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REGIONAL ANESTHESIA AND PAIN MEDICINE

Block of Neuronal Tetrodotoxin-Resistant Na+ Currents by Stereoisomers of Piperidine Local Anesthetics

Michael E. Bräu, PD Dr. med.^*, Pierre Branitzki, Andrea Olschewski, Dr. med.^*, Werner Vogel, Prof. Dr. rer. nat., and Gunter Hempelmann, Prof. Dr. med., Dr. h.c.^*

Departments of *Anesthesiology and Intensive Care Medicine and Physiology, Justus-Liebig-University, Giessen, Germany

Address correspondence and reprint requests to Michael E. Bräu, PD Dr. med, Abteilung Anaesthesiologie und Operative Intensivmedizin Justus-Liebig-Universität, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany. Address e-mail to meb{at}anesthesiology.de

	Abstract

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Tetrodotoxin (TTX)-sensitive Na⁺ channels in the peripheral nervous system are the major targets for local anesthetics. In the peripheral nociceptive system, a Na⁺ channel subtype resistant to TTX and with distinct electrophysiological properties seems to be of importance for impulse generation and conduction. A current through TTX-resistant Na⁺ channels displays slower activation and inactivation kinetics and has an increased activation threshold compared with TTX-sensitive Na⁺ currents and may have different pharmacological properties. We studied the effects of stereoisomers of piperidine local anesthetics on neuronal TTX-resistant Na⁺ currents recorded with the whole-cell configuration of the patch clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Stereoisomers of mepivacaine, ropivacaine, and bupivacaine reversibly inhibited TTX-resistant Na⁺ currents in a concentration and use-dependent manner. All drugs accelerated time course of inactivation. Half-maximal blocking concentrations were determined from concentration-inhibition relationships. Potencies for tonic and for use-dependent block increased with rising lipid solubilities of the drugs. Stereoselective action was not observed. We conclude that block of TTX-resistant Na⁺ currents may lead to blockade of TTX-resistant action potentials in nociceptive fibers and consequently may be responsible for pain suppression during local anesthesia.

Implications: Tetrodotoxin-resistant Na⁺ channels are important in peripheral nociception. During local anesthesia, these channels are blocked by mepivacaine, ropivacaine, and bupivacaine in a concentration and use-dependent manner, but not stereoselectively.

	Introduction

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Piperidine local anesthetics, such as mepivacaine, ropivacaine, and bupivacaine are widely used in clinical practice. These amide-linked drugs share a common basic structure, differing only in the length of the alkyl chain attached to the tertiary amine in molecule, and because of a chiral carbon in the piperidine ring, they exist as stereoisomers or enantiomers (Fig. 1). Despite similar nerve blocking potencies, the S(-)-forms of ropivacaine and bupivacaine have distinct advantages over the R(+)-forms or the racemic mixtures. This is mainly because of the decreased cardiovascular and central nervous system side effects of the S(-)-enantiomers (1) resulting in an increased therapeutic index. Because of these advantages, ropivacaine has only been introduced into clinical practice as the S(-)-enantiomer, and bupivacaine, used as the racemic mixture of both enantiomers, will soon be available in the S(-)-form known as levobupivacaine. The local anesthetic action of these drugs is achieved by Na⁺ channel blockade in the peripheral nervous system.

View larger version (12K): [in this window] [in a new window]   Figure 1. Basic structure of piperidine local anesthetics. Typical structural elements of local anesthetics can be identified, i.e., aromatic ring on the left, amide bond in the middle, and the tertiary amine in the piperidine ring on the right. The asterisk marks the chiral carbon of the structure. The alkyl chain (R) attached to the nitrogen (N) of the piperidine ring determines the lipid solubility of the drug and its local anesthetic and toxicological properties.

Compound action potentials in C-fibers are partly resistant to TTX, but depend on extracellular Na⁺ ions. This was demonstrated by using various preparations of different species including human sural nerves (2) and demonstrates the important role of TTX-resistant Na⁺ channels in conduction of nociceptive impulses. It further underlines the importance of blocking these channels during local anesthesia, which leads to blockade of TTX-resistant action potentials in nociceptive fibers and consequently to pain suppression during local anesthesia. TTX-resistant Na⁺ channels have also been cloned from human dorsal root ganglia (DRG)-neurons, which makes them putative targets for pain therapy in humans (4).

Because of its role in nociception, inhibition of neuronal TTX-resistant Na⁺ currents is relevant for blocking pain perception during local anesthesia. Therefore, we studied the blockade of neuronal TTX-resistant Na⁺ currents by stereoisomers of the piperidine local anesthetics, mepivacaine, ropivacaine, and bupivacaine.

	Methods

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Adult Wistar rats (200–300 g) were used for preparing the primary DRG-cell culture used in this work. Animals were killed by concussion and immediate cervical dislocation. The procedure was approved by the local veterinarian authority. Dorsal root ganglia were removed from the full length of the vertebral column and placed in calcium and magnesium-free phosphate buffered saline. After cleaning the ganglia from connective tissue, they were incubated for 30 min at 37°C in 2 mg/mL collagenase Worthington type CLS II (Biochrom, Berlin, Germany) and 2 mg/mL trypsin type III-S (Sigma, Deisenhofen, Germany) dissolved in phosphate buffered saline, in a shaking water bath. Afterward, the ganglia were washed three times with plating medium (described below) and transferred into 80 µg/mL DNAse, type IV and 100 µg/mL trypsin inhibitor, type I-S. Fire-polished pipettes with decreasing diameter were then used for mechanically dissociating the cells. After this procedure, the cells were plated out in 35 mm culture dishes and stored in plating medium under 95% O₂ and 5% CO₂ at room temperature until the experiment. Cells were used for the experiments 24–72 h after preparation. Significant changes in Na⁺ current properties, i.e., amplitude and time course of the currents, were not detected during this time period.

TTX-resistant Na⁺ currents were recorded by using the whole-cell patch clamp method. A culture dish containing the cells was placed on the stage of an inverted microscope and the plating medium was changed to low Na⁺ Tyrode. Experiments were conducted at 22°C.

Patch pipettes were pulled from glass capillaries (Type CEEBEE 101-PS, Chr. Bardram, Svendborg, Denmark) by using a Flaming/Brown Micropuller (Sutter Instrument Company, Science Products GmbH, Hofheim, Germany). The pipettes were fire polished before use and, when filled with internal solution, had a resistance of 0.8 to 1.2 M.

Current recordings were made by an Axopatch 200B patch clamp amplifier (Axon Instruments, Burlingame, CA) in the voltage-clamp mode. Analog data were filtered at 5 kHz, digitized at 20 kHz by using a 12-bit AD-converter (Labmaster TM-40 AD/DA board, Scientific Solutions, Solon, OH), and stored on the hard disk of a personal computer, which also served as the stimulus generator. All experiments were conducted with capacitance and series resistance compensation (60% to 70%). Leakage current correction was performed electronically with the patch clamp amplifier. PClamp 6.0 software (Axon Instruments) was used for acquisition and analysis of currents.

Low Na⁺ Tyrode used for the bath and control solution contained: 35 mM NaCl, 110 mM choline-chloride, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 6 mM glucose, and 10 mM HEPES. The pH was adjusted to 7.4 with tetraethylammoniumhydroxide. We added 100 nM TTX to suppress TTX-sensitive Na⁺ currents and 20 mM tetraethylammonium-chloride to block K⁺ currents. The low Na⁺ concentration was necessary to reduce the magnitude of Na⁺ currents to improve voltage-clamp conditions.

The internal solution, CsF_i, contained: 140 mM CsF, 10 mM NaCl, 3 mM EGTA, and 10 mM HEPES. The pH was adjusted to 7.2 with CsOH. Internal cesium fluoride was used to suppress potassium and calcium currents.

Plating medium was freshly made of 26 mL minimum essential medium, 3 mL fetal calf serum, 1000 IU penicillin, 1 mg streptomycin, and 0.6 mL L-glutamine (200 mM). All chemicals were obtained from Sigma, Deisenhofen, Germany. Local anesthetics were provided by ASTRA Pain control, Södertälje, Sweden.

Solution exchanges were performed with a multiple-barrel perfusion system. The barrels of the perfusion system were directly connected to syringes containing the control and test solutions. The syringes were constantly driven by a perfusion pump, giving a steady solution flow of 5 mL/h corresponding to a flow speed of 40 mm/min in each barrel. After formation of the whole-cell configuration, the cell was lifted up, still attached to the pipette tip and placed into the barrel containing the desired solution. The seal quality, as well as the signal/noise ratio, were not influenced by this procedure; solution exchanges were completed in <1 s.

Current-voltage relationships (I/E) were constructed by plotting the peak Na⁺ current (I_Na), elicited by 50 ms depolarizing pulses to different test potentials (E_t), after a 50 ms hyperpolarizing prepulse to -110 mV, against E_t. The variables describing the I/E relationship were evaluated by fitting the modified Boltzmann function

to the data points, where G_Na,max is the maximal possible Na⁺ conductance, E_50,a is the half-maximal activation potential, k_a is the slope factor of the activation curve, and E_Rev is the reversal potential of the current.

To determine blocking potencies for tonic and use-dependent block, concentration-inhibition curves were constructed from peak current inhibition by the drugs. For this, Na⁺ currents were elicited by a 50-ms depolarizing pulse to -10 mV, preceded by a 50-ms hyperpolarizing prepulse to -110 mV. The impulse protocol was applied in a train of 10 at a frequency of 2 Hz once in control solution, in different local anesthetic concentrations, and again in control solution to check reversibility. Fractional inhibition of the current was measured by dividing the peak current in the presence of drug by the peak current in the previous control solution during the first (tonic inhibition) and 10th pulse (use-dependent inhibition) of the train. Plotting fractional inhibition (f_i) against local anesthetic concentration (c) gave concentration-inhibition relationships for each drug for tonic and use-dependent (2 Hz) block. Nonlinear least-squares fitting of

to the data points was performed to evaluate half-maximal inhibiting concentrations (IC₅₀); h represents the Hill coefficient.

Fitting procedures, as well as the preparation of the figures were done with Fig.P 5.0 software (Biosoft, Cambridge, UK). Data points indicate mean ± SEM; given variables are fitted values ± SE of fit.

	Results

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In the absence of TTX, Na⁺ currents recorded from small and medium size (20–35 µm diameter) DRG-cells often had a mixture of varying proportions of fast and slow currents. Adding 100 nM TTX to the control solution completely blocked the fast Na⁺ current component, permitting the isolated investigation of the slow TTX-resistant Na⁺ current. In large neurons (>50 µm diameter), fast TTX-sensitive currents dominated, being the only Na⁺ current component in some cells. Traces of the fast TTX-sensitive Na⁺ current at various test potentials in a large cell without TTX are shown in Fig 2, A; and traces of the slow TTX-resistant current in a small cell after adding 100 nM TTX are shown in Fig 2, B. Plotting the peak current against test potential gave I/E-relationships of the currents, which were fitted with Equation 1 (Fig. 2 A and B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Traces of tetrodotoxin (TTX)-sensitive and TTX-resistant Na+ currents in dorsal root ganglion neurons and corresponding I/E relationships. Currents were elicited by depolarizing the membrane to different test potentials (inset). A, fast currents in a large cell (51 µm diameter) recorded without TTX. B, slow currents recorded from a medium size cell (26 µm diameter) in 100 nM TTX. The I/E relationship was fitted with Equation 1 (see text), giving E50 values of -42 ± 1 mV for the fast current and -21 ± 1 mV for the slow current (fitted values ± SE of fit). The I/E relationship is the peak Na+ current (INa), elicited by 50 ms depolarizing pulses to different test potentials (Et), after a 50 ms hyperpolarizing prepulse to -110 mV, against Et. nA = nano amperes.

View larger version (25K): [in this window] [in a new window]   Figure 3. Use-dependent block by piperidine stereoisomers. A, Traces of tetrodotoxin (TTX)-resistant currents in control solution and in stereoisomers of mepivacaine, ropivacaine, and bupivacaine. Dashed traces are recorded in control solution, solid traces represent trains of 10 recorded in 30 µM of the drug at a stimulation frequency of 2 Hz. B, Fractional current in 30 µM enantiomers in dependence on impulse number at 2 Hz stimulation. Current in control shows no or little detriment when applying the 10 Hz pulses (not shown). Impulse protocol is given above the traces. nA = nano amperes.

View larger version (19K): [in this window] [in a new window]   Figure 4. Concentration-inhibition curves for tonic and use-dependent block by the stereoisomers. Fractional inhibition of the peak current of the first (tonic, closed symbols) and 10th (use-dependent, open symbols) pulse in the 2 Hz train was plotted against concentration. Curves represent fits of Equation 2 to the data points, with the Hill coefficient h fixed to one (see text). Half-maximal blocking concentrations are presented in Table 1.

View larger version (19K): [in this window] [in a new window]   Figure 5. Half-maximal inhibiting concentrations (IC50) values for tonic (•) and use-dependent () block plotted against the number of C-atoms in the alkyl chain. Stereoisomers of mepivacaine, ropivacaine, and bupivacaine are marked by + or - in the symbol. For 1-pentyl-2,6-methyl-pipecoloxylidide (RAD393) and 1-octyl-2,6-methyl-pipecoloxylidide only the racemic mixtures were available. Correlation coefficients and slope factors of the regression lines were -0.97 and -0.22 for tonic and -0.98 and -0.34 for use-dependent block, respectively.

	Discussion

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Unfortunately, Na⁺ channels in nociceptive fibers cannot be investigated directly. However, in the somata of these fibers, channels are synthesized and incorporated into its membrane where they can be easily investigated with the patch clamp method. Current through neuronal TTX-resistant Na⁺ channels as measured in small and medium size DRG neurons has slower activation and inactivation kinetics, an increased activation threshold, and faster repriming kinetics (9) compared with TTX-sensitive Na⁺ currents. On the molecular level, it has become apparent that at least two distinct Na⁺ channels contribute to the TTX-resistant current in rat DRG neurons as shown by whole-cell experiments, single channel analysis (10), and cloning experiments (11). We did not discriminate between TTX-resistant channel subtypes, which are both important in nociceptive fibers of the peripheral nervous system (11). In fact, we do think that both channel types contribute to the TTX-resistant current we have measured. This is apparent from the voltage-dependent activation variables of the TTX-resistant current. The -21 mV we have measured as the half-maximal activation potential for the TTX-resistant current lies between those for the SNS/PN3 and SNS2/NaN Na⁺ channel subtype (11).

We investigated the effects of piperidine local anesthetics on TTX-resistant Na⁺ currents in small dorsal root ganglia neurons with respect to lipid solubility and to stereospecificity. The major findings are that the drugs produce state-dependent block; potency for tonic and use-dependent block increases with the length of the alkyl side chain of the drug, i.e., lipid solubility; and there is no significant stereoselectivity of current inhibition by mepivacaine, ropivacaine, or bupivacaine.

Local anesthetics produce state-dependent block. At least three major conformational states of the Na⁺ channel exist, i.e., resting, open, and inactivated. On short depolarization, the channel transits from the resting into the open state and subsequently, within a few milliseconds, into the inactivated state. Prolonged depolarizations put all types of voltage-gated Na⁺ channels into an additional slow inactivated state, the recovery from which, lies in the range of seconds. Use-dependent block of Na⁺ channels and leftward shift of availability curves are well known features of local anesthetics and are thought to result from state-dependent block, i.e., different channel states exhibit different affinities to the local anesthetic, as described by the modulated (5,6) or guarded (12) receptor hypothesis. In our experiments with TTX-resistant Na⁺ currents, a decreased inactivation time constant was observed in all drugs. This may either result from an increased rate from the open into the inactivated state of the channel, or from open channel block under the drug. In both cases, higher affinities of the drugs to channel states induced by depolarization may account for the observed phenomenon responsible for use-dependent block.

Ragsdale et al (13) could identify a putative local anesthetic receptor in rat brain IIa channels as two hydrophobic amino-acids residing in the IV-S6 segment of the -subunit. Because the cloned neuronal TTX-resistant Na⁺ channel, SNS/PN3, and SNS2/NaN have identical amino acids at corresponding positions, blockade via this specific receptor is also likely for these channels. Assuming that local anesthetics bind to these hydrophobic amino-acids in the channel pore, increased hydrophobicity of the alkyl moiety may lead via a tighter binding, i.e., increased affinity, to an increased potency of the drug. Because of the state-dependent higher affinity to the receptor, drugs with longer chains will stay longer at this receptor between stimuli (14–16), which may explain their enhanced use-dependent blocking ability at equal stimulating frequencies as observed in our experiments (Fig. 5). However, we cannot distinguish whether the overall lipophilicity of the drug molecule or the length of the alkyl side chain itself is relevant for the increased blocking potency.

Stereoselectivity is often regarded as evidence that a particular drug acts at a specific binding site. However, we cannot turn this around and say if there is no stereoselectivity, there is no specific receptor. The structural elements differing in the two stereoisomers may not be responsible for binding to the receptor.

Potencies of the investigated drugs correlate with their lipid solubilities. Lengthening the alkyl chain from CH₃ to C₈H₁₇ results in a 3000-fold increase of lipid solubility (partition coefficient) and leads to a 37-fold increase of potencies for tonic block and 245-fold for use-dependent block. In other preparations, similar results were obtained: Tonic and use-dependent inhibition of compound action potentials of A, B, and C-fibers in rabbit vagal nerves depend on lipid solubilities of the drugs (17). Likewise, tonic and use-dependent blockade of TTX-sensitive Na⁺ channels by amide local anesthetics correlates also with their lipid solubilities in peripheral nerve preparations (18,19).

Inhibition of the TTX-resistant Na⁺ current by piperidine local anesthetics is not stereoselective, neither for tonic nor for use-dependent block. The highest stereoselectivity factor was 1.4 for S(+)-mepivacaine over the R(-)-form for tonic block. Neuronal TTX-sensitive Na⁺ channels also show low stereoselectivity to piperidine local anesthetics. Tonic inhibition of Na⁺ currents in GH₃ cells displayed a stereoselectivity factor of 1.6 for R(+)- over S(-)-bupivacaine at -70 mV resting potential, which reversed to 0.8 when hyperpolarizing the membrane to -100 mV (20). Cardiac Na⁺ currents displayed no stereoselectivity in the resting state, but had a 1.7-fold stereopotency ratio for R(+)- over S(-)-bupivacaine in the inactivated state (21), which may account for stereospecificity of cardiotoxicity. Depression of compound action potentials by bupivacaine enantiomers is also not very stereoselective. In rabbit vagal nerves, no stereospecific difference has been observed (1). In vivo studies have demonstrated that local anesthetic potencies are similar for both bupivacaine enantiomers (1). The low stereoselectivity found in experimental studies is also observed for bupivacaine nerve block in clinical practice. Compared with racemic bupivacaine, the S(-)-bupivacaine has similar local anesthetic actions and no significant differences in nerve block are observed during epidural anesthesia (22) and brachial plexus blockade (23).

	Acknowledgments

 
Supported, in part, by the Deutsche Forschungsgemeinschaft, Grant Vo188/13 to WV and by the Förderkreis Anästhesie e.V. Giessen

We wish to thank Dr. Rune Sandberg of Astra Pain Control, Södertälje, Sweden, for providing us piperidine-enantiomers and Dr. Boris Safronov for critically reading the manuscript.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Bräu, M. E. Articles by Hempelmann, G. Search for Related Content PubMed PubMed Citation Articles by Bräu, M. E. Articles by Hempelmann, G.