Interaction of ropivacaine with cloned cardiac Kv4.3/KChIP2.2 complexes

BACKGROUND: Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels. METHODS: Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method. RESULTS: Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 microm (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 microm (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels. CONCLUSIONS:: The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.     

Inhibition of Kv4.3/KChIP2.2 Channels by Bupivacaine and Its Modulation by the Pore Mutation Kv4.3V401I

Background: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated.

Methods: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels.

Results: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 [mu]m, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2.     

Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I

BACKGROUND: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. METHODS: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. RESULTS: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. CONCLUSIONS: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.     

Long QT 1 Mutation KCNQ1A344V Increases Local Anesthetic Sensitivity of the Slowly Activating Delayed Rectifier Potassium Current

Background: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia.

Methods: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings.

Results: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 [mu]m, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 [mu]m, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication.     

Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

Background: Kv[beta]1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kv[beta]1.3 competes with them for a binding site at Kv1.5 channels.

Methods: Currents generated by the activation of Kv1.5 and Kv1.5 + Kv[beta]1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques.

Results: Block of Kv1.5, but not that produced on Kv1.5 + Kv[beta]1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 [mu]m (n = 15) and 39.8 +/- 8.2 [mu]m (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 [mu]m) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 [mu]m) in Kv1.5 + Kv[beta]1.3 channels. Therefore, the degree of stereoselectivity ([theta]) decreased from 9 to 4 in the presence of Kv[beta]1.3. The decrease in potency to block Kv1.5 + Kv[beta]1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kv[beta]1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kv[beta]1.3 binding) but not for L510A (part of Kv[beta]1.3 binding site).     

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current

BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. METHODS: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings. RESULTS: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 microM, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 microM, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. CONCLUSIONS: The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional anesthesia.     

Kvbeta1.3 reduces the degree of stereoselective bupivacaine block of Kv1.5 channels

BACKGROUND: Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. METHODS: Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. RESULTS: Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). CONCLUSIONS: The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.     

Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine

Background: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic.

Methods: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique.

Results: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 [mu]m, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 [mu]m) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential.     

Stereoselectivity of Bupivacaine in Local Anesthetic-sensitive Ion Channels of Peripheral Nerve

Background: The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)- and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel.

Methods: In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k-1) were determined.

Results: Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/-SEM) for tonic Na+ channel block were 29 +/- 3 [mu]M and 44 +/- 3 [mu]M, respectively. IC50 values for flicker K+ channel block were 0.15 +/- 0.02 [mu]M and 11 +/- 1 [mu]M, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k-1 were 0.83 +/- 0.13 x 106 M-1 [middle dot] s-1 and 0.13 +/- 0.03 s-1, respectively, for R(+)-bupivacaine and 1.90 +/- 0.20 x 106 M-1 [middle dot] s-1 and 8.3 +/- 1.0 s-1, respectively, for S(-)-bupivacaine.     

Enantioselective actions of bupivacaine and ropivacaine on coronary vascular resistance at cardiotoxic concentrations

The main concern with the use of the long-acting local anesthetics bupivacaine and ropivacaine is inadvertent IV injection, which exposes the heart to toxic drug concentrations. We tested the hypothesis that these chiral anesthetics exert enantioselective actions on coronary vascular tone, the regulation of which does not involve voltage-gated Na(+) channels. Coronary perfusion pressure (CPP) was continuously measured in isolated hearts perfused via the aorta at a constant flow rate. This method provides a sensitive assay of coronary vascular resistance in the intact heart. In parallel experiments, we examined the effects of bupivacaine and ropivacaine on intracellular [Ca(2+)] in coronary endothelial cells. In addition, the effect of bupivacaine on mitochondrial membrane potential was assessed using isolated ventricular myocytes. Racemic bupivacaine and R(+)-bupivacaine produced similar dose-dependent decreases in CPP. However, S(-)-bupivacaine, S(-)-ropivacaine and R(+)-ropivacaine increased CPP. In contrast to adenosine triphosphate, neither racemic bupivacaine nor S(-)-ropivacaine changed endothelial intracellular [Ca(2+)], suggesting that these clinically used drugs do not modulate endothelial nitric oxide synthase. We also showed that the putative uncoupler bupivacaine did not depolarize mitochondria in intact ventricular myocytes. In conclusion, the long-acting local anesthetics have enantioselective actions on coronary resistance vessels. Racemic bupivacaine and R(+)-bupivacaine are coronary vasodilators, whereas S(-)-bupivacaine, S(-)-ropivacaine and, to a lesser extent, R(+)-ropivacaine all induce coronary vasoconstriction.     

Local Anesthetic Inhibition of Baseline Potassium Channels with Two Pore Domains in Tandem

Background: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels.

Methods: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques.

Results: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 [micro sign]M mepivacaine, 222 [micro sign]M lidocaine, 51 [micro sign]M R(+)-ropivacaine, 53 [micro sign]M S(-)-ropivacaine, 668 [micro sign]M tetracaine, 41 [micro sign]M bupivacaine, and 39 [micro sign]M etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22 +/- 6 mV vs. 7 +/- 1 mV, respectively, and bupivacaine 31 +/- 7 mV vs. 6 +/- 4 mV).     

Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem

BACKGROUND: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels. METHODS: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques. RESULTS: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 microM mepivacaine, 222 microM lidocaine, 51 microM R(+)-ropivacaine, 53 microM S(-)-ropivacaine, 668 microM tetracaine, 41 microM bupivacaine, and 39 microM etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22+/-6 mV rs. 7+/-1 mV, respectively, and bupivacaine 31+/-7 mV vs. 6+/-4 mV). CONCLUSIONS: Local anesthetics inhibit tandem pore domain baseline potassium channels, and they could depolarize the resting membrane potential of cells expressing these channels. Whether inhibition of these channels contributes to conduction blockade or to the adverse effects of local anesthetics remains to be determined.     

Differences in Cardiotoxicity of Bupivacaine and Ropivacaine Are the Result of Physicochemical and Stereoselective Properties

Background: Ropivacaine is believed to have a lower incidence of clinical cardiac side effects than bupivacaine. The aim of this study was to compare the direct cardiac effects of the optically pure S (-)-ropivacaine isomer and its nonclinically used R (+)-isomer with both optically pure bupivacaine isomers in isolated hearts. The hypothesis was that differences in direct cardiac effects are distinguished not only by stereoselective actions of local anesthetic molecules to specific receptors, but also by physicochemical differences triggered by replacing the butyl- by a propyl-residual on pipecoloxylide.

Methods: Guinea pig hearts (n = 31) were excised and perfused by the Langendorff method. Atrial and ventricular bipolar electrodes were placed to measure heart rate and atrioventricular conduction time. Left ventricular pressure, coronary flow, and oxygen tensions were measured. Twelve hearts were perfused with increasing concentrations (0.5, 1.0, 5.0, and 10 [mu]m) of both isomers of bupivacaine, and 13 hearts were perfused with the same concentrations of ropivacaine isomers. Six hearts were perfused with higher concentrations (20, 30, 40, and 50 [mu]m) of both isomers of ropivacaine. The order of isomers and anesthetic chosen were randomized.

Results: Both anesthetics had negative inotropic and chronotropic effects without evidence of stereoselectivity. Equal concentrations of both isomers of bupivacaine had negative inotropic effects greater than that of ropivacaine isomers. Atrioventricular conduction time was prolonged by both anesthetics in a concentration-dependent manner, but bupivacaine isomers increased atrioventricular conduction time more than ropivacaine isomers. In contrast to other variables, atrioventricular conduction time showed evident stereoselectivity for bupivacaine at the lowest concentration (0.5 [mu]m) but only at higher concentrations for ropivacaine (> 30 [mu]m). The R (+)-isomer was more potent than the S (-)-isomer on increasing atrioventricular conduction time for both bupivacaine and ropivacaine.     

Structural Requirements of Human Ether-a-go-go-related Gene Channels for Block by Bupivacaine

Background: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine.

Methods: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels.

Results: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 [mu]m (n = 33). Bupivacaine (300 [mu]m) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 [mu]m) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001).     

Mechanisms underlying the QT interval-prolonging effects of sevoflurane and its interactions with other QT-prolonging drugs

BACKGROUND: Sevoflurane prolongs ventricular repolarization in patients, but the mechanisms are not fully characterized. The effects of sevoflurane on many cloned human cardiac ion channels have not been studied, and the interactions between sevoflurane and other drugs that prolong cardiac repolarization have not been detailed. METHODS: The effects of sevoflurane on action potentials and L-type Ca channels in guinea pig myocytes were examined. Sevoflurane's effects on cloned human cardiac K channels and the cloned human cardiac Na channel were studied. The consequences of combining sevoflurane and the class III antiarrhythmic drugs sotalol or dofetilide on action potential duration were also examined. RESULTS: Sevoflurane produced an increase in action potential duration at concentrations of 0.3-1 mm. Contrary to most drugs that delay ventricular repolarization, sevoflurane was without effect on the human ether-a-go-go-related gene cardiac potassium channel but instead produced a reduction in KvLQT1/minK K channel currents and inhibited the Kv4.3 K channel by speeding its apparent rate of inactivation. Sevoflurane had little effect on Na and Ca channel currents at concentrations of 1 mm or less. When the authors coadministered sevoflurane with sotalol or dofetilide, synergistic effects on repolarization were observed, resulting in large increases in action potential duration (up to 66%). CONCLUSION: Prolonged ventricular repolarization observed with administration of sevoflurane results from inhibition of KvLQT1/minK and Kv4.3 cardiac K channels. Combining sevoflurane with class III antiarrhythmic drugs results in supra-additive effects on action potential duration. The results indicate that sevoflurane, when administered with this class of drug, could result in excessive delays in ventricular repolarization. The results suggest the need for further clinical studies.     

Amitriptyline versus Bupivacaine in Rat Sciatic Nerve Blockade

Background: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro.

Methods: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated.

Results: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mm, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mm, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mm, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mm) and 656 +/- 27 min (10 mm) versus 155 +/- 9 min for bupivacaine (15.4 mm). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 [mu]m) at -150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 [mu]m) at -60 mV. High-frequency stimulation at 3 [mu]m caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline.     

Retigabine stimulates human KCNQ2/Q3 channels in the presence of bupivacaine

BACKGROUND: Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic. METHODS: KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique. RESULTS: Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential. CONCLUSIONS: The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.     

Stereospecific Interaction of Ketamine with Nicotinic Acetylcholine Receptors in Human Sympathetic Ganglion-like SH-SY5Y Cells

Background: Surprising clinical evidence suggests a block of sympathetic transmission by ketamine. The action of ketamine on nicotinic acetylcholine receptors (nAChRs) in human ganglions is unknown. Because ganglionic transmission depends on nAChRs, such information may help to clarify whether ketamine impairs ganglionic transmission in men. Because racemic ketamine as well as S (+)-ketamine are used clinically, the authors investigated stereospecific effects on human ganglionic nAChRs. Stereospecific psychomimetic effects have been attributed to voltage-dependent Kv channel inhibition; therefore the effects on nAChRs were compared with those on Kv channels present in the same cells.

Methods: Whole-cell currents through nAChRs and K channels were measured in SH-SY5Y cells with the patch-clamp technique by application of acetylcholine (1 mm, nAChRs) or by a step depolarization from a holding potential of -80 mV to +40 mV (K channels). Electrolyte conditions were identical for both currents.

Results: Racemic ketamine and the isomers inhibited nAChRs and K channels in a concentration-dependent and reversible manner. Racemic ketamine inhibited nAChRs and K channels, with the anesthetic concentration inducing the half-maximal effect being 1.4 and 300 [mu]m, respectively. Only inhibition of the nAChRs was stereoselective. The half-maximal concentrations were 0.8 and 3.6 [mu]m for S (+)- and R (-)-ketamine. The K channels were 350 and 70 times less sensitive to the effects of S (+)- and R (-)-ketamine.     

Stereoselectivity of bupivacaine in local anesthetic-sensitive ion channels of peripheral nerve.

BACKGROUND: The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)-and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel. METHODS: In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k(-1)) were determined. RESULTS: Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/- SEM) for tonic Na+ channel block were 29+/-3 microM and 44+/-3 microM, respectively. IC50 values for flicker K+ channel block were 0.15+/-0.02 microM and 11+/-1 microM, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k(-1) were 0.83+/-0.13x10(6) M(-1) x S(-1) and 0.13+/-0.03 s(-1), respectively, for R(+)-bupivacaine and 1.90+/-0.20x10(6) M(-1) x s(-1) and 8.3+/-1.0 s(-1), respectively, for S(-)-bupivacaine. CONCLUSIONS: Bupivacaine block of Na+ channels shows no salient stereoselectivity. Block of flicker K+ channels has the highest stereoselectivity ratio of bupivacaine action known so far. This stereoselectivity derives predominantly from a difference in k(-1), suggesting a tight fit between R(+)-bupivacaine and the binding site. The flicker K+ channel may play an important role in yet unknown toxic mechanisms of R(+)-bupivacaine.     

Isoflurane Decreases ATP Sensitivity of Guinea Pig Cardiac Sarcolemmal KATP Channel at Reduced Intracellular pH

Background: Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (KATP) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role KATP channel modulators play in this effect. Because pH is one of the regulators of KATP channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac KATP channel.

Methods: The effects of isoflurane on sarcolemmal KATP channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts.

Results: At pHi 7.4, intracellular ATP (1-1,000 [mu]m) inhibited KATP channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC50 of 8 +/- 1.5 [mu]m, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC50 shifting from 8 +/- 1.2 to 45 +/- 5.6 [mu]m. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC50 to 110 +/- 10.0 [mu]m.