THE actions of local anesthetics on the nervous system are reportedly related to their effects on K+as well as Na+channels in neurons.1Importantly, among these K+channels in the nervous system, a voltage-insensitive flickering K+channels has been found to be more sensitive than the Na+channel to lipophilic, amide-linked local anesthetics, especially to the piperidine derivatives bupivacaine and ropivacaine.2,3The flickering K+channel was mostly found in thin, myelinated nerve fibers, and it is a possible candidate for generating the resting potential of these fibers.4Therefore, these results indicate that the inhibition of K+channels contributes to the action of bupivacaine and ropivacaine on the nervous system.
Cumulative findings have demonstrated that K+channels play crucial roles in physiologic and pathophysiologic vasodilation.5–7Although S (−)-bupivacaine is less toxic on cardiac function or the central nervous system than racemic bupivacaine,8,9the effects of bupivacaine enantiomers on K+channels of vascular smooth muscle have not been studied. In addition, whether the S (−)-enantiomer ropivacaine affects these channels of vascular smooth muscle has been unknown.
Therefore, the current study was designed to determine the potency of amide-linked long-acting local anesthetic drugs on K+channels of vascular smooth muscle, by examining whether bupivacaine enantiomers as well as ropivacaine modify vasorelaxation induced by an adenosine triphosphate (ATP)–sensitive K+channel opener in the isolated rat aorta.
Materials and Methods
The institutional animal care and use committee (Wakayama, Japan) approved this study. Male Wistar rats (weight, 250–350 g) were anesthetized with inhalation of 3% halothane. Under this anesthetic condition, the rats were killed by exsanguination, and thoracic aortas were harvested. Thoracic aortic rings of 2.5 mm in length were studied in modified Krebs-Ringer’s bicarbonate solution (control solution) of the following composition: 119 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.17 mm MgSO4, 1.18 mm KH2PO4, 25 mm NaHCO3, and 11 mm glucose. In some rings, the endothelium was removed mechanically, and the endothelial removal or preservation was confirmed by the absence or the presence of the relaxation in response to acetylcholine (10−5m), respectively. Several rings cut from the same artery were studied in parallel. Each ring was connected to an isometric force transducer and suspended in an organ chamber filled with 10 ml control solution (37°C, pH 7.4) bubbled with 95% O2and 5% CO2. The artery was gradually stretched to the optimal point of its length–tension curve as determined by the contraction to phenylephrine (3 × 10−7m). In most of the studied arteries, optimal tension was achieved at approximately 1.5 g. During submaximal contraction to phenylephrine, the concentration–response curve to levcromakalim or diltiazem was obtained. Some rings were treated with glibenclamide, S (−)- or R (+)-bupivacaine, or ropivacaine, which was given 15 min before addition of phenylephrine (3 × 10−7m). The vasorelaxation was expressed as a percentage of the maximal relaxation in response to papaverine (3 × 10−4m), which was added at the end of experiments to produce the maximal relaxation (100%) of arteries.
The following pharmacologic agents were used: diltiazem, dimethyl sulfoxide, glibenclamide, and phenylephrine (Sigma, St. Louis, MO). Levcromakalim and S (−)-bupivacaine, R (+)-bupivacaine, and ropivacaine were gifts from GlaxoSmithKline plc (Greenford, United Kingdom) or AstraZeneca Pharmaceutical Co. (Södertälje, Sweden), respectively. Drugs, except for levcromakalim and glibenclamide, were dissolved in distilled water such that volumes of less than 60 μl were added to the organ chambers. Stock solutions of levcromakalim (10−5m) and glibenclamide (10−5m) were prepared in dimethyl sulfoxide (3 × 10−4m).
Data are expressed as mean ± sd. Statistical analysis was performed using repeated-measures analysis of variance, followed by the Scheffé F test for multiple comparisons. Differences were considered to be statistically significant when P was less than 0.05.
During submaximal contraction to phenylephrine (3 × 10−7m), the selective ATP-sensitive K+channel opener levcromakalim (10−8to 10−5m) produced vasorelaxation of the rat aorta with or without endothelium in a concentration-dependent fashion (fig. 1). This relaxation was abolished by a selective ATP-sensitive K+channel antagonist glibenclamide (10−5m) (fig. 1). In the aortas with endothelium, R (+)-bupivacaine (10−6to 10−5m) and S (−)-bupivacaine (3 × 10−6to 10−5m) inhibited vasorelaxation in response to levcromakalim in a concentration-dependent fashion, whereas ropivacaine did not affect this vasorelaxation (fig. 2A). In the aortas without endothelium, R (+)-bupivacaine (3 × 10−6to 10−5m) inhibited vasorelaxation to levcromakalim in a concentration-dependent fashion, whereas S (−)-bupivacaine reduced the relaxation only in the highest concentration used, and ropivacaine did not alter this vasorelaxation (fig. 2B).
The highest concentration of each compound (10−5m) did not affect vasorelaxation in response to the voltage-dependent Ca2+channel antagonist diltiazem (10−8to 3 × 10−4m) (fig. 3).
Levcromakalim is a selective ATP-sensitive K+channel opener in the rat aorta, suggesting that this preparation is a suitable model by which we can evaluate the role of ATP-sensitive K+channels in vascular smooth muscle.7In the rat aortas with and without endothelium, R (+)-bupivacaine caused the rather augmented inhibitory effect on the vasorelaxation via ATP-sensitive K+channels, compared with S (−)-isomer, although both R (+)- and S (−)-bupivacaine inhibited the vasorelaxation in a concentration-dependent fashion. These results support the following conclusions. First, the effects of bupivacaine enantiomers on vasorelaxation via ATP-sensitive K+channels are mostly mediated by their effects on these channels on vascular smooth muscle cells, because inhibitory actions of bupivacaine enantiomers were similar between the aortas with and without endothelium. Second, bupivacaine seems to stereoselectively reduce the vasorelaxation mediated by ATP-sensitive K+channels. Previous studies on rat cardiac myocytes, bovine adrenal zona fasciculata cells, and native Xenopus oocytes demonstrated that racemic bupivacaine, containing R (+)- and S (−)-bupivacaine, reduces ATP-sensitive K+currents.10–12These results obtained from studies performed using tissues other than blood vessels are certainly in agreement with our findings that bupivacaine enantiomers reduced vasorelaxation mediated by ATP-sensitive K+channels. In the rat aorta, the pure S (−)-enantiomer ropivacaine did not alter vasorelaxation caused by an ATP-sensitive K+channel opener. Considering the above findings regarding the inhibitory effects of bupivacaine enantiomers, it is likely that S (−)-enantiomers of local anesthetics show less potent effects on vasorelaxation mediated by ATP-sensitive K+channels. In addition, ropivacaine, compared with S (−)-bupivacaine, seems to have less impact on these K+channels of vascular smooth muscle cells.
The ATP-sensitive K+channel is a complex of two proteins: the sulfonylurea receptor and Kir6.1 or 6.2, which belongs to the inward rectifier K+channel family.13Because the sulfonylurea receptor of ATP-sensitive K+channel is reportedly a primary target of the openers of this channel, it is most likely that bupivacaine enantiomers modify vasorelaxation in response to an ATP-sensitive K+channel opener via the effect on the sulfonylurea receptor of these channels.14However, a recent study has found that racemic bupivacaine inhibits G protein–gated inward rectifier K+channels by antagonizing the interaction of phosphatidylinositol 4,5-bisphosphate with the channel.15Therefore, we cannot rule out the possibility that bupivacaine may act on the compartment of inward rectifier K+channel family in ATP-sensitive K+channels. In any case, it is highly possible that bupivacaine, which is a lipophilic anesthetic, directly affects some channel compartments because recent studies have already reported such direct action of bupivacaine on voltage-dependent K+channel proteins.16,17
Each compound evaluated in the current study, even in the highest concentration used, did not affect vasorelaxation in response to a voltage-dependent Ca2+channel antagonist diltiazem. These results may support the concept that bupivacaine does not inhibit vasodilator responses in general. Our findings that neither bupivacaine enantiomers nor ropivacaine affect contraction in response to phenylephrine and maximal relaxation induced by papaverine (data not shown) also neglect the possibility that the inhibitory effect of bupivacaine on vasorelaxation mediated by ATP-sensitive K+channels is due to its vasoconstrictor effect on the aorta.
Thresholds of a free plasma concentration in humans for central nervous toxicity were reported up to 1.5 × 10−6m and 2.6 × 10−6m for racemic bupivacaine and ropivacaine, respectively.18A recent study has found that free plasma concentrations higher than 1.5 × 10−6m are seen in infants during epidural infusion of bupivacaine because of a low α-1 acid glycoprotein concentration.19Therefore, our results suggest that in clinical situations, bupivacaine enantiomers, especially R (+)-bupivacaine, impair vasodilation mediated by ATP-sensitive K+channels, whereas ropivacaine does not alter the vasodilation.
During hypoxia, acidosis, and ischemia, ATP-sensitive K+channels are activated, resulting in arterial dilation or increased tolerance of tissues to ischemia or both.6,20,21Although it is still unclear whether our results have relevance to vasodilation in resistance blood vessels, during pathophysiologic situations, bupivacaine enantiomers, especially R (+)-bupivacaine, but not ropivacaine, may impair vasodilator effects induced by activation of ATP-sensitive K+channels, which play an important role in regulation of circulation.
In conclusion, this is the first study evaluating the effects of amide-linked long-acting local anesthetics, including bupivacaine enantiomers and ropivacaine, on K+channels of vascular smooth muscle. From our results, S (−)-enantiomers of amide-linked local anesthetics seem to show less potent effects on vasorelaxation mediated by ATP-sensitive K+channels. In these S (−)-isomers, ropivacaine may not affect these K+channels of vascular smooth muscle cells.