| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
| ||||||||||||||
| ||||||||||||||
|
|
|
|
||||||||||||
|
||||||||||||||
From the Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Address correspondence to Dr. Patrick Friederich, Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Address e-mail to patrick.friederich{at}kh-bogenhausen.de.
Abstract
BACKGROUND: Kv1.1 and Kv7.2/7.3 channels control excitability of neuronal cells. As hyperexcitability is a sign of neuropathic pain, epilepsy, and anxiety disorders, these channels may be important molecular targets of amitriptyline that cause pharmacological as well as toxicological effects by altering neuronal excitability. Since the molecular mechanisms underlying these effects of amitriptyline have not been fully elucidated, we aimed to characterize the interaction of amitriptyline with human Kv1.1 and Kv7.2/7.3 channels. We also intended to establish the interaction of amitriptyline with the Kv7.2/7.3 channel opener, retigabine.
METHODS: Kv1.1 and Kv7.2/7.3 channels were expressed in human embryonic kidney cells and in Chinese hamster ovary cells. The effects of amitriptyline and retigabine were studied with the patch-clamp technique.
RESULTS: Amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels in a concentration-dependent and reversible manner. The IC50-value was 22 ± 3 µM (n = 33) and 10 ± 1 µM (n = 40), respectively. Deactivating inward currents of Kv7.2/7.3 channels were inhibited with an IC50-value of 4.2 ± 0.6 µM (n = 32). Inhibition of Kv7.2/7.3 channels by amitriptyline reversibly depolarized the resting membrane potential. Retigabine reversed both the inhibitory action of amitriptyline on Kv7.2/7.3 channels as well as the depolarization of the membrane potential.
CONCLUSIONS: Since amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels only at toxicologically relevant plasma concentrations, our results suggest a role for these channels in the neuroexcitatory side effects of amitriptyline. As the inhibitory effects of amitriptyline were reversed by retigabine, a combination of amitriptyline and retigabine could be of additional benefit in the therapy of neuropathic pain.
The delayed rectifier potassium channels Kv1.1 and Kv7.2/7.3 play a major role in controlling the excitability of neuronal cells (1–3). They are prominently expressed in regions of the nervous system that are altered in neuropathic pain (1,3,4) and in regions involved in the generation of epilepsy (5). As hyperexcitability is a typical sign of neuropathic pain (1,6), epilepsy (7,8), and anxiety disorders (9), Kv1.1 and Kv7.2/7.3 channels may be molecular targets of drugs such as amitriptyline and retigabine.
Amitriptyline is a tricyclic antidepressant with analgesic and sedative properties (10) that provides analgesia in various neuropathic pain conditions such as diabetic neuropathy (11), and postherpetic neuralgia (12). Although serious side effects such as grand mal seizures can occur during routine therapy with tricyclic antidepressants (13), they are usually associated with intentional overdose (14). Despite the widespread use of amitriptyline and its well-established role in pain therapy, the molecular mechanisms underlying its analgesic and toxicologic actions have not been fully elucidated.
Amitriptyline analgesia has been suggested to result mainly from reuptake inhibition of serotonin and norepinephrine (15,16). In addition, alterations of voltage-gated ion channels and various receptors seem to be a factor contributing to the complex pharmacologic actions of amitriptyline (17–21). Apart from sodium channels (17,21), voltage-dependent neuronal potassium channels are involved in amitriptyline-induced analgesia (22,23). It has, for example, been reported that antisense oligonucleotides directed against the mRNA of Kv1.1 channels produce a dose-dependent inhibition of amitriptyline analgesia in mice (22).
Retigabine is a novel anticonvulsant compound that opens Kv7.2/7.3 channels (24,25). It effectively reduces the seizure activity in a wide variety of animal models (26) as well as in patients with refractory epilepsy (27). As neuronal Kv7 channels serve to stabilize the membrane potential and control neuronal excitability, the opening of these channels by retigabine seems useful in the treatment of diseases associated with neuronal hyperexcitability, such as epilepsy, neuropathic pain, and anxiety disorders (1,6,9,26–28). The current literature (1,6,9,24–28) allowed us to hypothesize that combining amitriptyline with retigabine for the treatment of neuropathic pain may be beneficial for several reasons. First, amitriptyline and retigabine may act synergistically by their differing analgesic mechanisms. Second, the antiepileptic action of retigabine could be of additional benefit in the therapy of neuropathic pain. However, this may only hold true if retigabine still stimulates Kv7.2/7.3 channels in the presence of amitriptyline.
Despite the well-established role of Kv1.1 and Kv7.2/7.3 channels in signaling pathways and their pathophysiological role in states of neuronal hyperexcitation, neither the direct effects of amitriptyline nor the interaction of amitriptyline and retigabine on Kv potassium channels have been studied. The aim of the present study, therefore, was to characterize the effects of amitriptyline as well as the interaction of amitriptyline and retigabine on human Kv1.1 and Kv7.2/7.3 channels.
METHODS
Cell Culture
Kv1.1 channels were stably expressed in human embryonic kidney (HEK 293) cells and Kv7.2/7.3 channels were transiently transfected in Chinese hamster ovary (CHO) cells. Cells were grown as nonconfluent monolayers according to standard culture protocols (29,30) and were cultured at 37°C in a humidified atmosphere (95% air, 5% CO2). Before the electrophysiological experiments the cells were subcultured in monodishes (35-mm diameter, NUNC, Roskilde, Denmark) at densities between 2 and 3 x 104 cells per dish. CHO cells were transiently transfected at least 24 h before recording with constructs containing cDNA encoding human Kv7.2 and Kv7.3 channels (GenBank Accession No. AF110020; AF033347) using lipofectamine (Invitrogen Life Technologies, Karlsruhe, Germany) according to the manufacturer's recommendation. For the expression of homo- and heteromultimers, equal amounts of Kv7.2 and Kv7.3 cDNA (1 µg) were used. Cotransfection with a construct for enhanced green fluorescent protein (BD Biosciences Clontech, Heidelberg, Germany) was used to detect positive cells.
Patch-Clamp Recordings
Whole cell currents and membrane potentials were measured with the voltage-clamp and current-clamp methods of the patch-clamp technique (31) using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) and Pulse software 8.53 (HEKA Elektronik, Lambrecht, Germany). The currents were filtered at 1 kHz and series resistance was actively compensated by at least 80%. The patch electrodes were fabricated from borosilicate glass capillary tubes with filament (World Precision Instruments, Saratoga, FL, USA) using a Sutter P-97 puller (Sutter Instrument Company, Novato, CA, USA). The pipettes had a resistance of 2 to 4 M
when filled with a solution containing the following electrolyte concentrations: KCl 160 mM, MgCl2 0.5 mM, HEPES 10 mM, Na2ATP 2 mM, pH 7.25, adjusted with KOH. The extracellular solution consisted of: NaCl 135 mM, KCl 5 mM, CaCl2 2 mM, MgCl2 2 mM, HEPES 5 mM, Sucrose 10 mM, Phenol red 0.01 mg/mL, pH 7.4, adjusted with NaOH. All experiments were performed at room temperature. Amitriptyline was dissolved in the extracellular solution prepared from a stock solution of 1 mM (all chemicals were purchased by Sigma, Deissenhofen, Germany). Retigabine was provided by Viatris GmbH Radebeul, Dresden, Germany. Retigabine (100 mM) was dissolved in dimethylsulfoxide (DMSO) to yield a stock solution. At the highest concentration of retigabine (10 µM) used in these experiments the DMSO concentration did not exceed 0.01%. At this concentration DMSO had no effect on Kv7.2/7.3 currents (n = 3). Test solutions containing the drugs were superperfused on the cells by a perfusion system driven by hydrostatic pressure with Teflon tubing.
Stimulation Protocols and Data Analysis
The holding potential during all experiments was –80 mV. Kv1.1 and Kv7.2/7.3 whole-cell currents were elicited by different protocols. The current– voltage relationship was established by depolarizing the cell membrane in steps of 10 mV for 100 ms between the membrane potentials –70 and +60 mV in case of Kv1.1 channels and for 1500 ms in case of Kv7.2/7.3 channels. The whole cell conductance was calculated using the following formula: Gmax = Imax/ (Vm – EK), where Imax is the maximum current of each test potential, Vm is the membrane potential and EK the Nernst potential for potassium, that was –87.5 mV under our experimental conditions. The conductance– voltage relationship was mathematically described by a Boltzmann equation (I = Imax/[1 + exp ((Vmid – Vm)/k)], where Vmid is the voltage of half-maximal activation, Vm is the membrane potential, and k is the slope factor). For the analysis of the activation shift individual conductance–voltage curves for each cell were constructed and the Vmid values were averaged. In case of Kv1.1 channels two Boltzmann equations were multiplied and Vmid was graphically estimated. The fitting procedure was done with Kaleidagraph software (Synergy-Software, Reading, PA, USA). For characterizing the concentration-dependent effects of amitriptyline, outward currents of Kv1.1 and Kv7.2/7.3 channels were measured that were elicited with a rectangle pulse-protocol which depolarized the membrane from the holding potential to +30 mV for 100 and 1200 ms, respectively. In case of Kv7.2/7.3 channels, the effects of amitriptyline on inward tail-currents at a membrane potential of –80 mV were also analyzed. The inhibition of the in- and outward currents by amitriptyline was measured as the inhibition of the maximum current and calculated by the following formula: The ratio of the maximum current (I) under influence of the tricyclic antidepressant and the mean of maximum current under control and washout conditions were subtracted from one (inhibition = 1 – (Idrug/((Icontrol + Iwashout)/2)). The data of the concentration-response curves were mathematically described by a Hill function (f = 1/[1 + (IC50/c)h]; where IC50-value is the concentration of the half-maximal inhibition, c is the drug concentration, and h is the Hill coefficient) using Kaleidagraph software. Standard errors of calculated Hill parameters were used as defined by Kaleidagraph. For analyzing, the effects of retigabine and amitriptyline on Kv7.2/7.3 outward currents were measured that were elicited with a rectangle pulse-protocol, which depolarized the membrane from the holding potential to +30 mV for 1200 ms. The effects were quantified as the reduction or stimulation of maximum current compared to control values. To analyze the pharmacological effects on the deactivation kinetics of Kv7.2/7.3 channels, tail currents were analyzed after a prepulse of 1000 ms length to a membrane potential of +30 mV and subsequent test pulses with a duration of 300 ms to membrane potentials increasing from –100 mV to –80 mV in 10 mV steps. The time constants were determined by fitting current decay with a monoexponential function using Pulsefit software (HEKA Elektronik, Lambrecht, Germany).
Statistical Analysis
Statistical significance was tested using ANOVA (Analysis of variance) and Tukey–Kramer Multiple Comparisons Test (Graph Pad Prism, San Diego, CA, USA) or two-sided paired Student's t-test as appropriate (Excel, Microsoft, Redmond, WA, USA). Data points are given as mean ± sd unless stated otherwise; n values indicate the number of experiments.
RESULTS
Original current recordings of Kv1.1 and Kv7.2/7.3 channels showed typical delayed rectifier currents (Fig. 1A and B). Only Kv1.1 channels exhibited slow inactivation. The current–voltage relationship demonstrated a threshold for activation between –40 and –30 mV for Kv1.1 channels and between –50 and –40 mV for Kv7.2/7.3 channels. The inhibitory effect of amitriptyline on Kv1.1 and Kv7.2/7.3 channels was therefore quantified as the reduction of the maximum outward current at a membrane potential of +30 mV (Fig. 1A and B). Amitriptyline inhibited the channels in a concentration-dependent and reversible manner. Under the influence of amitriptyline, the current traces of Kv1.1 channels exhibited inactivation-like behavior, whereas current traces of Kv7.2/7.3 channels did not show any inactivation-like behavior under the influence of the drug. The concentration-response data for inhibition of Kv1.1 and Kv7.2/7.3 channels were mathematically described by Hill equations (Fig. 1C). The IC50-values for the inhibition of amitriptyline was 22 ± 3 µM for Kv1.1 channels (mean ± sem, n = 4–7 experiments for each concentration) and 10 ± 1 µM for Kv7.2/7.3 channels (mean ± sem, n = 4–9 for each concentration). The corresponding Hill coefficients were 0.8 ± 0.1 and 0.9 ± 0.1 (mean ± sem).
|
The effects of amitriptyline on deactivation of Kv7.2/7.3 channels were also analyzed (Fig. 2A). Inward tail-currents of these channels were inhibited by amitriptyline to a larger extent than outward currents. The concentration-response curve yielded an IC50-value of 4.2 ± 0.6 µM and the Hill coefficient was 0.9 ± 0.1 (mean ± sem, n = 2–8 for each concentration; Fig. 2B). The time constants of deactivation were not significantly altered by amitriptyline (15.3 ± 3.2 ms vs. 16.1 ± 2.2 ms at –100 mV, 18.0 ± 4.0 ms vs. 19.8 ± 3.1 ms at –90 mV and 16.7 ± 6.1 ms vs. 18.8 ± 6.7 ms at –80 mV, n = 10 paired experiments, P > 0.05; Fig. 2C).
|
Kv7.2 and Kv7.3 channels are thought to form the heteromeric channel underlying the M-current (32,33). However, both subunits can also form homomeric channels with M-like currents but with smaller amplitudes than the heteromeric channels (Fig. 3A). As homomeric Kv7.2 and Kv7.3 channels have distinct pharmacological properties, we tested whether Kv7.2 and Kv7.3 channels also differ in their sensitivity to amitriptyline. A possible difference in the sensitivity to amitriptyline was tested at a concentration close to the IC50-value for the inhibition of heteromeric Kv7.2/7.3 channels (10 µM). This concentration was chosen because a potential difference would best be detected at the steepest part of the concentration-response curve. The results demonstrated that amitriptyline (10 µM) inhibited Kv7.2 channels to the same extent as Kv7.3 channels (48 ± 9%, n = 6 vs. 53 ± 9%, n = 5; P > 0.05; Fig. 3B).
|
To analyze the influence of amitriptyline on the conductance–voltage relationship of Kv1.1 and Kv7.2/7.3 channels, the maximum outward currents were converted to conductances, and the obtained conductance–voltage data were fitted with Boltzmann functions. In the case of Kv1.1 channels, amitriptyline (30 µM) shifted the voltage-dependence of channel activation in the hyperpolarizing direction (Vmid was –21.1 ± 2.0 mV under control and washout conditions and it was –25.1 ± 1.9 mV during the action of amitriptyline; n = 7, P < 0.05; Fig. 4A and C). The voltage-dependence of Kv7.2/7.3 channel activation remained unchanged (Vmid was –27.9 ± 7.6 mV under control and washout conditions and it was –28.4 ± 4.3 mV during the action of amitriptyline (10 µM), n = 7, P > 0.05; Fig. 4B and C). For establishing possible voltage-dependence of inhibition, maximum outward current (Imax) inhibition by amitriptyline was analyzed at potentials ranging from –30 mV to +30 mV. Whereas Kv1.1 channels were inhibited in a voltage-dependent manner (Inhibition of Imax at –30 mV was 14 ± 5% vs. 53 ± 7% at +30 mV, n = 7 paired experiments, P < 0.05, Fig. 4D), inhibition of Kv7.2/7.3 channels was voltage-independent (Inhibition of Imax at –30 mV was 39 ± 16% vs. 37 ± 4% at +30 mV, n = 7 paired experiments, P > 0.05, Fig. 4E).
|
After characterizing the inhibitory effects of amitriptyline on Kv7.2/7.3 channels the interaction of the Kv7.2/7.3 opener retigabine and amitriptyline was investigated. For this purpose, the following experimental sequence was performed (Fig. 5A–C): First currents through Kv7.2/7.3 channels were recorded under drug-free conditions. Then amitriptyline was applied on the cells at a concentration of 10 µM. As a next step, different concentrations of retigabine (300 nM, 1 µM, and 10 µM) were simultaneously applied with amitriptyline (10 µM) on the cells, followed by washout of the drugs. As predicted from the concentration-response curve (Fig. 1C), amitriptyline (10 µM), when given alone, reduced the maximum current carried by Kv7.2/7.3 channels by 45% ± 8% (Fig. 5A–C, n = 15; P < 0.01). Simultaneous application of retigabine and amitriptyline (10 µM) yielded current amplitudes significantly larger than during exclusive application of amitriptyline. Retigabine still stimulated Kv7.2/7.3 channels in the presence of amitriptyline. The effect of retigabine increased with the concentration of the Kv7.2/7.3 channel opener. It was already present at the lowest concentration of retigabine (300 nM) tested. Stimulation of Kv7.2/7.3 channels by retigabine in the presence of amitriptyline amounted to 14% ± 3% at 300 nM (n = 5 paired experiments; P < 0.05; Fig. 5A), 31% ± 4% at 1 µM (n = 5 paired experiments; P < 0.05; Fig. 5B) and 34% ± 4% at 10 µM (n = 5 paired experiments; P < 0.05; Fig. 5C).
|
Inhibition of Kv7.2/7.3 channels depolarizes neuronal cell membranes, whereas activation of Kv7.2/7.3 channels by retigabine consequently hyperpolarizes the membrane potential of neuronal cells. Therefore, in our model system, we tested the effects of amitriptyline and retigabine on the membrane potential of CHO cells expressing Kv7.2/7.3 channels. The original recordings of the membrane potential demonstrated that amitriptyline depolarized the membrane potential of CHO cells expressing Kv7.2/7.3 channels. The depolarizing action of amitriptyline was reversed by retigabine (Fig. 6A). Under control conditions, the cells expressing Kv7.2/7.3 had a resting membrane potential of –59.7 ± 6.1 mV (n = 8). Amitriptyline (10 µM) depolarized the membrane potential to –55.5 ± 6.0 mV, (n = 8; P < 0.05, Fig. 6B). The simultaneous application of amitriptyline (10 µM) and retigabine (10 µM) resulted in an immediate hyperpolarization of the membrane potential to –75.9 ± 6.4 mV (n = 8; P < 0.05, Fig. 6B).
|
DISCUSSION
Kv1.1 channels stably expressed in HEK293 cells and Kv7.2/7.3 channels transiently expressed in CHO cells gave rise to currents that exhibited typical features of delayed rectifier currents. Kv1.1 and Kv7.2/7.3 channels were inhibited by amitriptyline in a reversible and concentration-dependent manner. Kv1.1 channels were less sensitive to the inhibitory effect of amitriptyline than Kv7.2/7.3 channels. Inhibition of Kv1.1 channels was voltage-dependent. The tricyclic antidepressant induced inactivation-like behavior, and voltage-dependent activation was shifted in the hyperpolarizing direction. Voltage dependence of inhibition and inactivation-like behavior induced by amitriptyline are compatible with an open channel block of Kv1.1 channels (34,35). In contrast, inhibition of Kv7.2/7.3 channels was voltage-independent and the tricyclic antidepressant did not induce any inactivation-like behavior. Furthermore, gating of Kv7.2/7.3 channels was not modified by amitriptyline, as activation and deactivation were not altered. Deactivating inward currents of Kv7.2/7.3 channels were more sensitive to the inhibitory action of amitriptyline than the respective activating outward currents. This may imply a competition of amitriptyline with the outwardly permeating potassium ions (35). Taken together, these results may suggest that the observed differences in amitriptyline inhibition of Kv1.1 and Kv7.2/7.3 channels may result from different blocking mechanisms.
The deactivating inward currents of Kv7.2/7.3 channels were inhibited by amitriptyline with high potency. The concentration-response curve yielded an IC50-value of 4 µM. Almost the same potency is reported for specific blockers of Kv7.2/7.3 channels, such as linopirdine which inhibits Kv7.2/7.3 channels expressed in CHO cells with an IC50-value of 3.5 µM (36). Kv7.2 and Kv7.3 channels differ in their sensitivity to externally applied tetraethylammonium due to different amino acid residues at the corresponding position 284 (Kv7.2) and 323 (Kv7.3) (33). If amitriptyline, similar to externally applied tetraethylammonium, would bind to either of these positions of Kv7 channels, homomeric Kv7.2 and Kv7.3 channels may differ in their sensitivity to amitriptyline. This seems not to be the case, as amitriptyline sensitivity was not different between the homomers.
Tricyclic antidepressants have strong analgesic properties, and are often administered to alleviate neuropathic pain (10). A typical feature of pain is hyperexcitability of neurons (1,6). Kv1.1 and Kv7.2/7.3 channels play a major role in the regulation of excitability in nociceptive pathways and pain-sensing neurons (1–4). Whereas K+ channel blockers prevent amitriptyline analgesia in mice (22,23), the specific activation of Kv7.2/7.3 channels by retigabine attenuates neuropathic pain (1,28). Consistent with the idea that neuronal hyperexcitability is a typical feature of neuropathic pain (1,6), and also of epilepsy (7,8), inhibition of these channels by amitriptyline would result in an increase, rather than a decrease, of neuronal excitability.
Amitriptyline is clinically used in submicromolar plasma concentrations ranging between 0.36 and 0.90 µM. (21) Toxic plasma concentrations up to 4 µM have been reported (13). Voltage gated sodium channels (Nav1.4) demonstrate a highly state-dependent affinity for amitriptyline with IC50-values ranging from 0.26 µM for the open state to 0.51 µM for the inactivated state and 33 µM for the resting state (21). Compared to the resting state of Nav1.4 channels, Kv1.1 and Kv7.2/7.3 channels are more sensitive to amitriptyline. As calculated from the concentration-response curve, Kv1.1 and Kv7.2/7.3 outward currents would be inhibited at a concentration of 4 µM amitriptyline by 21% and 31%, respectively. Deactivating inward currents of Kv7.2/7.3 channels would be blocked by 50%. Amitriptyline furthermore depolarized the membrane potential of CHO cells expressing Kv7.2/7.3 channels. As sodium channels in their resting state are less sensitive than Kv7.2/7.3 channels, it may be hypothesized that blocking of Kv1.1 and Kv7.2/7.3 currents at the resting membrane potential leads to a depolarization of the neuronal membrane potential, and thus to an initial increase in excitability. As inhibition of Kv7.2/7.3 channels by as little as 25% causes seizures in humans (37), our results suggest a role of potassium channel inhibition in the neuroexcitatory side effects of amitriptyline, such as seizures (13).
Patients with neuropathic pain are often difficult to treat; therefore, different pharmacotherapeutic agents with different modes of action are typically combined in the current approach. Retigabine, by opening Kv7.2/7.3 channels (24,25), attenuates neuropathic pain in different animal models (1,28). As we showed in our model system, retigabine still activates Kv7.2/ 7.3 channels in the presence of amitriptyline; therefore, the combination of amitriptyline and retigabine may offer therapeutic advantages. First, they may act synergistically by their differing analgesic mechanisms. Furthermore, retigabine, by opening Kv7.2/7.3 channels, will not only exhibit analgesic properties but as an antiepileptic drug (26,27) it may increase the therapeutic safety of amitriptyline and could be of additional benefit in the therapy of neuropathic pain.
In summary the results of this study demonstrate that amitriptyline inhibits Kv1.1 and Kv7.2/7.3 channels in a concentration-dependent and reversible manner. Kv7.2/7.3 channels are more sensitive to amitriptyline than Kv1.1 channels. The difference in sensitivity may result from different blocking mechanisms. Deactivating inward currents of Kv7.2/7.3 channels were inhibited by amitriptyline with an IC50-value of 4 µM. Amitriptyline also depolarized the membrane potential of CHO cells expressing Kv7.2/7.3 channels. This may be of pathophysiological significance, since inhibition of these channels close to the neuronal resting potential would depolarize the cell membrane and would hence increase neuronal excitability. Our results thus suggest a role for potassium channel inhibition in the neuroexcitatory side effects of amitriptyline. The Kv7.2/7.3 opener, retigabine, still stimulates Kv7.2/7.3 channels in the presence of amitriptyline. It may therefore be hypothesized that the analgesic effects of amitriptyline and retigabine combine and may be beneficial in the therapy of neuropathic pain.
ACKNOWLEDGMENTS
We thank Andrea Zaisser for cell culture and Drs. Dirk Isbrandt and Howard Christian Peters, Institute of Neuronal Signal Transduction, Center for Molecular Neurobiology, University of Hamburg, Germany, for providing clones of Kv7.2 and Kv7.3 channels and Cornelia Siebrands, Institute of Neuronal Signal Transduction, for critically reading the manuscript. Retigabine was a kind gift from Viatris GmbH, Radebeul, Germany. The authors are very grateful for the support of Professor Olaf Pongs, Director, Institute of Neuronal Signal Transduction, Center for Molecular Neurobiology, University of Hamburg, Germany.
Footnotes
Accepted for publication January 15, 2007.
Supported by the European Society of Anaesthesiologists, Brussels, Belgium (Research Award).
Presented in part at the Wissenschaftliche Arbeitstage der DGAI, Würzburg, Germany (February 18–19, 2005).
REFERENCES
-adrenoceptors play an integral role in the antinociceptive mechanism of action of antidepressant compounds? Eur J Pharmacol 1999;378: 161–8.[ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
