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Anesthesiology:
Laboratory Investigations

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

Kawano, Takashi M.D.*; Oshita, Shuzo M.D.†; Takahashi, Akira M.D.‡; Tsutsumi, Yasuo M.D.*; Tomiyama, Yoshinobu M.D.§; Kitahata, Hiroshi M.D.∥; Kuroda, Yasuhiro M.D.#; Nakaya, Yutaka M.D.**

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Abstract

Background: Sarcolemmal adenosine triphosphate–sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits.
Methods: The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs.
Results: Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 μm) stereoselectively (levobupivacaine, IC50 = 168 μm; ropivacaine, IC50 = 249 μm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2ΔC36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics’ affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit.
Conclusions: Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.
ACCIDENTAL overdose of bupivacaine, a widely used local anesthetic, can cause serious cardiovascular collapse and ventricular arrhythmias, often refractory to resuscitation.1,2 One of the primary mechanisms of bupivacaine-induced cardiotoxicity is that it blocks cardiac Na+ channels, which it binds preferentially in the inactivated channel state.3 Blockade of voltage-gated K+ (Kv) channels by bupivacaine may also contribute to the bupivacaine effect by prolonging the duration of cardiac action potential.4,5 The cardiotoxicity of bupivacaine is greatly enhanced and potentiated by multiple factors, including hypoxia, acidosis, and hyperkalemia.6,7 Under these metabolic stress conditions, sarcolemmal adenosine triphosphate (ATP)–sensitive potassium (KATP) channels in cardiomyocytes and vascular smooth muscles are activated, causing shortening of the action potential duration and vasodilatation, respectively.8–11 It is therefore possible that sarcolemmal KATP channels in the cardiovascular system could be a target for bupivacaine-induced cardiotoxicity. It has been reported that bupivacaine-induced atrioventricular conduction block can be reversed by KATP channel openers in isolated guinea pig hearts.12
Bupivacaine contains an asymmetric carbon, and it is used clinically as a racemic mixture. The inhibitory effects of R(+)-bupivacaine on Na+ channels and hKv 1.5 channels are more potent than S(−)-bupivacaine (levobupivacaine), indicating stereoselective channel block,13,14 and the cardiotoxicity of R(+)-bupivacaine is more potent than that of S(−)-bupivacaine.15,16 Gonzalez et al.17 analyzed and compared the effects of R(+)-bupivacaine and S(−)-bupivacaine on HERG K+ channels expressed in CHO cells, and revealed that the inhibitory effects of R(+)-bupivacaine were lower than those induced by S(−)-bupivacaine, indicating stereoselective channel block. They also compared the effects of levobupivacaine, ropivacaine, and bupivacaine on HERG K+ channels and revealed that, similar to the relations between R(+)-bupivacaine and S(−)-bupivacaine, the inhibitory effects induced by racemic bupivacaine were lower than those induced by levobupivacaine. Both levobupivacaine and ropivacaine are the pure S(−)-enantiomers of N-butyl-pipecoloxylidide and N-propyl-pipecoloxylidide, i.e., they only differ in the length of the N-substituent, and are less cardiotoxic alternatives to racemic bupivacaine. These results suggest that small changes in the chemical structure of local anesthetics may cause different effects and that it would be possible to estimate at least a part of stereoselective effects of local anesthetics on sarcolemmal KATP channels in the cardiovascular system.
In the current study, to evaluate the stereoselectivity and tissue specificity of the effects of local anesthetics on cardiovascular KATP channels, we analyzed and compared the electrophysiologic effects and molecular mechanisms of bupivacaine, levobupivacaine, and ropivacaine on sarcolemmal KATP channel activities in both cardiomyocytes and vascular smooth muscle cells. We used the patch clamp method to study the effects of local anesthetics on KATP channels containing wild-type, mutant, or chimeric sulfonylurea receptors expressed in COS-7 cells.
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Materials and Methods

Molecular Biology
In the cardiovascular system, KATP channels are present in both cardiomyocytes and vascular smooth muscles and have a range of electrophysiologic properties because of their diverse molecular composition.9,18 It was recently found that the cardiac KATP channel isoform is composed of sulfonylurea receptor (SUR) 2A and inward-rectifier K+ channel subunit (Kir) 6.2 (SUR2A/Kir6.2), whereas the vascular KATP channel isoform may be a complex of SUR2B/Kir6.1.18,19
The human Kir6.2, rat Kir6.1, rat SUR1, rat SUR2A, and rat SUR2B cDNAs were kindly provided by Susumu Seino, M.D., Ph.D. (Professor and Chairman, Department of Cellular and Molecular Medicine, Chiba University, Chiba, Japan). A truncated form of human Kir6.2 lacking the last 36 amino acids at the C terminus was obtained by polymerase chain reaction amplification as previous described.20 Site-directed mutagenesis was carried out using a Site-Directed Mutagenesis system (Invitrogen Corp., Carlsbad, CA). Chimeric SURs (SUR1–2A, SUR1–2B, and SUR2–1) were made by exchanging the 42 C-terminal amino acids of SUR1 or SUR2A with those of SUR2A, SUR2B, and SUR1 with the overlap extension polymerase chain reaction method. All DNA products were sequenced by using a BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 377 DNA sequencer (Applied Biosystems) to confirm the sequence.
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Cell Culture and Transfection
COS-7 cells were plated at a density of 3 × 105 per 35-mm dish and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. A full-length Kir complementary DNA (cDNA) and a full-length SUR cDNA were subcloned into the mammalian expression vector pCMV6c. For electrophysiologic recordings, either wild-type or mutated pCMV6c Kir alone (1 μg), or pCMV6c Kir (1 μg) plus pCMV6c SUR (1 μg), were transfected into COS-7 cells with green fluorescent protein cDNA (pEGFP) as a reporter gene using the Lipofectamine and Opti-MEM 1 reagents (Life Technologies Inc., Rockville, MD) according to the manufacturer’s instructions. After transfection, cells were cultured for 48–72 h before being subjected to electrophysiologic recordings.
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Electrophysiologic Measurements
Membrane currents were recorded in the inside-out configurations using a patch clamp amplifier as described previously.20,21 Transfected cells were identified by detecting the green fluorescence under a fluorescence microscope. The intracellular solution contained 140 mm KCl, 2 mm EGTA, 2 mm MgCl2, and 10 mm HEPES (pH 7.3). The pipette solution contained 140 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 7.4). Recordings were made at 36 ± 0.5°C. Patch pipettes were pulled with an electrode puller (PP-830; Narishige, Tokyo, Japan). The resistance of pipettes filled with internal solution and immersed in the bath solution was 3–7 MΩ. The sampling frequency of the single-channel data was 5 KHz with a low-pass filter (1 KHz).
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Electrophysiologic Data Analysis
Equation (Uncited)
Equation (Uncited)
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Channel currents were recorded with a patch clamp amplifier (CEZ 2200; Nihon Kohden, Tokyo, Japan) and stored in a personal computer (Aptiva; International Business Machine Corporation, Armonk, NY) with an analog-to-digital converter (DigiData 1200; Axon Instruments, Foster, CA). pClamp version 7 software (Axon Instruments) was used for data acquisition and analysis. The open probability (Po) was determined from current amplitude histograms and was calculated as follows:
where tj is the time spent at current levels corresponding to j = 0, 1, 2, N channels in the open state; Td is the duration of the recording; and N is the number of channels active in the patch. Recordings of 2–3 min were analyzed to determine Po. The channel activities were expressed as NPo. Changes of NPo in the presence of drugs were calculated as the relative NPo between the values obtained before and after drug treatment. When the concentration-dependent effects of bupivacaine, levobupivacaine, and ropivacaine were studied, these drugs were injected into the cell bath using a glass syringe to the final five concentrations in a cumulative manner (total volume injected was approximately 10–20 μl).
Equation (Uncited)
Equation (Uncited)
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The drug concentration needed to induce half-maximal inhibition of the channels (IC50) and the Hill coefficient were calculated as follows:
where y is the relative NPo, [D] is the concentration of drug, Ki is IC50, and nH is the Hill coefficient.
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Drugs
The following drugs were used: levobupivacaine (S(−)-bupivacaine; Aldrich Chemical Co., Milwaukee, WI), ropivacaine (AstraZeneca, Osaka, Japan), bupivacaine, glibenclamide, diazoxide, and pinacidil (Sigma-Aldrich Japan, Tokyo, Japan). Bupivacaine, levobupivacaine, and ropivacaine were dissolved in distilled deionized water to yield stock solutions of 1 m from which further dilutions were made to obtain the desired final concentrations. Glibenclamide and pinacidil were dissolved in dimethyl sulfoxide (the final concentration of solvent was 0.01%); dimethyl sulfoxide at a concentration of 0.02% did not affect KATP channel currents.
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Statistics
All data are presented as mean ± SD. Differences between data sets were evaluated either by repeated-measures one-way analysis of variance followed by Scheffé F test or by Student t test. P < 0.05 was considered significant.
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Results

Single-channel Characteristics of Recombinant KATP Channel Currents
Fig. 1
Fig. 1
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As in a previous study,20 inwardly rectifying currents were obtained from excised inside-out patches in COS-7 cells transiently expressing two types of KATP channel isoforms, the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, with single-channel conductances of 70 ± 4 pS (n = 25) and 28 ± 3 pS (n = 24), respectively (fig. 1A). Figure 1B shows representative examples of these currents. When the patch was excised into a nucleotide-free solution, the SUR2A/Kir6.2 channel showed marked current increases that were blocked by 1 mm ATP, whereas the SUR2B/Kir6.1 channel was not spontaneously activated. Both channels were stimulated by 100 μm pinacidil and inhibited by 3 μm glibenclamide (fig. 1B).
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Effect of Anesthetics on Recombinant KATP Channels
Fig. 2
Fig. 2
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Fig. 3
Fig. 3
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Fig. 4
Fig. 4
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To assess the effects of bupivacaine, levobupivacaine, and ropivacaine on recombinant KATP channels, we measured single-channel currents on inside-out patches in the presence of these drugs. Application of 1 mm bupivacaine, levobupivacaine, or ropivacaine to the intracellular membrane surface inhibited the SUR2A/Kir6.2 channel currents, with relative NPo decreasing to 0.16 ± 0.08, 0.29 ± 0.08, and 0.31 ± 0.07, respectively (fig. 2). The SUR2B/Kir6.1 currents were also inhibited by bupivacaine, levobupivacaine, and ropivacaine at 1 mm, with relative NPo decreasing to 0.35 ± 0.07, 0.48 ± 0.12, and 0.53 ± 0.14, respectively (fig. 3). The inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on KATP NPo were reversible because the channel currents recovered after washout (figs. 2, 3). The effects were also concentration dependent (fig. 4).
Table 1
Table 1
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To further probe the mechanism of these anesthetics, we next tested their effects on the SUR2B/Kir6.2 currents. The IC50s and Hill coefficients of bupivacaine, levobupivacaine, and ropivacaine for the SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.1 channels are summarized in table 1. Bupivacaine inhibited the SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.2 channel activities with higher potency than levobupivacaine, indicating that the inhibitory effect of bupivacaine on KATP channels might be stereoselective. In addition, all of the anesthetics inhibited the SUR2A/Kir6.2 channels with higher potency than the SUR2B/Kir6.2 and SUR2B/Kir6.1 channels, indicating that the inhibition is dependent on the SUR subtype. The blockades by bupivacaine, levobupivacaine, and ropivacaine did not significantly change the conductance of the recombinant KATP channels in any case (data not shown).
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Effect of Anesthetics on Kir6.2ΔC36 Channel Activity
Wild-type Kir6.2 alone does not show functional channel activity, whereas a C-terminal truncated pore-forming subunit of Kir6.2 (Kir6.2ΔC36) lacking the last 36 amino acids is capable of forming a functional channel in the absence of SUR.22 This has proved to be a useful tool for discriminating the site at which various agents act on KATP channels.
Fig. 5
Fig. 5
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Adenosine triphosphate–sensitive inwardly rectifying currents were recorded from COS-7 cells transiently expressing the Kir6.2ΔC36 channel (fig. 5A), as was done previously.20 Figure 5A also shows that bupivacaine at 1 mm reversibly inhibited the Kir6.2ΔC36 currents, with relative NPo decreasing to 0.34 ± 0.12. The concentration-dependent effects of bupivacaine, levobupivacaine, and ropivacaine on the Kir6.2ΔC36 currents are shown in figure 5B. These observations suggest that the high-affinity site as well as the stereoselective mechanism of bupivacaine is located on the Kir6.0 subunit. The IC50 and Hill coefficients of bupivacaine, levobupivacaine, and ropivacaine for Kir6.2ΔC36 currents (table 1) indicate that the inhibitory effect of these anesthetics on the Kir6.0 subunit was enhanced by coexpression with SUR2A but not with SUR2B.
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Effect of Anesthetics on Mutated Kir6.2 Molecules
Fig. 6
Fig. 6
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We next used several site-specific mutations of Kir6.2ΔC36 channels to identify the regions of Kir6.2 that play critical roles in the bupivacaine-mediated inhibition of Kir6.2ΔC36 channel activity. Kir6.0 subunits possess two transmembrane domains, TM1 and TM2, linked by a pore loop, H5 (fig. 6A). The N and C termini are intracellular and contain the binding sites for intracellular ligands involved in ATP inhibition (arginine 50 [R50] and lysine 185 [K185]),23 pH sensitivity (histidine 175 [H175]),24 and channel gating (cysteine 166 [C166] and threonine 171 [T171])23,25 (fig. 6B).
The effects of 1 mm bupivacaine and 1 mm levobupivacaine, which inhibit wild-type Kir6.2ΔC36 currents in a stereoselective manner, were tested on ATP-insensitive mutants (R50G, K185Q), a pH-insensitive mutant (H175A), and mutants that change the channel gating (C166S, T171A). Bupivacaine and levobupivacaine block Kir6.2ΔC36-R50G, -K185Q, and -H175A currents as effectively as Kir6.2ΔC36 currents (fig. 6C), indicating that the bupivacaine-inhibitory site is not identical to that for ATP (R50 and K185) and protons (H175). In contrast, both bupivacaine affinity and stereoselectivity were decreased by the Kir6.2ΔC36-C166S and -T171A mutations. This indicates that the cytosolic end of the second transmembrane domain of Kir6.2 may play an important role in both affinity and stereoselectivity for bupivacaine binding.
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Effect of the C-terminal Tails of SUR2 Molecules on Anesthesia Sensitivity
Fig. 7
Fig. 7
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Sulfonylurea receptor 2A and SUR2B are splicing isoforms generated from a single gene that differ from each other only in the 42 amino acid segment at their C-terminal ends.26 The C-terminal segment may therefore be responsible for the differential effects of bupivacaine on the KATP channels containing these SUR2 isoforms. The C-terminal 42 amino acids (C42) of SUR2B possess only approximately 30% homology with those of SUR2A but approximately 70% with those of SUR1. Therefore, to clarify the role of C42, we examined the effect of the anesthetics on SUR1 and several chimeric molecules of SUR1 and SUR2 (fig. 7A).
Sulfonylurea receptor 1 and the chimeras SUR2-1, SUR1-2A, and SUR1-2B formed a fully functional KATP channels with Kir6.2. The bupivacaine sensitivity of the Kir6.2ΔC36 channels was not enhanced by coexpression with SUR1 and SUR2-1 at almost similar concentrations to the SUR2B/Kir6.2 channel (fig. 7B). In contrast, SUR1-2A and SUR2A enhanced the bupivacaine sensitivity of Kir6.2ΔC36 channels, although SUR1-2B did not (fig. 7B). These results strongly indicate that SUR2A-C42 plays a specific functional role in the control of the inhibitory effect of bupivacaine on the KATP channel.
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Discussion

In the current study, we expressed reconstituted SUR2A/Kir6.2 and SUR2B/Kir6.1 channels in COS-7 cells and demonstrated that bupivacaine, levobupivacaine, and ropivacaine inhibit these channels in a concentration-dependent manner. Bupivacaine was approximately threefold more potent than levobupivacaine and ropivacaine in blocking both channels. Furthermore, the potency of the local anesthetics to block SUR2A/Kir6.2 channels was approximately eightfold higher than the ability to block SUR2B/Kir6.1 channels. These results suggest that bupivacaine enantiomers and ropivacaine act in a stereoselective and tissue-specific way to inhibit sarcolemmal KATP channels in the cardiovascular system.
Ropivacaine and levobupivacaine, both S(−) pure enantiomer local anesthetics, exhibit a common chemical structure; they differ only in the length of the alkyl substituent of the tertiary nitrogen (position 1), which is a propyl group for ropivacaine and a butyl group for levobupivacaine. Several studies have shown that the length of this side chain is a structural determinant of the ability of these local anesthetics to inhibit cardiac Na+ channels, transient outward K+ channels, and the human Kv1.5 channels4,27,28: the longer the side chain, the higher the potency of the drug. In the current study, ropivacaine blockade of recombinant KATP channel currents is of lower potency than that of levobupivacaine, suggesting that the ability of bupivacaine-type local anesthetics to block KATP channels is also related to the length of their N-substituent.
The KATP channel is a hetero-octamer composed of two subunits: the pore-forming subunit Kir6.0 (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1, SUR2A, or SUR2B).9 Distinct from other inward-rectifier K+ channels, Kir6.0 subunits do not express functional channels by themselves. However, removal of the last 26 or 36 C-terminal amino acids of Kir6.2 (Kir6.2ΔC26 or Kir6.2ΔC36) results in channels that show significant currents in the absence of SUR.22 Our study shows that bupivacaine, levobupivacaine, and ropivacaine inhibit the Kir6.2ΔC36 channels as well as the reconstituted sarcolemmal KATP channels. In addition, the Hill coefficient for the inhibitory effects of bupivacaine was close to unity for SUR2A/Kir6.2, SUR2B/Kir6.1, SUR2B/Kir6.2, and Kir6.2ΔC36 channels, indicating that only a single anesthetic molecule needs to bind for the channel to close. These results also suggest that the high-affinity site of bupivacaine is located on the Kir6.0 subunit. In addition, bupivacaine blockade of Kir6.2ΔC36 currents is more potent than blockade by levobupivacaine and ropivacaine. Therefore, the stereoselectivity of the blockade of KATP channels by bupivacaine strongly indicates that it results from binding to a specific binding site on the Kir6.0 subunit.
It is well known that the intracellular domains of the Kir6.2 subunit possess binding sites for ATP (R50, K185) and protons (H175).23,24 The bupivacaine inhibitory site, however, is not identical to the ATP- and proton-binding sites because mutations that markedly decrease the sensitivity to ATP (R50G, K185Q) and protons (H175A) were without significant effect on bupivacaine block.
Recent electrophysiologic studies have indicated that the second transmembrane domain of the Kir6.0 channel lines the intracellular mouth of the pore, because mutations within this region affect the channel gating kinetics.23,25 The residues at cytosolic end of the second transmembrane domain of Kir6.2 are critical for the gating of Kir6.2ΔC36 because most mutations at this position, including C166S and T171A, substantially affected the single-channel kinetics; dramatic decrease occurred in the frequency of the long close channel state.23,25 Mutations in this region, C166S and T171A, reduced the inhibitory effects of bupivacaine and levobupivacaine, which may result from changing the channel gating kinetics rather than altering the affinity of these anesthetics for its binding site. These mutations also completely abolished the stereoselective block observed in wild-type Kir6.2ΔC36 channels. An equivalent region (the cytosolic end of the sixth transmembrane segments) has been shown to participate in the gating of both Kv channels and cyclic nucleotide-gated channels.29–31 Furthermore, mutations within this region of Kv channels have a similar effect on bupivacaine, markedly decreasing both the affinity and stereoselectivity of bupivacaine binding.14 The bupivacaine binding site located at the intracellular end of the pore may therefore be a common feature of K+ channels.
Our IC50 values for bupivacaine, levobupivacaine, and ropivacaine show that the SUR2A/Kir6.2 channels are more sensitive than the Kir6.2ΔC36 channels. These results indicate that although the primary site at which these anesthetics inhibit the KATP channel resides on Kir6.2, the presence of SUR2A enhances the blockade. In contrast, the IC50 values for the SUR2B/Kir6.1 and SUR2B/Kir6.2 channels are similar to that for Kir6.2ΔC36 despite differences in the Kir6.0 subunit, suggesting that SUR2B does not regulate the sensitivity to anesthetics. SUR2A and SUR2B are generated from a single gene and differ only in their 42 amino acid residues at the C-terminal tail (C42; amino acids 1505–1546).26 Therefore, these anesthetic-mediated inhibitions of the Kir6.0 subunit might be enhanced by the SUR2A tail but not affected by the SUR2B tail. The experiments using the SUR1 chimera whose C-terminal segment was replaced with that of either SUR2A or SUR2B strongly support this possibility. Therefore, the tissue specificity of the effects of these anesthetics on KATP channels is likely due to the presence of different SURs.
High concentrations of bupivacaine act on the cardiovascular system as a depressant; the primary site of action is the cardiomyocytes, but arteriolar vasodilatation is also produced.1,2 The cardiotoxic effects of bupivacaine are primarily explained by the blockade of voltage-sensitive Na+ channels.3 Bupivacaine binds much more potently to Na+ channels during the action potential plateau when the Na+ channels are inactivated.3 Furthermore, hypoxia, acidosis, and hyperkalemia commonly occur during local anesthetic toxicity and greatly potentiate the cardiotoxicity of bupivacaine.6,7 These conditions result in partial depolarization,32 which increases the fraction of voltage-sensitive Na+ channels in the inactivated state during diastole. Shortening of action potential duration and hyperpolarization of resting membrane potential have been suggested as potentially effective approaches in the treatment of bupivacaine-induced cardiotoxicity.4 Sarcolemmal KATP channels in cardiomyocytes, silent under physiologic conditions, are activated during metabolic stress, such as ischemia, acidosis, and hypoxia.8,9 The opening of these channels results in membrane hyperpolarization and a shortening of the action potential duration,33 resulting in a decrease in Ca2+ influx through voltage-dependent Ca2+ channels. Thus, bupivacaine-induced blockade of sarcolemmal KATP channels, especially in the presence of hypoxemia and acidosis, leads to a prolongation of the action potential duration, which might be expected to enhance the severity of bupivacaine-induced cardiotoxicity. This is confirmed by the fact that the KATP channel openers pinacidil and bimakalim attenuate the bupivacaine-induced atrioventricular conduction block.12 In addition to cardiac effects, high concentrations of local anesthetics produce vasodilatation, which may enhance the bupivacaine-induced cardiovascular collapse.1,2 In vascular smooth muscle cells, activation of KATP channels, which leads to an increase in K+ conductance and hyperpolarization of the cell membrane, results in vascular relaxation.26 Therefore, the inhibitory effects of bupivacaine on sarcolemmal KATP channels in vascular smooth muscle indicate that these channels are not involved in the vasodilatation effects of bupivacaine.
An experimental study performed in sheep demonstrated that intravascular injection of bupivacaine can result in transient plasma concentrations of the free form of bupivacaine of 4–12 μg/ml (12–36 μm),34 assuming a blood-to-plasma concentration ratio of 0.73, which caused a serious depression of cardiac conduction that was difficult to reverse. The threshold concentrations at which bupivacaine (> 10 μm), levobupivacaine (> 30 μm), and ropivacaine (> 30 μm) inhibit reconstituted SUR2A/Kir6.2 channels are within this range. Some types of Kv channels in cardiomyocytes are also inhibited by these local anesthetics.4,5 Therefore, the suppression of sarcolemmal SUR2A/Kir6.2 channel activities together with that of other K+ channels, especially during metabolic stress, would be expected to be accompanied by prolongation of the action potential, which can lead to higher bupivacaine cardiotoxicity. On the other hand, the concentrations of these local anesthetics that inhibited vascular KATP channel activities in the current study are very high (> 100 μm), suggesting that sarcolemmal SUR2A/Kir6.2 channels but not SUR2B/Kir6.1 channels may contribute to local anesthetic-induced cardiovascular toxicity.
Our study has several limitations. First, because one of the aims of the current study was to obtain an information about the structural determinants of stereoselective inhibition of KATP channels using clinically available local anesthetics, we compared the inhibitory effects of racemic bupivacaine and S(−)-bupivacaine on KATP channel activities. Based on the report of Gonzalez et al.17 showing that the inhibitory effects of both R(+)-bupivacaine and racemic bupivacaine on HERG K+ channel activities were lower than those induced by S(−)-bupivacaine, we expected it would be possible to estimate at least a part of stereoselective effects of bupivacaine on sarcolemmal KATP channels in the cardiovascular system. However, the racemic mixture may exhibit properties that are not shared by either of the isomers, as a result of interactions between the isomers. Therefore, it should be more appropriate to compare the effects of R(+)-bupivacaine and S(−)-bupivacaine to know stereoselective inhibition of bupivacaine on KATP channel activities. Second, as we discussed above, the threshold concentrations at which bupivacaine inhibited reconstituted SUR2A/Kir6.2 channels in the current study (>10 μm) are within plasma concentrations of bupivacaine (12–36 μm) obtained after intravascular injection of bupivacaine in sheep.34 Because we used inside-out patch clamp configurations, the threshold concentrations of bupivacaine obtained in the current study should be compared with intracellular bupivacaine concentrations. However, we are unaware of any report evaluating intracellular bupivacaine concentrations especially in an accidental overdose of bupivacaine.
In conclusion, the Kir6.0 subunit mediates the inhibition of reconstituted sarcolemmal SUR2A/Kir6.2 and SUR2B/Kir6.1 channel activities by bupivacaine, levobupivacaine, and ropivacaine. These inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are stereoselective, with bupivacaine more potent than levobupivacaine and ropivacaine, and tissue specific, with local anesthetics blocking SUR2A/Kir6.2 channels more potently than SUR2B/Kir6.1 channels. Our results further suggest that determinant domains are located at the intracellular pore mouth of Kir6.0 subunit for stereoselectivity and in the 42 amino acids at the C-terminal tail of SUR2A subunit for tissue specificity.
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References

1.Albright GA: Cardiac arrest following regional anesthesia with etidocaine or bupivacaine. Anesthesiology 1979; 51:285–7

2.Marx GF: Cardiotoxicity of local anesthetics: The plot thickens. Anesthesiology 1984; 60:3–5

3.Clarkson CW, Hondeghem LM: Mechanism for bupivacaine depression of cardiac conduction: Fast block of sodium channels during the action potential with slow recovery from block during diastole. Anesthesiology 1985; 62:396–405

4.Castle NA: Bupivacaine inhibits the transient outward K+ current but not the inward rectifier in rat ventricular myocytes. J Pharmacol Exp Ther 1990; 255:1038–46

5.Valenzuela C, Delpon E, Tamkun MM, Tamargo J, Snyders DJ: Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J 1995; 69:410–27

6.Moore DC, Thompson GE, Crawford RD: Long-acting local anesthetic drugs and convulsions with hypoxia and acidosis. Anesthesiology 1982; 56:230–2

7.Rosen MA, Thigpen JW, Shnider SM, Foutz SE, Levinson G, Koike M: Bupivacaine-induced cardiotoxicity in hypoxic and acidotic sheep. Anesth Analg 1985; 64:1089–96

8.Noma A: ATP-regulated K+ channels in cardiac muscle. Nature 1983; 305:147–148

9.Ashcroft FM, Ashcroft SJH: Properties and functions of ATP-sensitive K-channels. Cell Signaling 1990; 2:197–214

10.Daut J, Maier-Rudolph W, von Beckerath N, Mehrke G, Gunther K, Goede-Meinen L: Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels. Science 1990; 247:1341–4

11.Wang X, Wu J, Li L, Chen F, Wang R, Jiang C: Hypercapnic acidosis activates KATP channels in vascular smooth muscles. Circ Res 2003; 92:1225–32

12.Boban M, Stowe DF, Gross GJ, Pieper GM, Kampine JP, Bosnjak ZJ: Potassium channel openers attenuate atrioventricular block by bupivacaine in isolated hearts. Anesth Analg 1993; 76:1259–65

13.Valenzuela C, Snyders DJ, Bennett PB, Tamargo J, Hondeghem LM: Stereoselective block of cardiac sodium channels by bupivacaine in guinea pig ventricular myocytes. Circulation 1995; 92:3014–24

14.Franqueza L, Longobardo M, Vicente J, Delpon E, Tamkun MM, Tamargo J, Snyders DJ, Valenzuela C: Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels. Circ Res 1997; 81:1053–64

15.Mazoit JX, Decaux A, Bouaziz H, Edouard A: Comparative ventricular electrophysiologic effect of racemic bupivacaine, levobupivacaine, and ropivacaine on the isolated rabbit heart. Anesthesiology 2000; 93:784–92

16.Santos AC, DeArmas PI: Systemic toxicity of levobupivacaine, bupivacaine, and ropivacaine during continuous intravenous infusion to nonpregnant and pregnant ewes. Anesthesiology 2001; 95:1256–64

17.Gonzalez T, Arias C, Caballero R, Mereno I, Deplon E, Tamargo J, Valenzuela C: Effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels: Stereoselective bupivacaine block. Br J Pharmacol 2002; 137:1269–79

18.Yamada M, Isomoto S, Matsumoto S, Kondo C, Shindo T, Horio Y, Kurachi Y: Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel. J Physiol 1997; 499:715–20

19.Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, Seino S: A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 1996; 16:1011–7

20.Kawano T, Oshita S, Takahashi A, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Nakaya Y: Molecular Mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate–sensitive potassium channels. Anesthesiology 2004; 100:338–46

21.Kawano T, Oshita S, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Takahashi A, Nakaya Y: Clinically relevant concentrations of propofol have no effect on adenosine triphosphate–sensitive potassium channels in rat ventricular myocytes. Anesthesiology 2002; 96:1472–7

22.Tucker SJ, Gribble FM, Zhao C, Trapp S, Ashcroft FM: Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 1997; 387:179–83

23.Tucker SJ, Gribble FM, Proks P, Trapp S, Ryder TJ, Haug T, Reimann F, Ashcroft FM: Molecular determinants of KATP channel inhibition by ATP. EMBO J 1998; 17:3290–6

24.Xu H, Wu J, Cui N, Abdulkadir L, Wang R, Mao J, Giwa LR, Chanchevalap S, Jiang C: Distinct histidine residues control the acid-induced activation and inhibition of the cloned KATP channel. J Biol Chem 2001; 276:38690–6

25.Trapp S, Proks P, Tucker SJ, Ashcroft FM: Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP. J Gen Physiol 1998; 112:333–49

26.Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, Matsuzawa Y, Kurachi Y: A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel. J Biol Chem 1996; 271:24321–4

27.Courtney KR: Structure-activity relations for frequency-dependent sodium channel block in nerve by local anesthetics. J Pharmacol Exp Ther 1980; 213:114–9

28.Longobardo M, Delpon E, Caballero R, Tamargo J, Valenzuela C: Structural determinants of potency and stereoselective block of hKv1.5 channels induced by local anesthetics. Mol Pharmacol 1998; 54:162–9

29.Liu Y, Holmgren M, Jurman ME, Yellen G: Gated access to the pore of a voltage-dependent K+ channel. Neuron 1997; 19:175–84

30.Loukin SH, Vaillant B, Zhou XL, Spalding EP, Kung C, Saimi Y: Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1. EMBO J 1997; 16:4817–25

31.Zong X, Zucker H, Hofmann F, Biel M: Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels. EMBO J 1998; 17:353–62

32.Bethell HW, Vandenberg JI, Smith GA, Grace AA: Changes in ventricular repolarization during acidosis and low-flow ischemia. Am J Physiol 1998; 275:H551–61

33.Nichols CG, Ripoll C, Lederer WJ: ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction. Circ Res 1991; 68:280–7

34.Kotelko DM, Shnider SM, Dailey PA, Brizgys RV, Levinson G, Shapiro WA, Koike M, Rosen MA: Bupivacaine-induced cardiac arrhythmias in sheep. Anesthesiology 1984; 60:10–8

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