Title Effects of α2-adrenoceptor agonists on tetrodotoxin-resistantNa+ channels in rat dorsal root ganglion neurons( 本文(Fulltext) )
Author(s) ODA, A.; IIDA, H.; TANAHASHI, S.; OSAWA, Y.;YAMAGUCHI, S.; DOHI, S.
Citation [European Journal of Anaesthesiology] vol.[24] no.[11] p.[934]-[941]
Issue Date 2007-11
Rights Copyright (C)2007 Cambridge University Press
Version 出版社版 (publisher version) postprint
URL http://hdl.handle.net/20.500.12099/26865
doi: 10.1017/S0265021507000543
Original Article
Effects of a
2-adrenoceptor agonists on tetrodotoxin-resistant
Na
1channels in rat dorsal root ganglion neurons
*A. Oda, H. Iida, S. Tanahashi, Y. Osawa, S. Yamaguchi, S. Dohi
Gifu University Graduate School of Medicine, Department of Anesthesiology and Pain Medicine, Gifu, Japan
Summary
Background and objective: When intrathecally or epidurally administered, a2-adrenoceptor agonists produce
potent antinociception by affecting the activity of primary afferent fibres and spinal cord neurons. Recent reports have indicated that in dorsal root ganglion neurons, tetrodotoxin-resistant Na1 channels play important roles in the conduction of nociceptive sensation. We therefore investigated the effects of a2
-adrenoceptor agonists on tetrodotoxin-resistant Na1 currents. Methods: Using the whole-cell patch-clamp technique, we recorded tetrodotoxin-resistant Na1 currents from rat dorsal root ganglion neurons. Results: Both clonidine and dexmedetomidine reduced the peak amplitude of the tetrodotoxin-resistant Na1 current concentration- and use-dependently. The concentration required for a half-maximal effect was sig-nificantly lower for dexmedetomidine (58.0 6 10.2 mmol) than for clonidine (257.2 6 30.9 mmol) at holding potential 270 mV. The current inhibitions induced by these agonists were not prevented by 1 mmol yohimbine, an a2-adrenoceptor antagonist. Both clonidine and dexmedetomidine shifted the inactivation
curve for the tetrodotoxin-resistant Na1current in the hyperpolarizing direction. The combinations clonidine with lidocaine and dexmedetomidine with lidocaine produced an additive blockade-type interaction on the tetrodotoxin-resistant Na1current. Conclusions: The results suggest that a direct inhibition of tetrodotoxin-resistant Na1channels may contribute to the antinociceptive effects of clonidine and dexmedetomidine when used as additives to regional anaesthesia.
Keywords: ION CHANNELS; SODIUM CHANNELS; ANAESTHESIA GENERAL, mechanism of actions of anaesthetics; ADRENERGIC ALPHA AGONISTS, dexmedetomidine, clonidine.
Introduction
For some years, clonidine and dexmedetomidine, a2-adrenoceptor agonists, have been in general use in the fields of anaesthesia, intensive care and pain management. Clonidine has been used for epidural
or intrathecal anaesthesia in combination with opioids or local anaesthetics [1] and has a potent antinociceptive effect when administered epidurally
[2,3] or intrathecally [4] as the sole drug. Report-edly, a2-adrenoceptor agonists produce an
anti-nociceptive effect through several antianti-nociceptive mechanisms at the spinal level [5]. The main mechanism is the inhibition of cyclic adenosine 30,50-monophosphate (cAMP) formation via G-protein. As a result, the calcium conductance of primary nerve fibres is decreased through an inhibition of voltage-gated calcium channels, whereupon the release of substance P from such fibres is reduced. In addition, potassium channels in dorsal horn neurons
*
Presented in part at the annual meeting of the International Anesthesia Research Society, Ft. Lauderdale, Florida, USA (2001).
Correspondence to: Hiroki Iida, Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan. E-mail: iida@cc.gifu-u.ac.jp; Tel: 181 58 230 6404; Fax: 181 58 230 6405
Accepted for publication 9 March 2007 EJA 4026 First published online 14 June 2007
might open, followed by cell hyperpolarization and a decrease in impulse transmission [5]. Previous studies have revealed that in peripheral neurons, tetrodotoxin-resistant (TTX-R) Na1channels are key elements in the transmission of nociceptive sensory information[6,7]. We therefore designed the present study in order to elucidate the potential direct effects of a2-adrenoceptor agonists on TTX-R Na
1
channels in rat dorsal root ganglion (DRG) neurons (using the whole-cell patch-clamp technique).
Methods
Preparation of cells
DRG neurons were isolated as previously described
[8]. Briefly, adult Sprague–Dawley rats (200–250 g, n 5 40) were anaesthetized using intraperitoneal pentobarbital, then killed by decapitation and DRGs were rapidly removed along the cervical, thoracic and lumbar sections of the spinal cord. The DRGs were incubated at 378C for 23–30 min in Tyrode solution (for composition, see below) containing 2 mg mL21 collagenase (Type 1; Sigma, St Louis, MO, USA) and 5 mg mL21 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 filtering 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 378C for 2–8 h before being used for
patch-clamp experiments. Recording of membrane currents
A coverslip with cells was placed in a small organ bath (0.8 mL) on the stage of an inverted microscope (TMD; Nikon, Tokyo, Japan). Two to six cells were studied from each animal. Recordings of whole-cell membrane currents were made at an experimental temperature of 23 6 28C, n 5 65) using standard patch-clamp techniques [9]. Patch pipettes were made from glass capillaries using a four-step puller (P-97; Sutter Instrument Company, CA, USA), their tips being fire-polished using a microforge (MF-830; Narishige, Tokyo, Japan) to give a final resistance of 1.0–2.0 MV. Membrane currents were amplified using a current amplifier (Axon Instruments, CA, USA), and signals were digitized by means of a 12-bit analogue-to-digital converter (Digidata 1200B; Axon Instruments). The current signals were filtered at a cut-off frequency of 5 kHz, digitized at a sampling rate of 20 kHz using PClamp v.8.0 software (Axon Instruments) and stored on a personal computer.
Series resistance was compensated as far as possible (by 90–100%). A P11/4P protocol[10]was used for leak subtraction. Data analysis and preparation of figures were performed using Origine v.6 software (Microcal Software Inc., MA, USA). Potassium channel currents were suppressed by the inclusion of Cs1in the pipette solution and tetraethylammonium (TEA) in the external solution. Calcium channel currents were suppressed by the inclusion of F2in the pipette solution and Mg21in the external solution.
DRG neurons were held under a voltage clamp at 2120 mV after the whole-cell patch-clamp con-figuration had been achieved, then Na1 currents were evoked by depolarizing pulses (50 ms in duration) to 210 mV. We observed TTX-R Na1 currents under external solution containing 0.2 mmol TTX. Small DRG neurons play an important role in nociceptive transmission [6] and we have reported that rat DRG neurons of smaller size (,30 mm) preferentially express TTX-R Na1 currents [8]. We therefore recorded from DRG neurons with a diameter of <30 mm.
Solutions and drugs
The Tyrode solution was of the following composi-tion (mmol): NaCl 140.0, KCl 4.0, MgCl2 2.0,
glucose 10.0 and N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES) 10.0, and it was adjus-ted to pH 7.4 with NaOH. As reporadjus-ted elsewhere
[11], the pipette solution was of the following com-position (mmol): CsF 135.0, NaCl 10.0 and HEPES 5.0 (adjusted to pH 7.0 with CsOH). The external solution was: NaCl 25.0, tetramethylammonium chloride 75.0, TEA chloride 20.0, CsCl 5.0, CaCl2
1.8, MgCl2 1.0, glucose 25.0 and HEPES 5.0
(adjusted to pH 7.4 with TEA-OH).
The drugs used were clonidine hydrochloride, yohimbine hydrochloride, lidocaine hydrochloride and tetrodotoxin (Sigma, St Louis, MO, USA). Dexmedetomidine hydrochloride was provided by Abbott Laboratories (Abbott Park, IL, USA). Application of drugs
Extracellular application of drugs was achieved by replacing the bath solution in the recording cham-ber (0.8 mL) with drug-containing solution 7–10 times within 20 s.
Analysis
Analyses were performed as previously described
[8]. Inactivation curves were drawn according to the Boltzmann equation:
INa= max INa ¼ 1=f1 þ exp½ðVh Vh0:5Þ=khg; ð1Þ
Effects of a2-agonists on Na 1
channels 935
where max INa is the maximal value for INa, Vh is
the membrane potential achieved using a 150 ms prepulse (conditioning) potential, Vh0.5 is the
potential at which INa is half of max INa and kh is
the slope factor.
The dose–response curves for the blocking actions of drugs on TTX-R Na1 currents were fitted to the Hill equation:
% inhibition ¼ 100=f1 þ ðIC50=½DrugÞhg; ð2Þ
where IC50 is the half-maximum concentration for
the inhibitory action of a given drug,[Drug]is the drug concentration and h is the Hill coefficient.
The dissociation constants for the binding of drugs to the TTX-R Na1 channels in the inacti-vated state were calculated using the equation: Ki ¼ ½Drug=fð1 þ ½Drug=KrÞ expðV0:5=khÞ 1g;
ð3Þ where [Drug] is the concentration of a given drug, Kr the dissociation constant for drug binding to the
TTX-R Na1 channels in the closed-available state, V0.5 the drug-induced shift in amplitude in the
voltage-dependent inactivation curve and kh the
slope factor for the inactivation curve. Statistical analysis
All values are expressed as means 6 SD. Statistical significance was assessed using a Student’s paired or unpaired t-test, differences being considered significant at P , 0.05.
Results
Concentration–response relationships for the blocking actions of clonidine and dexmedetomidine on TTX-R Na1 currents were obtained at two different holding potentials (HPs), 2120 and 270 mV, with stepping to 210 mV in each case. At HP 5 2120 mV, the IC50 values obtained for
clonidine and dexmedetomidine were significantly higher than the corresponding values obtained at HP 5 270 mV (Fig. 1a, Table 1).
The effects of these a2-adrenoceptor agonists on
the voltage dependence of the steady-state inacti-vation were investigated using a conventional double-pulse protocol (HP 5 2120 mV, duration of the prepulse 5150 ms) (Fig. 2a, b). We selected concentrations of 300 and 1000 mmol for clonidine, and 100 and 300 mmol for dexmedetomidine since these inhibited the TTX-R Na1 currents by approximately 30% and 70%, respectively (Fig. 1a). As shown in Figure 2a, b and Table 1, both cloni-dine and dexmedetomicloni-dine shifted Vh0.5 (the
potential at which the peak amplitude of the Na1
current is half-maximal) in the hyperpolarizing direction. Drug effects on the voltage dependence of the steady-state inactivation were reversible during wash out. Using Eq. (3), we calculated the Kivalues
for clonidine and dexmedetomidine by entering, respectively, values of 1000 and 300 for [Drug], 611.8 and 152.3 for Kr [the IC50 values for
cloni-dine and dexmedetomicloni-dine for the TTX-R Na1 current at an HP of 2120 mV, which can be used if we assume that the Na1 channels are all in the closed-available state (Fig. 2)], 4 and 8.5 for V0.5,
and 5.4 and 5.91 for kh. The Kivalues for clonidine −6.0 −5.5 −5.0 −4.5 −4.0 −3.5 −3.0 −2.5 −2.0 −1.5 0 20 40 60 80 100 −7.0 −6.5 −6.0 −5.5 −5.0 −4.5 −4.0 −3.5 −3.0 −2.5 −2.0 0 20 40 60 80 100 (a) (b) clonidine HP-120 clonidine HP-70 dexmedetomidine HP-120 dexmedetomidine HP-70 % Inhibition % Inhibition log [Drugs] (M) log [Drugs] (M) clonidine dexmedetomidine lidocaine yohimbine Figure 1.
(a) Concentration–response curves for the blocking actions of clonidine and dexmedetomidine on TTX-R Na1 currents. Currents were evoked by stepping (for 50 ms) from 2120 mV or 270 mV to 210 mV. (b) Concentration–response curves for the blocking actions of clonidine, dexmedetomidine, lidocaine and yohimbine on TTX-R Na1 currents at HP 270 mV. Currents were evoked by stepping (for 50 ms) from 270 mV to 210 mV. Abscissae: log molar concentration of drugs. Ordinates: percentage inhibition of peak current amplitude (the peak amplitudes elicited in the absence of drugs were given the value 0%). Each data point represents the mean 6 SD of six measurements from each cell. HP 5 holding potential. See Table 1 for IC50 values.
and dexmedetomidine are thereby calculated to be 220.9 and 26.3 mmol, respectively.
To examine the effect of yohimbine, an a2
antagonist, on the inhibitory effects of clonidine and dexmedetomidine on the TTX-R Na1current, we added 1 mmol yohimbine to 300 mmol clonidine or 100 mmol dexmedetomidine (concentrations that inhibited the TTX-R Na1 peak current by approximately 30%) (Fig. 3a, b). We chose the concentration of 1 mmol for yohimbine at HP 2120 mV because yohimbine itself inhibited the TTX-R Na1 current when used at concentrations above 1 mmol (three experiments from each cell: data not shown). We also determined the IC50value
for yohimbine at HP 270 mV (Fig. 4). Yohimbine 1 mmol did not alter the inhibition of the TTX-R Na1 peak current induced by either clonidine or dexmedetomidine.
To examine possible interactions of clonidine and dexmedetomidine with lidocaine, we administered each agent at its IC50 concentration: lidocaine
at 72.6 mmol (Table 1) either with clonidine at 257.2 mmol (Fig. 4a) or with dexmedetomidine at 58.0 mmol (Fig. 4b), in each case at an HP of 270 mV. The percentage inhibitions of the peak current were 68.1 6 4.8% for lidocaine with clo-nidine (six experiments from each cell) and 68.4 6 4.7% for lidocaine with dexmedetomidine (six experiments from each cell).
Both clonidine and dexmedetomidine caused a use-dependent inhibition of the TTX-R Na1 cur-rent. Currents were evoked repeatedly by stepping (for 10 ms) to 210 mV from 270 mV at one of the
three frequencies (0.2, 5 or 20 Hz). The peak amplitude of the current evoked by the first-step pulse was normalized as 100% in the absence or presence of 100 mmol clonidine or 30 mmol dexmedetomidine (concentrations that induced approximately 20230% inhibition of the peak current). Approximately 1 min was allowed to elapse between one pulse train and the next at each frequency. The percentages of first pulse inhibition were 22.7 6 8.6% for clonidine and 31.3 6 17.4% for dexmedetomidine (n 5 18 experiments from each cell). At frequencies of 5 and 20 Hz, there were significant differences (*P , 0.05) in peak ampli-tude when the current evoked by the 15th pulse was compared between the absence and presence of drugs (clonidine or dexmedetomidine) (Table 2). Discussion
The present results demonstrate that both clonidine and dexmedetomidine block the TTX-R Na1current in rat DRG neurons in a concentration-dependent manner, and that the IC50 for dexmedetomidine
(58 6 10 mmol) is 4.43 times lower than that for clonidine (257 6 31 mmol) at HP 5 270 mV. Yohimbine did not prevent these blocking effects of clonidine and dexmedetomidine, but at a higher dose yohimbine itself induced a significant reduc-tion in this current. Clonidine and dexmedetomi-dine shifted the voltage-dependent inactivation curve for the TTX-R Na1 channels in the hyper-polarizing direction, and also produced a use-dependent block of these channels. Combinations of
Table 1. IC50values and inactivation parameters for TTX-R Na1channels in rat DRG cell.
Holding potential (mV) Hill coefficient IC50for TTX-R (mmol)
Clonidine (n 5 6 cells for each HP) 2120 0.95 6 0.11 611.8 6 192
270 1.15 6 0.14 257.2 6 30.9*
Dexmedetomidine (n 5 6 cells for each HP) 2120 1.38 6 0.21 152.3 6 44.4
270 1.30 6 0.120 58.0 6 10.2y
Lidocaine (n 5 6 cells) 270 1.18 6 0.100 72.6 6 7.44
Yohimbine (n 5 6 cells) 270 0.89 6 0.140 2.59 6 0.69
Inactivation parameters (mmol) Vh0.5(mV) kh (mV)
Clonidine (n 5 6 cells for each concentration) 0 233.7 6 3.74 5.40 6 0.55
300 235.6 6 3.57 5.43 6 0.53
1000 237.7 6 3.70 6.25 6 0.50
Dexmedetomidine (n 5 6 cells for each concentration) 0 234.9 6 2.31 5.91 6 0.34
100 238.8 6 2.76 5.70 6 1.11
300 243.4 6 4.00 6.42 6 0.66
DRG, dorsal root ganglion; TTX-R, tetrodotoxin-resistant.
*P , 0.05 vs. IC
50value for clonidine HP 2120 mV. y
P , 0.05 vs. IC50value for dexmedetomidine HP 2120 mV.
Vh0.55 potential at which peak current amplitude is half the maximal value.
kh5 slope factor.
Values are mean 6 SD.
Effects of a2-agonists on Na 1
channels 937
clonidine with lidocaine and dexmedetomidine with lidocaine produced an additive blockade-type inter-action on the TTX-R Na1current.
Both clonidine and dexmedetomidine produced a use-dependent blockade. In our previous study[12], 30 mmol lidocaine inhibited the relative 15th peak current amplitude by 62.8% 6 12.0 at 5 Hz and by 37.4 6 9.1% at 20 Hz (using the same protocol as
that used here). This inhibition by lidocaine was much more powerful than those induced by cloni-dine and dexmedetomicloni-dine in the present study (Table 2). Thus, both clonidine and dexmedetomi-dine would seem to produce a much weaker open-channel blockade than lidocaine.
a2-adrenoceptor agonists, such as clonidine and dexmedetomidine, produce antinociception when injected epidurally or intrathecally [1,13–15]. The mechanisms underlying their antinociceptive effects have been explained by a2-adrenoceptor activation [5]. Since both dexmedetomidine and clonidine induced TTX-R Na1-current suppression in the present study (although with different potencies), their actions might seem likely to result from acti-vation of a2-adrenoceptors. However, since
yohim-bine did not block the Na1-current suppressions induced by these two drugs, and since yohimbine itself completely blocked this Na1 current when we used a dose that also blocks the antinociceptive
−100 −80 −60 −40 −20 0 0.0 0.2 0.4 0.6 0.8 1.0 control clonidine 1000 µmol clonidine 300 µmol ---clonidine washout Relative Amplitude of Na + Current Relative Amplitude of Na + Current Conditioning potential (mV) −100 −80 −60 −40 −20 0 0.0 0.2 0.4 0.6 0.8 1.0 control dexmedetomidine 100 µmol dexmedetomidine 300 µmol ---dexmedetomidine washout Conditioning potential (mV) (a) (b) Figure 2.
Effects of clonidine and dexmedetomidine on voltage-dependent inactivation of TTX-R Na1 channels. Conditioning pulses stepped (for 150 ms) from 2120 to 210 mV in 10 mV increments were followed by a 5-ms test pulse stepped to 210 mV from various prepulse potentials. (a) Relative peak amplitude of the Na1 current plotted against the membrane potential attained by use of a given conditioning pulse in the absence (control, ’; wash out, B) or presence of clonidine 300 mmol (J) or 1000 mmol (>). (b) Same as in (a), but for dexmedetomidine 100 mmol (n) or 300 mmol (m). The peak amplitude of the TTX-R Na1current evoked by a test pulse without any preceding conditioning pulse was normalized as 1.0 both in the absence and presence of the drug. Data points were fitted by the Boltzmann equation. Each data point represents the mean 6 SD of six measurements from each cell. The voltage dependence of the current inactivation was shifted by clonidine and dexmedetomidine in the negative membrane-potential direction. 40 20 0 −2500 −2000 −1500 −1000 −500 0 500 ms pA pA control
clonidine 300 µmol + yohimbine 1 µmol clonidine 300 µmol washout 40 20 0 −4000 −3000 −2000 −1000 0 1000 ms control dexmedetomidine 100 µmol
dexmedetomidine 100 µmol + yohimbine 1 µmol washout
(a)
(b)
Figure 3.
Effects of yohimbine on the blocking actions of clonidine and dexmedetomidine. Currents were evoked by stepping (for 50 ms) from 2120 to 210 mV. The presence of 1 mmol yohimbine did not alter the inhibitions of the TTX-R Na1 current that were induced by clonidine 300 mmol (a) and dexmedetomidine 100 mmol (b).
effects of epidural dexmedetomidine and clonidine
[3], it is possible that the suppression of Na1currents is due to a direct blocking effect on TTX-R Na1 currents that is independent of the
adrenoceptor-mediated, G-protein-coupled mechanisms. Syner-gism with lidocaine in the clinical setting might be explained by different mechanisms of action (a-adrenoceptor vs. sodium channel).
Both clonidine and dexmedetomidine were found to inhibit the TTX-R Na1 current even in the presence of yohimbine, and a combination of either of these drugs with lidocaine produced an additive inhibition of the TTX-R Na1current. Previous rat tail-flick studies have indicated that intrathecal clonidine and lidocaine act synergistically to reduce the nociceptive response [16,17]. It is possible that a difference in the sites of action between a2
-adrenergic agonists and lidocaine might contribute to this synergistic interaction because a synergistic interaction can occur when drugs affect different critical points along a common antinociceptive pathway [18]. It was demonstrated some years ago in an animal study that after an intrathecal injection of 300 mg clonidine (which produced a near-maximal antinociceptive effect after intrathecal administration in an experiment employing mech-anical stimulation), its cerebrospinal fluid (CSF) concentration was roughly 100–10 000 ng mL21 over the 4 h for which an analgesic effect was observed [14]. Those concentrations (equivalent to 0.5–50 mmol) are much lower than the IC50 value
we obtained for clonidine (257.2 mmol) at an HP of 270 mV. In a human study of epidural clonidine, the half-maximal effective concentration of cloni-dine in the CSF was found to be 80 6 6 ng mL21 (approximately 0.75 mmol)[19]. On the other hand, intrathecal injection of 100 mg dexmedetomidine (same antinociceptive potency as 300 mg intrathecal clonidine) produced almost the same concentration range as that seen after 300 mg intrathecal clonidine (around 0.5–50 mmol) [13], the upper end of 20 0 −2500 −2000 −1500 −1000 −500 0 500 ms control dexmedetomidine + lidocaine washout 20 0 −2500 −2000 −1500 −1000 −500 0 500 pA pA ms control clonidine + lidocaine washout (a) (b) Figure 4.
Interactions of clonidine with lidocaine (a) and of dexmedetomi-dine with lidocaine (b) (each agent was applied at its IC50 concentration). Currents were evoked by stepping (for 50 ms) from 270 to 210 mV.
Table 2. Use-dependent block of Na1channels by clonidine and dexmedetomidine. Frequency
(Hz)
Concentration (mmol)
Relative peak amplitude of 15th current (%) Clonidine (n 5 6 cells for each frequency) 0.2 0 98.9 6 0.62
100 96.4 6 4.5
5 0 89.0 6 1.9
100 85.4 6 4.3*
20 0 79.0 6 4.6
100 73.6 6 6.3*
Dexmedetomidine (n 5 6 cells for each frequency) 0.2 0 96.9 6 3.5
30 93.5 6 3.1
50 0 88.5 6 5.4
30 84.4 6 6.3*
200 0 78.1 6 8.0
30 72.3 6 7.5*
Values are mean 6 SD.
*
P , 0.05 vs. control 15th value at same frequency.
Effects of a2-agonists on Na 1
channels 939
which is close to the IC50value we obtained here for
dexmedetomidine (58.0 mmol at HP 5 270 mV). When 300 mg clonidine or 100 mg dexmedetomi-dine is given epidurally, the CSF concentrations are about the same or slightly less than those measured after intrathecal administration, but much greater than those measured after intravenous administration (,1 ng mL21) [13,19]. Thus, we suggest that a therapeutic dose of dexmedetomidine may produce antinociception in part via an inhibi-tion of TTX-R Na1 channels in DRG like local anaesthetic effects after its epidural or intrathecal administration.
Dexmedetomidine shifted the inactivation curve more strongly in the hyperpolarizing direction than clonidine (Fig. 2). This difference may be attributed to these a2-agonists having different affinities for
the TTX-R Na1channels in a given channel state. Judging from their Kivalues, affinities of clonidine
and dexmedetomidine for the inactivated state would be 2.8 and 5.8 times higher than those for the close-available state of the TTX-R Na1 chan-nels. Thus, dexmedetomidine has about two times higher potency than clonidine for the TTX-R Na1 channels in the inactivation state. Rat DRG cells obtained from a chronic constriction-injury neuro-pathic-pain model reportedly show a shift the voltage dependence of activation and inactivation of the TTX-R Na1currents to a more negative value
[20]. Since the proportion of TTX-R Na1channels in the inactivation state might increase in such a neuropathic-pain model, dexmedetomidine may be more effective than clonidine at blocking TTX-R Na1channels under neuropathic-pain conditions.
In conclusion, both clonidine and dexmedeto-midine block TTX-R Na1 channels in rat DRG neurons in a dose-dependent and use-dependent manner. The mechanism underlying such blocking effects seems likely to be due to a direct action on TTX-R Na1 channels, and not a2-adrenoceptor
activation. Such direct inhibitions of TTX-R Na1 channels may contribute to the antinociceptive effects of clonidine and dexmedetomidine when used as additives to regional anaesthesia.
Acknowledgements
The authors thank Drs S Komori and T Unno Laboratory of Pharmacology, Department of Veterinary Science, Faculty of Agriculture, Gifu University Graduate School of Medicine, Gifu, Japan for stimulating discussion throughout this work. This work was supported by Grant-in-Aid for Scientific Research Nos. 14207059 and 18591697 from the Ministry of Education, Science and Cul-ture, Tokyo, Japan.
References
1. Eisenach JC, De Kock M, Klimscha W. Alpha(2)-adrenergic agonists for regional anesthesia. A clinical review of clonidine (1984–1995). Anesthesiology 1996; 85: 655–674.
2. Mendez R, Eisenach JC, Kashtan K. Epidural clonidine analgesia after cesarean section. Anesthesiology 1990; 73: 848–852.
3. Asano T, Dohi S, Ohta S, Shimonaka H, Iida H. Antinociception by epidural and systemic alpha(2)-adrenoceptor agonists and their binding affinity in rat spinal cord and brain. Anesth Analg 2000; 90: 400–407. 4. Filos KS, Goudas LC, Patroni O, Polyzou V. Intrathecal
clonidine as a sole analgesic for pain relief after cesarean section. Anesthesiology 1992; 77: 267–274.
5. Smith H, Elliott J. Alpha2 receptors and agonists in pain management. Curr Opin Anaesthesiol 2001; 14: 513–518.
6. Akopian AN, Souslova V, England S et al. The tetrodotoxin-resistant sodium channel SNS has a specia-lized function in pain pathways. Nat Neurosci 1999; 2: 541–548.
7. Sangameswaran L, Delgado SG, Fish LM et al. Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons. J Biol Chem 1996; 271: 5953–5956.
8. Oda A, Ohashi H, Komori S, Iida H, Dohi S. Characteristics of ropivacaine block of Na1 channels in rat dorsal root ganglion neurons. Anesth Analg 2000; 91: 1213–1220.
9. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981; 391: 85–100.
10. Bezanilla F, Armstrong CM. Inactivation of the sodium channel. I. Sodium current experiments. J Gen Physiol 1977; 70: 549–566.
11. Song JH, Huang CS, Nagata K, Yeh JZ, Narahashi T. Differential action of riluzole on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J Pharmacol Exp Ther 1997; 282: 707–714.
12. Osawa Y, Oda A, Iida H, Tanahashi S, Dohi S. The effects of class Ic antiarrhythmics on tetrodotoxin-resistant Na1 currents in rat sensory neurons. Anesth Analg 2004; 99: 464–471 table of contents.
13. Eisenach JC, Shafer SL, Bucklin BA, Jackson C, Kallio A. Pharmacokinetics and pharmacodynamics of intraspinal dexmedetomidine in sheep. Anesthesiology 1994; 80: 1349–1359.
14. Castro MI, Eisenach JC. Pharmacokinetics and dynamics of intravenous, intrathecal, and epidural clonidine in sheep. Anesthesiology 1989; 71: 418–425.
15. Aveline C, Metaouna ES, Masmoudi A, Boelle PY, Bonnet F. The effect of clonidine on the minimum local analgesic concentration of epidural ropivacaine during labor. Anesth Analg 2002; 95: 735–740.
16. Kawamata T, Omote K, Kawamata M, Iwasaki H, Namiki A. Antinociceptive interaction of intrathecal alpha2-adrenergic agonists, tizanidine and clonidine, with lidocaine in rats. Anesthesiology 1997; 87: 436–448.
17. Hao S, Takahata O, Iwasaki H. Antinociceptive interac-tion between spinal clonidine and lidocaine in the rat formalin test: an isobolographic analysis. Anesth Analg 2001; 92: 733–738.
18. Berenbaum MC. What is synergy? Pharmacol Rev 1989; 41: 93–141.
19. Eisenach J, Detweiler D, Hood D. Hemodynamic and analgesic actions of epidurally administered clonidine. Anesthesiology 1993; 78: 277–287.
20. Kral MG, Xiong Z, Study RE. Alteration of Na1currents in dorsal root ganglion neurons from rats with a painful neuropathy. Pain 1999; 81: 15–24.
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channels 941
