Direct Vasocontractile Activities of Bupivacaine Enantiomers on the Isolated Rat Thoracic Aorta

Background. In vitro studies with isolated arteries have shown direct vasoactivity of racemic bupivacaine. However, there is little information on the direct vasoactivities of bupivacaine enantiomers, S(−)- and R(+)-bupivacaine. Methods. We performed functional examinations using isolated intact thoracic aortic rings from male Wistar rats. Changes in ring tension produced by S(−)-, R(+)-, or racemic bupivacaine were measured in Krebs solution. Results. S(−)-bupivacaine produced the strongest contraction of the three agents. R(+)-bupivacaine showed limited vasoconstriction. The effects of racemic bupivacaine were located between these two. Conclusion. Each bupivacaine enantiomer showed specific vasocontractile activity, which affects the activity of racemic bupivacaine.


Introduction
The local anesthetic bupivacaine is a racemic mixture of S(−)-and R(+)-enantiomers. Racemic bupivacaine has biphasic vasoactivities, namely, vasoconstriction at a low concentration and vasodilatation at a high concentration [1]. Since 1976, when Aps and Reynolds showed this vasoactivity in a double-blind trial with forearm skin color changes of 31 volunteers [1], these vasoactivities have been further demonstrated using various in vivo methods with various animals or humans, such as television microscopy in rat cremaster muscle microvasculatures [2], intravital microscopy through a spinal window in dog pial vasculatures [3], laser Doppler imaging in human skin [4], as well as other techniques. Although inhibition of sympathetic nerves innervating arteries by racemic bupivacaine could not be ignored in in vivo studies, some in vitro studies with isolated preparations from human umbilical arteries [5][6][7], rat uterine arteries [8], and human uterine arteries [9] have confirmed that the vasoactivities are produced by direct actions of racemic bupivacaine itself on the arteries. S(−)-bupivacaine was developed as an alternative longacting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a lower potential for producing systemic toxicity [10]. S(−)-bupivacaine also has biphasic vasoactivities similar to those of racemic bupivacaine, which has been shown in in vivo studies [4,[11][12][13]. Furthermore, some of the in vivo studies have shown that R(+)-bupivacaine produced a dose-dependent vasodilatation [11,13]. However, there is little information on the direct vasoactivities of S(−)-and R(+)-bupivacaine based on in vitro study. In this study, we investigated the vasocontractile activities of these agents using isolated rat aorta.

Animals.
The experimental protocol was approved by the institutional animal care committee of Asahi University. Male Wistar rats weighing 240-280 g were used.

Functional Experiments.
Rats were killed by decapitation under sevoflurane anesthesia, and the thoracic aorta was isolated and removed [14]. The thoracic aorta was placed in Krebs-Henseleit solution (mM; NaCl 118, KCl 4.7, NaHCO 3 25, KH 2 PO 4 1.2, MgSO 4 1.2, CaCl 2 2.5, and glucose 10; pH 7.4). Aortic rings were carefully prepared under a dissecting microscope, and then each intact ring was carefully pulled by wires in an organ chamber containing 5 mL Krebs-Henseleit solution bubbled with 95% O 2 and 5% CO 2 at 37 • C. After a resting tension of 1.0 g was applied during one-hour equilibration period, changes in the tension were recorded isometrically when S(−)-bupivacaine, R(+)-bupivacaine, or racemic bupivacaine was cumulatively applied. Contractions were expressed as mg contractile tension.

Statistical Analysis.
The results are expressed as mean ± SD. The maximum response (E max ) and the concentration producing a half-maximal response (EC 50 ) were determined by Finley's probit analysis. Significance of differences was analyzed by Kruskal-Wallis test and Scheffé method as a post hoc comparison for multiple comparisons at a significance level of 0.05.

Results
S(−)-bupivacaine, R(+)-bupivacaine, and racemic bupivacaine produced a biphasic response in the aortic rings, namely, concentration-dependent contraction from 10 μM to 1 mM and relaxation at higher concentrations (n = 8, Figure 1.) The vasocontractile responses produced by S(−)bupivacaine and racemic bupivacaine were significantly stronger than those by R(+)-bupivacaine. Furthermore, there were significant differences among E max or EC 50 of each bupivacaine (Table 1).

Discussion
As there has been only limited study of the direct vasoactivities of two bupivacaine enantiomers, S(−)-and R(+)bupivacaine, we compared the vasocontractile effects of these two agents with that of racemic bupivacaine in this study. S(−)-bupivacaine showed the strongest E max of the three agents, while R(+)-bupivacaine showed limited vasoconstriction. Although racemic bupivacaine produced as much E max as S(−)-bupivacaine statistically (Table 1), the activity level of racemic bupivacaine was located between those of S(−)-bupivacaine and R(+)-bupivacaine graphically ( Figure 1). R(+)-bupivacaine, which produced small vasoconstriction even at high concentration, may interfere with vasoconstriction by S(−)-bupivacaine in racemic bupivacaine, which consists of the two enantiomers. Several in vitro studies have been done on racemic bupivacaine's direct vasoactivities. It has been reported that racemic bupivacaine contracted isolated rat uterine arteries [8], human uterine arteries [9], human umbilical arteries [5][6][7], and veins [6] in various degrees. Different vasocontractile activities of the enantiomers among the vessels might be, at least, a cause of the variety. S(−)-bupivacaine produced the strongest vasoconstriction of the three bupivacaines in our study. Considering that the clinical use of S(−)-bupivacaine is increasing because of its lower toxicity [10], it is important to note that it may produce greater vasoconstriction than racemic bupivacaine does. Bupivacaine administered for epidural anesthesia raises the intrathecal and plasma concentration [16] and might contract several important vessels, including the pial, epidural, uterine, umbilical arteries, as well as others, with subsequent decrease of blood flow.