Free
Meeting Abstracts  |   March 1997
Effects of Ropivacaine on a Potassium Channel (hKv1.5) Cloned from Human Ventricle
Author Notes
  • (Valenzuela, Delpon) Assistant Professor of Pharmacology.
  • (Franqueza, Pilar) Postdoctoral Research Fellow in Pharmacology.
  • (Snyders) Associate Professor of Medicine and Pharmacology.
  • (Tamargo) Professor of Pharmacology.
  • Received from the Institute of Pharmacology and Toxicology, CSIC, School of Medicine, Madrid, Spain, and the Departments of Medicine and Pharmacology, Vanderbilt University, School of Medicine, Nashville, Tennessee. Submitted for publication May 10, 1996. Accepted for publication December 3, 1996. Supported by grants from Fondo de Investigaciones Sanitarias 95/0318, Comision Interministerial de Ciencia y Tecnologia (SAF96–0042), Comunidad Autonoma de Madrid (157/92), and National Institutes of Health (HL47599).
  • Address reprint requests to Dr. Valenzuela: Institute of Pharmacology and Toxicology, CSIC, School of Medicine, Universidad Complutense 28040 Madrid, Spain.
Article Information
Meeting Abstracts   |   March 1997
Effects of Ropivacaine on a Potassium Channel (hKv1.5) Cloned from Human Ventricle
Anesthesiology 3 1997, Vol.86, 718-728. doi:
Anesthesiology 3 1997, Vol.86, 718-728. doi:
Some local anesthetics contain an asymmetric carbon atom and can, therefore, be separated into optical isomers, although they are usually used clinically as racemic mixtures. If individual enantiomers display distinct pharmacologic or toxicologic properties, then the clinically used form effectively represents the administration of two distinct drugs. Significant differences have been demonstrated in the effects of distinct enantiomers, not only on the nerve-blocking potency, but also in their toxicologic effects on the central nervous and cardiovascular systems. [1–3] One of the more cardiotoxic local anesthetics is bupivacaine, which is clinically used as a racemic mixture. [3–7] In vivo studies have demonstrated that the potency and duration of the anesthesia were equal or even larger for S(-)-bupivacaine than the R(+)-enantiomer. [1,2,8] More importantly, the LD50for R(+)-bupivacaine was approximately 30–40% lower than for s(-)-bupivacaine. [1,8] Although the higher potency of R(+)-bupivacaine (1.6-fold) over s(-)-bupivacaine to inhibit the fast inward Na sup + current (INa)[9] could, in part, explain its higher cardiotoxicity, several studies have also shown a prolongation of the QT interval of the electrocardiogram in anesthetized dogs [10–12] and human volunteers [13] receiving high doses of bupivacaine. In some cases, this was accompanied by torsades de pointes, [14] suggesting that block of K sup + channels is also involved in bupivacaine cardiotoxicity. We have recently shown that the potency of R(+)-bupivacaine to block a cardiac K sup + channel cloned from human ventricle (hKv1.5) is sevenfold higher than that exhibited by s(-)-bupivacaine (KD= 4.7 micro Meter and 27.3 micro Meter, respectively). [15] This could explain the observed differences between both enantiomers in toxicologic studies, and the resulting action potential prolongation would further exacerbate Na sup +-channel block.
Ropivacaine (S-[-]-1-Propyl-2',6'-pipecoloxylidide hydrochloride monohydrate) is the S(-)-enantiomer of AL381, [16] a local anesthetic chemically related to bupivacaine (Figure 1) that was synthesized as a less toxic alternative to bupivacaine. Although ropivacaine has been shown to be less cardiotoxic than bupivacaine in intact animal studies, [13,16,17] it has also been demonstrated that ropivacaine is able to induce the appearance of early after-depolarizations in isolated preparations, [18] probably secondary to a blockade of K sup + channels. In human cardiomyocytes, K sup + currents activated by depolarization have been identified as transient outward current currents (ITO), [19–22] and delayed ractifier currents with rapid (IKr) and slow (IKs) components. [23] More recently, a very rapidly activating delayed rectifier current (IKur) was observed in adult human atria. [24,25] The functional and pharmacologic characteristics of IKurare similar to those reported for the cloned cardiac human Kv1.5 channel. [24–28] Selective block of this current affects action potential duration in human atria, [24] indicating that hKv1.5 current represents one of the molecular targets for (class III) antiarrhythmic agents.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
×
The purpose of the current study was to determine the mechanism of action of ropivacaine on hKv1.5 channels expressed in a mammalian cell line (Ltk sup -) without the complications of overlapping currents. Preliminary results of the current study have been published previously in abstract form. [29] 
Materials and Methods
Cell Culture and Solutions
We used a cell line stably expressing hKv1.5, as described previously. [27,28] Transfected cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% horse serum and 0.25 mg/ml G418, under a 5% CO2atmosphere. The cultures were passed every 3–5 days, using a brief trypsin treatment. Before experimental use, subconfluent cultures were incubated with 2 micro Meter dexamethasone for 24 h to induce the expression of hkv1.5 channels (which is driven by a dexamethasone inducible promoter). The cells were removed from the dish with a rubber policeman, a procedure that left the vast majority of the cells intact. The cell suspension was stored at room temperature (21–23 degrees Celsius) and used within 12 h for all the experiments reported here.
The intracellular pipette filling solution contained (in mM): K-aspartate 80, phosphocreatine 3, KCl 50, KH2PO410, MgATP 5, HEPES 10, EGTA 5, and was adjusted to pH 7.25 with KOH. The bath solution contained (in mM): NaCl 130, KCl 4, CaCl21.8, MgCl21, HEPES 10, and glucose 10, and was adjusted to pH 7.35 with NaOH. Ropivacaine (a gift from Chiroscience, London, England) was dissolved in distilled deionized water to yield stock solutions of 10 mM. Further dilutions in external solution were made to obtain the desired final concentration.
Electrical Recording
Experiments were performed in a small volume (0.5-ml) bath mounted on the stage of an inverted microscope (Nikon model TMS, Garden City, NY) continuously perfused at a flow rate of 0.5–1.0 ml/min. The hKv1.5 currents were recorded at room temperature (21–23 degrees Celsius) using the whole-cell voltage-clamp configuration of the patch-clamp technique with an Axopatch-1C patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 2 kHz (4 pole Bessel filter) and sampled at 4 kHz. Data acquisition and command potentials were controlled by the PCLAMP 5.5.1. software (Axon Instruments).
Micropipettes were pulled from borosilicate glass capillary tubes (GD-1, Narishige, Tokyo, Japan) on a programmable horizontal puller (Sutter Instrument, San Rafael, CA) and heat polished with a microforge (Narishige, Tokyo, Japan). When filled with the intracellular solution and immersed into the bath (external) solution, the pipette resistance ranged between 1 and 3 M Omega. The micropipettes were gently lowered onto the cells to obtain a gigaohm seal (16 +/- 6 G Omega) after applying suction. After seal formation, cells were lifted from the bottom of the perfusion bath, and the membrane patch was ruptured with brief additional suction. The capacitive transients elicited by symmetrical 10-mV steps from -80 mV were recorded at 50 kHz (filtered at 10 kHz) for subsequent calculation of capacitative surface area, access resistance, and input impedance. Thereafter, capacitance and series resistance compensation were optimized, and 80% compensation of the effective access resistance was usually obtained.
Pulse Protocol and Analysis
After control data were obtained, bath perfusion was switched to drug-containing solution. Drug infusion or removal was monitored with test pulses from -80 mV to +30 mV and applied every 30 s until steady-state was obtained (within 10–15 min). The holding potential was maintained at -80 mV. The cycle time within each protocol was 10 s, to avoid accumulation of block or incomplete deactivation of the current.
The protocol to obtain current-voltage (I-V) relations and activation curves consisted of 250-ms pulses imposed in 10-mV increments between -80 and +60 mV, with additional interpolated pulses to yield 5-mV increments between -30 and +10 mV (i.e., the activation range of the hKv1.5 channels). [28] The “steady-state” I-V relations were obtained by measuring the current at the end of the 250-ms depolarizations. Between -80 and -40 mV, only passive linear leak was observed and least squares fits to these data were used for passive leak correction. Deactivating “tail” currents were recorded at -40 mV. The activation curve was obtained from the tail current amplitude immediately after the capacitive transient. Measurements were performed using the CLAMPFIT program of pCLAMP 5.5.1. and by a custom-made analysis program.
Activation curves were fitted with a Boltzmann equation:Equation 1in which s represents the slope factor, E the membrane potential, and Ehthe voltage at which 50% of the channels are open. The time course of tail currents and the slow inactivation were fitted with the sum of exponentials. The activation kinetics was determined with the dominant time constant of activation approach in which a single exponential was fitted to the latter 50% of activation. [28,30,31] The curve fitting procedure used a nonlinear least-squares (Gauss-Newton) algorithm; results were displayed in linear and semilogarithmic format, together with the difference plot. Goodness of the fit was judged by the chi squared criterion and by inspection for systematic nonrandom trends in the difference plot.
A first-order blocking scheme was used to describe drug-channel interaction. Apparent affinity constant, KD, and Hill coefficient, n sub H, were obtained from fitting of the fractional block, f, at various drug concentrations [D]:Equation 2and apparent rate constants for binding (k) and unbinding (l) were obtained from solving:Equation 3(a,b)
Voltage dependence of block was determined as follows: leak-corrected current in the presence of drug was normalized to matching control to yield the fractional block at each voltage (f = 1 - Idrug/Icontrol). The voltage dependence of block was fitted to:Equation 4where z, F, R and T have their usual meaning in thermodynamics, delta represents the fractional electrical distance [32] (i.e., the fraction of the transmembrane electrical field sensed by a single charge at the receptor site), and KDsup * represents the apparent dissociation constant at the reference potential (0 mV).
Statistical Methods
Results are expressed as mean +/- SEM. Paired Student's t test was used to compare the effects of ropivacaine with the control values. Statistical significance was taken as P < 0.05.
Results
Dose-dependent Block of hKv1.5 Induced by Ropivacaine
(Figure 2) shows original records of hKv1.5 current expressed in mouse Ltk sup - cells obtained in the absence and in the presence of 100 micro Meter ropivacaine after the application of 250 ms depolarizing pulses from -80 mV to different test potentials between -60 and +60 mV. Under control conditions, the hKv1.5 current rose rapidly with a sigmoidal time course to a peak and then declined slowly (slow and partial inactivation). Outward tail currents were observed after repolarization to -40 mV. Activation time constants ranged from 25.2 +/- 1.3 ms at -10 mV (n = 20) to 2.0 +/- 0.4 ms at +60 mV (n = 20), as described previously. [27,28,33] Development of block after switching of the bath perfusion with ropivacaine exhibited a time constant of 3–4 min, which was approximately 5 times slower than the effect of changing extracellular K sup + concentration at similar flow rates. This delay suggested an intramembrane or intracellular site of action and, therefore, 10–15 min of equilibration were allowed before assessment of drug effects. Without modifying the initial activation time course of the current, 100 micro Meter ropivacaine accelerated the decline of hKv1.5 current to the reduced steady-state level with a time constant of 12.2 +/- 0.6 ms. At the end of a 250-ms step to +60 mV, this concentration of ropivacaine reduced the hKv1.5 current by 56 +/- 2%(n = 18). The amplitude of the current was restored to 90% after 20 min perfusion of the cells with drug-free solution in the experiment shown. On average, the current was restored to 88 +/- 1.5%(n = 50) of its initial control value after a 20-min washout.
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
×
(Figure 3) shows the concentration dependence of ropivacaine block of hKv1.5. To allow for the slower kinetics at lower concentrations (described later), we used suppression of current at the end of 250-ms depolarizations to +60 mV as an index of steady-state inhibition [fractional block = 1 -(Idrug/Icontrol)]. In the presence of 10, 50, and 500 micro Meter ropivacaine, hKv1.5 inhibition averaged 6 +/- 1%(n = 4), 36 +/- 3%(n = 4), and 86 +/- 2%(n = 5), respectively. A nonlinear least-squares fit of the concentration-response equation (eq. 2, see Materials and Methods) yielded an apparent KDof 80 +/- 4 micro Meter and a Hill coefficient of 1.17 +/- 0.08. The dashed line in Figure 3illustrates a fit of the same data, with the Hill coefficient fixed at 1. The apparent KDvalue obtained for ropivacaine was similar to that obtained without constraining the Hill coefficient (78 +/- 6 micro Meter; P > 0.05). By either analysis, the results suggest that binding of a single ropivacaine molecule is sufficient to block the hKv1.5 channel.
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
×
Voltage-dependent Block of hKv1.5 Channels by Ropivacaine
(Figure 4(A)) shows the effects of 100 micro Meter ropivacaine on the steady state current-voltage (I-V) relation for the hKv1.5 channel. The I-V relation under control conditions was quasilinear for depolarizations positive to +10 mV, whereas the sigmoidicity between -30 and +10 mV reflects the voltage-dependence of channel gating. [28] In the presence of 100 micro Meter ropivacaine, the curve displayed a downward curvature at test potentials positive to 0 mV, which suggested that ropivacaine produced more extensive block at very positive depolarizations. To quantitate the voltage dependence of hKv1.5 block, we determined the relative current (Idrug/Icontrol) as a function of membrane potential. Figure 4(B) shows that hKv1.5 block increased steeply between -30 and 0 mV, which corresponded to the voltage range of channel opening (dashed line). This steep increase in block coinciding with the activation range of the channel strongly suggests that channels need to open (or activate) before ropivacaine can bind and block them. Therefore, ropivacaine appears to act as an open-state blocker of hKv1.5 channels. Between 0 mV and +60 mV, block continued to increase, but with a more shallow voltage dependence, although the voltage-dependence of channel opening has saturated over this voltage range. The percentage of block induced by 100 micro Meter ropivacaine significantly increased, from 51.9 +/- 2.3% at 0 mV to 58.8 +/- 2.5% at +60 mV (n = 9; P < 0.01). Ropivacaine is a weak base and has a tertiary amine group with pKa= 8.1; therefore, it is predominantly present in its charged form at the intracellular pH of 7.2. Therefore, this shallow component in the voltage dependence of block could result from the influence of the transmembrane electrical field on the interaction between cationic ropivacaine and the channel receptor, as has been described for quinidine and bupivacaine. [15,27] This was quantified using a Boltzmann relation based on the Woodhull model (eq. 4 in Materials and Methods). The solid line in Figure 4(B) represents the fit of this Boltzmann equation to the data points positive to 0 mV. The latter restriction was required to quantify this effect independent from the voltage dependence of channel opening. In the experiment shown, we obtained a delta value of 0.16 for the fractional electrical distance (i.e., the fraction of the membrane electrical field sensed by a single charge at the receptor site). [32] In the presence of 100 micro Meter ropivacaine, the delta value averaged 0.153 +/- 0.007 (n = 6).
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
×
Concentration Dependence of Time Course of Channel Block
If ropivacaine can only gain access to its binding site when the channel is open, then the inhibition of the potassium current would only develop after the channels start to open, and development of block should be conspicuous if the blocking rate is slower than the opening rate. Figure 5(A) shows superimposed recordings of 250 ms depolarizations from -80 to +60 mV under control conditions and in the presence of two different concentrations of ropivacaine, 100 micro Meter and 200 micro Meter, respectively. Under control conditions, the potassium current reached its maximum level within 10 ms and then declined slowly, with a time constant of 160 ms. In the presence of ropivacaine, the peak current was decreased in a concentration-dependent manner, and the subsequent time course displayed an additional rapid exponential component superimposed on the slow inactivation. As illustrated in Figure 5(A), the time course of this decline was concentration-dependent. The time constants of the fast component in the presence of 100, 200, and 500 micro Meter ropivacaine were 12.2 +/- 0.6 (n = 12), 7.0 +/- 0.6 (n = 5), and 4.3 +/- 0.2 ms (n = 5), respectively. These time constants (tauB) were at least eight times faster than those of the slow inactivation. Therefore, we used the tauBvalues as an approximation of bimolecular drug-channel interaction kinetics, similar to the approach used previously for the interaction of other drugs with this channel. [15,27] Figure 5(B) shows the apparent rate of block (1/tauB) versus ropivacaine concentration (n = 22 experiments). The straight line is the least squares fit to the relation 1/tauB= k x [D]+ l, which yielded apparent association (k) and dissociation (l) rate constants of (0.45 +/- 0.03) x 106M sup -1 s sup -1 and (37.2 +/- 13.8) s sup -1, respectively.
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
×
After a depolarizing step to +60 mV, the hKv1.5 currents deactivated completely on return to -40 mV, with a time constant of 44 +/- 7 ms (n = 6), as illustrated in Figure 6. This time constant reflects mainly the virtually irreversible closing of the channel. If ropivacaine binds only to the open state of hKv1.5 channels, then the dissociation of the drug from the blocked channel should result transiently in an open channel (which could subsequently close). Blocked channels are not conducting, and the conversion to open channels may, therefore, result initially in a rising phase of the tail current. Subsequently, the tail current may display a slower decline because some fraction of the open channels become blocked again, rather than closing irreversibly. [15,27,34,35] Figure 6shows the superposition of the tail currents obtained at -40 mV after 250 ms depolarization to +60 mV under control conditions and in the presence of 100 micro Meter ropivacaine. After exposure to the drug, the initial amplitude of the tail current was reduced, and the tail current displayed a rising phase before the maximum tail peak was reached. The subsequent decline of the tail current was slower than in control conditions (44 +/- 7 vs. 142 +/- 22 ms, in control and in the presence of 100 micro Meter ropivacaine, respectively; P <0.01, n = 6), resulting in a “crossover” phenomenon. These results further support an open-channel interaction between ropivacaine and hKv1.5 channels.
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
×
Open Channel Block Model
The experimental results obtained in this study were interpreted and simulated using a simplified kinetic state diagram similar to that used for the related Shaker and RCK1 channels [36] and previously used to describe drug-induced block of the hKv1.5 channel. [15,27] To incorporate the interaction of ropivacaine into this diagram, we assumed that this drug binds only to the open state of the channel. Figure 7shows the mathematical simulation of the effects of 100 and 200 micro Meter ropivacaine, based on this open-channel block model using the experimentally derived association and dissociation rate constants (Figure 5(B)). Although this model only assumes an open channel interaction, it reproduced the important experimental findings:(1) the dose-dependent initial suppression of peak outward hKv1.5 current, which is due to the fast open channel block at concentrations greater than 100 micro Meter;(2) the steady state block at the end of 250 ms depolarizing pulses;(3) the kinetics of block induced by ropivacaine; and (4) the slower deactivation process in the presence of ropivacaine. Therefore, a simple open channel binding model appears sufficient to explain the effects of ropivacaine on the hKv1.5 channel both qualitatively and quantitatively.
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
×
Discussion
In this study, we analyzed the effects of the pure S(-)-enantiomer of ropivacaine on hKv1.5 channels stably expressed in a mammalian cell line. Our main findings are that:(1) the charged form of ropivacaine blocks the hKv1.5 channel after it opens;(2) binding occurs within the transmembrane electrical field; and (3) unbinding is required before the channel can close. In addition, the potency of ropivacaine to block hKv1.5 is 3 and 20 times less compared with S(-)-bupivacaine and R(+)-bupivacaine, respectively. [15] This could explain, at least in part, the lower cardiotoxicity of ropivacaine versus bupivacaine.
Ropivacaine Acts As an Open State Blocker of hKv1.5 Channels
Ropivacaine inhibited hKv1.5 current in a time-dependent manner. After depolarization from -80 mV to +60 mV, the activation time course of the current was not modified, but the drug decreased the peak current amplitude and induced a fast initial decline during the depolarizing step, which reached steady state at the end of a 250-ms depolarizing pulse. This time-dependent decline of the current could be attributed to several mechanisms, but is consistent with an open-channel interaction. Another argument for open (or activated) state interactions derives from the voltage-dependence of block. As shown in Figure 3, block of hKv1.5 channels by ropivacaine exhibited two components of voltage-dependence:(1) a very steep one, which coincides with the range of membrane potentials at which the channels open, and (2) a shallower one for membrane potentials positive to 0 mV. The first component strongly indicates that ropivacaine needs the channel to open before it can bind to it. The further increase in block observed at potentials positive to 0 mV was consistent with a value of electrical distance (delta) of 0.153 +/- 0.007 (n = 6). This delta value indicates that charged ropivacaine senses 15% of the applied transmembrane electrical field, referenced to the cytoplasmic membrane side. This analysis assumes that ropivacaine gains access to the receptor of the hKv1.5 channel from the inside of the cell. These results are consistent with the proposed open channel block mechanism. Another finding that supports the open state block induced by ropivacaine is the “crossover” observed between the deactivating tail currents recorded at -40 mV under control conditions and in the presence of ropivacaine (Figure 6). In the absence of drug, channel deactivation after repolarization is fast and virtually irreversible (4 beta >> alpha). However, if a large fraction of channels is blocked by ropivacaine during the preceding depolarizing step and the unbinding rate (l) is fast enough, then the tail current may display a rising phase that will reflect the unblocking process and the redistribution to the lower level of block observed at -40 mV. In addition, a mathematical simulation in which we assumed that ropivacaine only binds to the open state of the channel reproduced the experimental results obtained in the current study. Therefore, the data strongly indicate that ropivacaine blocks hKv1.5 channels by binding to their open state (or a closely associated activated state).
Comparison of the Effects of Ropivacaine and S(-)-Bupivacaine on hKv1.5 Channels
Ropivacaine, a new amide type local anesthetic agent, is a S(-)-pure enantiomer, chemically related to bupivacaine, which was synthesized as a less toxic alternative to bupivacaine. [13,16,17] The difference between both is that S(-)-bupivacaine exhibits a butyl substituent on the tertiary nitrogen (position 1), whereas ropivacaine has a propyl group at this position (Figure 1). Qualitatively, the results observed with ropivacaine in the current study are similar to those previously reported for S(-)-bupivacaine in hKv1.5 channels [15] : both act as open channel blockers with a similar intrinsic voltage-dependence, which suggests that they bind at the same binding site. Therefore, the 3-fold difference in potency between S(-)-bupivacaine (KD= 27 micro Meter)[15] and ropivacaine (KD= 80 micro Meter) to block hKv1.5 channels appears to be related to the length of the chain at position 1 of the molecule (butyl vs. propyl). The values for the apparent dissociation constant (KD) can be converted into apparent binding energies (Delta G) relative to a 1 M standard concentration using the relation Delta G =-RT x ln [l(mol/L)/KD], where R is the universal gas constant and T is the absolute temperature. The change in free energy indicates the relative stability of the drug-bound channel in the presence of the drug. The value for Delta G for ropivacaine was -5.39 Kcal/mole. Compared with the value previously obtained for S(-)-bupivacaine (-6.17 Kcal/mole), [15] we get a difference in Delta G of 0.78 Kcal/mole. This value is somewhat larger than that obtained for addition of methylene groups in a series of small bisquaternary ammonium K sup +-channel blockers (0.30–0.31 Kcal/mol), [37,38] but similar to that obtained in the same studies for longer alkyl derivatives (0.7–0.72 Kcal/mol). [37,38] Importantly, these values are in the range of that expected for the classical hydrophobic effect: Delta G approximately 0.8 Kcal/mol per methyl transferred from aqueous to hydrophobic phase. [38] This suggests that the addition of one methyl group increases the stability of the drug channel interaction through a hydrophobic interaction. Consistent with this, we found that the dissociation rate constant was slower for S(-)-bupivacaine (24 s sup -1)[15] than for ropivacaine (37 s sup -1).
Putative Receptor Site for Ropivacaine in hKv1.5 Channels
The voltage dependence observed in the presence of ropivacaine strongly suggests that the binding site is located within the membrane electrical field (i.e., it is located in the membrane spanning regions of the hKv1.5 channel). Tetraethylammonium (TEA) blocks ShakerB K sup + channels with a voltage dependence described by an electrical distance of 0.15, [34,39] similar to that previously described for quinidine (delta = 0.18), [27,40] R(+)-, and S(-)-bupivacaine (delta = 0.16), [15] and for ropivacaine in the current experiments. These findings suggest the existence of a common receptor site for TEA, local anesthetics, and antiarrhythmic drugs both in Shaker and hKv1.5 channels. In addition, the functional similarity between ropivacaine-induced hKv1.5 block and that induced by TEA on Shaker channels, as well as the high structural similarity of the ion pores, would suggest that the T477 residue (equivalent of T441 Sh) in hKv1.5 channels may be involved in ropivacaine block. We recently reported that this residue is involved in the binding of bupivacaine enantiomers to hKv1.5 channels, [41] although it does not determine its stereoselective interactions with the channel, which seems to be related to the T505 residue in the S6 segment. [41] In addition, Yeola et al. [42] demonstrated that T505 and V512 are major molecular determinants for quinidine binding to these K sup + channels, whereas mutations of residues affecting TEA block had little effect. The latter suggests some degree of selectivity for binding of open channel blockers. Furthermore, the affinities of TEA-derivatives in Shaker and that of quinidine in Kv1,5 were enhanced by substitutions that increased the hydrophobicity of the binding pocket. As discussed above, the difference in affinity between S(-)-bupivacaine and ropivacaine is consistent with an hydrophobic effect. Further experiments are needed to elucidate whether the molecular determinants for ropivacaine binding are distinct from those for quinidine or bupivacaine.
Practical Implications Regarding Toxicity
The toxic effects of local anesthetics on brain and heart provided the initial stimulus to develop ropivacaine. Animal [16–18,43–45] and clinical studies [13,46] have demonstrated that ropivacaine displays less toxicity in the central nervous and cardiovascular systems compared with bupivacaine. In addition, ropivacaine possesses a greater margin of safety than bupivacaine when comparing the dose to induce convulsions, to depress intracardiac conduction and contractility or to cause cardiovascular collapse. [13,16,18,47] 
In healthy human volunteers, the mean peak plasma concentrations (Cmax) at the end of the intravenous infusion of ropivacaine and bupivacaine up to a maximal dose of 150 mg averaged 1.50 +/- 0.36 micro gram [center dot] ml sup -1, [48] and 1.7 +/- 0.5 micro gram [center dot] ml sup -1, respectively, [13] and 1.06 +/- 0.36 micro gram [center dot] ml sup -1 after bilateral intercostal blockade of T5-T11 with 140 mg. [49] In clinical studies, the Cmaxof ropivacaine achieved were quite similar after axillary plexus block (1.48 +/- 0.25 micro gram [center dot] ml sup -1)[50] when administered epidurally (from 0.53 +/- 0.19 micro gram [center dot] ml sup -1 to 1.53 +/- 0.60 micro gram [center dot] ml sup -1)[51,52] and after extradural administration (between 0.76 and 0.93 micro gram [center dot] ml sup -1). [53] In all these studies, no adverse cardiac effects were observed at these plasma concentrations. This finding may be explained because the Cmaxvalues were lower than the threshold toxic plasma concentrations of ropivacaine required for the development of seizure activity (> 4 micro gram [center dot] ml sup -1) in awake dogs. [47] In fact, the Cmaxin patients undergoing extradural anesthesia in whom intravenous injection of 0.75% ropivacaine was given was suspected to be 3.7 micro gram [center dot] ml sup -1. [54] 
In the experiments shown in the current study, the threshold concentration at which ropivacaine inhibited hKv1.5 channels was approximately 10 micro Meter (2.86 micro gram [center dot] ml sup -1), a concentration greater than the Cmaxreported in most clinical trials. [13,49–53] In addition, the KDvalue (80 micro Meter) is even greater than the plasma concentrations that produce cardiovascular collapse and death in conscious dogs. [47] Therefore, it seems that the absence of changes in the QTc interval of the electrocardiogram can be explained because the Cmaxafter the recommended dose in clinical practice is less than that needed to block hKv1.5 channels, thereby leaving an acceptable safety margin in therapy.
However, it is difficult to relate the in vivo plasma concentrations of drugs perfusing in vitro preparations, particularly with drugs like ropivacaine, which are highly bound (94%) to plasma proteins. [48,51] Considering the effect of protein binding in vivo, the threshold concentration at which ropivacaine blocks hKv1.5 channels is even greater. Assuming only 90% protein binding, 10 micro Meter in vitro should be equivalent to approximately 100 micro Meter in vivo, or 28.6 micro gram [center dot] ml sup -1. Feldman et al. [47] showed that in vivo plasma concentrations of 10–70 micro gram [center dot] ml sup -1 are associated with profound central nervous system and cardiovascular toxicity. Therefore, although bupivacaine and ropivacaine share a common mechanism of action in terms of hKv1.5 block, the concentrations needed to exert extensive block of this channel are much greater than those observed during routine clinical use, and even during accidental intravascular injection, suggesting that the clinical toxicity of ropivacaine may not solely be related to its effects on hKv1.5 channels. However, to the extent that hKv1.5 is involved in the control of cardiac repolarization, [24] these results can explain, at least in part, the reduced cardiac toxicity of ropivacaine versus bupivacaine.
REFERENCES
Aberg G: Toxicological and local anaesthetic effects of optically active isomers of two local anaesthetic compounds. Acta Pharmacol Toxicol 1972; 31:273-86.
Aps C, Reynolds F: An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. Br J Clin Pharmacol 1978; 6:63-8.
Covino B: Toxicity and systemic effects of local anesthetic agents, Local Anesthetics Handbook of Experimental Pharmacology, Vol. 81. Edited by Strichartz G. Heidelberg, Springer-Verlag, 1987, pp 187-212.
Nath S, Haggmark S, Johansson G, Reiz S: Differential depressant and electrophysiologic cardiotoxicity of local anesthetics: An experimental study with special reference to lidocaine and bupivacaine. Anesth Analg 1986; 65:1263-70.
Albright G: Cardiac arrest following regional anesthesia with etidocaine or bupivacaine. Anesthesiology 1979; 51:285-7.
Buffington C: The magnitude and duration of direct myocardial depression following intracoronary local anesthetics: A comparison of lidocaine and bupivacaine. Anesthesiology 1989; 70:280-7.
Prentiss J: Cardiac arrest following caudal anesthesia. Anesthesiology 1979; 50:51-3.
Luduena F, Bogado E, Tullar B: Optical isomers of mepivacaine and bupivacaine. Arch Int Pharmacodyn Ther 1972; 200:359-69.
Valenzuela C, Snyders D, Bennett P, Tamargo J, Hondeghem L: Stereoselective block of cardiac sodium channels by bupivacaine in guinea pig ventricular myocytes. Circulation 1995; 92:3014-24.
Avery P, Redon D, Schaenzer G, Rusy B: The influence of serum potassium on the cerebral and cardiac toxicity of bupivacaine and lidocaine. Anesthesiology 1984; 61:134-8.
Wheeler D, Bradley E, Woods W: The electrophysiologic actions of lidocaine and bupivacaine in the isolated, perfused canine heart. Anesthesiology 1988; 68:201-12.
Solomon D, Bunegin L, Albin M: The effect of magnesium sulfate administration on cerebral and cardiac toxicity of bupivacaine in dogs. Anesthesiology 1990; 72:341-6.
Scott B, Lee A, Fagan D, Bowler G, Bloomfield P, Lundh R: Acute toxicity of ropivacaine compared with that of bupivacaine. Anesth Analg 1989; 69:563-9.
Kasten G, Martin S: Bupivacaine cardiovascular toxicity: Comparison of treatment with bretylium and lidocaine. Anesth Analg 1985; 64:911-6.
Valenzuela C, Delpon E, Tamkun M, Tamargo J, Snyders D: Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J 1995; 69:418-27.
Akerman B, Hellberg I-B, Trossvik C: Primary evaluation of the local anaesthetic properties of the amino amide agent ropivacaine (LEA 103). Acta Anaesthesiol Scand 1988; 32:571-8.
Pitkanen M, Feldman H, Arthur G, Covino B: Chronotropic and inotropic effects of ropivacaine, bupivacaine, and lidocaine in the spontaneously beating and electrically paced isolated, perfused rabbit heart. Reg Anesth 1992; 17:183-92.
Moller R, Covino B: Cardiac electrophysiologic properties of bupivacaine and lidocaine compared with those of ropivacaine, a new amide local anesthetic. Anesthesiology 1990; 72:322-9.
Wettwer E, Amos G, Gath J, Hauser G, Mewes T, Reidemeister J, Ravens U: Transient outward current in human and rat ventricular cardiomyocytes. Cardiovasc Res 1993; 27:1662-9.
Beuckelmann D, Nabauer M, Erdmann E: Alterations of K sup + currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res 1993; 73:379-85.
Escande D, Coulombe A, Faivre J, Deroubaiz E, Coraboeuf E: Two types of transient outward currents in adult human atrial cells. Am J Physiol 1987; 252:H142-8.
Shibata E, Drury T, Refsum H, Aldrete V, Giles W: Contributions of a transient outward current to repolarization in human atrium. Am J Physiol 1989; 257:H1773-81.
Wang Z, Fermini B, Nattel S: Delayed rectifier outward current and repolarization in human atrial myocytes. Circ Res 1993; 73:276-85.
Wang Z, Fermini B, Nattel S: Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed K sup + current similar to Kv1.5 cloned channel currents. Circ Res 1993; 73:1061-76.
Fedida D, Wible B, Wang Z, Fermini B, Faust F, Nattel S, Brown A: Identity of a novel delayed rectifier current from human heart with a cloned K sup + channel current. Circ Res 1993; 73:210-6.
Tamkun M, Knoth K, Walbridge J, Kroemer H, Roden D, Glover D: Molecular cloning and characterization of two voltage-gated K sup + channel cDNAs from human ventricle. FASEB J 1991; 5:331-7.
Snyders D, Knoth K, Roberds S, Tamkun M: Time-, state- and voltage-dependent block by quinidine of a cloned human cardiac channel. Mol Pharmacol 1992; 41:332-9.
Snyders D, Tamkun M, Bennett P: 1993. A rapidly activating and slowly inactivating potassium channel cloned from human heart. J Gen Physiol 1993; 101:513-43.
Valenzuela C, Delpon E, Franqueza L, Gay P, Perez O, Tamargo J: Effects of (S)-ropivacaine on human cardiac delayed rectifier (Kv1.5) channels. Methods Find Exp Clin Pharmacol 1995; 17(suppl A):51.
White M, Bezanilla F: Activation of squid axon K sup + channels: Ionic and gating current studies. J Gen Physiol 1985; 85:539-54.
Valenzuela C, Sanchez-Chapula J, Delpon E, Elizalde A, Perez O, Tamargo J: Imipramine blocks rapidly activating and delays slowly activating K sup + current activation in guinea pig ventricular myocytes. Circ Res 1994; 74:687-99.
Woodhull A: Ionic blockade of sodium channels in nerve. J Gen Physiol 1973; 61:687-708.
Philipson L, Hice R, Schaeffer K, LaMendola J, Bell G, Nelson D, Steiner D: Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel. Proc Natl Acad Sci U S A 1991; 88:53-7.
Choi K, Mossman C, Aube J, Yellen G: The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron 1993; 10:533-41.
Carmeliet E: Use-dependent block and use-dependent unblock of the delayed rectifier K sup + current by almokalant in rabbit ventricular myocytes. Circ Res 1993; 73:857-68.
Zagotta W, Aldrich R: Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle. J Gen Physiol 1990; 95:29-60.
Miller C: Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K sup + channel. J Gen Physiol 1982; 79:869-91.
Villarroel A, Alvarez O, Oberhauser A, Latorre R: Probing a Ca sup 2+ -activated K sup + channel with quaternary ammonium ions. Pflugers Arch 1988; 413:118-26.
Yellen G, Jurman M, Abramson T, MacKinnon R: Mutations affecting internal TEA blockade identify the probable pore-forming region of a K sup + channel. Science 1991; 251:939-42.
Snyders D, Yeola S: Determinants of antiarrhythmic drug action. Electrostatic and hydrophobic components of block of the human cardiac hKv1.5 channel. Circ Res 1995; 77:575-83.
Franqueza L, Delpon E, Gay P, Snyders D, Tamargo J, Valenzuela C: Molecular determinants for stereoselective bupivacaine binding in hKv1.5 channels. Biophys J 1996; 70:A400.
Yeola S, Rich T, Uebele V, Tamkun M, Snyders D: Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K sup + channel. Role of S6 in antiarrhythmic drug binding. Circ Res 1996; 78:1105-14.
Reiz S, Haggmark G, Johansson G, Nath S: Cardiotoxicity of ropivacaine-A new amide local anaesthetic agent. Acta Anaesthesiol Scand 1989; 33:93-8.
Arthur G, Feldman H, Covino B: Comparative pharmacokinetics of bupivacaine and ropivacaine, a new amide local anesthetic. Anesth Analg 1988; 67:1053-8.
Reiz S, Nath S: Cardiotoxicity of local anaesthetic agents. Br J Anaesth 1986; 58:736-46.
McClure J: Ropivacaine. Br J Anaesth 1996; 76:300-7.
Feldman H, Arthur G, Covino B: Comparative systemic toxicity of convulsant and supraconvulsant doses of intravenous ropivacaine, bupivacaine and lidocaine in the conscious dog. Anesth Analg 1989; 69:794-801.
Lee A, Fagan D, Lamont M, Tucker G, Halldin M, Scott D: Disposition kinetics of ropivacaine in humans. Anesth Analg 1989; 69:736-8.
Kopacz D, Emanuelsson B-M, Thompson G, Carpenter R, Stephenson C: Pharmacokinetics of ropivacaine and ropivacaine for bilateral intercostal blockade in healthy male volunteers. Anesthesiology 1994; 81:1139-48.
Vainionpaa V, Haavisto E, Huha T, Korpi K, Nuutinen L, Hollmen A, Jozwiak H, Magnusson A: A clinical and pharmacokinetic comparison of ropivacaine and bupivacaine in axillary plexus block. Anesth Analg 1995; 81:534-8.
Concepcion M, Arthur G, Steel S, Bader A, Covino B: A new local anesthetic ropivacaine. Its epidural effects in humans. Anesth Analg 1990; 70:80-5.
Katz J, Bridenbaugh P, Knarr D, Helton S, Denson D: Pharmacodynamics and pharmacokinetics of epidural ropivacaine in humans. Anesth Analg 1990; 70:16-21.
Morrison L, Emanuelsson B, McClure J, Pollok A, McKeown D, Brockway M, Jozwiak H, Wildsmith J: Efficacy of extradural ropivacaine: Comparison with bupivacaine. Br J Anaesth 1994; 72:164-9.
Brockway M, Bannister J, McClure J, Mckeown D, Wildsmith J: Comparison of extradural ropivacaine and bupivacaine. Br J Anesth 1991; 66:31-7.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
Figure 1. Chemical structure of bupivacaine and ropivacaine. The asterisk indicates the asymmetric carbon in the molecule.
×
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
Figure 2. Effects of ropivacaine on hKv1.5 currents. Currents are shown for depolarizations from -80 mV to voltages between -60 and +60 mV in steps of 20 mV. Tail currents were obtained on return to -40 mV. Effects of ropivacaine (100 micro Meter) on hKv1.5. Traces were obtained in control conditions (top panel) and in the presence of 100 micro Meter ropivacaine (bottom panel). Cell capacitance, 22 pF. Data filtered at 2 kHz (four-pole Bessel) and digitized at 10 kHz; additional digital filtering at 1 kHz.
×
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
Figure 3. Concentration dependence of ropivacaine-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from -80 mV to +60 mV was used as index of block. Data are mean +/- SEM of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/{1 +(KD/[D])nH}. For comparison, the dashed line represents the fit for a Hill coefficient (nH) of 1.
×
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
Figure 4. Voltage dependence of hKv1.5 block by ropivacaine (100 micro Meter). Panel A: Current-voltage relation (250 ms isochronal) in control conditions [round bullet, filled] and in the presence of 100 micro Meter ropivacaine [circle, open]. Panel B: Relative current expressed as IRopivacaine/Icontrolfrom data shown in panel A. The dashed line represents the activation curve of the hKv1.5 channel for this experiment. Block increased steeply between -20 mV and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Materials and Methods) and yielded delta = 0.16.
×
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
Figure 5. Kinetics of block induction by ropivacaine. Left panel: Superimposed traces for steps from -80 mV to +60 mV and tail currents recorded on return to -40 mV under control conditions (0) and in the presence of 100 and 200 micro Meter ropivacaine. In the presence of ropivacaine, the current activated initially as under control conditions but reached a lower peak and subsequently declined more quickly. Right panel: Rate of block as a function of drug concentration. The time constant of ropivacaine-induced fast component (tauB) was obtained from biexponential fits to the falling phase of the tracings shown in left panel. The inverse of tauBwas plotted versus ropivacaine concentration. For a first-order blocking scheme, a linear relation is expected: 1/tauB= k x [D]+ l. The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained.
×
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
Figure 6. Tail current crossover. Currents recorded in control conditions and in the presence of 100 micro Meter ropivacaine were superimposed. Tail currents were obtained at -40 mV after a 250-ms depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of ropivacaine with those recorded under control conditions.
×
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
Figure 7. Mathematical simulation of ropivacaine-hKv1.5 interaction. The open-channel block model was used with the following rate constants: at +60 mV, alpha = 400 s sup -1, beta = 1 s sup -1, k = 0.45 (micro Meter) sup -1 x s sup -1, and l = 37 s sup -1; at -40 mV, alpha = 0.1 s sup -1, beta = 7 s sup -1, k = 0.275 (micro Meter) sup -1 x s sup -1 and l = 60 s sup -1. For depolarization, simulations for control and for 100 and 200 micro Meter ropivacaine are displayed; for the tails, control and 100 micro Meter ropivacaine are shown. Arrow shows crossover. Currents for step and tail were scaled, to reflect the difference in driving force.
×