European Journal of Pharmacology 740 (2014) 560–564
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Dexmedetomidine produced analgesic effect via inhibition of HCN currents Ying-cong Yang, Qing-tao Meng, Xia Pan, Zhong-yuan Xia n, Xiang-dong Chen Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 April 2013 Received in revised form 18 June 2014 Accepted 19 June 2014 Available online 8 July 2014
The purpose of this study was to investigate the mechanism by which systemic dexmedetomidine exerts analgesic effect and examine effect of dexmedetomidine on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels currents. The experiments were performed on C57BL/6 J and HCN1 knockout mice. The analgesic effects of intraperitoneal dexmedetomidine (10–40 μg/kg) were measured by a tail-flick test. Whole-cell clamp recordings were used to examine the properties of cloned HCN subunit currents expressed in HEK 293 cells under control condition and dexmedetomidine administration (0.1–10 μM). Injection of dexmedetomidine caused a clear time and dose-related increase in the tail-flick latency of both wild type and knockout mice. Compared with the wild type group, the MPE (maximum possible effect) of tail-flick latency induced by 30 μg/kg and 40 μg/kg dexmedetomidine in knockout mice was significantly lower. The α2-adrenergic receptor antagonist yohimbine (5 μg/kg) reduced the MPE of dexmedetomidine (30 μg/kg) both in wild type and knockout mice. Dexmedetomidine(0.1–10 μM) inhibited HCN1 and HCN2 channel currents in HEK 293 cells, caused a decrease of maximal currents, an increase of inhibition rate of hyperpolarization-activated currents (Ih), and a negative shift in V1/2. We conclude that dexmedetomidine produces a dose-dependently analgesic effect, and the effect is likely due to the inhibition of HCN currents. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dexmedetomidine HCN currents α2-adrenergic receptor agonist Analgesic effect Chemical compounds studied in this article: Dexmedetomidine (Chem CID: 5311068)
1. Introduction Dexmedetomidine, a potent and highly selective agonist of the
α2-adrenergic receptors has been widely used as an analgesic and antinociceptive adjuvant (Gerresheim and Schwemmer, 2013). Animal experiments showed that peripheral application of dexmetetomidine and other α2-adrenergic receptor agonist improved the threshold of mechanical and thermal pain. Dexmedetomidine has been shown to enhance effects of local anesthetic bupivacaine and ropivacaine for sciatic nerve blocks and increase duration of analgesia dose-dependently (Brummett et al., 2008, 2009, 2010). Clinical study also showed that systemically administered dexmedetomidine exerted moderate analgesic effect in human ( Jaakola et al.,1991; Hall et al., 2000). α2-adrenergic receptors are widely distributed throughout the peripheral and central nervous system, and their activation produces a variety of effects. However, the mechanism of these effects is not yet very clear. Clonidine,another α2-adrenergic receptor agonist, has been shown to effectively inhibit hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels currents in
n
Corresponding author. Tel.: þ 86 27 88041911; fax: þ 86 27 88042292. E-mail address: [email protected] (Z.-y. Xia).
http://dx.doi.org/10.1016/j.ejphar.2014.06.031 0014-2999/& 2014 Elsevier B.V. All rights reserved.
different neuronal types, including dorsal root ganglion (Yagi and Sumino, 1998), pyramidal neurons of the prefrontal cortex (Carr et al., 2007) and dopamine neurons of the ventral tegmental area (Inyushin et al., 2010). These studies suggest that the effects of α2-adrenergic receptors agonist are closely related to the HCN channels. The aim of the present study is to investigate the mechanism by which systemic dexmedetomidine exerts analgesic effect, and examine effect of dexmedetomidine on HCN currents.
2. Material and methods 2.1. Animals The experiments were performed on C57BL/6J and HCN1 knockout mice weighing 19–25 g obtained from Center of Experimental Animals in Wuhan University, China. Animals were housed five per cage in a room maintained at 227 1 1C with an alternating 12 h dark/12 h light cycles. The HCN1 knockout mice were originally prepared and characterized by the Kandel laboratory (Nolan et al., 2003); they have been deposited for distribution by Jackson Labs (stock #005034). Mice homozygous for the targeted mutation are viable, fertile,
Y.-c. Yang et al. / European Journal of Pharmacology 740 (2014) 560–564
561
normal in size and longevity and they do not display any gross physical or behavioral abnormalities. The treatment and care of the experimental animals was conducted according to the Animal Care and Use guideline of Wuhan University and was approved by the Institutional Ethics Committee at Renmin Hospital of Wuhan University, China.
Statistical evaluations were performed using SPSS software and Graphpad prism, and a difference was considered statistically significant at P o0.05.
2.2. Drugs and chemicals
3.1. Effects of i.p. administration dexmedetomidine in tail-flick test
Dexmedetomidine hydrochloride injection (Abbott Laboratories) and yohimbine (Sigma Chemical) were diluted in physiological saline (0.9% NaCl) for animal experiments. All drugs and chemicals were diluted in bath solution for patch clamp recording.
Injection of dexmedetomidine caused a clear time and doserelated increase in the tail-flick latency of both wild type and knockout mice (Fig. 1). Effect of 10 μg/kg and 20 μg/kg dexmedetomidine in wild type mice was apparent within 15 min, lasting until 60 min and 75 min respectively. The duration of analgesia at 30 μg/kg and 40 μg/kg of dexmedetomidine was up to 120 min (Fig. 1Aa). Effect of 10 μg/kg and 20 μg/kg dexmedetomidine in knockout mice was apparent within 30 min, lasting until 45 min and 60 min respectively. The duration of analgesia at 30 μg/kg dose was up to 90 min whereas at 40 μg/kg, it was 120 min (Fig.1Ab) The MPE in wild type mice and knockout mice after application of dexmedetomidine are presented in Fig. 1B. In wild type mice, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine was 33.26 7 1.90% and 53.85 79.69% respectively. In knockout mice, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine was 22.06 73.63% and 37.18 74.63% respectively. Compared with the wild type group, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine in knockout mice was significantly lower. The MPE of 10 μg/kg and 20 μg/kg dexmedetomidine was no obvious difference between groups.
2.3. Tail-flick test The analgesia response against thermal stimuli was assessed with the tail-flick test. Mice were gently held with the tail positioned in the tail-flick apparatus (MK-330B; Muromachi Kikai Co., Ltd., Tokyo, Japan) for radiant thermal stimulation of the dorsal surface of the tail. The intensity of the thermal stimulus was adjusted to cause the animal to flick its tail within 2.5 to 3 s as the baseline. The tail-flick latency was measured before and 15, 30, 45, 60, 75, 90, 105 and 120 min after the i.p administration of dexmedetomidine and dexmedetomidine plus yohimbine. Control mice received saline injection. Examines were terminated if the animals did not respond within 15 s in order to minimize tissue damage. The tail flick latency was converted to represent the percent maximum possible effect (MPE), according to the following formula: MPE¼100 (postdrug latency-predrug latency)/(15-predrug latency) (D’Amour and Smith, 1941). Peak maximum percent effect (MPE) values of individual animals from the time–response data were pooled for each dose to construct the dose–response curves. 2.4. Recordings from cloned HCN channels in HEK 293 cells HEK 293 cells were cultured using standard procedures and transiently transfected with HCN channel constructs, together with an enhanced GFP plasmid (Clontech, Mountain View, CA), using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Recordings were obtained 24 to 48 h after transfection. Wholecell recordings were obtained using 3 to 5 MΩ patch pipettes and an Axopatch 200B amplifier at room temperature. Bath solution was perfused continuously ( 2 ml/min) and contained the following (in mM): 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH 7.3; internal solution contained the following (in mM):120 KCH4SO3, 4 NaCl, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 10 EGTA, 3 Mg-ATP, and 0.3 GTP-Tris, pH 7.2 (Himes et al., 1977). 2.5. Data acquisition and analysis Data were acquired using pCLAMP software and a Digidata 1322 A digitizer (Axon Instruments). For voltage-clamp recording, time-dependent hyperpolarization-activated currents (Ih) were evoked with incrementing (Δ 10 mV) hyperpolarizing pulses (3–4 s) from a holding potential of 40 mV, followed immediately by a step to fixed potential ( 90 mV) to obtain tail currents. Current amplitude at each potential was taken as the timedependent current at the end of hyperpolarizing voltage steps; maximal available current was determined at 120 mV. Tail currents were normalized, plotted as a function of the preceding hyperpolarization step voltage, and fitted with Boltzmann curves for derivation of activation parameters (V1/2) by using Origin software (Origin Lab). All data are presented as means 7S.E.M. Statistical tests included one- and two-way ANOVA, Student's t test, as indicated.
3. Results
3.2. Effects of i.p. administration of dexmedetomidineþyohimbine in tail-flick test As shown in Fig. 2, application of yohimbine, the MPE of wild type and knockout mice was 9.46 72.77% and 2.72 72.55% respectively. Yohimbine (5 μg/kg) significantly reduced the MPE of 30 μg/kg dexmedetomidine by 23.80% and 19.34% in wild type and knockout mice (P o0.05 vs demedetomidine alone, n ¼10), respectively. 3.3. Dexmedetomidine inhibits HCN1 and HCN2 channel currents expressed in HEK 293 cells. In HEK 293 cells expressing HCN1 subunits, dexmedetomidine caused a decrease in HCN1 currents, from 627.53 7 200.86 to 204.27 727.95 pA . The effects of dexmedetomidine (0.1–10 μM) were concentration-dependent (Fig. 3B). In the presence of 0.1 μM, 1 μM, 10 μM dexmedetomidine, the inhibition rates of HCN currents were 18.82 71.72%, 38.4375.39% and 46.48 76.66% respectively. Similar to HCN1, dexmedetomidine (0.1–10 μM) also decreased HCN2 currents , from 593.87 756.64 to 199.99 7 24.72 pA, in concentration-dependent manner in cells expressing HCN2 subunits(Fig. 3B). In the presence of 0.1 μM, 1 μM, 10 μM dexmedetomidine, the inhibition rate of HCN2 currents was 23.857 2.59%, 41.14 7 4.82% and 58.19 75.22% respectively. In the same concentration of dexmedetomidine, the inhibition rate of HCN1 and HCN2 currents has no significant difference (Fig. 3C). To examine the effect of dexmedetomidine on the voltagedependent activation of Ih, an activation curve was constructed by a tail current analysis. The Ih activation curve fitted with the Boltzmann equation is shown in Fig. 3D. In HEK 293 cells expressing either HCN1 or HCN2 subunits, the application of dexmedetomidine (0.1–10 μM) produced a dose-related shift to the left of the Ih activation curve. V1/2 of HCN1 shifted from 81.80 71.52 to 103.09 76.14 mV by 10 μM dexmedetomidine, and V1/2 of HCN2 shifted from 102.61 71.16 to 120.00 7
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Fig. 1. Time and dose-dependent analgesia effects induced by dexmedetomidine(10, 20, 30, 40 μg/kg, i.p.) in the tail-flick test. Data are presented as means 7 S.E.M. The drug is administered after the baseline value is determined, and control group is treated with saline. (A) Time course of analgesia effects induced by dexmedetomidine (a, wild type mice; b, knockout mice). nPo 0.05 and nnPo 0.01 vs saline-treated controls, n¼ 6–9. (B) Dose-analgesia response curves to dexmedetomidine in the tail-flick test. Responses are expressed as %MPE. %MPE values at the time point at which peak analgesia responses are observed for wild type and knockout mice are used to plot the curve (*Po 0.05, wild type vs knockout, n¼ 6–9).
Fig. 2. Antagonism of dexmedetomidine-induced analgesia response by yohimbine. Application of 30 μg/kg dexmedetomidine results in small MPE values in the presence of 5 μg/kg yohimbine both in wild type and knockout mice (nP o0.01, dex vs yoh þ dex, n¼ 10).
1.56 mV by 10 μM dexmedetomidine. These data revealed that dexmedetomidine (0.1–10 μM) produced a significant shift in the V1/2 to a more hyperpolarized potential (P o0.05, n ¼ 5–8). However, the slope values were not altered by dexmedetomidine (P 40.05, n ¼5–8).
4. Discussion Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a family of six transmembrane domain, single poreloop, hyperpolarization activated, non-selective cation channels. HCN channels are activated at negative membrane potentials and repolarization following action potential firing resulting in a depolarizing current influencing the threshold for subsequent action potential generation. Consequently, HCN currents play
a critical role in regulating neuronal resting membrane potential and excitability (Maher et al., 2009; Dunlop et al., 2009). The HCN channel family comprises four distinct subtypes (HCN1–4). They are widely distributed in the nervous system and the heart. HCN1 is the subtype with the fastest kinetics, mainly expressed in the peripheral nervous system, sense organs and central nervous system associated with fine sensory. HCN2 is distributed nearly throughout most brain regions and spinal nerve. In contrast, HCN3 is expessed at very low levels in the central nervous system. HCN4 is mainly distributed in the cardiac conduction system (Biel et al., 2009). Therefore, we choose HCN1 and HCN2 to study the analgesic mechanism of dexmedetomidine. In the present experiments, we found that knocking out HCN1 channels significantly reduced the analgesic effects induced by systemically administered dexmedetomidine. Under whole cell voltage clamp conditions in HEK 293 cells expressing HCN subunits, we demonstrated that dexmedetomidine inhibits voltage-dependent HCN channel currents. For channels expressing either HCN1 or HCN2 subunits, dexmedetomidine caused a decrease of maximal currents and a negative shift in V1/2 of Ih. These data suggest that analgesic effect produced by dexmedetomidine is likely due to the inhibition of HCN currents. The autonomic beating of the heart and a considerable number of rhythmic activities in the nervous system are controlled by Ih (Pape, 1996; Hagiwara et al., 1988). Dexmedetomidine dosedependently cause a decrease in Ih that was due primarily to inhibition of the maximal current amplitude and a statistically significant hyperpolarizing shift in the voltage dependence of Ih activation. The effects was similar to that observed in rat hypoglossal motoneurons induced by clonidine(Parkis and Berger, 1997). The attenuation of dexmedetomidine-induced analgesia response(both wild type and knockout mice) by yohimbine supports the existence of an α2-adrenergic receptor mechanism. This is consistent with other central neurons relevant to clonidine(Yagi
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Fig. 3. Dexmedetomidine inhibits HCN channel currents in HEK 293 cells. (A) Sample currents from HEK 293 cells expressing mHCN1 or mHCN2 channel constructs evoked by hyperpolarizing voltage steps from 40 to 130 mV before and during exposure to dexmedetomidine (10 μM); voltage steps are followed by a step to 90 mV for tail current analysis. (B) Steady state I–V curves from currents at the end of the voltage steps under control conditions, during exposure to dexmedetomidine for mHCN1 or mHCN2 constructs (n¼ 5–8). (C) Dose-dependent inhibition of Ih by dexmedetomidine (nP o 0.05, 0.1 μM vs 1 μM,10 μM; ΔP40.05, HCN1 vs HCN2; n¼ 5–8). (D) Activation curves are determined from tail currents (nPo 0.05, vs control, n¼ 5–8).
and Sumino, 1998; Parkis and Berger, 1997; Adachi et al., 2005). Dexmedetomidine binds to α2-adrenoceptors on the cell membrane of neurons, which leads to activation of the G protein-coupled
inwardly rectifying K þ channels and inhibition of Ih channels, resulting in hyperpolarization of the membrane. Yohimbine blocked the Ih suppression by dexmedetomidine (Shirasaka et al., 2007). The
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α2 receptor selective agonists, clonidine and UK14304, were found to decrease Ih amplitude and to slow its rate of activation indicating a negative shift in the current's voltage dependence. Two α2 receptor antagonists, yohimbine and RS79948, prevented the effects of α2 receptor activation. The second messenger system associated with the α2 receptor revealed that Ih inhibition is independent of cyclic adenosine monophosphate (cAMP) and resulted from the activation of protein kinase C (Inyushin et al., 2010). In our experiment, yohimbine only partially reduced the analgesic effect of dexmedetomidine, but not all. Especially in wild type mice, after administration of yohimbine, dexmedetomidine still showed some analgesic effect. This finding indicates that dexmedetomidine elicits its analgesic effect in mice not only via α2-adrenergic receptors, but also a direct channel inhibition via α2-independent mechanism. Dexmedetomidine added to ropivacaine for sciatic nerve block in rats caused an increase in the duration of analgesia, which was reversed by pretreatment with an enhancer of Ih current (forskolin). The analgesic effect of dexmedetomidine was not reversed by an α2-adrenoceptor antagonist (Brummett et al., 2011). However, the research on dexmedetomidine is inadequate. Clonidine and dexmedetomidine both are α2-receptor agonist, with a similar chemical structure and pharmacological properties. Research on clonidine might contribute to our understanding of the mechanism of dexmedetomidine. Clonidine caused significant bradycardic effects in vivo and in isolated atria of α2ABC-KO mice. Experiments with the HEK 293 cells expressing HCN1, HCN2 and HCN4 subunits, as well as with SAN cells isolated from wild type and α2ABC-KO mice, clonidine was shown to directly inhibit HCN currents(Knaus et al., 2007). It is also conceivable that HCN channels ubiquitously expressed in the central nervous system may be similarly inhibited by clonidine or dexmedetomidine. A significant body of molecular and pharmacological evidence is now emerging to support a role for HCN channels in the function of sensory neurons and pain sensation (Maher et al., 2009; Dunlop et al., 2009). HCN channel is expressed in a variety of sensory neurons that respond to painful stimuli. It is believed that inhibition of HCN current in sensory neurons decreases neuronal excitability, reduces pain sensation and affects pain conduction (Grosu and Lavand’homme , 2010). Our findings suggest a specific mechanism that dexmedetomidine produces analgesic effect by inhibition of Ih. However, research in this area is still insufficient. Further study will be done in future. Learning about more dexmedetomidine pharmacological mechanism will help it become a new therapeutic approach in the treatment of pain. References Adachi, T., Robinson, D.M., Miles, G.B., Funk, G.D., 2005. Noradrenergic modulation of XII motoneuron inspiratory activity does not involve α2-receptor inhibition of the Ih current or presynaptic glutamate release. J. Appl. Physiol. 98, 1297–1308.
Biel, M., Wahl-Schott, C., Michalakis, S., Zong, X., 2009. Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89, 847–885. Brummett, C.M., Norat, M.A., Palmisano, J.M., Lydic, R., 2008. Perineural administration of dexmedetomidine in combination with bupivacaine enhances sensory and motor blockade in sciatic nerve block without inducing neurotoxicity in rat. Anesthesiology 109, 502–511. Brummett, C.M., Padda, A.K., Amodeo, F.S., Welch, K.B., Lydic, R., 2009. Perineural dexmedetomidine added to ropivacaine causes a dose-dependent increase in the duration of thermal antinociception in sciatic nerve block in rat. Anesthesiology 111, 1111–1119. Brummett, C.M., Amodeo, F.S., Janda, A.M., Padda, A.K., Lydic, R., 2010. Perineural dexmedetomidine provides an increased duration of analgesia to a thermal stimulus when compared with a systemic control in a rat sciatic nerve block. Reg. Anesth. Pain Med. 35, 427–431. Brummett, C.M., Hong, E.K., Janda, A.M., Amodeo, F.S., Lydic, R., 2011. Perineural dexmedetomidine added to popivacaine for sciatic nerve block in rats prolongs the duration of analgesia by blocking the hyperpolarization-activated cation current. Anesthesiology 115 (4), 836–843. Carr, D.B., Andrews, G.D., Glen, W.B., Lavin, A., 2007. Alpha2-Noradrenergic receptors activation enhances excitability and synaptic integration in rat prefrontal cortex pyramidal neurons via inhibition of HCN currents. J. Physiol. 584, 437–450. D’Amour, F.E., Smith, D.L., 1941. A method determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74–79. Dunlop, J., Vasilyev, D., Lu, P., Cummons, T., Bowlby, M.R., 2009. Hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels and pain. Curr. Pharm Des. 15 (15), 1767–1772. Gerresheim, G., Schwemmer, U., 2013. Dexmedetomidine. Anaesthesist 62 (8), 661–674. Grosu, I., Lavand’homme, P., 2010. Use of dexmedetomidine for pain control. F1000 Med. Rep. 2, 90. Hagiwara, N., Irisawa, H., Kameyama, M., 1988. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sinoatrial node cells. J. Physiol. 395, 233–253. Hall, J.E., Uhrich, T.D., Barney, J.A., Arain, S.R., Ebert, T.J., 2000. Sedative, amnestic, and analgesic properties of small-dose dexmedetomidine infusions. Anesth. Analg. 90, 699–705. Himes Jr, R.S., DiFazio, C.A., Burney, R.G., 1977. Effects of lidocaine on the anesthetic requirements for nitrous oxide and halothane. Anesthesiology 47, 437–440. Inyushin, M.U., Arencibia-Albite, F., Vázquez-Torres, R., Vélez-Hernández, M.E., Jiménez-Rivera, C.A., 2010. Alpha-2 noradrenergic receptor activation inhibits the hyperpolarization-activated cation current (Ih) in neurons of the ventral tegmental area. Neuroscience 167 (2), 287–297. Jaakola, M.L., Salonen, M., Lehtinen, R., Scheinin, H., 1991. The analgesic action of dexmedetomidine – a novel alpha2-adrenoceptor agonist – in healthy volunteers. Pain 46, 281–285. Knaus, A., Zong, X.G., Beetz, N., Jahns, R., Lohse, M.J., Biel, M., Hein, L., 2007. Direct inhibition of cardiac hyperpolarization-activated cyclic nucleotide-gated pacemaker channels by clonidine. Circulation 115, 872–880. Maher, M.P., Wu, N.T., Guo, H.Q., Dubin, A.E., Chaplan, S.R., Wickenden, A.D., 2009. HCN channels as targets for drug discovery. Comb. Chem. High Throughput Screen. 12 (1), 64–72. Nolan, M.F., Malleret, G., Lee, K.H., Gibbs, E., Dudman, J.T., Santoro, B., Yin, D., Thompson, R.F., Siegelbaum, S.A., Kandel, E.R., Morozov, A., 2003. The hyperpolarization-activated HCN1 channel is important formotor learning and neuronal integration by cerebellar Purkinje cells. Cell 115, 551–564. Pape, H.C., 1996. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu. Rev. Physiol. 58, 299–327. Parkis, M.A., Berger, A.J., 1997. Clonidine reduces hyperpolarization-activated inward current (Ih) in rat hypoglossal motoneurons. Brain Res. 769, 108–118. Shirasaka, T., Kannan, H., Takasaki, M., 2007. Activation of a G protein–coupled inwardly rectifying K� current and suppression of Ih contribute to dexmedetomidine-induced inhibition of rat hypothalami paraventricular nucleus neurons. Anesthesiology 107, 605–615. Yagi, J., Sumino, R., 1998. Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons. J. Neurophysiol. 80 (3), 1094–1104.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Dexmedetomidine produced analgesic effect via inhibition of HCN currents Ying-cong Yang, Qing-tao Meng, Xia Pan, Zhong-yuan Xia n, Xiang-dong Chen Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 April 2013 Received in revised form 18 June 2014 Accepted 19 June 2014 Available online 8 July 2014
The purpose of this study was to investigate the mechanism by which systemic dexmedetomidine exerts analgesic effect and examine effect of dexmedetomidine on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels currents. The experiments were performed on C57BL/6 J and HCN1 knockout mice. The analgesic effects of intraperitoneal dexmedetomidine (10–40 μg/kg) were measured by a tail-flick test. Whole-cell clamp recordings were used to examine the properties of cloned HCN subunit currents expressed in HEK 293 cells under control condition and dexmedetomidine administration (0.1–10 μM). Injection of dexmedetomidine caused a clear time and dose-related increase in the tail-flick latency of both wild type and knockout mice. Compared with the wild type group, the MPE (maximum possible effect) of tail-flick latency induced by 30 μg/kg and 40 μg/kg dexmedetomidine in knockout mice was significantly lower. The α2-adrenergic receptor antagonist yohimbine (5 μg/kg) reduced the MPE of dexmedetomidine (30 μg/kg) both in wild type and knockout mice. Dexmedetomidine(0.1–10 μM) inhibited HCN1 and HCN2 channel currents in HEK 293 cells, caused a decrease of maximal currents, an increase of inhibition rate of hyperpolarization-activated currents (Ih), and a negative shift in V1/2. We conclude that dexmedetomidine produces a dose-dependently analgesic effect, and the effect is likely due to the inhibition of HCN currents. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dexmedetomidine HCN currents α2-adrenergic receptor agonist Analgesic effect Chemical compounds studied in this article: Dexmedetomidine (Chem CID: 5311068)
1. Introduction Dexmedetomidine, a potent and highly selective agonist of the
α2-adrenergic receptors has been widely used as an analgesic and antinociceptive adjuvant (Gerresheim and Schwemmer, 2013). Animal experiments showed that peripheral application of dexmetetomidine and other α2-adrenergic receptor agonist improved the threshold of mechanical and thermal pain. Dexmedetomidine has been shown to enhance effects of local anesthetic bupivacaine and ropivacaine for sciatic nerve blocks and increase duration of analgesia dose-dependently (Brummett et al., 2008, 2009, 2010). Clinical study also showed that systemically administered dexmedetomidine exerted moderate analgesic effect in human ( Jaakola et al.,1991; Hall et al., 2000). α2-adrenergic receptors are widely distributed throughout the peripheral and central nervous system, and their activation produces a variety of effects. However, the mechanism of these effects is not yet very clear. Clonidine,another α2-adrenergic receptor agonist, has been shown to effectively inhibit hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels currents in
n
Corresponding author. Tel.: þ 86 27 88041911; fax: þ 86 27 88042292. E-mail address: [email protected] (Z.-y. Xia).
http://dx.doi.org/10.1016/j.ejphar.2014.06.031 0014-2999/& 2014 Elsevier B.V. All rights reserved.
different neuronal types, including dorsal root ganglion (Yagi and Sumino, 1998), pyramidal neurons of the prefrontal cortex (Carr et al., 2007) and dopamine neurons of the ventral tegmental area (Inyushin et al., 2010). These studies suggest that the effects of α2-adrenergic receptors agonist are closely related to the HCN channels. The aim of the present study is to investigate the mechanism by which systemic dexmedetomidine exerts analgesic effect, and examine effect of dexmedetomidine on HCN currents.
2. Material and methods 2.1. Animals The experiments were performed on C57BL/6J and HCN1 knockout mice weighing 19–25 g obtained from Center of Experimental Animals in Wuhan University, China. Animals were housed five per cage in a room maintained at 227 1 1C with an alternating 12 h dark/12 h light cycles. The HCN1 knockout mice were originally prepared and characterized by the Kandel laboratory (Nolan et al., 2003); they have been deposited for distribution by Jackson Labs (stock #005034). Mice homozygous for the targeted mutation are viable, fertile,
Y.-c. Yang et al. / European Journal of Pharmacology 740 (2014) 560–564
561
normal in size and longevity and they do not display any gross physical or behavioral abnormalities. The treatment and care of the experimental animals was conducted according to the Animal Care and Use guideline of Wuhan University and was approved by the Institutional Ethics Committee at Renmin Hospital of Wuhan University, China.
Statistical evaluations were performed using SPSS software and Graphpad prism, and a difference was considered statistically significant at P o0.05.
2.2. Drugs and chemicals
3.1. Effects of i.p. administration dexmedetomidine in tail-flick test
Dexmedetomidine hydrochloride injection (Abbott Laboratories) and yohimbine (Sigma Chemical) were diluted in physiological saline (0.9% NaCl) for animal experiments. All drugs and chemicals were diluted in bath solution for patch clamp recording.
Injection of dexmedetomidine caused a clear time and doserelated increase in the tail-flick latency of both wild type and knockout mice (Fig. 1). Effect of 10 μg/kg and 20 μg/kg dexmedetomidine in wild type mice was apparent within 15 min, lasting until 60 min and 75 min respectively. The duration of analgesia at 30 μg/kg and 40 μg/kg of dexmedetomidine was up to 120 min (Fig. 1Aa). Effect of 10 μg/kg and 20 μg/kg dexmedetomidine in knockout mice was apparent within 30 min, lasting until 45 min and 60 min respectively. The duration of analgesia at 30 μg/kg dose was up to 90 min whereas at 40 μg/kg, it was 120 min (Fig.1Ab) The MPE in wild type mice and knockout mice after application of dexmedetomidine are presented in Fig. 1B. In wild type mice, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine was 33.26 7 1.90% and 53.85 79.69% respectively. In knockout mice, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine was 22.06 73.63% and 37.18 74.63% respectively. Compared with the wild type group, the MPE of 30 μg/kg and 40 μg/kg dexmedetomidine in knockout mice was significantly lower. The MPE of 10 μg/kg and 20 μg/kg dexmedetomidine was no obvious difference between groups.
2.3. Tail-flick test The analgesia response against thermal stimuli was assessed with the tail-flick test. Mice were gently held with the tail positioned in the tail-flick apparatus (MK-330B; Muromachi Kikai Co., Ltd., Tokyo, Japan) for radiant thermal stimulation of the dorsal surface of the tail. The intensity of the thermal stimulus was adjusted to cause the animal to flick its tail within 2.5 to 3 s as the baseline. The tail-flick latency was measured before and 15, 30, 45, 60, 75, 90, 105 and 120 min after the i.p administration of dexmedetomidine and dexmedetomidine plus yohimbine. Control mice received saline injection. Examines were terminated if the animals did not respond within 15 s in order to minimize tissue damage. The tail flick latency was converted to represent the percent maximum possible effect (MPE), according to the following formula: MPE¼100 (postdrug latency-predrug latency)/(15-predrug latency) (D’Amour and Smith, 1941). Peak maximum percent effect (MPE) values of individual animals from the time–response data were pooled for each dose to construct the dose–response curves. 2.4. Recordings from cloned HCN channels in HEK 293 cells HEK 293 cells were cultured using standard procedures and transiently transfected with HCN channel constructs, together with an enhanced GFP plasmid (Clontech, Mountain View, CA), using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Recordings were obtained 24 to 48 h after transfection. Wholecell recordings were obtained using 3 to 5 MΩ patch pipettes and an Axopatch 200B amplifier at room temperature. Bath solution was perfused continuously ( 2 ml/min) and contained the following (in mM): 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH 7.3; internal solution contained the following (in mM):120 KCH4SO3, 4 NaCl, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 10 EGTA, 3 Mg-ATP, and 0.3 GTP-Tris, pH 7.2 (Himes et al., 1977). 2.5. Data acquisition and analysis Data were acquired using pCLAMP software and a Digidata 1322 A digitizer (Axon Instruments). For voltage-clamp recording, time-dependent hyperpolarization-activated currents (Ih) were evoked with incrementing (Δ 10 mV) hyperpolarizing pulses (3–4 s) from a holding potential of 40 mV, followed immediately by a step to fixed potential ( 90 mV) to obtain tail currents. Current amplitude at each potential was taken as the timedependent current at the end of hyperpolarizing voltage steps; maximal available current was determined at 120 mV. Tail currents were normalized, plotted as a function of the preceding hyperpolarization step voltage, and fitted with Boltzmann curves for derivation of activation parameters (V1/2) by using Origin software (Origin Lab). All data are presented as means 7S.E.M. Statistical tests included one- and two-way ANOVA, Student's t test, as indicated.
3. Results
3.2. Effects of i.p. administration of dexmedetomidineþyohimbine in tail-flick test As shown in Fig. 2, application of yohimbine, the MPE of wild type and knockout mice was 9.46 72.77% and 2.72 72.55% respectively. Yohimbine (5 μg/kg) significantly reduced the MPE of 30 μg/kg dexmedetomidine by 23.80% and 19.34% in wild type and knockout mice (P o0.05 vs demedetomidine alone, n ¼10), respectively. 3.3. Dexmedetomidine inhibits HCN1 and HCN2 channel currents expressed in HEK 293 cells. In HEK 293 cells expressing HCN1 subunits, dexmedetomidine caused a decrease in HCN1 currents, from 627.53 7 200.86 to 204.27 727.95 pA . The effects of dexmedetomidine (0.1–10 μM) were concentration-dependent (Fig. 3B). In the presence of 0.1 μM, 1 μM, 10 μM dexmedetomidine, the inhibition rates of HCN currents were 18.82 71.72%, 38.4375.39% and 46.48 76.66% respectively. Similar to HCN1, dexmedetomidine (0.1–10 μM) also decreased HCN2 currents , from 593.87 756.64 to 199.99 7 24.72 pA, in concentration-dependent manner in cells expressing HCN2 subunits(Fig. 3B). In the presence of 0.1 μM, 1 μM, 10 μM dexmedetomidine, the inhibition rate of HCN2 currents was 23.857 2.59%, 41.14 7 4.82% and 58.19 75.22% respectively. In the same concentration of dexmedetomidine, the inhibition rate of HCN1 and HCN2 currents has no significant difference (Fig. 3C). To examine the effect of dexmedetomidine on the voltagedependent activation of Ih, an activation curve was constructed by a tail current analysis. The Ih activation curve fitted with the Boltzmann equation is shown in Fig. 3D. In HEK 293 cells expressing either HCN1 or HCN2 subunits, the application of dexmedetomidine (0.1–10 μM) produced a dose-related shift to the left of the Ih activation curve. V1/2 of HCN1 shifted from 81.80 71.52 to 103.09 76.14 mV by 10 μM dexmedetomidine, and V1/2 of HCN2 shifted from 102.61 71.16 to 120.00 7
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Fig. 1. Time and dose-dependent analgesia effects induced by dexmedetomidine(10, 20, 30, 40 μg/kg, i.p.) in the tail-flick test. Data are presented as means 7 S.E.M. The drug is administered after the baseline value is determined, and control group is treated with saline. (A) Time course of analgesia effects induced by dexmedetomidine (a, wild type mice; b, knockout mice). nPo 0.05 and nnPo 0.01 vs saline-treated controls, n¼ 6–9. (B) Dose-analgesia response curves to dexmedetomidine in the tail-flick test. Responses are expressed as %MPE. %MPE values at the time point at which peak analgesia responses are observed for wild type and knockout mice are used to plot the curve (*Po 0.05, wild type vs knockout, n¼ 6–9).
Fig. 2. Antagonism of dexmedetomidine-induced analgesia response by yohimbine. Application of 30 μg/kg dexmedetomidine results in small MPE values in the presence of 5 μg/kg yohimbine both in wild type and knockout mice (nP o0.01, dex vs yoh þ dex, n¼ 10).
1.56 mV by 10 μM dexmedetomidine. These data revealed that dexmedetomidine (0.1–10 μM) produced a significant shift in the V1/2 to a more hyperpolarized potential (P o0.05, n ¼ 5–8). However, the slope values were not altered by dexmedetomidine (P 40.05, n ¼5–8).
4. Discussion Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a family of six transmembrane domain, single poreloop, hyperpolarization activated, non-selective cation channels. HCN channels are activated at negative membrane potentials and repolarization following action potential firing resulting in a depolarizing current influencing the threshold for subsequent action potential generation. Consequently, HCN currents play
a critical role in regulating neuronal resting membrane potential and excitability (Maher et al., 2009; Dunlop et al., 2009). The HCN channel family comprises four distinct subtypes (HCN1–4). They are widely distributed in the nervous system and the heart. HCN1 is the subtype with the fastest kinetics, mainly expressed in the peripheral nervous system, sense organs and central nervous system associated with fine sensory. HCN2 is distributed nearly throughout most brain regions and spinal nerve. In contrast, HCN3 is expessed at very low levels in the central nervous system. HCN4 is mainly distributed in the cardiac conduction system (Biel et al., 2009). Therefore, we choose HCN1 and HCN2 to study the analgesic mechanism of dexmedetomidine. In the present experiments, we found that knocking out HCN1 channels significantly reduced the analgesic effects induced by systemically administered dexmedetomidine. Under whole cell voltage clamp conditions in HEK 293 cells expressing HCN subunits, we demonstrated that dexmedetomidine inhibits voltage-dependent HCN channel currents. For channels expressing either HCN1 or HCN2 subunits, dexmedetomidine caused a decrease of maximal currents and a negative shift in V1/2 of Ih. These data suggest that analgesic effect produced by dexmedetomidine is likely due to the inhibition of HCN currents. The autonomic beating of the heart and a considerable number of rhythmic activities in the nervous system are controlled by Ih (Pape, 1996; Hagiwara et al., 1988). Dexmedetomidine dosedependently cause a decrease in Ih that was due primarily to inhibition of the maximal current amplitude and a statistically significant hyperpolarizing shift in the voltage dependence of Ih activation. The effects was similar to that observed in rat hypoglossal motoneurons induced by clonidine(Parkis and Berger, 1997). The attenuation of dexmedetomidine-induced analgesia response(both wild type and knockout mice) by yohimbine supports the existence of an α2-adrenergic receptor mechanism. This is consistent with other central neurons relevant to clonidine(Yagi
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Fig. 3. Dexmedetomidine inhibits HCN channel currents in HEK 293 cells. (A) Sample currents from HEK 293 cells expressing mHCN1 or mHCN2 channel constructs evoked by hyperpolarizing voltage steps from 40 to 130 mV before and during exposure to dexmedetomidine (10 μM); voltage steps are followed by a step to 90 mV for tail current analysis. (B) Steady state I–V curves from currents at the end of the voltage steps under control conditions, during exposure to dexmedetomidine for mHCN1 or mHCN2 constructs (n¼ 5–8). (C) Dose-dependent inhibition of Ih by dexmedetomidine (nP o 0.05, 0.1 μM vs 1 μM,10 μM; ΔP40.05, HCN1 vs HCN2; n¼ 5–8). (D) Activation curves are determined from tail currents (nPo 0.05, vs control, n¼ 5–8).
and Sumino, 1998; Parkis and Berger, 1997; Adachi et al., 2005). Dexmedetomidine binds to α2-adrenoceptors on the cell membrane of neurons, which leads to activation of the G protein-coupled
inwardly rectifying K þ channels and inhibition of Ih channels, resulting in hyperpolarization of the membrane. Yohimbine blocked the Ih suppression by dexmedetomidine (Shirasaka et al., 2007). The
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α2 receptor selective agonists, clonidine and UK14304, were found to decrease Ih amplitude and to slow its rate of activation indicating a negative shift in the current's voltage dependence. Two α2 receptor antagonists, yohimbine and RS79948, prevented the effects of α2 receptor activation. The second messenger system associated with the α2 receptor revealed that Ih inhibition is independent of cyclic adenosine monophosphate (cAMP) and resulted from the activation of protein kinase C (Inyushin et al., 2010). In our experiment, yohimbine only partially reduced the analgesic effect of dexmedetomidine, but not all. Especially in wild type mice, after administration of yohimbine, dexmedetomidine still showed some analgesic effect. This finding indicates that dexmedetomidine elicits its analgesic effect in mice not only via α2-adrenergic receptors, but also a direct channel inhibition via α2-independent mechanism. Dexmedetomidine added to ropivacaine for sciatic nerve block in rats caused an increase in the duration of analgesia, which was reversed by pretreatment with an enhancer of Ih current (forskolin). The analgesic effect of dexmedetomidine was not reversed by an α2-adrenoceptor antagonist (Brummett et al., 2011). However, the research on dexmedetomidine is inadequate. Clonidine and dexmedetomidine both are α2-receptor agonist, with a similar chemical structure and pharmacological properties. Research on clonidine might contribute to our understanding of the mechanism of dexmedetomidine. Clonidine caused significant bradycardic effects in vivo and in isolated atria of α2ABC-KO mice. Experiments with the HEK 293 cells expressing HCN1, HCN2 and HCN4 subunits, as well as with SAN cells isolated from wild type and α2ABC-KO mice, clonidine was shown to directly inhibit HCN currents(Knaus et al., 2007). It is also conceivable that HCN channels ubiquitously expressed in the central nervous system may be similarly inhibited by clonidine or dexmedetomidine. A significant body of molecular and pharmacological evidence is now emerging to support a role for HCN channels in the function of sensory neurons and pain sensation (Maher et al., 2009; Dunlop et al., 2009). HCN channel is expressed in a variety of sensory neurons that respond to painful stimuli. It is believed that inhibition of HCN current in sensory neurons decreases neuronal excitability, reduces pain sensation and affects pain conduction (Grosu and Lavand’homme , 2010). Our findings suggest a specific mechanism that dexmedetomidine produces analgesic effect by inhibition of Ih. However, research in this area is still insufficient. Further study will be done in future. Learning about more dexmedetomidine pharmacological mechanism will help it become a new therapeutic approach in the treatment of pain. References Adachi, T., Robinson, D.M., Miles, G.B., Funk, G.D., 2005. Noradrenergic modulation of XII motoneuron inspiratory activity does not involve α2-receptor inhibition of the Ih current or presynaptic glutamate release. J. Appl. Physiol. 98, 1297–1308.
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