gree时ec他0 ofhe IFFF EMBS Endothelium-Independent Vasorelaxant Effect of Lidocaine in Rat Aortic Rings O-X.Shan.D-S.Lin.H-F.Jin.O.Gao.Y.Lu.O.Xia II METHODOLOOY ae KH-P0.1 e5 L INTRODUCTION n at 37 with Ring or KC 0.7803-49-304S20.09204IFFE 3753
Abstract—In the present study, lidocaine relaxed, in a concentration-dependent manner, the contractions induced by either phenylephrine or a high concentration of KCl (60 mM) in endothelium-intact rat aortic rings. Mechanical removal of endothelium did not significantly modify lidocaine-induced vasorelaxation. In endothelium-denuded aortic rings depolarized by 60 mM KCl, lidocaine inhibited Ca2+-induced contraction. Lidocaine also reduced the transient contraction elicited by phenylephrine in Ca2+-free medium. Pretreatment of endothelium-denuded aorta with tetraethylammonium, a nonspecific K+ channel blocker, had no effect on the relaxant effect of lidocaine. These results indicate that lidocaine induces an endothelium-independent relaxation in rat aortic rings. The main mechanisms may include suppression in Ca2+ influx through the voltage-sensitive Ca2+ channels and inhibition of intracellular Ca2+ release in the vascular smooth muscle cells. Keywords—Lidocaine, relaxation, Ca2+ influx, Ca2+ release, aortic rings I. INTRODUCTION Lidocaine is commonly used as a local anesthetic or one of the class IB antiarrhythmic drugs. However, an overdose of lidocaine or inadvertent intravenous injection during regional anesthesia can result in serious complications, including cardiovascular toxicity. In contrast to a consistent myocardial depression, an increase [1], decrease [2] or minimal change [3] in peripheral vascular resistance induced by local anesthetic agents has been reported. The direct vascular effect of lidocaine also remains controversial. In the arterioles of an in vivo rat cremaster muscle preparation [4] and in in vitro human radial arterial rings [5] or epicardial porcine coronary arteries [6], lidocaine elicited a biphasic dose-dependent response, with vasoconstriction at low concentrations and relaxation at high concentrations. In isolated human hand veins, lidocaine increased the contractile response to K+ and produced a contraction by itself at high concentrations [7]. In rabbit thoracic aorta, lidocaine itself evoked an endothelium-independent contraction, but relaxed phenylephrine-precontracted aortic rings with or without endothelium [8]. Also, it has been shown that lidocaine could inhibit endothelium-dependent vasodilatation induced by receptor- or nonreceptor-mediated vasodilators [9], as well as the endothelium-independent relaxations [8;10;11]. The reasons for these different findings are unclear. A discrepancy in species and tissue preparations or in the concentration of lidocaine cannot be excluded. Based on these controversial data, we investigated the effect of lidocaine on isolated rat thoracic aortic rings and proceeded further to characterize the mechanisms underlying its effect in the present study. II. METHODOLOGY Male Sprague-Dawley (SD) rats (250-300 g) were obtained from Zhejiang Institute of Medical Science (Hangzhou, Zhejiang) and treated in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang University School of Medicine. Preparation of rat aortic rings SD rats were killed by stunning and subsequent cervical dislocation. Thoracic aortae were rapidly removed and stored in cold Kreb’s solution of the following composition (in mM): NaCl 118, NaHCO3 25, glucose 10, KH2PO4 1.2, MgSO4 1.2, KCl 4.7, and CaCl2 1.25. Aortae were cleaned of fat and connective tissues and cut into 3 mm rings. In some experiments, the endothelium was mechanically removed by inserting a forcep into the lumen of an artery and gently rolling the preparation. Rings were suspended in organ chambers containing 10 ml Krebs solution at 37°C, bubbled with 95% O2 and 5% CO2. After equilibration under no tension for 15 min, the preparations were passively stretched to an optimal tension of 2 g. Rings were equilibrated for 1 h and the Kreb’s solution was changed every 15 min. Changes in isometric tension were recorded by force transducers connected to a data acquisition system (MacLab, ADInstruments). Before each experiment, rings were stimulated with 60 mM KCl at least 3 times until a reproducible contractile response was obtained. The presence of functional endothelium was verified by the ability of acetylcholine (10-5 M) to induce more than 70% relaxation of aortic rings precontracted with phenylephrine (10-6 M). Experimental protocols In the first set of experiments, after phenylephrine (10-6 M) or KCl (60 mM) elicited a steady contraction of rat aortic rings with or without endothelium, lidocaine (10-7 ~3×10-2 M) was added cumulatively. Tension was expressed as a percentage of phenylephrine- or KCl-induced contraction. In the experiment examining the involvement of K+ channels, rings without functional endothelium were incubated with tetraethylammonium (5 mM), a nonspecific K+ channel blocker [12;13], for 30 min before phenylephrine application. Endothelium-Independent Vasorelaxant Effect of Lidocaine in Rat Aortic Rings Q-X. Shan, D-S. Lin, H-F. Jin, Q. Gao, Y. Lu, Q. Xia Department of Physiology, Zhejiang University School of Medicine, Hangzhou 310031, P. R. China E-mail: qiqixianxian@yahoo.com
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In the second group of experiments, aortic rings were incubated in Ca2+-free solution containing 50 µM EGTA and 60 mM KCl for 20 min. Ca2+ was then added cumulatively to obtain a concentration-response curve. 10-3 M lidocaine was added 10 min before the addition of CaCl2. Contraction was expressed as a percentage of the maximal contraction in response to 60 mM KCl in standard Krebs solution. In the third group of experiments, the rings were exposed to Ca2+-free medium with 50 µM EGTA for 15 min before the application of 10-6 M phenylephrine to induce the first transient contraction. The rings were then washed with normal Krebs solution three times and incubated for at least 40 min for refilling of the intracellular stores. Subsequently, the medium was rapidly replaced with Ca2+-free solution and the rings were incubated for 15 min. The second contraction was then induced by phenylephrine in the absence and presence of 10-3 M lidocaine, which was added 10 min before phenylephrine application. The ratio of the second contraction to the first contraction in Ca2+-free medium was calculated. Similarly, the ratio of the responses to phenylephrine in the normal Ca2+-containing medium was also calculated. Data analysis and statistical Data were expressed as mean ± S.E.M. Comparisons were made by unpaired student’s t test between two groups. A P<0.05 was considered significant. III. RESULTS A Vasorelaxant effect of lidocaine After 10-6 M phenylephrine induced a steady contraction, lidocaine was added accumulatively. Lidocaine induced a concentration-dependent relaxation in phenylephrineprecontracted aortic rings. Lidocaine caused complete relaxation of endothelium-intact and -denuded aortic rings. Functional removal of the endothelium did not affect the lidocaine-induced relaxation (Fig. 1A). Similarly, lidocaine produced a concentration-dependent reduction of high K+ - induced contraction either in the endothelium-intact or in the endothelium-denuded arteries (Fig. 1B). B Effect of lidocaine on Ca2+-induced contraction Ca2+ induced a concentration-dependent contraction of rat aortic rings without endothelium depolarized by 60 mM KCl. Incubation of the aortic rings with 10-3 M lidocaine significantly inhibited Ca2+-induced contraction (Fig. 2). C Effect of lidocaine on phenylephrine-induced contraction in Ca2+-free medium In endothelium-denuded rings, a transient contractile response in Ca2+-free medium was elicited by 10-6 M phenylephrine. A second contraction was then induced again by phenylephrine in the absence or presence of 10-3 M -7 -6 -5 -4 -3 0 20 40 60 80 100 120 +Endothelium - Endothelium Lidocaine,log (M) A) % of PE-induced contraction -7 -6 -5 -4 -3 0 20 40 60 80 100 120 B) Lidocaine,log (M) % of KCl-induced contraction Fig. 1 Concentration-dependent relaxation of lidocaine (10-7~3×10-2 M) in phenylephrine (PE)- (10-6 M) (A) or KCl- (60 mM) (B) preconstricted rat aortic rings with (+Endothelium) or without endothelium (-Endothelium). Data are expressed as mean ± S.E.M of n observations, n=5~10. 0 1 2 3 4 5 6 0 20 40 60 80 100 control + Lidocaine **** ** ** ** Ca2+ (mM) % of KCl-induced contraction Fig. 2 Effect of lidocaine (10-3 M) on Ca2+-induced contraction of rat aortic rings without endothelium depolarized with high-K+ . Results are expressed as mean ± S.E.M. of 7~8 observations. ** p<0.01, compared with control. lidocaine. The ratio of the second contraction to the first contraction was calculated. Pretreatment of the aortic rings with 10-3 M lidocaine for 10 min significantly reduced this
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ratio of the responses to phenylephrine in Ca2+-free medium (Fig. 3). When the same protocol was repeated in normal Ca2+-containing medium, lidocaine-induced decrease in this ratio also existed. 0 60 120 ** ** Ca2+-containing medium Ca2+-free medium Control + Lidocaine Ratio (%) Fig. 3 Effect of lidocaine (10-3 M) on ratio of the contractile responses to phenylephrine of rat aortic rings without endothelium in Ca2+-containing or Ca2+-free medium. Results are expressed as mean ± S.E.M. of n observations (n=5~11). ** p<0.01, compared with control. 0 20 40 60 80 100 120 Control + TEA lidocaine, log (M) -4 -3 -2 % of PE-induced contraction Fig. 4 Effect of tetraethylammonium (TEA) (5 mM) on lidocaineinduced vasorelaxation in endothelium-denuded rat aortic rings precontracted with phenylephrine (PE). Results are expressed as mean ± S.E.M. of n observations (n=6 for both control and +TEA groups). IV. DISCUSSION Although lidocaine is widely used clinically as a local anesthetic or an antiarrhythmic drug, its vascular effect and the underlying mechanisms remain a controversial issue. A potent vasorelaxant effect of lidocaine was found in the present study. It almost completely relaxed the rat aortic rings precontracted by either phenylephrine or high concentration of KCl. This lidocaine-induced vasorelaxation was observed in rat aorta with or without functional endothelium, suggesting that its vasorelaxant effect is endothelium-independent. It is well known that KCl-induced contraction mainly results from Ca2+ influx upon depolarization of the cell membrane, which activates voltage-dependent L-type Ca2+ channels [14]. Lidocaine inhibited KCl-induced vasoconstriction in endothelium-denuded rat aorta, suggesting that lidocaine exerts its vasorelaxant effect, at least in part, by blocking the L-type Ca2+ channel. Furthermore, lidocaine significantly reduced the Ca2+- induced contraction in aortic rings depolarized by KCl. A decreased sensitivity to Ca2+ under depolarizing conditions suggests that there is either a suppression of Ca2+ channels or a decreased sensitivity to Ca2+ at the level of the contractile proteins. Therefore, in addition to its main effect on L-type Ca2+ channel, an inhibitory effect of lidocaine on Ca2+- sensitivity of the contractile proteins in vascular smooth muscle cannot be excluded. Lidocaine elicited a significant inhibition of phenylephrine-induced contraction. This inhibitory effect was observed when lidocaine was added either before or after the induced contraction. Phenylephrine is an α1- adrenoceptor agonist. It causes vasoconstriction by activating phospholipase C and primarily triggering Ca2+ release from the sarcoplasmic reticulum followed by sustained Ca2+ entry [15]. To test the hypothesis that the vasorelaxant effects of lidocaine might be due to inhibition of the intracellular Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle, we then performed the experiment in a calcium-free medium. Under this condition, phenylephrine-induced transient contraction mainly results from Ca2+ release from the sarcoplasmic reticulum [16]. Our results showed that lidocaine inhibited the contractile response induced by phenylephrine in the absence of extracellular Ca2+ in endothelium-denuded aorta, suggesting an inhibitory effect of lidocaine on intracellular Ca2+ release within the vascular smooth muscle. However, the effect of phenylephrine in vascular smooth muscle is far more complicated [15]. It cannot be excluded that lidocaine may inhibit non-voltage-sensitive Ca2+ channels and decrease Ca2+-sensitivity of the contractile proteins in the vascular smooth muscle. K+ channels play an important role in the regulation of muscle contractility and vascular tone [17]. Direct activation of K+ channels in vascular smooth muscle cells leads to membrane hyperpolarization and subsequent reduction in Ca2+ influx through L-type Ca2+ channels. To test the hypothesis that lidocaine-induced vasodilation could be mediated by membrane hyperpolarization by opening K+ channel in vascular smooth muscle, we pretreated the endothelium-denuded rings with tetraethylammonium, a nonspecific K+ channel blocker. However, tetraethylammonium had no effect on lidocaine-induced relaxation, suggesting that activation of K+ channel is not involved in the vasorelaxant effect of lidocaine. In summary, lidocaine induced a concentrationdependent, endothelium-independent relaxation in rat aortic rings preconstricted by either phenylephrine or KCl. The
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primary mechanisms may include both reduction in Ca2+ influx through the voltage-dependent Ca2+ channels and inhibition of intracellular Ca2+ release in the vascular smooth muscle cells. ACKNOWLEDGMENT This work was supported by the Student Research Training Program (SRTP) of ZJU and 2004 Seeding Foundation of Zhejiang University for Young Investigators. REFERENCES [1] Nystrom, E. U., Heavner, J. E., and Buffington, C. W., "Blood pressure is maintained despite profound myocardial depression during acute bupivacaine overdose in pigs," Anesth.Analg., vol. 88, no. 5, pp. 1143-1148, May1999. [2] Covino, B. G., "Toxicity of local anesthetic agents," Acta Anaesthesiol.Belg., vol. 39, no. 3 Suppl 2, pp. 159-164, 1988. [3] Liu, P., Feldman, H. S., Covino, B. M., Giasi, R., and Covino, B. G., "Acute cardiovascular toxicity of intravenous amide local anesthetics in anesthetized ventilated dogs," Anesth.Analg., vol. 61, no. 4, pp. 317-322, Apr.1982. [4] Johns, R. A., DiFazio, C. A., and Longnecker, D. E., "Lidocaine constricts or dilates rat arterioles in a dosedependent manner," Anesthesiology, vol. 62, no. 2, pp. 141- 144, Feb.1985. [5] Jernbeck, J. and Samuelson, U. E., "Effects of lidocaine and calcitonin gene-related peptide (CGRP) on isolated human radial arteries," J.Reconstr.Microsurg., vol. 9, no. 5, pp. 361- 365, Sept.1993. [6] Perlmutter, N. S., Wilson, R. A., Edgar, S. W., Sanders, W., Greenberg, B. H., and Tanz, R., "Vasodilatory effects of lidocaine on epicardial porcine coronary arteries," Pharmacology, vol. 41, no. 5, pp. 280-285, 1990. [7] Arner, M. and Hogestatt, E. D., "Actions of some vasodilators on isolated human hand veins," Acta Physiol Scand., vol. 130, no. 4, pp. 671-677, Aug.1987. [8] Turan, N. N., Demiryurek, A. T., and Celebi, H., "Effects of lidocaine on rabbit isolated thoracic aorta," Pharmacol.Res., vol. 42, no. 5, pp. 453-458, Nov.2000. [9] Johns, R. A., "Local anesthetics inhibit endotheliumdependent vasodilation," Anesthesiology, vol. 70, no. 5, pp. 805-811, May1989. [10] Kinoshita, H., Ishikawa, T., and Hatano, Y., "Differential effects of lidocaine and mexiletine on relaxations to ATPsensitive K+ channel openers in rat aortas," Anesthesiology, vol. 90, no. 4, pp. 1165-1170, Apr.1999. [11] Minamoto, Y., Nakamura, K., Toda, H., Miyawaki, I., Kitamura, R., Vinh, V. H., Hatano, Y., and Mori, K., "Suppression of acetylcholine-induced relaxation by local anesthetics and vascular NO-cyclic GMP system," Acta Anaesthesiol.Scand., vol. 41, no. 8, pp. 1054-1060, Sept.1997. [12] Gupte, S. A., Li, K. X., Okada, T., Sato, K., and Oka, M., "Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels," J.Pharmacol.Exp.Ther., vol. 301, no. 1, pp. 299-305, Apr.2002. [13] Cadorette, C., Sicotte, B., Brochu, M., and St Louis, J., "Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats," Am.J.Physiol Heart Circ.Physiol, vol. 278, no. 2, pp. H567-H576, Feb.2000. [14] Cauvin, C., Loutzenhiser, R., and Van Breemen, C., "Mechanisms of calcium antagonist-induced vasodilation," Annu.Rev.Pharmacol.Toxicol., vol. 23 pp. 373-396, 1983. [15] Karaki, H., Ozaki, H., Hori, M., Mitsui-Saito, M., Amano, K., Harada, K., Miyamoto, S., Nakazawa, H., Won, K. J., and Sato, K., "Calcium movements, distribution, and functions in smooth muscle," Pharmacol.Rev., vol. 49, no. 2, pp. 157-230, June1997. [16] Ko, W. H., Yao, X. Q., Lau, C. W., Law, W. I., Chen, Z. Y., Kwok, W., Ho, K., and Huang, Y., "Vasorelaxant and antiproliferative effects of berberine," Eur.J.Pharmacol., vol. 399, no. 2-3, pp. 187-196, July2000. [17] Nelson, M. T. and Quayle, J. M., "Physiological roles and properties of potassium channels in arterial smooth muscle," Am.J.Physiol, vol. 268, no. 4 Pt 1, pp. C799-C822, Apr.1995