuptake system inhibition, which would result in greater extracellular availability of this catecholamine and, as a consequence, in activation of a1-adrenoreceptos for a longer time. On the other hand, high ropivacaine concentrations have induced decreased vas reactivity to norepinephrine, characterized by decreased Emax of this agonist in preparations without reserpine, probably due to its direct effect depressing smooth muscle and/or membrane stabilizing effect as a consequence of calcium channels block, since smooth muscle tonic contraction is dependent on external calcium 17,20.
In terms of chemical structure, although there is no correlation between ropivacaine and cocaine planar structures and in spite the fact that cocaine is an ester and ropivacaine an amide, both have shown the same response in our experimental model.
Results obtained in our experimental model suggest that, similarly to cocaine, ropivacaine also blocks neuronal norepinephrine reuptake by sympathetic nervous terminals.
We acknowledge Universidade Federal do Maranhão, Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq and Astrazeneca for supplying ropivacaine.
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Submitted for publica January 26, 2005
Accepted for publication June 10, 2005
* Received from Laboratório de Farmacologia do Departamento de Ciências Fisiológicas da Universidade Federal do Maranhão, UFMA. São Luís, MA