Levobupivacaine induces vasodilatation, but not vasoconstriction, in rat mesenteric artery

Levobupivacaína induz vasodilatação, mas não vasoconstrição em artéria mesentérica de rato

Liciane dos Santos MENEZES Liane Maciel de Almeida SOUZA Márcio Roberto Viana dos SANTOS Patrícia Santos Cunha MENDONÇA Ítalo José Alves MOREIRA Allan Carlos Araújo de OLIVEIRA About the authors

Abstract

Introduction

Levobupivacaine (LEVO) can replace analgesia because it exhibits low toxicity and causes minor vasoconstriction, enabling its use in patients in whom vasoconstrictors are contraindicated.

Objective

We aimed to evaluate the effects of LEVO in isolated rat superior mesenteric artery by using the vascular reactivity technique and compare its effect to that of lidocaine.

Material and method

Arterial rings were obtained from the mesenteric artery of male Wistar rats and kept in organ baths. For recording isometric contractions, each ring was suspended by cotton threads from a force transducer, which was connected to a data acquisition system.

Result

Both lidocaine and LEVO did not show a vasoconstrictor effect on the basal tone of the arterial rings with functional endothelium. However, when the rings were pre-contracted with phenylephrine, both drugs were able to induce concentration-dependent vasodilatation. The vasodilator effect induced by LEVO did not change after removal of the endothelium, or with the addition of tetraethylammonium (1 mM), a non-selective K+ channel blocker. In the rings without functional endothelium, which were pre-contracted with depolarizing Tyrode’s solution (KCl 80 mM), LEVO-induced vasodilatation was not significantly different from that observed in the rings pre-contracted with phenylephrine. Moreover, it did not show a significant additional vasodilator effect compared to the maximal vasodilator effect of nifedipine.

Conclusion

This study demonstrated that LEVO produces a vasodilator effect in the rat superior mesenteric artery in an endothelium-independent manner. This effect seems to be mediated via Ca2+ channel blockade in the vascular smooth muscle cells.

Descritores:
Lidocaína; artéria mesentérica; vasodilatação

Resumo

Introdução

Levobupivacaína pode ser uma nova alternativa para analgesia por apresentar baixa toxicidade e vasoconstrição, permitindo sua utilização em pacientes que apresentam contra-indicação no uso de vasoconstritores.

Objetivo

Avaliar os efeitos da levobupivacaína utilizando a técnica de reatividade vascular em artéria mesentérica isolada de rato e comparar este efeito à lidocaína.

Material e método

Anéis foram obtidos da artéria mesentérica de ratos machos Wistar e foram mantidos em cubas. Para o registro das contrações isométricas, cada anel foi suspenso por linhas de algodão fixadas a um transdutor de força acoplado a um sistema de aquisição.

Resultado

Tanto a lidocaína como a levobupivacaína não apresentaram efeito vasocontritor sobre o tônus basal em anéis com endotélio functional. No entanto, quando os anéis foram pré-contraídos com fenilefrina, ambas as drogas induziram um vasorrelaxamento concentração-dependente. O efeito vasorrelaxante causado pela levobupivacaína não foi diferente após a remoção do endotélio, ou com o tetraetilamônio (1mM), um bloqueador não seletivo dos canais para. K+. Em anéis sem endotélio funcional e pré-contraídos com solução despolarizante de Tyrode (KCl 80mM), o vasorelaxamento induzido pela levobupivacaína não foi significativamente diferente daquele observado em anéis pré-contraídos com fenilefrina e não apresentou um efeito adicional significativo sobre o relaxamento máximo da nifedipina.

Conclusão

Este estudo demonstrou que a levobupivacaína produz efeito vasorrelaxante em artéria mesentérica de rato, que é endotélio independente. Este efeito parece envolver os bloqueadores de canais para Ca2+ em célula muscular vascular lisa.

Descritores:
Lidocaína; artéria mesentérica; vasodilatação

INTRODUCTION

Advances in operation techniques and concerns about pain control have supported and sustained several pharmacological and clinical studies on local anesthetics11 Carvalho RWF, Pereira CU, Anjos ED, Laureano JR Fo, Vasconcelos BCE. Anestésicos locais: como escolher e prevenir complicações sistêmicas. Rev Port Estomatol Med Dent Cir Maxilofac. 2010 Abr-Jun;51(2):113-20.. Local anesthetics are widely used in dentistry to minimize pain, in order to provide a safe and effective dental treatment22 Vasconcelos RJH, Nogueira RVB, Leal AKR, Oliveira CTV, Bezerra JGB. Alterações sistêmicas decorrentes do uso da lidocaína e prilocaína na prática odontológica. Rev Cir Traumat Buco-Maxilo-Facial. 2002 Jan-Jun;1(2):13-9..

Local anesthetics are classified according to their chemical structure into ester and amide-types, the latter being used more frequently in clinical settings. Amide-type anesthetics include prilocaine, lidocaine, mepivacaine, articaine, ropivacaine, and bupivacaine (BUPI)33 Ramacciato JC, Motta RHL, Groppo FC, Volpato MC, Ranali J. Anestésicos locais. Rev Assoc Paul Cir Dent. 2007;61(4):486-7.. BUPI is a long-acting anesthetic, which can be used in long surgeries without the need for anesthetic complementation. However, it exhibits a certain degree of cardiotoxicity, caused by the dextrorotatory enantiomer in the racemic mixture (S50-R50), which encouraged researchers to develop an anesthetic with a lower toxic potential44 Albright GA. Cardiac arrest following regional anesthesia with etidocaine or bupivacaine. Anesthesiology. 1979 Oct;51(4):285-7. http://dx.doi.org/10.1097/00000542-197910000-00001. PMid:484889.
http://dx.doi.org/10.1097/00000542-19791...
,55 Ohmura S, Kawada M, Ohta T, Yamamoto K, Kobayashi T. Systemic toxicity and resuscitation in bupivacaine-, levobupivacaine-, or ropivacaine-infused rats. Anesth Analg. 2001 Sep;93(3):743-8. http://dx.doi.org/10.1097/00000539-200109000-00039. PMid:11524350.
http://dx.doi.org/10.1097/00000539-20010...
.

Thus, levobupivacaine (LEVO), a local anesthetic with properties similar to those of BUPI but with less central and cardiac toxicity, was introduced. This difference is due to its predominant levorotatory enantiomer (S75-R25)55 Ohmura S, Kawada M, Ohta T, Yamamoto K, Kobayashi T. Systemic toxicity and resuscitation in bupivacaine-, levobupivacaine-, or ropivacaine-infused rats. Anesth Analg. 2001 Sep;93(3):743-8. http://dx.doi.org/10.1097/00000539-200109000-00039. PMid:11524350.
http://dx.doi.org/10.1097/00000539-20010...

6 Fawcett JP, Kennedy JM, Kumar A, Ledger R, Kumara GM, Patel MJ, et al. Comparative efficacy and pharmacokinetics of racemic bupivacaine and S-bupivacaine in third molar surgery. J Pharm Pharm Sci. 2002 May-Aug;5(2):199-204. PMid:12207874.
-77 Bajwa SJ, Kaur J. Clinical profile of levobupivacaine in regional anesthesia: a systematic review. J Anaesthesiol Clin Pharmacol. 2013 Oct;29(4):530-9. http://dx.doi.org/10.4103/0970-9185.119172. PMid:24249993.
http://dx.doi.org/10.4103/0970-9185.1191...
.

LEVO is considered as a new alternative to analgesia, which is associated with low toxicity when applied in the dental area and with minor vasoconstriction88 Choi YS, Jeong YS, Ok SH, Shin IW, Lee SH, Park JY, et al. The direct effect of levobupivacaine in isolated rat aorta involves lipoxygenase pathway activation and endothelial nitric oxide release. Anesth Analg. 2010 Feb;110(2):341-9. http://dx.doi.org/10.1213/ANE.0b013e3181c76f52. PMid:19955508.
http://dx.doi.org/10.1213/ANE.0b013e3181...

9 Mukozawa M, Takakura K, Mizogami M. Direct vasocontractile activities of bupivacaine enantiomers on the isolated rat thoracic aorta. Anesthesiol Res Pract. 2010;2010:820186. http://dx.doi.org/10.1155/2010/820186. PMid:20981258.
http://dx.doi.org/10.1155/2010/820186...

10 Baik JS, Sohn JT, Ok SH, Kim JG, Sung HJ, Park SS, et al. Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent. Can J Physiol Pharmacol. 2011 Jul;89(7):467-76. http://dx.doi.org/10.1139/y11-046. PMid:21812525.
http://dx.doi.org/10.1139/y11-046...

11 Kiliçaslan A, Duman A, Sahin AS. In vitro vasoactive effects of levobupivacaine and ropivacaine on the isolated human umbilical artery and vein. Balkan Med J. 2011;28(2):164-8.
-1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7.. Thus, it is a satisfactory option for subjects in whom the use of vasoconstrictors is contraindicated1313 Barros EG, Marquez IM, Zanetta-Barbosa D. Avaliação comparativa da latência e da duração do cloridrato de levobupivacaína 0, 5 por cento sem e com vasoconstrictor em anestesia terminal infiltrativa. Rev Odontol UNESP. 2006 Abr-Jun;35(2):165-70..

The present study aimed to evaluate the possible vasoconstrictor effect of LEVO in isolated rat superior mesenteric artery using the technique of vascular reactivity. The superior mesenteric artery is a resistant artery, which can reflect some possible effects on the vascular reactivity in smaller blood vessels such as those present in the area treated by local anesthetics in dentistry. We also compared the effect of LEVO to that of lidocaine (LIDO).

MATERIAL AND METHOD

Animals

Thirteen male Wistar normotensive rats (250-300 g) were obtained from the Department of Physiology in the Federal University of Sergipe, Sergipe, Brazil. They were maintained in a large cage under controlled conditions of temperature and light (lights on: 06:00-18:00), and were fed with rodent diet and tap water ad libitum. All procedures were approved by the Animal Research Ethics Committee of the Federal University of Sergipe (Protocol number 27/2012) and were carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996).

Drugs

LEVO in enantiomeric excess 50% and lidocaine (LIDO) were purchased from Cristália and L-phenylephrine chloride (Phe), acetylcholine chloride (Ach), nifedipine (NIF), and tetraethylammonium (TEA) were purchased from Sigma-Aldrich.

Tissue Preparation

Rats were sacrificed by exsanguination under anesthesia and the superior mesenteric artery was removed, cleaned from the connective and fat tissues, and sectioned in rings (1-2 mm). These rings were suspended in organ baths containing 10 mL of Tyrode’s solution, gassed with carbogen, and maintained at 37 °C under a resting tension of 0.75 g for 60 min (stabilization period). The isometric tension was recorded by a force transducer (Letica, Model TRI210, Barcelona, Spain) coupled to an amplifier-recorder (AECAD 0804, AVS, São Paulo – SP, Brasil). When necessary, the endothelium was removed with a fine steel wire and its functionality was assessed based on the ability of Ach (1 µM) to reduce more than 75% of the tone pre-induced by Phe (1 µM). The absence of Ach-induced relaxation was considered as evidence that the endothelium of the arterial rings became non-functional.

Characterization of the Effect of LIDO and LEVO on the Basal Tone of the Arterial Rings

After confirming the presence of functional endothelium and the complete recovery of the basal tone, LIDO at concentrations of 3 x 10–7-3 x 10–4 M (n = 4) and LEVO at concentrations of 3 x 10–7-3 x 10–4 M (n = 9) were cumulatively and separately added to the bath in order to construct a control concentration-response curve.

Characterization of the Effect of LIDO and LEVO on the Pre-contracted Arterial Rings

After verifying the presence of functional endothelium, intact arterial rings were pre-contracted again with Phe (1 µM). Then, during the tonic phase of contraction, LIDO at concentrations of 3 x 10–7-3 x 10–4 M (n = 5) or LEVO at concentrations of 3 x 10–7-3 x 10–4 M (n = 6) was cumulatively added to the bath to construct a concentration-response curve.

Assessment of the Role of the Vascular Endothelium in the Responses Induced by LEVO

After verifying the absence of functional endothelium, the rings were pre-contracted with Phe (1 µM) and during tonic phase of contraction, increasing concentrations of LEVO (3 x 10–7-3 x 10–4 M; n = 4) were cumulatively added to the bath. The concentration-response curve obtained from this experiment was compared with that obtained from the experiment conducted on arterial rings with functional endothelium.

Assessment of the Role of Ca2+ in the Responses Induced by LEVO

The possible effect of LEVO on Ca2+ channels was investigated using the concentration-response curve for LEVO (3 x 10–7-3 x 10–4 M; n = 4) in arterial rings without endothelium in the presence of a high concentration of potassium. In this protocol, the normal Tyrode’s solution was replaced by a K+ depolarizing Tyrode’s solution containing 80 mM of KCl and the tissues were immersed in this solution until the end of the experiment.

The involvement of dihydropyridine-sensitive voltage-operated calcium channels (Cavs) was assessed via testing the response of the rings without endothelium, pre-contracted with Phe (1 μM) to the anesthetic (3 x 10–4 M; n = 6) in the absence or presence of NIF (10 μM), a selective blocker of the dihydropyridine-sensitive Cavs1414 Hagiwara S, Mitsui M, Karaki H. Effects of felodipine, nifedipine and verapamil on cytosolic Ca and contraction in vascular smooth muscle. 2+Eur J Pharmacol. 1993 Mar;234(1):1-7. http://dx.doi.org/10.1016/0014-2999(93)90698-H. PMid:7682512.
http://dx.doi.org/10.1016/0014-2999(93)9...
.

Assessment of the Contribution of K+ Channels to the Responses Induced by LEVO

The possible effect of LEVO on K+ channels was investigated using the concentration-response curve for LEVO (3 x 10–7-3 x 10–4 M; n = 7) in arterial rings without endothelium incubated with 1 mM of TEA for 30 min, which is a non-selective K+ channel blocker at this concentration1515 Cook NS. Effect of some potassium channel blockers on contractile responses of the rabbit aorta. J Cardiovasc Pharmacol. 1989 Feb;13(2):299-306. http://dx.doi.org/10.1097/00005344-198902000-00019. PMid:2468961.
http://dx.doi.org/10.1097/00005344-19890...
. The concentration-response curve for this experimental condition was compared with that obtained in the absence of the blocker.

Statistical Analysis

Values were expressed as mean ± S.E.M. The results were analyzed with one or two-way ANOVA followed by Bonferroni post-hoc test. All analyses were performed using GraphPad Prism™ 5.0. A value of p<0.05 was considered significant.

RESULT

As shown in Figure 1A, both LIDO and LEVO were unable to induce vasoconstriction of the blood vessel during basal tone. However, in rings with functional endothelium, which was pre-contracted with phenylephrine, both LIDO and LEVO were capable of inducing concentration-dependent relaxation (Emax = 74.5 ± 11%; n = 5 and 91.13 ± 9.8%; n = 6, respectively) (Figures 1B and 2).

Figure 1
Original registration showing the effect of LIDO and LEVO in isolated arterial rings of the rat superior mesenteric artery on the basal tone (A) and in isolated arterial rings of the rat superior mesenteric artery pre-contracted with 1 µM of Phe (B).
Figure 2
Concentration-response curves for LIDO (3 x 10–7-3 x 10–4 M; n = 9) and LEVO (3 x 10–7-3 x 10–4 M; n = 4) on the basal tone of isolated rings of the rat superior mesenteric artery with functional endothelium (A) and in isolated rings of the rat superior mesenteric artery with functional endothelium, pre-contracted with 1 µM Phe (B).Values are expressed as mean ± S.E.M. The data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test.

The concentration-dependent relaxation induced by LEVO in arterial rings without functional endothelium, which were pre-contracted with Phe (1 µM), was not significantly different from that obtained in rings with functional endothelium (Emax = 91.13 ± 9.8%; n = 6 and 90.4 ± 2.8%; n = 4, respectively) (Figure 3A).

Figure 3
Concentration-response curves for LEVO (3 x 10–7-3 x 10–5 M) in isolated rings of the rat superior mesenteric artery with (n = 6) and without (n = 4) functional endothelium, pre-contracted with 1 µM Phe (A), and in isolated rings of the rat superior mesenteric artery without functional endothelium after TEA (1 mM) (n = 7) (B). Values are expressed as mean ±S.E.M. The data were analyzed with two-way ANOVA followed by Bonferroni post-hoc test. *p < 0.05 and *** p < 0.001 vs. rings without the endothelium pre-contracted with Phe.

The possible involvement of Ca2+ channels was investigated using the curves, in which the rings were pre-contracted with depolarizing Tyrode’s solution (KCl 80 mM). LEVO was capable of inducing vasodilatation similar to that achieved in the rings without functional endothelium, which were pre-contracted with Phe (1 µM) (Emax = 92.5 ± 1.0%; n = 4 and 90.4 ± 2.8%; n = 4, respectively), as shown in Figure 4A. LEVO (3 x 10–4 M) and NIF (10 µM) were able to induce vasodilatation in the rings without endothelium, pre-contracted with Phe (89.3 ± 6.1%; n = 5 and 80.0 ± 4.6%; n = 6, respectively). However, LEVO did not induce a significant additional effect in the rings preincubated with NIF (97.0 ± 3.15%, n=6) (Figure 4B).

Figure 4
Concentration-response curves for LEVO (3 x 10–7- 3 x 10–5 M; n = 6) in isolated rings of the rat superior mesenteric artery without functional endothelium pre-contracted with Phe (n = 4) or KCL 80 mM (n = 4) (A), and the vasodilator effect of NIF (10 μM), LEVO (3 x 10–4 M), LEVO after the maximum relaxation of NIF and NIF after the maximum relaxation of LEVO in the rings of the rat mesenteric artery without endothelium pre-contracted with Phe (1 μM) (B). Values are expressed as mean ± S.E.M. The data were analyzed with one or two-way ANOVA followed by Bonferroni post-hoc test.

To assess the possible contribution of K+-channels to the vasodilator effect induced by LEVO, rings without functional endothelium were pre-incubated with 1 mM of TEA. At this concentration, TEA was able to non-selectively block K+ channels1515 Cook NS. Effect of some potassium channel blockers on contractile responses of the rabbit aorta. J Cardiovasc Pharmacol. 1989 Feb;13(2):299-306. http://dx.doi.org/10.1097/00005344-198902000-00019. PMid:2468961.
http://dx.doi.org/10.1097/00005344-19890...
. In this protocol, the vasodilator effect induced by LEVO was found to be significantly higher as compared to that induced by the drug in the absence of the inhibitor (Emax = 90.4 ± 2.8%; n = 4 vs 123.7 ± 3.6%; n = 7) (Figure 3B).

DISCUSSION

Studies showed that LEVO in 50% enantiomeric excess induced certain intrinsic vasoconstriction in vitro88 Choi YS, Jeong YS, Ok SH, Shin IW, Lee SH, Park JY, et al. The direct effect of levobupivacaine in isolated rat aorta involves lipoxygenase pathway activation and endothelial nitric oxide release. Anesth Analg. 2010 Feb;110(2):341-9. http://dx.doi.org/10.1213/ANE.0b013e3181c76f52. PMid:19955508.
http://dx.doi.org/10.1213/ANE.0b013e3181...

9 Mukozawa M, Takakura K, Mizogami M. Direct vasocontractile activities of bupivacaine enantiomers on the isolated rat thoracic aorta. Anesthesiol Res Pract. 2010;2010:820186. http://dx.doi.org/10.1155/2010/820186. PMid:20981258.
http://dx.doi.org/10.1155/2010/820186...
-1010 Baik JS, Sohn JT, Ok SH, Kim JG, Sung HJ, Park SS, et al. Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent. Can J Physiol Pharmacol. 2011 Jul;89(7):467-76. http://dx.doi.org/10.1139/y11-046. PMid:21812525.
http://dx.doi.org/10.1139/y11-046...
,1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7.,1616 Bouaziz H, Iohom G, Estèbe JP, Campana WM, Myers RR. Effects of levobupivacaine and ropivacaine on rat sciatic nerve blood flow. Br J Anaesth. 2005 Nov;95(5):696-700. http://dx.doi.org/10.1093/bja/aei242. PMid:16183680.
http://dx.doi.org/10.1093/bja/aei242...
and in vivo1717 Aps C, Reynolds F. An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. Br J Clin Pharmacol. 1978 Jul;6(1):63-8. http://dx.doi.org/10.1111/j.1365-2125.1978.tb01683.x. PMid:666948.
http://dx.doi.org/10.1111/j.1365-2125.19...

18 Newton DJ, McLeod GA, Khan F, Belch JJ. The effect of adjuvant epinephrine concentration on the vasoactivity of the local anesthetics bupivacaine and levobupivacaine in human skin. Reg Anesth Pain Med. 2004 Jul-Aug;29(4):307-11. http://dx.doi.org/10.1097/00115550-200407000-00002. PMid:15305248.
http://dx.doi.org/10.1097/00115550-20040...
-1919 Ergil J, Akkaya T, Gozaydin O, Gunsoy B, Alicura S, Aladag E, et al. Vasoconstrictive and analgesic efficacy of locally infiltrated levobupivacaine in tonsillectomy patients. Int J Pediatr Otorhinolaryngol. 2012 Oct;76(10):1429-33. http://dx.doi.org/10.1016/j.ijporl.2012.06.016. PMid:22776810.
http://dx.doi.org/10.1016/j.ijporl.2012....
. Therefore, this study aimed to assess the vasoactivity of LEVO in in vitro experiments using arterial rings prepared from isolated rat superior mesenteric artery, a resistant artery, which can reflect the possible effects on the vascular reactivity in smaller blood vessels such as those present in the area treated by local anesthetic in clinical dentistry. Furthermore, this effect was compared to that of lidocaine, a standard local anesthetic used in dentistry.

In this study, it was shown that both LEVO and LIDO has no contractile effect on basal tone in the rat mesenteric artery. These results corroborate the findings of Chang et al.2020 Chang HC, Chen SY, Huang YF, Liu FL, Cherng YG, Wang HW. Effects of levobupivacaine on isolated rat tracheal smooth muscle. J Anesth. 2015 Oct;29(5):809-12. http://dx.doi.org/10.1007/s00540-015-2026-8. PMid:25995060.
http://dx.doi.org/10.1007/s00540-015-202...
, who demonstrated that LEVO did not exhibit a contractile effect on the rat tracheal smooth muscles. In contrast, Iida et al.2121 Iida H, Ohata H, Iida M, Nagase K, Uchida M, Dohi S. The differential effects of stereoisomers of ropivacaine and bupivacaine on cerebral pial arterioles in dogs. Anesth Analg. 2001 Dec;93(6):1552-6. http://dx.doi.org/10.1097/00000539-200112000-00046. PMid:11726442.
http://dx.doi.org/10.1097/00000539-20011...
and Mukozawa et al.99 Mukozawa M, Takakura K, Mizogami M. Direct vasocontractile activities of bupivacaine enantiomers on the isolated rat thoracic aorta. Anesthesiol Res Pract. 2010;2010:820186. http://dx.doi.org/10.1155/2010/820186. PMid:20981258.
http://dx.doi.org/10.1155/2010/820186...
,1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7. have shown that LEVO was able to induce vasoconstriction in studies on dog cerebral arterioles and rat thoracic aorta, respectively. These controversial results may be attributed to the differences in the structure of the chemical agents, the vascular bed, the experimental conditions, or the species used.

In addition, these opposite effects induced by LEVO can be justified because some anesthetics, including LEVO and BUPI, exhibit a biphasic activity depending on the concentration used. It was observed that these anesthetics cause vasodilatation at high concentrations, and vasoconstriction at smaller concentrations, both in vivo and in vitro1616 Bouaziz H, Iohom G, Estèbe JP, Campana WM, Myers RR. Effects of levobupivacaine and ropivacaine on rat sciatic nerve blood flow. Br J Anaesth. 2005 Nov;95(5):696-700. http://dx.doi.org/10.1093/bja/aei242. PMid:16183680.
http://dx.doi.org/10.1093/bja/aei242...

17 Aps C, Reynolds F. An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. Br J Clin Pharmacol. 1978 Jul;6(1):63-8. http://dx.doi.org/10.1111/j.1365-2125.1978.tb01683.x. PMid:666948.
http://dx.doi.org/10.1111/j.1365-2125.19...

18 Newton DJ, McLeod GA, Khan F, Belch JJ. The effect of adjuvant epinephrine concentration on the vasoactivity of the local anesthetics bupivacaine and levobupivacaine in human skin. Reg Anesth Pain Med. 2004 Jul-Aug;29(4):307-11. http://dx.doi.org/10.1097/00115550-200407000-00002. PMid:15305248.
http://dx.doi.org/10.1097/00115550-20040...
-1919 Ergil J, Akkaya T, Gozaydin O, Gunsoy B, Alicura S, Aladag E, et al. Vasoconstrictive and analgesic efficacy of locally infiltrated levobupivacaine in tonsillectomy patients. Int J Pediatr Otorhinolaryngol. 2012 Oct;76(10):1429-33. http://dx.doi.org/10.1016/j.ijporl.2012.06.016. PMid:22776810.
http://dx.doi.org/10.1016/j.ijporl.2012....
.

Since no vasoconstrictor activity was observed on the basal tone, experiments were conducted to evaluate the possible vasodilator effect of LEVO and compare it to that of LIDO. The vasodilatation induced by LEVO was similar to that caused by the lidocaine.

Furthermore, we assessed the underlying mechanism responsible for the vasodilator effect of LEVO. Since the endothelium is an important regulator of the vascular tone via releasing endothelium-derived relaxing factors, mainly nitric oxide (NO) and products derived from the activation of cyclooxygenase (COX) enzyme, such as prostacyclins, experiments were carried out to assess the role of the endothelium in this response2222 Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980 Nov;288(5789):373-6. http://dx.doi.org/10.1038/288373a0. PMid:6253831.
http://dx.doi.org/10.1038/288373a0...
. However the endothelium probably does not contribute to vasodilatador effect of LEVO.

In accordance with the results of the present study, LEVO showed an endothelium-independent effect on the rat thoracic aorta1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7. as well as on isolated human umbilical artery and vein1111 Kiliçaslan A, Duman A, Sahin AS. In vitro vasoactive effects of levobupivacaine and ropivacaine on the isolated human umbilical artery and vein. Balkan Med J. 2011;28(2):164-8.. However, it was shown that the effect of LEVO on rat aorta was dependent on nitric oxide released by the endothelium88 Choi YS, Jeong YS, Ok SH, Shin IW, Lee SH, Park JY, et al. The direct effect of levobupivacaine in isolated rat aorta involves lipoxygenase pathway activation and endothelial nitric oxide release. Anesth Analg. 2010 Feb;110(2):341-9. http://dx.doi.org/10.1213/ANE.0b013e3181c76f52. PMid:19955508.
http://dx.doi.org/10.1213/ANE.0b013e3181...
. These findings reinforce that the vascular effects of LEVO may differ depending on some variables, such as the vascular bed used.

We also evaluated whether the effects induced by LEVO are mediated via another endothelium-independent signaling pathway, such as Ca2+-channel blockade. It is known that increased external K+ concentration induces smooth muscle contraction through the activation of Cavs and subsequent release of calcium from the sarcoplasmic reticulum. Contractions induced by high concentrations of K+ are inhibited by Ca2+ -channel blockers or by the removal of Ca2+ from the external environment because they are totally dependent on Ca2+ influx2323 Karaki H, Weiss GB. Calcium release in smooth muscle. Life Sci. 1988;42(2):111-22. http://dx.doi.org/10.1016/0024-3205(88)90674-1. PMid:2447464.
http://dx.doi.org/10.1016/0024-3205(88)9...
.

LEVO induced vasodilatation on pre-contracted rings with depolarizing Tyrode’s solution (KCl 80 mM) and did not promote an additional vasodilator effect compared to the maximum effect induced by NIF, suggesting that LEVO may act mainly in a similar way as NIF, i.e., blocking dihydropyridine-sensitive Cavs.

Choi et al.88 Choi YS, Jeong YS, Ok SH, Shin IW, Lee SH, Park JY, et al. The direct effect of levobupivacaine in isolated rat aorta involves lipoxygenase pathway activation and endothelial nitric oxide release. Anesth Analg. 2010 Feb;110(2):341-9. http://dx.doi.org/10.1213/ANE.0b013e3181c76f52. PMid:19955508.
http://dx.doi.org/10.1213/ANE.0b013e3181...
, Baik et al.1010 Baik JS, Sohn JT, Ok SH, Kim JG, Sung HJ, Park SS, et al. Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent. Can J Physiol Pharmacol. 2011 Jul;89(7):467-76. http://dx.doi.org/10.1139/y11-046. PMid:21812525.
http://dx.doi.org/10.1139/y11-046...
and Mukozawa et al.1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7. demonstrated that LEVO-induced vasoconstriction in the aorta is mediated mainly by the intracellular calcium and by Ca2+ influx through Cavs88 Choi YS, Jeong YS, Ok SH, Shin IW, Lee SH, Park JY, et al. The direct effect of levobupivacaine in isolated rat aorta involves lipoxygenase pathway activation and endothelial nitric oxide release. Anesth Analg. 2010 Feb;110(2):341-9. http://dx.doi.org/10.1213/ANE.0b013e3181c76f52. PMid:19955508.
http://dx.doi.org/10.1213/ANE.0b013e3181...
,1010 Baik JS, Sohn JT, Ok SH, Kim JG, Sung HJ, Park SS, et al. Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent. Can J Physiol Pharmacol. 2011 Jul;89(7):467-76. http://dx.doi.org/10.1139/y11-046. PMid:21812525.
http://dx.doi.org/10.1139/y11-046...
,1212 Mukozawa M, Takakura K, Obata Y, Shimo K, Shigemi K. Levobupivacaine induces vasoconstriction via Ca -dependent and -independent mechanisms in isolated rat thoracic aorta. 2+Circulation Control in Medicine. 2013;34:71-7.. However, our results suggest that LEVO acts via inhibition of Ca2+ influx through these channels. These differences might be related to the different vascular beds used and different experimental conditions. Nevertheless, additional studies are needed for a better understanding of the underlying mechanism of action.

Moreover, there are reports in the literature that K+ channels also play an important role in the regulation of the vascular tone2424 Akata T. Cellular and molecular mechanisms regulating vascular tone. Part 2: regulatory mechanisms modulating Ca mobilization and/or myofilament Ca sensitivity in vascular smooth muscle cells. 2+2+J Anesth. 2007;21(2):232-42. http://dx.doi.org/10.1007/s00540-006-0488-4. PMid:17458653.
http://dx.doi.org/10.1007/s00540-006-048...
. K+ channel opening in the smooth muscle cell membrane causes K+ efflux, resulting in hyperpolarization that closes the Cavs, resulting in decreased influx of Ca2+ ions and thus, relaxation of the smooth muscles2525 Ko EA, Han J, Jung ID, Park WS. Physiological roles of K+ channels in vascular smooth muscle cells. J Smooth Muscle Res. 2008 Apr;44(2):65-81. http://dx.doi.org/10.1540/jsmr.44.65. PMid:18552454.
http://dx.doi.org/10.1540/jsmr.44.65...
. LEVO was able to induce vasodilatation in rings without functional endothelium with TEA, which suggests that these channels are not involved in LEVO-induced vasodilator effect.

CONCLUSION

The results of this study demonstrated that LEVO did not induce vasoconstriction of arterial rings prepared from rat superior mesenteric artery, however, it produced a vasodilator effect. This LEVO-induced vasodilatation was endothelium and K+ channel-independent, however, it could be mediated via the blockade of Cavs in the vascular smooth muscle cells. We suggest that LEVO exhibits several vascular effects depending on the vascular bed used. Although LEVO did not display the expected vasoconstriction in our study, LEVO remains beneficial and can be widely used in dentistry as it is less cardiotoxic and less neurotoxic than BUPI, and it also provides long-lasting anesthesia.

ACKNOWLEDGEMENTS

We are grateful to the Federal University of Sergipe and Cristália Laboratory, for their cooperation, support, and encouragement to the research. We are also thankful to CNPq/Brazil and FAPITEC/SE/Brazil for their financial support.

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Publication Dates

  • Publication in this collection
    01 Sept 2016
  • Date of issue
    Sep-Oct 2016

History

  • Received
    23 Dec 2015
  • Accepted
    22 June 2016
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