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Anesth Analg 2003;96:1019-1026
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Blockade of Voltage-Operated Neuronal and Skeletal Muscle Sodium Channels by S(+)- and R(-)-Ketamine

Gertrud Haeseler, MD*, Diana Tetzlaff*, Johannes Bufler, MD{dagger}, Reinhard Dengler, MD{dagger}, Sinikka Münte, MD*, Hartmut Hecker, PhD{ddagger}, and Martin Leuwer, MD§

*Anesthesiology, {dagger}Neurology and Neurophysiology, and {ddagger}Biometrics, Hannover Medical School, Hannover, Germany; and §University Department of Anaesthesia, The University of Liverpool, Liverpool, United Kingdom

Address correspondence and reprint requests to Gertrud Haeseler, MD, Department of Anesthesiology, OE8050 Hannover Medical School, D-30623 Hannover, Germany. Address e-mail to Haeseler.Gertrud{at}MH-Hannover.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Besides its general anesthetic effect, ketamine has local anesthetic-like actions. We studied the voltage- and use-dependent interaction of S(+)- and R(-)-ketamine with two different isoforms of voltage-operated sodium channels, with a special emphasis on the difference in affinity between resting and inactivated channel states. Rat brain IIa and human skeletal muscle sodium channels were heterologously expressed in human embryonic kidney 293 cells. S(+)- and R(-)-ketamine reversibly suppressed whole-cell sodium inward currents; the 50% inhibitory concentration values at -70 mV holding potential were 240 ± 60 µM and 333 ± 93 µM for the neuronal isoform and 59 ± 10 µM and 181 ± 49 µM for the skeletal muscle isoform. S(+)-ketamine was significantly more potent than R(-)-ketamine in the skeletal muscle isoform only. Ketamine had a higher affinity to inactivated than to resting channels. However, the estimated difference in affinity between inactivated and resting channels was only 8- to 10-fold, and the time course of drug equilibration between inactivated and resting channels was too fast to cause use-dependent block at 10 Hz up to a concentration of 300 µM. These results suggest that ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated channel state.

IMPLICATIONS: Blockade of sodium channels by ketamine shows voltage dependency, an important feature of local anesthetic action. However, ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated state. Because it does not elicit phasic blockade at small concentrations, its ability to reduce the firing frequency of action potentials may be small.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Ketamine has been in clinical use as an analgesic and, in larger concentrations, as a dissociative anesthetic for almost 30 yr. The two enantiomers of ketamine, S(+)- and R(-)-ketamine, have distinct pharmacological properties (1). Although a wide variety of molecular target sites have been identified for ketamine (2), it is generally acknowledged that its general anesthetic actions are mediated primarily via blockade of the N-methyl-D-aspartate receptor (3). Additionally, when applied intrathecally in large concentrations, ketamine has clinically relevant local anesthetic properties (4). The molecular mechanism that most likely accounts for this local anesthetic-like action is its blockade of voltage-operated sodium channels (5–9) . Ketamine interacts with sodium channels in a local anesthetic-like fashion. Its in vitro potency in blocking sodium channels in the resting state is of the same order of magnitude as that of lidocaine (9). This special effect profile, i.e., the combination of a local anesthetic-like action with N-methyl-D-aspartate receptor-blocking effects, which modulate pain processing (10), has led to a renewed clinical interest in the use of ketamine for epidural or spinal application in the prevention of neuropathic or postoperative chronic pain states (11–13) . However, epidural ketamine in small concentrations as the sole drug did not provide a local anesthetic effect sufficient for adequate postoperative pain relief (14–16) . The aim of this in vitro study was to characterize the blocking effects of both ketamine enantiomers to define the concentrations required for blockade of sodium currents at different holding potentials, with a special emphasis on the difference in affinity between resting and inactivated channel states because preferential drug binding to inactivated channels and stabilization of the inactivated state is a prominent feature of traditional local anesthetics, i.e., lidocaine (17). Moreover, we were interested in a potential stereoselectivity or possible isoform-specific differences of the effects. We investigated the rat brain IIa form, which is most abundant in the adult brain (18), and hSkM1, the principle voltage-gated sodium channel expressed in the adult human skeletal muscle (19).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The {alpha} subunits of rat brain IIa and hSkM1 sodium channels were heterologously expressed in a mammalian cell line (human embryonic kidney [HEK] 293). Stably transfected HEK 293 cell lines expressing either the {alpha} subunit of rat brain IIa or hSkM1 sodium channels were a gift from Professor Lehmann-Horn, Ulm, Germany. The expression vector pRc/CMV (Invitrogen, San Diego, USA) was used for mammalian transfection. Transfection was performed with calcium phosphate precipitation. Permanent expression was achieved by selection for resistance to the aminoglycoside antibiotic Geneticin (G418) (Life Technology, Eggenstein, Germany) (20). Successful channel expression was verified electrophysiologically.

S(+)- and R(-)-ketamine were kindly provided by Waltraud Lesch (Pfizer GmbH, Freiburg, Germany). Ketamine was dissolved directly in a bath solution immediately before the experiments. The bath solution contained (mM) NaCl 140, MgCl2 1, KCl 4, CaCl2 2, HEPES 5, and dextrose 5. Patch electrodes contained [mM] CsCl2 130, MgCl2 2, EGTA 5, and HEPES 10. All solutions were adjusted to 290 mOsm by the addition of mannitol and to pH 7.4 by the addition of Cs(OH)2.

Standard whole-cell voltage-clamp experiments (21) were performed at 20°C. Each experiment consisted of test recordings with the drug present at only one concentration and of drug-free control recordings before and after the test. All test experiments were performed within 5 min of patch rupture. During the first 5 min of whole-cell recording, time-dependent hyperpolarizing shifts in control conditions were less than -2 mV (22). At least three different experiments were performed at each concentration, and the data were averaged to establish concentration-response plots.

For data acquisition and further analysis, we used the EPC9 digitally controlled amplifier in combination with Pulse and Pulse Fit software (HEKA Electronics, Lambrecht, Germany). The EPC9 provides automatic subtraction of capacitive and leakage currents by means of a prepulse protocol. The data were filtered at 10 kHz and digitized at 20 µs per point. The input resistance of the patch pipettes was at 2.0–3.5 M{Omega}, and cell capacitances ranged between 9 and 15 pF; the residual series resistance (after 50% compensation) was 1.2–2.5 M{Omega}. Experiments with an increase in series resistance were rejected. Voltage-activated currents were studied by applying different voltage-clamp protocols (described in Results or in figure legends).

Drug effects on the peak current amplitude were assessed at a holding potential close to the normal resting potential of nerve and muscle in physiological conditions (-70 mV) (23,24) and at hyperpolarized membrane potentials (-100 and -150 mV). The residual sodium current (INa+) in the presence of drug (with respect to the current amplitude in control solution) was plotted against the applied concentration of the drug [C].

Concentration dependence of the peak current suppression was fitted by using the Hill equation (Equation 1), which yielded the concentration for half-maximum channel blockade (IC50) and the Hill coefficient nH.


Statistical analysis of the variables of the Hill curves was performed to reveal isoform- or stereospecific differences in ketamine block by using the program "Nonlinear Least Squares Regression" of S-PLUS (S- PLUS 2000 Professional Release 1; MathSoft, Inc., Cambridge, MA), as described previously (25). The null hypothesis of no variable difference was rejected at P <= 0.05. All data are presented as means ± SD.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
A total of 94 cells were included in the study. Average currents in the control experiments after depolarization from -100 to 0 mV were -3.0 ± 1.3 nA (n = 47) for the skeletal muscle isoform and -2.9 ± 1.6 nA (n = 47) for the neuronal isoform. Maximum inward currents elicited by 40-ms pulses from -150, -100, or -70 mV to 0 mV were reversibly suppressed by S(+)- and R(-)-ketamine in a concentration-dependent manner. Representative current traces derived from four different experiments are depicted in Figure 1.



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  		Figure 1. Representative current traces after a 40-ms depolarization from -100 to 0 mV in the absence (control and washout) and presence of 2 different concentrations of S(+)-ketamine. Ketamine reversibly suppressed the peak inward current in both isoforms. Because each cell was exposed to one drug concentration only, the current traces depicted stem from four different experiments. The control currents were all scaled to the same size. The numerical amount of the peak inward current (nA) in the respective control experiment is indicated at the lower left of each row.

 
Blockade was rapid in onset and readily reversible on washout. The amount of block achieved depended on the holding potential from which the depolarization was started. Although concentration-response curves were almost superimposable at -150 and -100 mV holding potential, sensitivity to ketamine was increased in both isoforms when the membrane was kept at -70 mV. The IC50 values for S(+)- and R(-)-ketamine derived from Hill fits to the data at -150 and -70 mV are depicted in Figure 2. S(+)-ketamine was significantly more potent than R(-)-ketamine at all holding potentials in the skeletal muscle isoform only. Isoform-specific differences (higher sensitivity for blocking effects in the skeletal muscle isoform with respect to the neuronal isoform) were detected only for S(+)-ketamine. Calculated values for the Hill coefficients (nH) ranged between 0.5 and 0.8 for both stereoisomers and both channel isoforms at all holding potentials, indicating that ketamine binding shows no cooperativity (26).



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  		Figure 2. Tonic block achieved by both ketamine enantiomers in both channel isoforms at -70, -100, and -150 mV holding potential. Depolarizing pulses to 0 mV were started from -150 mV ({image}), -100 mV ({blacktriangleup}), or -70 mV ({blacksquare}). Each symbol represents the mean value of the residual sodium currents in the presence of drug with respect to the corresponding control experiments (Itest/Icontrol; mean ± SD), derived from at least three different experiments for each concentration tested. Although concentration-response plots were almost superimposable at -150 and -100 mV, sensitivity was increased at -70 mV holding potential. The solid lines are Hill fits (nH) (Equation 1) to the data with the indicated variables at -70 and -150 mV holding potential, respectively. *Significant difference in the 50% inhibitory concentration (IC50) values with respect to R(-)-ketamine and a significant difference in the IC50 values for voltage-gated human skeletal muscle versus rat type IIa neuronal sodium channels.

 
The increase in blocking potency at a holding potential of -70 mV, where a definite fraction of the channels is inactivated, compared with -150 mV, where all channels are expected to be in the resting state, suggests that the amount of block achieved by ketamine depends on the membrane potential and is increased with an increased fraction of inactivated channels with respect to resting channels. The voltage-dependence of the block and the affinity for the inactivated state was further assessed by applying a double-pulse protocol. After brief depolarizations, sodium channels enter a fast-inactivated state, from which they cannot readily reopen. Currents elicited by test pulses (Itest) starting from varying prepulse potentials (from -150 to -5 mV), normalized to the current elicited at the most hyperpolarized prepotential (-150 mV), represent the relative fraction of channels that have not been inactivated during the 50-ms inactivating prepulse. Boltzmann fits to the resulting current-voltage plots yield the membrane potential at half-maximum channel availability (V0.5) and the slope factor k, reflecting the voltage sensitivity of inactivation gating (see Equation 2).


In control conditions, the variables of the Boltzmann fits reflect the voltage dependence of the distribution between resting and fast-inactivated channels. Summarized control data showed that half of the channels were unavailable at -43.8 ± 7 mV (neuronal isoform) and at -56.6 ± 4.1 mV (skeletal muscle isoform) because of fast inactivation. The slope factors k were 7.9 ± 1.1 and 7.4 ± 1.0, respectively. With exposure to ketamine, V0.5 was shifted in the direction of more negative prepulse potentials; the degree of alteration showed concentration dependence. Drug effects on the voltage dependence of channel availability were reversible during washout, taking into account that a small hyperpolarizing shift may occur as a function of the recording time until completion of the washout experiments. Figures 3B and 4B plot normalized test pulse currents as a function of prepulse voltage in the absence and presence of 100 µM S(+)-ketamine in the neuronal and skeletal muscle isoforms, respectively.



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  		Figure 3. Effects of ketamine on fast-inactivated channels assessed by shifts in the steady-state availability curve in the neuronal isoform. A, Representative current traces in the control and in 100 µM S(+)-ketamine during short (4-ms) test pulses to 0 mV, after a 50-ms prepulse from -150 mV to -150 mV (first row of traces), -70 mV (second row of traces), or a membrane potential close to the potential for half-maximum channel inactivation for the respective channel isoform in the controls (-45 mV in case of the neuronal isoform and -55 mV in case of the skeletal muscle isoform). There is no current activation during the 50-ms prepulse from -150 mV to the indicated prepulse potentials. All current traces elicited by the test pulses are scaled to maximum value at a -150-mV prepulse potential, indicated at the left of the fist row. The traces reveal a slight increase in the peak current suppression achieved by S(+)-ketamine at more depolarized prepulse potentials. B, Steady-state availability curves assessed by a two-pulse protocol in the absence (control, •; washout, {image}) and presence of 100 µM S(+)-ketamine ({triangleup}). Each symbol represents the mean fractional current derived from at least 3 different experiments, elicited by a 4-ms test pulse to 0 mV, after a 50-ms inactivating prepulse from -150 mV to the indicated prepulse potential. Currents were normalized to maximum value (in each series at -150 mV prepotential); solid lines represent the best Boltzmann fit (Equation 2) to the data with the indicated variable V0.5 (mV) (voltage of half-maximum channel availability) for the respective control and test experiments. The indicated errors are standard deviations. Currents were normalized either to maximum value in the presence of drug (filled symbols) or to maximum value in the controls (empty symbols). Vertical arrows illustrate the increase in the peak current suppression induced by 100 µM S(+)-ketamine at more depolarized holding potentials versus hyperpolarized holding potentials. This reduction in channel availability at depolarized prepotentials resulted in a voltage shift in the midpoints of the availability curve ({Delta}V0.5), as indicated by the horizontal arrows. C, Concentration dependence of the drug-induced negative shifts in the midpoints ({Delta}V0.5 [mV]) of the steady-state availability plots relative to the starting values for S(+)- and R(-)-ketamine. Each symbol represents the mean value derived from at least three different experiments each; error bars are standard deviations. The solid line is a least-squares fit of the model of Bean et al. (17) (Equation 3) to the averaged data. Variables inserted into the equation are printed in italics; the slope factor k was derived from Boltzmann fits to the control data, and the 50% inhibitory concentration (IC50) at -150 mV holding potential was derived from the concentration-response curves depicted in Figure 2. The respective dissociation constants (KdI) from the inactivated channel state for S(+)- and R(-)-ketamine derived from that fit to the averaged data were 88 and 74 µM for the neuronal isoform.

 


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  		Figure 4. Effects of ketamine on fast-inactivated channels assessed by shifts in the steady-state availability curve in the skeletal muscle isoform. A, Representative current traces in the control and in 100 µM S(+)-ketamine during short (4-ms) test pulses to 0 mV, after a 50-ms prepulse from -150 mV to -150 mV (first row of traces), -70 mV (second row of traces), or a membrane potential close to the potential for half-maximum channel inactivation for the respective channel isoform in the controls (-45 mV in case of the neuronal isoform and -55 mV in case of the skeletal muscle isoform). There is no current activation during the 50-ms prepulse from -150 mV to the indicated prepulse potentials. All current traces elicited by the test pulses are scaled to maximum value at a -150-mV prepulse potential, indicated at the left of the fist row. The traces reveal a slight increase in the peak current suppression achieved by S(+)-ketamine at more depolarized prepulse potentials. B, Steady-state availability curves assessed by a two-pulse protocol in the absence (control, •; washout, {image}) and presence of 100 µM S(+)-ketamine ({triangleup}). Each symbol represents the mean fractional current derived from at least 3 different experiments, elicited by a 4-ms test pulse to 0 mV, after a 50-ms inactivating prepulse from -150 mV to the indicated prepulse potential. Currents were normalized to maximum value (in each series at -150 mV prepotential); solid lines represent the best Boltzmann fit (Equation 2) to the data with the indicated variable V0.5 (mV) (voltage of half-maximum channel availability) for the respective control and test experiments. The indicated errors are standard deviations. Currents were normalized either to maximum value in the presence of drug (filled symbols) or to maximum value in the controls (empty symbols). Vertical arrows illustrate the increase in the peak current suppression induced by 100 µM S(+)-ketamine at more depolarized holding potentials versus hyperpolarized holding potentials. This reduction in channel availability at depolarized prepotentials resulted in a voltage shift in the midpoints of the availability curve ({Delta}V0.5), as indicated by the horizontal arrows. C, Concentration dependence of the drug-induced negative shifts in the midpoints ({Delta}V0.5 [mV]) of the steady-state availability plots relative to the starting values for S(+)- and R(-)-ketamine. Each symbol represents the mean value derived from at least three different experiments each; error bars are standard deviations. The solid line is a least-squares fit of the model of Bean et al. (17) (Equation 3) to the averaged data. Variables inserted into the equation are printed in italics; the slope factor k was derived from Boltzmann fits to the control data, and the 50% inhibitory concentration (IC50) at -150 mV holding potential was derived from the concentration-response curves depicted in Figure 2. The respective dissociation constants (KdI) from the inactivated channel state for S(+)- and R(-)-ketamine derived from that fit to the averaged data were 22 and 48 µM for the skeletal muscle isoform.

 
The drug-induced hyperpolarizing shifts reflect an additional reduction of channel availability in the voltage range of channel inactivation compared with -150 mV. The concentration dependence of this effect for both ketamine stereoisomers is depicted in Figures 3C and 4C.

To estimate the dissociation constant (KdI) of S(+)- and R(-)-ketamine for the fast-inactivated state of both channel isoforms, we used a model developed by Bean et al. (17) for lidocaine effects on Purkinje fibers. The model is based on the assumption that the higher amount of channel block achieved with consecutive membrane depolarization is determined by the apportionment of channels between resting and fast-inactivated states, as well as by the different binding affinities of the blocking drug for the two channel states (Equation 3). However, the model should be regarded only as an approximation, yielding estimates for the difference between resting and inactivated state affinity. Because the Hill coefficients nH were approximately 1 at -150 mV holding potential, the dissociation constant for the resting state (KdR) was represented by the concentration for half-maximum current inhibition at -150 mV holding potential (IC50-150).


{Delta}V0.5 is the shift in the midpoint in each drug concentration (mean, n > 3), and k is the mean value for the slope factor derived from Boltzmann fits to the current-voltage plots (Equation 2). Figures 3C and 4C show the respective model fits to the averaged data. For S(+)- and R(-)-ketamine, the estimated values of KdI derived from that fit were 88 and 74 µM for the neuronal and 22 and 48 µM for the skeletal muscle isoform.

The time of membrane repolarization required for the recovery of sodium channels from depolarization-induced nonconducting states was examined at -100 mV by a two-pulse recovery protocol with varying time intervals between the 30-ms inactivating prepulse and the test pulse to 0 mV. The time constants of recovery ({tau}rec) were derived from exponential fits to the fractional current after recovery from inactivation, plotted against the time interval between the inactivating prepulse and the test pulse (Equation 4).


In the absence of the drug, both rat IIa and hSkM1 recovery time courses were described by a monoexponential function ({tau}rec1, 4.1 ± 3.1 and 3.5 ± 2.3 ms). S(+)-and R(-)-ketamine introduced a second, slower component ({tau}rec2) of 27–30 ms to the recovery time course, which is assumed to reflect the time course of drug dissociation from inactivated channel states (27). The amplitude of this slow component of recovery of approximately 30 ms increased from <10% of the total recovery amplitude in the 100 µM concentrations to 15%–18% in 300 µM S(+)- or R(-)-ketamine, respectively. This means that, up to a concentration of 300 µM, recovery from inactivated channel block at a hyperpolarized holding potential (-100 mV) should be too fast to accumulate relevant use-dependent block at stimulating frequencies lower than 10 Hz.

Use-dependent or phasic block was defined as the additional reduction in INa+ for the last pulse relative to the first pulse, assessed in a 10-Hz test train of 10-ms depolarizing pulses from -100 to 0 mV. As expected, ketamine up to a concentration of 300 µM did not induce use-dependent block >8% at 10 Hz.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study we observed blocking effects of ketamine on heterologously expressed voltage-operated sodium channels at concentrations larger than 10 µM. Differences in potency between S(+)- and R(-)-ketamine were small and reached statistical significance in the skeletal muscle isoform only with S(+)-ketamine being more potent than R(-)-ketamine. The IC50 values for tonic block of sodium channel {alpha} subunits expressed in HEK 293 cells corresponded to the IC50 values reported for ketamine block of sodium channels expressed in native tissue (6–8) . On the one hand, qualitatively, sodium channel blockade by ketamine shares important features with blockade of sodium channels by lidocaine-like local anesthetics (17,28) , including a leftward shift in the voltage dependency of channel availability and a prolongation of recovery from inactivated channel block at large concentrations (>300 µM) (9). All these phenomena can be explained by the assumption that the drug binds more tightly to the inactivated form of the channel, thereby stabilizing the inactivated state (17,27) . On the other hand, a quantitative analysis of the voltage dependency of ketamine block reveals important differences between ketamine and lidocaine-like local anesthetics.

Although tonic block at hyperpolarized holding potentials occurs in the same concentration range for both ketamine enantiomers as for lidocaine (28), the difference in affinity between resting and inactivated channel states derived from the voltage shift in the availability curve was approximately 8- to 10-fold with ketamine in our study, as opposed to approximately 50-fold in the case of lidocaine (28). This relatively small difference in affinity between inactivated and resting channels seen with ketamine might explain why mechanistic studies of ketamine‘s actions on sodium channels led to conflicting results. Some authors did find a shift in the inactivation curve (9), whereas others did not (5). For lidocaine-like local anesthetics, it has been shown that differences in the blocking effects between the tissue-specific sodium channel isoforms are determined by the fraction of inactivated channels, which in turn depends on the respective membrane potential, on the one hand, and the isoform-specific voltage dependence of inactivation, on the other (29). This explanation apparently does not hold for ketamine, because isoform-specific differences in the sensitivity to S(+)-ketamine were detected at all holding potentials. Our control data revealed that the voltage of half-maximum channel inactivation was more negative in the skeletal muscle isoform than in the neuronal isoform. At -70 mV holding potential, approximately 20% of the skeletal muscle channels undergo inactivation, yet only 5% of neuronal sodium channels do so. If the observed higher sensitivity of the skeletal muscle isoform to S(+)-ketamine were determined primarily by the fraction of inactivated channels at a given membrane potential, the difference between the skeletal muscle and the neuronal isoform would have been apparent at -70 mV only and not at -150 mV, where all channels are expected to be in the resting state.

Major differences between ketamine and lidocaine-like local anesthetics refer not only to the reduced voltage dependency of ketamine block, but also to the time course of drug equilibration between resting and inactivated channel states. After membrane repolarization, recovery from inactivated channel block in the presence of ketamine is much faster compared with the time course of recovery of lidocaine-bound channels. The slow component of recovery of approximately 30 ms comprised <10% of the recovery amplitude in 100 µM ketamine in our study, whereas in the presence of 100 µM lidocaine, the slow component of recovery was >100 ms and comprised ~50% of the recovery amplitude (28). This rapid recovery from inactivated channel block explains the lack of use-dependent block seen with smaller ketamine concentrations in our study. The fact that ketamine is far less effective than lidocaine in inducing use-dependent blockade was reported recently (9). In the case of ketamine, use-dependent block apparently occurs only in a large-concentration range, which causes considerable tonic block at the same time (7,9) . Additionally, differences between studies concerning the amount of phasic block induced by ketamine (6,7,9) may be attributed to differences in the experimental protocol.

The functional importance of use-dependent block was revealed by current-clamp experiments, where small concentrations of lidocaine or bupivacaine did not suppress the single action potential in dorsal root ganglion neurons but clearly reduced the firing frequency of action potentials (30). These results indicate that the IC50 values for tonic block in the case of lidocaine and bupivacaine may underestimate the channel sensitivity to these local anesthetics, because smaller anesthetic concentrations may be sufficient for block of sodium channels during repetitive firing in neurons. Because ketamine is less potent in phasic blockade, larger concentrations of the compound might be required for a comparable local anesthetic effect.

For this study, we used {alpha} subunits of rat brain IIa and hSkM1 sodium channels expressed in a mammalian cell line (HEK 293). However, native sodium channels are not homomeric. They are heteromultimeric proteins consisting of {alpha}, ß1, and ß2 subunits. Despite the lack of the ß subunit, the suitability of our preparation for studying channel-gating kinetics has been verified experimentally. The {alpha} subunit is the primary pore-forming subunit of the channel and functions as an ion channel when expressed alone (31). When skeletal muscle or brain {alpha} subunits are expressed in oocytes, they have abnormally slow inactivation kinetics, which are accelerated by coexpression of the ß1 subunit (32). In contrast, when expressed in a mammalian cell line, {alpha} subunits of rat brain IIa and hSkM1 sodium channel {alpha} subunits show normal gating characteristics (like native channels) (33,34) . The type IIa sodium channel is the most prominent subtype present in adult brain, and the rat brain IIa subtype has more than 97% structural identity with the equivalent human brain sodium channel (35).

So far, clinical studies about epidural ketamine for postoperative analgesia involved doses up to 30 mg, corresponding to a 0.02% to 0.2% ketamine solution (1). In this small concentration range, epidural ketamine as the sole drug does not provide a local anesthetic effect sufficient for adequate postoperative pain relief (1). In a pharmacokinetic study using a dog model, a ketamine dose of 3 mg/kg was readily absorbed from the epidural space into the cerebrospinal fluid. Maximum concentrations of ketamine in the cerebrospinal fluid reached 5 µg/mL (20 µM) (36).

Our in vitro data reveal that this concentration may rather represent the threshold concentration for ketamine-induced sodium channel blockade. Thus, we hypothesize that larger ketamine doses (>3 mg/kg) may be required for an adequate local anesthetic effect via blockade of voltage-operated sodium channels. However, the clinical use of ketamine as a local anesthetic in larger concentrations might be limited by systemic effects because of its high bioavailability (36).


   Acknowledgments
 
We are indebted to Prof. Frank Lehmann-Horn, Ulm, Germany, for providing us with transfected cells; Birgitt Nentwig, Department of Anesthesiology, Hannover, Germany, for taking care of the cell culture; Dr. Hans-Peter Reiffen, Department of Anesthesiology, Hannover, Germany, for help with software problems; and Jobst Kilian and Andreas Niesel, Department of Neurology, Hannover, Germany, for technical support. S(+)- and R(-)-ketamine were kindly provided by Waltraud Lesch, Pfizer GmbH, Freiburg, Germany.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Schmid RL, Sandler AN, Katz J. Use and efficacy of low-dose ketamine in the management of acute postoperative pain: a review of current techniques and outcomes. Pain 1999; 82: 111–25.[Web of Science][Medline]
  2. Meller ST. Ketamine: relief from chronic pain through actions at the NMDA receptor? Pain 1996; 68: 435–6.[Web of Science][Medline]
  3. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–14.[Medline]
  4. Bion JF. Intrathecal ketamine for war surgery: a preliminary study under field conditions. Anaesthesia 1984; 39: 1023–8.[Web of Science][Medline]
  5. Benoit E. Effects of intravenous anesthetics on nerve axons. Eur J Anaesthesiol 1995; 12: 59–70.[Web of Science][Medline]
  6. Hara Y, Chugun A, Nakaya H, Kondo H. Tonic block of the sodium and calcium currents by ketamine in isolated guinea pig ventricular myocytes. J Vet Med Sci 1998; 60: 479–83.[Web of Science][Medline]
  7. Zhou ZS, Zhao ZQ. Ketamine blockage of both tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels of rat dorsal root ganglion neurons. Brain Res Bull 2000; 52: 427–33.[Web of Science][Medline]
  8. Bräu ME, Sander F, Vogel W, Hempelmann G. Blocking mechanisms of ketamine and its enantiomers in enzymatically demyelinated peripheral nerve as revealed by single-channel experiments. Anesthesiology 1997; 86: 394–404.[Web of Science][Medline]
  9. Wagner LE, Gingrich KJ, Kulli JC, Yang J. Ketamine blockade of voltage-gated sodium channels. Anesthesiology 2001; 95: 1406–13.[Web of Science][Medline]
  10. Ripamonti C, Dickerson ED. Strategies for the treatment of cancer pain in the new millennium. Drugs 2001; 61: 955–77.[Web of Science][Medline]
  11. Hartrick CT, Wise JJ, Patterson JS. Preemptive intrathecal ketamine delays mechanical hyperalgesia in the neuropathic rat. Anesth Analg 1997; 86: 557–60.
  12. Klimscha W, Horvath G, Szikszay M, et al. Antinociceptive effect of the S(+)-enantiomer of ketamine on carrageenan hyperalgesia after intrathecal administration in rats. Anesth Analg 1998; 86: 561–5.[Abstract]
  13. Himmelseher S, Ziegler-Pithamitsis D, Argiriadou H, et al. Small-dose S(+)-ketamine reduces postoperative pain when applied with ropivacaine in epidural anesthesia for total knee arthroplasty. Anesth Analg 2001; 92: 1290–5.[Abstract/Free Full Text]
  14. Kawana Y, Sato H, Shimada H, et al. Epidural ketamine for postoperative pain relief after gynecologic operations: a double-blind study and comparison with epidural morphine. Anesth Analg 1987; 66: 735–8.[Abstract/Free Full Text]
  15. Peat SJ, Bras P, Hanna MH. A double-blind comparison of epidural ketamine and diamorphine for postoperative analgesia. Anaesthesia 1989; 44: 555–8.[Web of Science][Medline]
  16. Ravat F, Dorne R, Baechle JP, et al. Epidural ketamine or morphine for postoperative analgesia. Anesthesiology 1987; 66: 819–22.[Web of Science][Medline]
  17. Bean BP, Cohen CJ, Tsien RW. Lidocaine block of cardiac sodium channels. J Gen Physiol 1983; 81: 613–42.[Abstract/Free Full Text]
  18. Beckh S, Noda M, Lübbert H, Numa S. Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development. EMBO J 1989; 8: 3611–6.[Web of Science][Medline]
  19. George A, Komisarof J, Kallen R, Barchi R. Primary structure of the adult human skeletal muscle voltage-dependent sodium channel. Ann Neurol 1992; 31: 131–7.[Web of Science][Medline]
  20. Mitrovic N, George AL, Heine R, et al. K+-aggravated myotonia: destabilization of the inactivated state of the human muscle sodium channel by the V1589M mutation. J Physiol 1994; 478: 395–402.[Abstract/Free Full Text]
  21. Hamill OP, Marty A, Neher E, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981; 391: 85–100.[Web of Science][Medline]
  22. Haeseler G, Petzold J, Hecker H, et al. Succinylcholine metabolite succinic acid alters steady-state activation in muscle sodium channels. Anesthesiology 2000; 92: 1385–92.[Web of Science][Medline]
  23. Hodgkin AL, Horowicz P. The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J Physiol 1959; 148: 127–60.
  24. Kandel ER, Schwartz JH, Jessel TM. Principles of neural science. 3rd ed. New York: Elsevier, 1991.
  25. Haeseler G, Störmer M, Bufler J, et al. Propofol blocks skeletal muscle sodium channels in a voltage-dependent manner. Anesth Analg 2001; 92: 1192–98.[Abstract/Free Full Text]
  26. Colquhoun D. Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 1998; 125: 923–47.
  27. Vedantham V, Cannon SC. The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels. J Gen Physiol 1999; 113: 7–16.[Abstract/Free Full Text]
  28. Fan Z, George AL, Kyle JW, Makielski JC. Two human paramyotonia congenita mutations have opposite effects on lidocaine block of Na+ channels expressed in a mammalian cell line. J Physiol 1996; 496: 275–86.[Abstract/Free Full Text]
  29. Wright SN, Wang SY, Kallen RG, Wang GK. Differences in steady-state inactivation between Na channel isoforms affect local anaesthetic binding affinity. Biophys J 1997; 73: 779–88.[Web of Science][Medline]
  30. Scholz A, Kuboyama N, Hempelmann G, Vogel W. Complex blockade of TTX-resistant Na+ currents by lidocaine and bupivacaine reduce firing frequency in DRG neurons. J Neurophysiol 1998; 79: 1746–54.[Abstract/Free Full Text]
  31. Catterall WA. Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 1992; 72 (Suppl 4): 15–48.
  32. Moran O, Nizzari M, Conti F. Endogenous expression of the ß1A sodium channel subunit in HEK-293 cells. FEBS Lett 2000; 473: 132–4.[Web of Science][Medline]
  33. Chahine M, Bennett P, George A, Horn R. Functional expression and properties of the human skeletal muscle sodium channel. Pflügers Arch 1994; 427: 136–42.[Web of Science][Medline]
  34. Sarkar SN, Adhikari A, Sikdar SK. Kinetic characterization of rat brain type IIA sodium channel {alpha}- subunit stably expressed in a somatic cell line. J Physiol 1995; 488: 633–45.[Abstract/Free Full Text]
  35. Ahmed CM, Ware DH, Lee SC, et al. Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain. Proc Natl Acad Sci U S A 1992; 89: 8220–4.[Abstract/Free Full Text]
  36. Pedraz JL, Calvo MB, Gascon AR, et al. Pharmacokinetics and distribution of ketamine after extradural administration in dogs. Br J Anaesth 1991; 67: 310–6.[Abstract/Free Full Text]
Accepted for publication November 27, 2002.




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