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Effects of ifenprodil on voltage-gated tetrodotoxin-resistant Na+ channels in rat sensory neurons

Tanahashi, S.*; Iida, H.*; Oda, A.*; Osawa, Y.*; Uchida, M.*; Dohi, S.*

European Journal of Anaesthesiology: September 2007 - Volume 24 - Issue 9 - p 782–788
doi: 10.1017/S0265021507000440
Original Article
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Background and objective: To examine a possible mechanism for the antinociceptive action of the N-methyl-d-aspartate receptor antagonist ifenprodil, we compared its effects with those of ketamine on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons, which play an important role in the nociceptive pain pathway.

Methods: Experiments were performed on dorsal root ganglion neurons from Sprague-Dawley rats, recordings of whole-cell membrane currents being made using patch-clamp technique.

Results: Both drugs blocked tetrodotoxin-resistant Na+ currents dose dependently, their half-maximal inhibitory concentrations being 145 ± 12.1 μmol (ketamine) and 2.6 ± 0.95 μmol (ifenprodil). Ifenprodil shifted the inactivation curve for tetrodotoxin-resistant Na+ channels in the hyperpolarizing direction and shifted the activation curve in the depolarizing direction. Use-dependent blockade of tetrodotoxin-resistant Na+ channels was more marked with ifenprodil than with ketamine. When paired with lidocaine, these drugs produced similar additive inhibitions of tetrodotoxin-resistant Na+ channel activity.

Conclusions: The observed suppressive effects on tetrodotoxin-resistant Na+ channel activity may, at least in part, underlie the antinociceptive effects of these N-methyl-d-aspartate receptor antagonists.

*Gifu University Graduate School of Medicine, Department of Anesthesiology and Pain Medicine, Gifu City, Gifu, Japan

Presented in part at the annual meeting of the American Society of Anesthesiologists, Orlando, Florida, 12-16 October 2002.

Correspondence to: Shuji Dohi, Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan. E-mail: shu-dohi@cc.gifu-u.ac.jp; Tel: +81 58 230 6404; Fax: +81 58 230 6405

Accepted for publication 17 March 2006

First published online 27 April 2007

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Introduction

Over the last two decades, our knowledge of the roles played by N-methyl-d-aspartate (NMDA) receptors within the central nervous system (CNS), especially in the transmission of nociceptive information [1,2], has increased tremendously. This has encouraged us to use NMDA-receptor antagonists clinically for the relief of chronic pain. It has been reported that blockade of NMDA receptors can produce antinociceptive effects in rat models of inflammatory and neuropathic pain [2], and that the antinociceptive effects of NMDA-receptor antagonists result from their binding to NMDA receptors within the CNS [1]. In addition, some experimental in vivo studies have indicated that NMDA-receptor antagonists (such as ketamine and ifenprodil) exert peripheral antinociceptive effects. It has been speculated on the basis of in vivo experiments that both intrathecal and systemic ketamine would block axonal conduction [3]. Indeed, ketamine injected subcutaneously into the region of the peripheral receptive field produces naloxone-reversible antinociception [4]. Moreover, ifenprodil has been reported to block the rabbit's corneal reflex by a local analgesic effect [5]. Thus, NMDA-receptor antagonists seem to act on neuronal structures at several levels. Ketamine reportedly inhibits tetrodotoxin-resistant (TTX-r) Na+ currents in rat dorsal root ganglion (DRG) neurons with a mode of action similar to that of local anaesthetics [6]. Since the TTX-r Na+ channels present in rat DRG cells and in C fibres within human peripheral nerves are implicated in nociceptive signal transmission, it has been suggested that NMDA antagonists may perform some roles in pain transmission within the peripheral nervous system [7,8]. However, no report is yet available to indicate whether ifenprodil affects TTX-r Na+ channels in rat DRG neurons. Consequently, in the present study we examined the effects of ifenprodil and ketamine on TTX-r Na+ channel activity in rat DRG neurons.

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Methods

Experiments were performed on DRG neurons isolated from male Sprague-Dawley rats (200-300 g). The experimental protocols were approved by our Institutional Committee for Animal Care. Rats were anaesthetized with sodium pentobarbital (200 g kg−1 intraperitoneally) and decapitated. Then, DRGs were rapidly removed along the cervical, thoracic and lumbar sections of the spinal cord, and placed in Tyrode solution (for composition, see below). The DRGs were incubated at 37°C for 35-40 min in Tyrode solution containing 2 mg mL−1 collagenase (Type 1; Sigma, St Louis, MO, USA) and 5 mg mL−1 dispase II (Boehringer Mannheim, Indianapolis, IN, USA). After washing three times with fresh, enzyme-free Tyrode solution, single neuronal cells were obtained by gentle agitation in Tyrode solution through a small-bore Pasteur pipette. After filtration of the cell suspension, the collected cells were resuspended in Tyrode solution, placed on glass coverslips and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 2-8 h before use in the patch-clamp experiments.

A coverslip carrying cells was placed in a small organ bath on the stage of an inverted microscope (IX70; Olympus, Tokyo, Japan). Recordings of whole-cell membrane currents were made at an average experimental temperature of 22-24°C by means of the patch-clamp technique. Patch pipettes were made from glass capillaries using a six-step vertical puller (P-97; Sutter Instruments, Novato, CA, USA) to give a final resistance of 1.5-2.0 MΩ. Membrane currents were amplified using a current amplifier (Axopatch 200B; Axon Instruments, Foster City, CA, USA). Data analysis was performed on an IBM-compatible personal computer using a data acquisition and analysis instrument (Origin 6.1J; OriginLab Co., Northampton, MA, USA). The current signals were filtered at a cutoff frequency of 5 kHz and digitized at a sampling rate of 20 kHz. Series resistance was compensated by more than 80%, and leak subtraction was performed by means of P/4 protocol. Experimental data were discarded when the voltage error exceeded 5 mV after compensation of the series resistance.

The Tyrode solution was of the following composition (mmol): NaCl 140.0, KCl 4.0, MgCl2 2.0, glucose 10.0, HEPES 10.0 (adjusted to pH 7.4 with NaOH) [9]. The pipette solution was of the following composition (mmol): CsF 135.0, NaCl 10.0, HEPES 5.0, EGTA 3.0 (adjusted to pH 7.0 with CsOH). The external solution was as follows: NaCl 25.0, tetramethylammonium chloride 75.0, tetraethylammonium chloride 20.0, CsCl 5.0, CaCl2 1.8, MgCl2 1.0, glucose 25.0, HEPES 5.0, LaCl3 3 μmol (adjusted to pH 7.4 with tetraethylammonium-OH).

Ketamine (Sigma, St Louis, MO, USA), ifenprodil (Aventis Pharma Ltd, Tokyo, Japan) and tetrodotoxin (Sigma, St Louis, MO, USA) were used. All drugs were dissolved in distilled water and added to the superfusion medium. Extracellular application of drugs was achieved by replacing the bath solution in the recording chamber (800 μL) with drug-containing solution 5-7 times within 20 s.

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Data analysis

Analyses were performed as described previously [10]. The dose-response curves for the blocking actions of ketamine and ifenprodil on TTX-r currents were fitted to the Hill equation:

where IC50 is the half-maximal concentration for the inhibitory action of ketamine or ifenprodil, [Drug] is the concentration of a given drug and h is the Hill coefficient.

From the peak Na+ currents (Ina), the Na+ conductance values (gna) were calculated, normalized to the maximum value obtained in control, and plotted as a function of the membrane potential. Activation curves and inactivation curves were drawn according to the Boltzmann equation:

where gnamax and maxIna are the maximal values for gna and Ina, Vg is the membrane potential achieved using a step pulse, Vh is the membrane potential achieved using a prepulse potential, Vg0.5 is the potential at which gna is half gnamax, Vh0.5 is the potential at which Ina is half maxIna, and kg and kh are the slope factors.

All values in the text are expressed as mean ± SD. The statistical significance of differences was assessed using a t-test. Differences were considered significant when P < 0.05.

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Results

Ketamine and ifenprodil each block TTX-r Na+ channel current dose dependently

Small DRG neurons play an important role specific to nociceptive transmission in physiologic and pathophysiologic sensory processing [11]. TTX-r Na+ channels are present mainly in small DRG neurons [6], and thus we selected small DRG neurons (diameter <30 μmol) for the present study. The short-lasting Na+ current present in DRG neurons is adequately blocked by 0.2 μmol TTX (Fig. 1a). A long-lasting Na+ current was found that was resistant to block by 0.2 μmol TTX (Fig. 1b), and this was considered to result from activation of TTX-r Na+ channels.

Figure 1.

Figure 1.

To examine the effects of ketamine and ifenprodil on TTX-r Na+ currents, whole-cell membrane Na+ currents were evoked every 10 s by stepping for 50 ms from a holding potential of −70 to −10 mV. The holding potential of −70 mV we chose is approximately the resting membrane potential of DRG neurons [10]. Each drug was applied with a stepwise increase in concentration every 2 min. The TTX-r Na+ current appeared as a brief inward current (activated and then inactivated within 5 ms), followed by a longer, inward current that persisted for 20 ms or more. The decrease in current amplitude induced by a given drug (see below) was reversed by removing the applied drug from the bath solution. We discarded data from any DRG cells that did not exhibit a recovery in peak current amplitude to 50% or more of the initial value at 5 min after removal of drug.

Ketamine and ifenprodil each blocked the peak TTX-r Na+ channel current in a dose-dependent manner (Fig. 1c,d). This inhibition recovered after washing out the drug. The IC50 for the TTX-r Na+ current were (holding potential, −70 mV) ketamine = 145 ± 12.1 μmol (n = 6) and ifenprodil = 2.6 ± 0.95 μmol (n = 6). The Hill coefficients were ketamine = 0.9 ± 0.04 (n = 6) and ifenprodil = 1.2 ± 0.02 (n = 6). There was a significant difference between the IC50 values (P < 0.05), but not between the Hill coefficients (Fig. 2a).

Figure 2.

Figure 2.

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Voltage-dependent kinetics of TTX-r Na+ channel currents induced by ketamine and ifenprodil

Voltage-dependent activation curves for TTX-r Na+ currents are shown in Figure 3. Upon ifenprodil application (3 μmol), V0.5 was changed by 4.9 mV (from −14.9 to −10.0 mV, n = 4), but it was not changed by application of 140 μmol ketamine (−16.4 mV, n = 4).

Figure 3.

Figure 3.

The voltage dependence of the steady-state inactivations of the TTX-r Na+ current was investigated using a conventional double-pulse protocol (Fig. 3). After separate applications of ketamine (140 μmol) and ifenprodil (3 μmol), Vin0.5 was shifted by −5.7 mV (from −32.3 to −38.0 mV) and −9.5 mV (from −33.4 to −42.9 mV), respectively (n = 6, P < 0.05). Ifenprodil also induced a significant change in Vin0.5 at the lower dose of 1 μmol (n = 6, P < 0.05). Although each drug shifted the inactivation curve for the TTX-r Na+ channel current in the hyperpolarizing direction, neither of the slope factors showed a significant change from control.

The dissociation constant for a drug's binding to Na+ channels in the inactivated state (Ki) can be calculated from the equation [12,13]: ΔV0.5 = Vs× ln{(1 + [D]/Ki)/(1 + [D]/Kr)}, where ΔV0.5 is the drug-induced shift in amplitude in the voltage-dependent inactivation curve, Vs is the slope factor of the inactivation curve, [D] is the concentration of drug, and Kr is the dissociation constant for a drug's binding to Na+ channels in the close-available state. Using the values of 5.7 mV for ΔV0.5 and 4.8 mV for Vs obtained with 140 μmol ketamine, and a value of 145 μmol for Kr, the Ki value calculated for ketamine was 26 μmol. However, the Ki value for ifenprodil is calculated to be 0.18 μmol.

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Use-dependent blockade of TTX-r Na+ channels by ketamine and ifenprodil

To test for use-dependent blockade of TTX-r Na+ channels by NMDA antagonists, trains of depolarizing pulses (to −10 mV from a holding potential of −70 mV) were applied at one of three frequencies (0.2, 5 or 20 Hz) in the absence or presence of NMDA-receptor antagonist (Fig. 4). Ketamine and ifenprodil each produced an approximately 20% inhibition of the peak current for TTX-r Na+ channels. Currents were evoked repeatedly in the presence of 0.2 μmol TTX by stepping for 10 ms to 0 mV from a holding potential of −70 mV at one of three different frequencies. The peak amplitude of the Na+ current evoked by the first step pulse was normalized as 100% in the absence or presence of each drug. The current amplitude evoked by the 15th stimulus at a given frequency in the presence of a given drug was compared with the corresponding value obtained in the absence of drug. The use-dependent blockade of TTX-r Na+ channels induced by each drug increased with the frequency. There was a significant difference in the current amplitude evoked by the 15th pulse between before and after ketamine and between before and after ifenprodil application at both 5 and 20 Hz (n = 6 each, P < 0.05; Fig. 4a,b), but the effects were more prominent with ifenprodil than with ketamine (P < 0.05; Fig. 4b). At a frequency of 0.2 Hz, however, no significant use-dependent blockade was observed with ketamine.

Figure 4.

Figure 4.

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Effects of combined applications of lidocaine with NMDA antagonists on TTX-r Na+ channels

The effects of single and combined drug applications on TTX-r Na+ channels were examined in the presence of 0.2 μmol TTX (Fig. 2b). Single treatment with lidocaine (60 μmol), ketamine (200 μmol) or ifenprodil (3 μmol) induced decreases in TTX-r Na+ currents. The percentage decrease in the TTX-r Na+ current from control was about 50% for each single treatment. Combination treatment with lidocaine plus ketamine (60 μmol, 200 μmol) or lidocaine plus ifenprodil (60 μmol, 3 μmol) caused about a 70% decrease in the current. The effects of combination treatments were significantly greater than those of the single treatments with NMDA antagonists (n = 4 each, P < 0.05).

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Discussion

The results of the present study indicate that ketamine and ifenprodil block TTX-r Na+ currents in small DRG neurons dose dependently. Ketamine has been demonstrated to cause TTX-r Na+ channel blockade in a whole-cell patch-clamp study [6]. From the difference in their IC50 values, the sensitivity of TTX-r Na+ channels for ifenprodil is likely to be about 40 times greater than that for ketamine. Small-diameter neurons are associated with the unmyelinated C-fibres in peripheral nerves [6,9,14]. TTX-r Na+ channels, which play an important role in the transmission of peripheral nociceptive information to the spinal cord, are found preferentially in sensory neurons of small diameter in DRG [9].

In the present study, ifenprodil shifted the activation curve for TTX-r Na+ channels in the depolarizing direction, and ketamine and ifenprodil each shifted the inactivation curve for these channels in the hyperpolarizing direction dose dependently (Fig. 3). The dissociation constant for a drug's binding to Na+ channels in the inactivated state can be calculated [12,13]. From this calculation, it is suggested that ketamine and ifenprodil have about a 5.7 times and 15 times higher affinity, respectively, for the inactivated state than for the close-available state of these Na+ channels. A rat inflammatory pain model exhibits increases both in the magnitude of the TTX-r Na+ current and in the rates of activation and inactivation of TTX-r Na+ channels in small DRG neurons [15]. Since chronically damaged nerve fibres have a resting membrane potential shifted in the more depolarizing direction at the injury site [16], the dose-dependent shift in the inactivation curve in the hyperpolarizing direction observed with both ketamine and ifenprodil in the present study may indicate that drug molecules decrease the rate of open-state TTX-r Na+ channels even at more depolarized membrane states [14]. These findings seem to suggest that ifenprodil reduces the excitability of DRG neurons more potently than ketamine.

Ketamine and ifenprodil each induced a use-dependent blockade of TTX-r Na+ channels that increased with the pulse frequency (Fig. 4a,b). A use-dependent blockade of TTX-r Na+ channels with a similar property has been observed with local anaesthetics [14]. In the case of the binding of local anaesthetics, the inactivation gate seems to retain channel blockers inside the channel pore during use-dependent blockade [17]. Moreover, local anaesthetics block C-fibres during high-frequency stimulation [18], and bind to open and inactivated Na+ channels during repetitive stimulation, and dissociate from channels slowly between stimuli. Thus, their use-dependent effects are likely to be enhanced by shortening the interpulse interval. Ketamine and ifenprodil each block TTX-r Na+ channel activity. When the repetitive firing rate of a peripheral nerve increases, such drugs may block or reduce the activity of those TTX-r Na+ channels transmitting the nociceptive information. The effects of drugs on Na+ channels vary with the charge (i.e. according to whether the drug is in the polarized or non-polarized state). According to the view that a drug in charged form can enter an ion channel in its open state, the magnitude of the use-dependent inhibition of an ion channel current is suggested to be related to the pKa value for a given drug [13]. Since the pKa values for ketamine and ifenprodil are 7.5 and 9.69, respectively, ifenprodil would be expected to induce a stronger use-dependent block than ketamine. At pH 7.4 in external solution, the ratio of polarized type to non-polarized type of ifenprodil is high. When most drugs are in the polarized state, their efficacy as an open-channel blocker becomes greater. Therefore, the drug action would be expected to show slow recovery from block, and the drug effects will become stronger. In such a state, a use-dependent block is observed even during low frequency stimulation (0.2 Hz), and it becomes stronger as the stimulus frequency is increased. Thus, it is likely that the use-dependent block observed with ifenprodil was stronger than that observed with ketamine because most of the ifenprodil was in the polarized state in the solution employed in the present experiment.

Ketamine, classified as a noncompetitive NMDA-receptor antagonist, could bind to the phencyclidine (PCP) site of the open state of the NMDA receptor complex. Ketamine also binds to a membrane-associated site that does not require the channel to be open [19]. Ifenprodil, classified as an NR2B-selective antagonist, binds to a specific site on the NR1 subunit, and potentially to the NR2B subunit, allosterically interacting with the proton sensor and polyamine binding site on the NR1 subunit [1]. NMDA-receptor antagonists have been shown to have differential affinity for NR1/NR2A vs. NR1/NR2B subunit-containing receptors (the affinities being ketamine, NR1/NR2A ≈ NR1/NR2B; ifenprodil, NR1/NR2A≪NR1/NR2B) [20].

Combined applications of ketamine or ifenprodil with lidocaine may have had additive effects on TTX-r Na+ channels in DRG neurons. Lidocaine binds to the S6 segment of domain 4 on the Na+ channel α-subunit, which is related to the activation and inactivation processes [21]. We assume that ketamine and ifenprodil reach the same binding site as lidocaine because they have similar electrophysiological characteristics to lidocaine, and the consequence of the increased concentrations of compounds acting on Na+ channels is that an additive effect may result. This additive effect may be useful for enhanced pain relief in clinical settings if lidocaine with an NMDA antagonist is used as combined antinociceptive therapy.

In conclusion, in our study ketamine and ifenprodil blocked TTX-r Na+ channels both dose and use dependently. Ifenprodil blocked the peak TTX-r Na+ channel current more potently than ketamine. Ifenprodil would reduce the activity of TTX-r Na+ channels on primary sensory neurons. The suppression of TTX-r Na+ channel activity by ifenprodil may be, at least in part, related to its antinociceptive effects in patients with chronic inflammatory pain.

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Acknowledgement

This work was supported by Grant-in-Aid for Scientific Research Nos. 14207059 and 13671570 (Ministry of Education, Science and Culture, Tokyo, Japan).

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Keywords:

RECEPTORS N-METHYL-d-ASPARTATE, antagonists, ifenprodil; KETAMINE; SODIUM CHANNELS, tetrodotoxin resistant; RAT; ANALGESICS, mechanism of action

© 2007 European Society of Anaesthesiology