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Involvement of ATP-Sensitive K+ Channels in the Peripheral Antinociceptive Effect Induced by the α2-Adrenoceptor Agonist Xylazine
Journal of Pharmacological Sciences
J Pharmacol Sci 111, 323 – 327 (2009)4
©2009 The Japanese Pharmacological Society
Full Paper
Involvement of ATP-Sensitive K+ Channels in the Peripheral Antinociceptive Effect Induced by the α2-Adrenoceptor Agonist Xylazine Thiago Roberto Lima Romero1 and Igor Dimitri Gama Duarte1,* 1
Department of Pharmacology, Institute of Biological Sciences (ICB), Federal University of Minas Gerais (UFMG), Av. Antônio Carlos, 6627-campus da Pampulha, CEP 31.270.100, Belo Horizonte, MG, Brazil
Received April 1, 2009; Accepted August 11, 2009
Abstract. Xylazine is an α2-adrenergic agonist extensively used in veterinary medicine and animal experimentation for producing antinociception, sedation, and muscle relaxation. The nitric oxide (NO) / cGMP / ATP-sensitive K+ (KATP) channel pathway has been proposed as the action mechanism of peripheral antinociception of several groups of drugs, including opioids and nonsteroidal analgesics. Considering the lack of knowledge regarding the mechanisms involved in xylazine effects, the present study investigated the contribution of K+ channels on peripheral antinociception induced by xylazine using the rat paw pressure test, in which hyperalgesia was induced by intraplantar injection of prostaglandin E2. Xylazine administered into the right hind paw elicited a local antinociceptive effect, since only much higher doses produced a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine was antagonized by glibenclamide, a specific blocker of KATP channels. In another experiment, tetraethylammonium, a voltage-dependent K+-channel blocker, and paxilline and dequalinium, which are selective blockers for the large- and small-conductance Ca2+-activated K+ channels, respectively, were ineffective at blocking xylazine antinociception. These results provide evidence that the peripheral antinociceptive effect of xylazine probably results from KATP-channel activation, while the voltage-dependent K+ channels, small- and large-conductance Ca2+-activated K+ channels, appear not to be involved in this mechanism. Keywords: xylazine, α2-adrenoceptor, K+ channel, peripheral antinociception different receptors, including α2-adrenoceptor agonists, at supraspinal and spinal sites is also dependent of K+ channels (6). Xylazine is an α2-adrenoceptor agonist (10, 11) that is extensively used in veterinary medical practice and animal experimentation for producing sedation, antinociception, and muscle relaxation (12). Although xylazine has been shown to promote peripheral antinociception by α2C-adrenoceptor activation (13), inducing nitric oxide synthase to produce nitric oxide with posterior increase of cGMP in the peripheral nociceptors (14), its mechanism of action has not yet been clearly elucidated. Thus the aim of the present study was to determine the involvement of K+ channels in peripheral antinociception induced by xylazine. For this purpose, the effects of glibenclamide, a specific KATP-channel blocker (15); tetraethylammonium, a nonselective voltage-dependent K+-channel blocker (16); dequalinium, a selective blocker of small-conductance Ca2+-activated
Introduction Nitric oxide (NO) can activate different types of K+ channels in different types of tissues by increasing cGMP (1 – 4). Potassium currents play an important role in the regulation of neuronal excitability by permitting an efflux of K+ ions through the membrane (injunction current), bringing the potential of membrane values closer to repolarization and further from the threshold that triggers action potential, leading to a decrease in neurotransmitter release (5, 6). Several studies have determined that opening of the ATP-sensitive K+ (KATP) channels is the final stage of the peripheral antinociceptive mechanism of morphine (7), dipyrone (8), and diclofenac (9). The antinociceptive action mechanism induced by various agonists of *Corresponding author. [email protected] Published online in J-STAGE doi: 10.1254 / jphs.09103FP
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K+ channel (17); and paxilline, a selective blocker of large-conductance Ca2+-activated K+ channels (18) were tested on the peripheral antinociceptive effect of xylazine in the hyperalgesic rat paw model. Materials and Methods Animals All experiments were performed on 160 – 200 g male Wistar rats [from Center for Laboratory Animal Facilities (CEBIO) of the Federal University of Minas Gerais (UFMG)]. The rats were housed in a temperaturecontrolled room (23 ± 1°C) on an automatic 12-h light/ dark cycle (06:00 – 18:00 h). All tests were conducted during the light phase (08:00 – 15:00 h). Food and water were freely available until the onset of the experiments. All animal procedures and protocols were approved by the Ethics Committee on Animal Experimentation (CETEA) of UFMG. Measurement of hyperalgesia Hyperalgesia was induced by subcutaneous injection of prostaglandin E2 (PGE2) (2 µg) into the plantar surface of the hind paw. Hyperalgesia was measured according to the paw pressure test described by Green and Young (19) and Randall and Sellito (20). An analgesimeter was used (Ugo-Basile, Comerio, Italy) with a cone-shaped paw-presser with a rounded tip, which applies a linearly increasing force to the hind paw. The weight in grams (g) required to elicit the nociceptive response of paw flexion was determined as the nociceptive threshold. A cutoff value of 300 g was used to reduce the possibility of damage to the paws. The nociceptive threshold was measured in the right paw and determined as the average of the three consecutive trials recorded before and 3 h after PGE2 injection. The threshold was calculated as the difference between these two averages (Δ of nociceptive threshold) and is expressed in grams. Δ of nociceptive threshold >0 means hyperalgesia by PGE2 injection and decrease of this value means antihyperalgesic effect by the tested drug. Drugs administration All drugs were administered using an injected volume of 50 μl/ paw with the exception of PGE2, where an injected volume of 100 μl/paw was used. Xylazine (Sigma, St. Louis, MO, USA), tetraethylammonium chloride (Sigma), dequalinium chloride (Calbiochem, La Jolla, CA, USA), and paxilline chloride (Sigma) were dissolved in isotonic saline; PGE2 (Sigma) was dissolved in 8% ethanol in saline; and glibenclamide (Sigma) was dissolved in 1% Tween 20 (Vetec, Belo Horizonte,
MG, Brazil). Experimental protocol Xylazine was administered subcutaneously in the right hind paw 2 h and 55 min after local injection of PGE2. In the protocol used to determine whether xylazine was acting outside the injection paw, PGE2 was injected into both hind paws, while xylazine was administered into the right paw, after which the nociceptive threshold was measured in both hind paws. Tetraethylammonium was administered 30 min prior to xylazine. Other K+-channel blockers were administered 5 min prior to xylazine. It should be noted that the protocols concerning dose and time of administration of each drug used in this study were obtained through literature data and pilot experiments. Statistical analysis The data were statistically analyzed by one-way analysis of variance (ANOVA) and the post-hoc Bonferroni test for multiple comparisons. Probabilities of less than 5% (P<0.05) were considered to be statistically significant. Results The administration of xylazine (25, 50, and 100 μg) into the right hind paw produced an antinociceptive response against the PGE2 hyperalgesia (2 μg/paw) in a dose-dependent manner (Fig. 1A), although this was not statistically significant for the dose of 25 μg/paw. When administered into the right paw, xylazine administration at a higher dose did not produce an antinociceptive effect in the left paw, indicating that at this dose, it only presented a peripheral site of action (Fig. 1B); moreover, this dose did not induce any effect in the non-hyperalgesic paws (Fig. 1A). The antinociceptive effect of the highest dose of xylazine used (100 μg/paw) was antagonized in a dosedependent manner by the KATP-channel blocker glibenclamide (20, 40, and 80 μg/paw), although the antagonism was not statistically significant for a dose of 20 μg/paw (Fig. 2). Glibenclamide alone did not alter the control groups (Fig. 2). Another experiment, shown in Fig. 3A, verified that the voltage-dependent K+-channel blocker tetraethylammonium (30 μg) injected into the paw did not significantly reduce peripheral antinociception induced by xylazine at 100 μg/paw in the PGE2-induced hyperalgesia model. Tetraethylammonium alone did not induce hyperalgesia or antinociception (data not shown). Figure 3B shows that neither dequalinium (50 μg /paw), a blocker selective for small-conductance Ca2+-
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Fig. 2. Antagonism induced by intraplantar administration of glibenclamide of peripheral antinociception produced by xylazine in hyperalgesic paws (PGE2, 2 μg). Glibenclamide (Gli, μg/ paw) was administered 5 min prior to xylazine (Xyl, μg/ paw). # and * indicate a significant difference compared to PGE2 + Veh 1 + Veh 2– and PGE2 + Veh 1 + Xyl 100–injected controls, respectively (P<0.05, ANOVA + the Bonferroni test). Veh 1 = 1% Tween 20, Veh 2 = Saline, Veh 3 = 8% ethanol in saline. Each column represents the mean ± S.E.M. (n = 4 – 5).
Discussion
Fig. 1. Effect of xylazine on PGE2-induced hyperalgesia in rats (A) and exclusion of outside paw antinociceptive effect of xylazine (B). A: Xylazine (Xyl, μg/paw) was administered 2 h and 55 min after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in Materials and Methods. # indicates a significant difference from the PGE2 + Veh 1–injected control (P<0.05, ANOVA + the Bonferroni test); Veh 1 = Saline, Veh 2 = Ethanol 8% in saline. B: PGE2 (2 μg) was administered in both hind paws, right (R) and left (L). Xylazine (100 μg/paw) was administered 2 h and 55 min after PGE2 in the right hind paw (Xyl 100, R paw). Antinociceptive responses were measured in both hind paws, as described in Materials and Methods. # indicates a significant difference from the PGE2 R paw + Veh R paw–injected group (P<0.05, ANOVA + the Bonferroni test); Veh = Saline, R paw = Right paw, L paw = Left paw. A and B: Each column represents the mean ± S.E.M. (n = 4 – 5).
activated K+ channels, nor paxilline (20 μg/paw), a blocker selective for large-conductance Ca2+-activated K+ channels, significantly modified the peripheral antinociception induced by xylazine at 100 μg/paw. Dequalinium and paxilline alone did not induce hyperalgesia or any overt behavioural effect (data not shown).
In present experiments, xylazine induced a peripheral dose-dependent antihyperalgesic effect against PGE2 injection. Using the strategy of evaluating the efficacy of ipsi versus contralateral paw, xylazine administration at the dose of 100 µg revealed local hyperalgesic action in the paw, so this dose was chosen for the other experiments. Even though that there are evidences that a direct inhibition of tetrodotoxin-resistant Na+ channels may contribute to the antinociceptive effect of the α2adrenergic agonist clonidine (21), numerous studies have been suggested that KATP-channel opening is one mechanism by which many α2-adrenergic agonists induce antinociceptive effects (6). Ocaña and Baeyens (22) reported that KATP channels were involved in the central antinociceptive mechanism of the α2-adrenergic agonist clonidine in the tail-flick test since glibenclamide antagonized this effect; and in the same work, voltage-dependent K+ channels participation was discarded since 4-aminopyridine and tetraethylammonium were unable to reverse the effect of clonidine. Besides clonidine, the antinociception of several other α2adrenergic agonists, including guanabenz, tizanidine, and dexmedetomidine, were antagonized by the sulphonylureas tolbutamide and glibenclamide, reaffirming the importance of KATP-channel activation regarding the antinociceptive action of α2-adrenergic agonists (23). The presence of α2-adrenoceptors in the dorsal root
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Fig. 3. Effect of intraplantar administration of tetraethylammonium (TEA) (A) or dequalinium (DQ) and paxilline (Pax) (B) on peripheral antinociception produced by xylazine in hyperalgesic paws (PGE2, 2 μg). A: TEA (μg/ paw) was administered 30 min prior to xylazine (μg/ paw). B: DQ (μg/ paw) and Pax (μg/paw) were administered 5 min prior to xylazine (μg/ paw). A and B: # indicates a significant difference from the PGE2 + Veh 1 + Veh 1–injected control (P<0.05, ANOVA + the Bonferroni test). Veh 1 = Saline. Each column represents the mean ± S.E.M. (n = 5).
ganglion in rats was described (24). In situ hybridization and immunohistochemistry was used to confirm that mRNA encoding for α2B and α2C appeared to be present in the majority of both large- and small-diameter neurons in the dorsal root ganglion and that α2A is present in the dorsal root ganglion neurons, but that the population expressing this mRNA is extremely small (24). The functional KATP channels were identified in both largeand small-diameter neurons in the dorsal root ganglion (25). The pore-forming Kir6.1 or Kir6.2 subunit determines ATP-sensitivity and unitary conductance, while
the SUR1 or SUR2 subunit confers responsiveness to KATP-channel openers and sulfonylureas (26). In the dorsal root ganglion, it was confirmed by immunohistochemistry that the Kir6.2 subunit, but not the Kir6.1 subunit, is present; and additionally, Kir6.2 and SUR subunits co-localize at the same membrane sites (25). Studies by our group have shown that the peripheral antinociceptive effect of several drugs was antagonized in a dose-dependent manner by KATP-channel blockers glibenclamide and tolbutamide. The peripheral antinociceptive effect of morphine (7), dipyrone (8, 27), and diclofenac (9) were blocked by these sulphonylureas, highlighting the involvement of these channels in peripheral antinociception. Moreover, in these works, the participation of voltage-dependent K+ channels, small-conductance Ca2+-activated K+ channels, and largeconductance Ca2+-activated K+ channels in peripheral antinociception was discarded. In the current work, the participation of K+ channels was evaluated regarding peripheral antinociception induced by xylazine in the rat paw PGE2-induced hyperalgesia test. The results obtained here demonstrated that glibenclamide dose-dependently blocked the peripheral antinociceptive effect of xylazine and revealed the inefficiency of tetraethylammonium, dequalinium, and paxilline in inhibiting the peripheral antinociceptive effect of xylazine. The opening of KATP channels by xylazine could be due to its ability to activate Gi /o proteins since both Gα and Gβγ subunits of these proteins are able to activate KATP channels (28, 29). Therefore, it was hypothesized that activation of the NO – cGMP pathway could induce antinociception through the opening of K+ channels. This hypothesis was confirmed when the antinociception induced by sodium nitroprusside (an NO donor) and dibutyryl-cGMP (a membrane permeable analogue of cGMP) was found to be antagonized by glibenclamide (30, 31) and potentiated by diazoxide (32). Since, our group showed that xylazine acts by the NO – cGMP pathway (14) and knowing that glibenclamide blocks KATP channels in a specific manner, without altering the action of Ca2+-activated or voltage-dependent K+ channels (15, 33, 34), the present data supports the notion that after the activation of the NO /cGMP pathway (14), KATP channels could be the final step of the peripheral antinociceptive mechanism of xylazine. Acknowledgments Research was supported by Conselho Nacional de Pesquisa (CNPq grants 476685 /2004-4) and fellowships from the Conselho Nacional de Pesquisa (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The entire text was revised by a Britishborn scientific text editor.
Xylazine Activating ATP-Sensitive K+ Channels
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J Pharmacol Sci 111, 323 – 327 (2009)4
©2009 The Japanese Pharmacological Society
Full Paper
Involvement of ATP-Sensitive K+ Channels in the Peripheral Antinociceptive Effect Induced by the α2-Adrenoceptor Agonist Xylazine Thiago Roberto Lima Romero1 and Igor Dimitri Gama Duarte1,* 1
Department of Pharmacology, Institute of Biological Sciences (ICB), Federal University of Minas Gerais (UFMG), Av. Antônio Carlos, 6627-campus da Pampulha, CEP 31.270.100, Belo Horizonte, MG, Brazil
Received April 1, 2009; Accepted August 11, 2009
Abstract. Xylazine is an α2-adrenergic agonist extensively used in veterinary medicine and animal experimentation for producing antinociception, sedation, and muscle relaxation. The nitric oxide (NO) / cGMP / ATP-sensitive K+ (KATP) channel pathway has been proposed as the action mechanism of peripheral antinociception of several groups of drugs, including opioids and nonsteroidal analgesics. Considering the lack of knowledge regarding the mechanisms involved in xylazine effects, the present study investigated the contribution of K+ channels on peripheral antinociception induced by xylazine using the rat paw pressure test, in which hyperalgesia was induced by intraplantar injection of prostaglandin E2. Xylazine administered into the right hind paw elicited a local antinociceptive effect, since only much higher doses produced a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine was antagonized by glibenclamide, a specific blocker of KATP channels. In another experiment, tetraethylammonium, a voltage-dependent K+-channel blocker, and paxilline and dequalinium, which are selective blockers for the large- and small-conductance Ca2+-activated K+ channels, respectively, were ineffective at blocking xylazine antinociception. These results provide evidence that the peripheral antinociceptive effect of xylazine probably results from KATP-channel activation, while the voltage-dependent K+ channels, small- and large-conductance Ca2+-activated K+ channels, appear not to be involved in this mechanism. Keywords: xylazine, α2-adrenoceptor, K+ channel, peripheral antinociception different receptors, including α2-adrenoceptor agonists, at supraspinal and spinal sites is also dependent of K+ channels (6). Xylazine is an α2-adrenoceptor agonist (10, 11) that is extensively used in veterinary medical practice and animal experimentation for producing sedation, antinociception, and muscle relaxation (12). Although xylazine has been shown to promote peripheral antinociception by α2C-adrenoceptor activation (13), inducing nitric oxide synthase to produce nitric oxide with posterior increase of cGMP in the peripheral nociceptors (14), its mechanism of action has not yet been clearly elucidated. Thus the aim of the present study was to determine the involvement of K+ channels in peripheral antinociception induced by xylazine. For this purpose, the effects of glibenclamide, a specific KATP-channel blocker (15); tetraethylammonium, a nonselective voltage-dependent K+-channel blocker (16); dequalinium, a selective blocker of small-conductance Ca2+-activated
Introduction Nitric oxide (NO) can activate different types of K+ channels in different types of tissues by increasing cGMP (1 – 4). Potassium currents play an important role in the regulation of neuronal excitability by permitting an efflux of K+ ions through the membrane (injunction current), bringing the potential of membrane values closer to repolarization and further from the threshold that triggers action potential, leading to a decrease in neurotransmitter release (5, 6). Several studies have determined that opening of the ATP-sensitive K+ (KATP) channels is the final stage of the peripheral antinociceptive mechanism of morphine (7), dipyrone (8), and diclofenac (9). The antinociceptive action mechanism induced by various agonists of *Corresponding author. [email protected] Published online in J-STAGE doi: 10.1254 / jphs.09103FP
323
324
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K+ channel (17); and paxilline, a selective blocker of large-conductance Ca2+-activated K+ channels (18) were tested on the peripheral antinociceptive effect of xylazine in the hyperalgesic rat paw model. Materials and Methods Animals All experiments were performed on 160 – 200 g male Wistar rats [from Center for Laboratory Animal Facilities (CEBIO) of the Federal University of Minas Gerais (UFMG)]. The rats were housed in a temperaturecontrolled room (23 ± 1°C) on an automatic 12-h light/ dark cycle (06:00 – 18:00 h). All tests were conducted during the light phase (08:00 – 15:00 h). Food and water were freely available until the onset of the experiments. All animal procedures and protocols were approved by the Ethics Committee on Animal Experimentation (CETEA) of UFMG. Measurement of hyperalgesia Hyperalgesia was induced by subcutaneous injection of prostaglandin E2 (PGE2) (2 µg) into the plantar surface of the hind paw. Hyperalgesia was measured according to the paw pressure test described by Green and Young (19) and Randall and Sellito (20). An analgesimeter was used (Ugo-Basile, Comerio, Italy) with a cone-shaped paw-presser with a rounded tip, which applies a linearly increasing force to the hind paw. The weight in grams (g) required to elicit the nociceptive response of paw flexion was determined as the nociceptive threshold. A cutoff value of 300 g was used to reduce the possibility of damage to the paws. The nociceptive threshold was measured in the right paw and determined as the average of the three consecutive trials recorded before and 3 h after PGE2 injection. The threshold was calculated as the difference between these two averages (Δ of nociceptive threshold) and is expressed in grams. Δ of nociceptive threshold >0 means hyperalgesia by PGE2 injection and decrease of this value means antihyperalgesic effect by the tested drug. Drugs administration All drugs were administered using an injected volume of 50 μl/ paw with the exception of PGE2, where an injected volume of 100 μl/paw was used. Xylazine (Sigma, St. Louis, MO, USA), tetraethylammonium chloride (Sigma), dequalinium chloride (Calbiochem, La Jolla, CA, USA), and paxilline chloride (Sigma) were dissolved in isotonic saline; PGE2 (Sigma) was dissolved in 8% ethanol in saline; and glibenclamide (Sigma) was dissolved in 1% Tween 20 (Vetec, Belo Horizonte,
MG, Brazil). Experimental protocol Xylazine was administered subcutaneously in the right hind paw 2 h and 55 min after local injection of PGE2. In the protocol used to determine whether xylazine was acting outside the injection paw, PGE2 was injected into both hind paws, while xylazine was administered into the right paw, after which the nociceptive threshold was measured in both hind paws. Tetraethylammonium was administered 30 min prior to xylazine. Other K+-channel blockers were administered 5 min prior to xylazine. It should be noted that the protocols concerning dose and time of administration of each drug used in this study were obtained through literature data and pilot experiments. Statistical analysis The data were statistically analyzed by one-way analysis of variance (ANOVA) and the post-hoc Bonferroni test for multiple comparisons. Probabilities of less than 5% (P<0.05) were considered to be statistically significant. Results The administration of xylazine (25, 50, and 100 μg) into the right hind paw produced an antinociceptive response against the PGE2 hyperalgesia (2 μg/paw) in a dose-dependent manner (Fig. 1A), although this was not statistically significant for the dose of 25 μg/paw. When administered into the right paw, xylazine administration at a higher dose did not produce an antinociceptive effect in the left paw, indicating that at this dose, it only presented a peripheral site of action (Fig. 1B); moreover, this dose did not induce any effect in the non-hyperalgesic paws (Fig. 1A). The antinociceptive effect of the highest dose of xylazine used (100 μg/paw) was antagonized in a dosedependent manner by the KATP-channel blocker glibenclamide (20, 40, and 80 μg/paw), although the antagonism was not statistically significant for a dose of 20 μg/paw (Fig. 2). Glibenclamide alone did not alter the control groups (Fig. 2). Another experiment, shown in Fig. 3A, verified that the voltage-dependent K+-channel blocker tetraethylammonium (30 μg) injected into the paw did not significantly reduce peripheral antinociception induced by xylazine at 100 μg/paw in the PGE2-induced hyperalgesia model. Tetraethylammonium alone did not induce hyperalgesia or antinociception (data not shown). Figure 3B shows that neither dequalinium (50 μg /paw), a blocker selective for small-conductance Ca2+-
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Fig. 2. Antagonism induced by intraplantar administration of glibenclamide of peripheral antinociception produced by xylazine in hyperalgesic paws (PGE2, 2 μg). Glibenclamide (Gli, μg/ paw) was administered 5 min prior to xylazine (Xyl, μg/ paw). # and * indicate a significant difference compared to PGE2 + Veh 1 + Veh 2– and PGE2 + Veh 1 + Xyl 100–injected controls, respectively (P<0.05, ANOVA + the Bonferroni test). Veh 1 = 1% Tween 20, Veh 2 = Saline, Veh 3 = 8% ethanol in saline. Each column represents the mean ± S.E.M. (n = 4 – 5).
Discussion
Fig. 1. Effect of xylazine on PGE2-induced hyperalgesia in rats (A) and exclusion of outside paw antinociceptive effect of xylazine (B). A: Xylazine (Xyl, μg/paw) was administered 2 h and 55 min after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in Materials and Methods. # indicates a significant difference from the PGE2 + Veh 1–injected control (P<0.05, ANOVA + the Bonferroni test); Veh 1 = Saline, Veh 2 = Ethanol 8% in saline. B: PGE2 (2 μg) was administered in both hind paws, right (R) and left (L). Xylazine (100 μg/paw) was administered 2 h and 55 min after PGE2 in the right hind paw (Xyl 100, R paw). Antinociceptive responses were measured in both hind paws, as described in Materials and Methods. # indicates a significant difference from the PGE2 R paw + Veh R paw–injected group (P<0.05, ANOVA + the Bonferroni test); Veh = Saline, R paw = Right paw, L paw = Left paw. A and B: Each column represents the mean ± S.E.M. (n = 4 – 5).
activated K+ channels, nor paxilline (20 μg/paw), a blocker selective for large-conductance Ca2+-activated K+ channels, significantly modified the peripheral antinociception induced by xylazine at 100 μg/paw. Dequalinium and paxilline alone did not induce hyperalgesia or any overt behavioural effect (data not shown).
In present experiments, xylazine induced a peripheral dose-dependent antihyperalgesic effect against PGE2 injection. Using the strategy of evaluating the efficacy of ipsi versus contralateral paw, xylazine administration at the dose of 100 µg revealed local hyperalgesic action in the paw, so this dose was chosen for the other experiments. Even though that there are evidences that a direct inhibition of tetrodotoxin-resistant Na+ channels may contribute to the antinociceptive effect of the α2adrenergic agonist clonidine (21), numerous studies have been suggested that KATP-channel opening is one mechanism by which many α2-adrenergic agonists induce antinociceptive effects (6). Ocaña and Baeyens (22) reported that KATP channels were involved in the central antinociceptive mechanism of the α2-adrenergic agonist clonidine in the tail-flick test since glibenclamide antagonized this effect; and in the same work, voltage-dependent K+ channels participation was discarded since 4-aminopyridine and tetraethylammonium were unable to reverse the effect of clonidine. Besides clonidine, the antinociception of several other α2adrenergic agonists, including guanabenz, tizanidine, and dexmedetomidine, were antagonized by the sulphonylureas tolbutamide and glibenclamide, reaffirming the importance of KATP-channel activation regarding the antinociceptive action of α2-adrenergic agonists (23). The presence of α2-adrenoceptors in the dorsal root
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Fig. 3. Effect of intraplantar administration of tetraethylammonium (TEA) (A) or dequalinium (DQ) and paxilline (Pax) (B) on peripheral antinociception produced by xylazine in hyperalgesic paws (PGE2, 2 μg). A: TEA (μg/ paw) was administered 30 min prior to xylazine (μg/ paw). B: DQ (μg/ paw) and Pax (μg/paw) were administered 5 min prior to xylazine (μg/ paw). A and B: # indicates a significant difference from the PGE2 + Veh 1 + Veh 1–injected control (P<0.05, ANOVA + the Bonferroni test). Veh 1 = Saline. Each column represents the mean ± S.E.M. (n = 5).
ganglion in rats was described (24). In situ hybridization and immunohistochemistry was used to confirm that mRNA encoding for α2B and α2C appeared to be present in the majority of both large- and small-diameter neurons in the dorsal root ganglion and that α2A is present in the dorsal root ganglion neurons, but that the population expressing this mRNA is extremely small (24). The functional KATP channels were identified in both largeand small-diameter neurons in the dorsal root ganglion (25). The pore-forming Kir6.1 or Kir6.2 subunit determines ATP-sensitivity and unitary conductance, while
the SUR1 or SUR2 subunit confers responsiveness to KATP-channel openers and sulfonylureas (26). In the dorsal root ganglion, it was confirmed by immunohistochemistry that the Kir6.2 subunit, but not the Kir6.1 subunit, is present; and additionally, Kir6.2 and SUR subunits co-localize at the same membrane sites (25). Studies by our group have shown that the peripheral antinociceptive effect of several drugs was antagonized in a dose-dependent manner by KATP-channel blockers glibenclamide and tolbutamide. The peripheral antinociceptive effect of morphine (7), dipyrone (8, 27), and diclofenac (9) were blocked by these sulphonylureas, highlighting the involvement of these channels in peripheral antinociception. Moreover, in these works, the participation of voltage-dependent K+ channels, small-conductance Ca2+-activated K+ channels, and largeconductance Ca2+-activated K+ channels in peripheral antinociception was discarded. In the current work, the participation of K+ channels was evaluated regarding peripheral antinociception induced by xylazine in the rat paw PGE2-induced hyperalgesia test. The results obtained here demonstrated that glibenclamide dose-dependently blocked the peripheral antinociceptive effect of xylazine and revealed the inefficiency of tetraethylammonium, dequalinium, and paxilline in inhibiting the peripheral antinociceptive effect of xylazine. The opening of KATP channels by xylazine could be due to its ability to activate Gi /o proteins since both Gα and Gβγ subunits of these proteins are able to activate KATP channels (28, 29). Therefore, it was hypothesized that activation of the NO – cGMP pathway could induce antinociception through the opening of K+ channels. This hypothesis was confirmed when the antinociception induced by sodium nitroprusside (an NO donor) and dibutyryl-cGMP (a membrane permeable analogue of cGMP) was found to be antagonized by glibenclamide (30, 31) and potentiated by diazoxide (32). Since, our group showed that xylazine acts by the NO – cGMP pathway (14) and knowing that glibenclamide blocks KATP channels in a specific manner, without altering the action of Ca2+-activated or voltage-dependent K+ channels (15, 33, 34), the present data supports the notion that after the activation of the NO /cGMP pathway (14), KATP channels could be the final step of the peripheral antinociceptive mechanism of xylazine. Acknowledgments Research was supported by Conselho Nacional de Pesquisa (CNPq grants 476685 /2004-4) and fellowships from the Conselho Nacional de Pesquisa (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The entire text was revised by a Britishborn scientific text editor.
Xylazine Activating ATP-Sensitive K+ Channels
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