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Involvement of different types of potassium channels in the antidepressant-like effect of tramadol in the mouse forced swimming test
European Journal of Pharmacology 613 (2009) 74–78
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Involvement of different types of potassium channels in the antidepressant-like effect of tramadol in the mouse forced swimming test Cristiano R. Jesse, Ethel A. Wilhelm, Nilda B.V. Barbosa, Cristina W. Nogueira ⁎ Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil
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Article history: Received 16 February 2009 Received in revised form 13 April 2009 Accepted 20 April 2009 Available online 3 May 2009 Keywords: Tramadol Potassium channels Antidepressant Mice (Mouse) Forced swimming test
a b s t r a c t Administration of tramadol elicited an antidepressant-like effect in the rat forced swimming test (FST) by a mechanism dependent on the inhibition of the L-arginine-nitric oxide (NO)-guanylate cyclase pathway. Since it has been reported that NO can activate different types of potassium (K+) channels in several tissues, the present study investigated the possibility of synergistic interactions between different types of K+ channel inhibitors and tramadol in the mouse FST. Intracerebroventricular pretreatment of mice with tetraethylammonium (TEA, a non-specific inhibitor of K+ channels, 25 pg/site), glibenclamide (an ATP-sensitive K+ channel inhibitor, 0.5 pg/site) or charybdotoxin (a large- and intermediate conductance calcium-activated K+ channel inhibitor, 25 pg/site) was able to produce a synergistic action of a subeffective dose of tramadol (1 mg/ kg, p.o.). Conversely, pretreatment with apamin (a small-conductance calcium-activated K+ channel inhibitor, 10 pg/site) did not modify the action of a subeffective dose of tramadol (1 mg/kg, p.o.). Administration of tramadol and the K+ channel inhibitors, alone or in combination, did not affect the number of crossings and rearings in the open field test (OFT). Reduction in the immobility time elicited by an active dose of tramadol (40 mg/kg, p.o.) in the FST was prevented by pretreatment of mice with cromakalim (a K+ channel opener, 10 µg/site, i.c.v.), without affecting the number of crossings and rearings in the OFT. Thus, our findings clearly suggest that oral acute administration of tramadol produces antidepressant-like effect on the FST in mice by a mechanism that involves the K+ channels. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tramadol is a clinically used, orally active, analgesic drug that is considered to act in the central nervous system (Klotz, 2003). One of the cellular mechanisms for effect of tramadol is the activation of µopioid receptors (Hennies et al., 1988; Raffa et al., 1992). It has been shown that tramadol possesses a non-opioid mechanism that contributes to its pharmacological actions (Rojas-Corrales et al., 2005). In accordance with the recognized implication of noradrenaline and serotonin in pharmacological action, tramadol has been shown to inhibit the re-uptake of noradrenaline and serotonin, thereby increasing the concentration of these two neurotransmitters in selected brain areas, thus raising the pain threshold (Driessen and Reimann, 1992). Conversely, the mechanism of action of tramadol remains unclear, because its binding affinity for opioid receptors appears to be too low to account for the pharmacological effect via this system, and the noradrenergic and serotonergic involvement is still not completely understood (Oliva et al., 2002). However, tramadol is not a single-mechanism analgesic and it may also be a regulator of ion channels (Raffa et al., 1992). For example, tramadol has been reported ⁎ Corresponding author. Tel.: +55 55 32208140; fax: +55 55 32208978. E-mail address: [email protected] (C.W. Nogueira). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.04.041
to inhibit Ca2+-activated Cl− current induced by 5-hydroxytryptamine (Shiraishi et al., 2001; Mert et al., 2003; Ogata et al., 2004). Tramadol has been studied in the forced swimming test (FST) in mice, a test developed to predict the antidepressant action of drugs. Reports have illustrated that tramadol has a putative antidepressant clinical effect in various depressive states (Spencer, 2000), including resistant depression (Shapira et al., 2001), these clinical results suggest that tramadol has a putative antidepressant clinical effect. In addition, the mechanism of action and structure of tramadol is very similar to that of some antidepressants such as venlafaxine. A study (Rojas-Corrales et al., 1998) has shown that, tramadol displays an antidepressant-like effect in mice, associated by the noradrenergic system rather than serotonergic or opioidergic pathway (Tejedor-Real et al., 1995). We have previously demonstrated that oral administration of tramadol produces an antidepressant-like effect in the rat FST by a mechanism that involves the inhibition of L-arginine-nitric oxide (NO) pathway (Jesse et al., 2008). It has been reported that both NO and guanosine cyclic monophosphate (cGMP), produced through the activation of soluble guanylate cyclase (sGC), can activate different types of K+ channels in several tissues (Bolotina et al., 1994; Jeong et al., 2001a,b). Different types of K+ channel inhibitors such as tetraethylammonium (TEA), apamin, charybdotoxin, gliquidone or
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glibenclamide were able to produce an antidepressant-like effect in the mouse FST (Galeotti et al., 1999; Budni et al., 2007; Kaster et al., 2005), whereas the K+ channel openers such as minoxidil or cromakalim increased the immobility time, evidencing the induction of a depressant-like effect (Kobayashi et al., 2004; Kaster et al., 2007). Based on the considerations above, the present study was performed to investigate whether tramadol administered by oral route causes antidepressant-like effect, employing the FST in mice. In addition, the present study attempts to investigate the involvement of different types of K+ channels in the antidepressant-like effect of tramadol in the mouse FST. 2. Materials and methods 2.1. Animals The behavioral experiments were conducted using male adults Swiss mice (25–35 g) maintained at 22–25 °C with free access to water and food, under a 12:12 hour light/dark cycle, with lights on at 6:00 a.m. All manipulations were carried out between 08.00 a.m. and 04.00 p.m. All experiments were performed on separate groups of animals and each animal was used only once in each test. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria, Brazil. All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
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cerebral ventricle, the brains were dissected and examined macroscopically after the test.
2.5. Drugs and treatment Tramadol (1RS,2RS)-2-[(dimethylamine)-methyl]-1-(3-methoxyphe cyclohexanol hydrochloride, was a gift from Cristália (São Paulo, Brazil). The following drugs were used: TEA, (Sigma Chemical Co, USA), charybdotoxin, cromakalim and glibenclamide (Tocris Cookson, Ballwin, MO, USA). Cromakalim was dissolved in saline with 10% Tween 80, whereas all the other drugs were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Appropriate vehicle-treated groups were simultaneously assessed. The drugs were administered by i.c.v. route, in a volume of 5 µl per mouse. To test the hypothesis that the antidepressant-like effect of tramadol is mediated through the inhibition of K+ channels, distinct groups of animals were treated with different classes of drugs. For this purpose animals were pretreated by i.c.v. route with subeffective doses of TEA (a non-specific inhibitor of K+ channels, 25 pg/site),
2.2. Forced swimming test (FST) The test was conducted using the method described by Porsolt et al. (1977). Briefly, mice were individually forced to swim in open cylinders (25 cm height× 10 cm diameter) containing 19 cm of water at 25 ± 1 °C. The duration of immobility was scored during the 6 min test period as described previously (Zomkowski et al., 2006). Each mouse was recorded as immobile when floating motionless or making only those movements necessary to keep its head above water. In order to assess the antidepressant-like effect of tramadol in the FST, this compound was orally administered (dose range: 1–40 mg/ kg, p.o.) 1 h before the FST or OFT. 2.3. Open-field test (OFT) To assess the possible effects of tramadol on the locomotor and exploratory activities, mice were evaluated in the OFT. The open-field was made of polywood and surrounded by walls 30 cm in height. The floor of the open field, 45 cm in length and 45 cm in width, was divided by masking tape markers into 09 squares (3 rows of 3). Each animal was placed individually at the center of the apparatus and observed for 6 min to record the locomotor (number of segments crossed with the four paws) and exploratory activities (expressed by the number of time rearing on the hind limbs) (Savegnago et al., 2008). 2.4. Intracerebroventricular injection technique Intracerebroventricular (i.c.v.) administration was performed under anesthesia. Briefly, a 0.4 mm external diameter hypodermic needle attached to a cannula, which was linked to a 25 µl Hamilton syringe, was inserted perpendicularly through the skull and no more than 2 mm into the brain of the mouse. A volume of 5 µl was then administered. The injection was given over 30 s, and the needle remained in place for another 30 s in order to avoid the reflux of the substances injected. The injection site was 1 mm to the right or left from the mid-point on a line drawn through to the anterior base of the ears. To ascertain that the drugs were administered exactly into the
Fig. 1. Effect of tramadol orally administered in the mouse FST (A) and in the number of crossing (B) and rearing (C) in the OFT in mice. Tramadol (1–40 mg/kg) was orally administered 1 h before the challenge tests. Values are expressed as means ± S.E.M. of 5–7 animals.⁎P b 0.05 and ⁎⁎P b 0.001 when compared to the vehicle treated group.
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site, i.c.v.) (Kaster et al., 2005) 30 min before tramadol (40 mg/kg, an effective dose) administration, and 1 h later the FST or OFT was carried out. 2.6. Statistical analysis All experimental results are given as the mean (s) ± S.E.M. Comparisons between experimental and control groups were performed by one-way (tramadol) or two-way ANOVA (inhibitors of K+ channels and/or a K+ channel opener X tramadol) followed by Newman– Keuls' test for post hoc comparison when appropriate. A value of P b 0.05 was considered to be significant. Main effects are presented only when interaction was not significant. 3. Results 3.1. Effect of tramadol orally administered in the mouse FST and OFT One-way ANOVA revealed a significant effect of tramadol in the FST (F(4,35) = 12.16, P b 0.001) (Fig. 1A). It can be seen that tramadol, given (1 h earlier) by oral route at the doses of 20 and 40 mg/kg, decreased immobility time in the FST. Tramadol given by p.o. route, at all doses tested, did not cause any change in the number of crossings (F(4,35) = 1.09, P b 0.127) (Fig. 1B) and rearings (F (4,35) = 1.00, P b 0.422) (Fig. 1C) in the mouse OFT. 3.2. Effect of K+ channel inhibitor on the antidepressant-like action of tramadol in mice evaluated in the FST The results depicted in Fig. 2A show the effect of TEA (25 pg/site, i.c.v.) in producing a synergistic effect with a subeffective dose of tramadol
Fig. 2. Effect of pretreatment of mice with TEA (25 pg/site, i.c.v.) (A), glibenclamide (0.5 pg/site, i.c.v.) (B) and charybdotoxin (25 pg/site, i.c.v.) (C) in the action of a subeffective dose of tramadol (1 mg/kg, p.o.) in the FST in mice. Values are expressed as means ± S.E.M. (n = 5–7 mice/group). Data were analyzed by Two Way Analysis of Variance (ANOVA) followed by Newman-Keuls test. ⁎⁎P b 0.01 compared to the vehicle group pretreated with TEA (A) and with charybdotoxin (C).⁎⁎⁎P b 0.001 compared to the vehicle group pretreated with glibenclamide.
glibenclamide (an ATP-sensitive K+ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calciumactivated K+ channel inhibitor, 25 pg/site) and apamin (a smallconductance calcium-activated K+ channel inhibitor, 10 pg/site) (Kaster et al., 2005). Fifteen minutes later the animals received a single oral administration of tramadol (1 mg/kg, p.o., a subeffective dose) and a further 1 h was allowed to elapse before the animals were tested in the FST. In order to rule out any psychostimulant effect of the interaction of K+ channel inhibitors and tramadol, mice were pretreated by i.c.v. route with TEA (25 pg/site), glibenclamide (0.5 pg/site), charybdotoxin (25 pg/site) or apamin (10 pg/site) 15 min before the administration of tramadol (1 mg/kg, p.o., a subeffective dose). After 1 h the OFT was carried out. To address the role played by the K+ channels in the antidepressant-like effect caused by tramadol in the FST, distinct groups of animals were treated with cromakalim (a K+ channel opener, 10 µg/
Fig. 3. Effect of pretreatment of mice with apamin (10 pg/site, i.c.v.) (A) in the action of a subeffective dose of tramadol (1 mg/kg, p.o.) and with cromakalim (10 µg/site, i.c.v.) (B) in the action of an effective dose of tramadol (40 mg/kg, p.o.) in the FST in mice. Values are expressed as means ± S.E.M. (n = 5–7 mice/group). Data were analyzed by Two Way Analysis of Variance (ANOVA) followed by Newman-Keuls test. #P b 0.01 compared to the vehicle group pretreated with cromakalim.
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(1 mg/kg, p.o.) in the FST (F(1,28)= 10.68, P b 0.002). No significant effects were observed in the number of crossings (F(1,28) = 0.66, Pb 0.424) and rearings (F(1,28) = 1.14, P b 0.293) in the OFT (data not shown). Fig. 2B shows that glibenclamide (0.5 pg/site, i.c.v.) produced a synergistic action with tramadol (1 mg/kg, p.o.) in the FST (F(1,28) = 11.10, P b 0.002). The number of crossings (F(1,28) = 0.55, P b 0.465) and rearings (F(1,28) = 0.84, P b 0.367) was unmodified by the administration of both drugs (data not shown). The results demonstrated in Fig. 2C show that a subeffective dose of tramadol (1 mg/kg, p.o.) produced a synergistic effect with charybdotoxin (25 pg/site, i.c.v.) (F(1,28) = 10.27, P b 0.003). The administration of tramadol with charybdotoxin did not modify the number of crossings (F(1,28) = 0.48, P b 0.496) and rearings (F(1,28) = 1.43, P b 0.245) in the OFT (data not shown). Fig. 3A demonstrates that apamin (10 pg/site, i.c.v.) was not able to produce a synergistic action with a subeffective dose of tramadol (1 mg/kg, p.o.) in the FST in mice (F(1,28) = 0.08, P b 0.77). The combined administration of tramadol with the K+ channel inhibitor, apamin (10 pg/site, i.c.v.), did not produce any effect on the number of crossings (F(1,28) = 0.05, P b 0.951) and rearings (F(1,28) = 1.03, P b 0.318) in the OFT (data not shown). 3.3. Effects of K+ channel opener on the antidepressant-like effect of tramadol in mice evaluated in the FST Fig. 3B shows the effect of pretreatment with cromakalim (10 µg/ site, i.c.v.) in the antidepressant-like effect of tramadol (40 mg/kg, p.o.) in the mouse FST. Cromakalim was able to reverse the reduction of the immobility time produced by tramadol (40 mg/kg, p.o.) (F(1,28) = 21.34, P b 0.001). No significant differences were observed in the number of crossings (F(1,28) = 0.02, P b 0.931) and rearings (F(1,28) = 0.14, P b 0.708) in the OFT (data not shown). 4. Discussion The present study, for the first time, demonstrates that the inhibition of different types of K+ channels augments the antidepressant-like effect of oral administration of tramadol, a synthetic opioid, in the mouse FST, a widely used animal model of antidepressant-like activity (Cryan et al., 2002). Treatment of mice with pharmacological compounds able to block different types of K+ channels, such as TEA, glibenclamide and charybdotoxin was able to cause a synergistic effect with a subeffective dose of tramadol in the FST. In addition, pretreatment of mice with the K+ channel opener, cromakalim, prevented the decrease in the immobility time (antidepressant-like effect) induced by an effective dose of tramadol in the FST. The FST is the most widely employed screening test for antidepressants in rodents (Cryan et al., 2002; Porsolt et al., 1977). Rodents when forced to swim in a cylinder from which they cannot escape will, after an initial period of vigorous activity, display a characteristic immobile posture which can be readily identified and is said to reflect a state of ‘behavioural despair’. Moreover, it has some drawbacks represented by the possibility of obtaining some false positives or negatives (Borsini and Meli, 1988). Drugs enhancing motor activity may give a “false” positive effect in the forced swimming test. Therefore, this test would not be as a good test for antidepressants such as bupropion, nomifensine, and amineptine, since these agents increase motor activity (Borsini and Meli, 1988). In the present study, active doses of tramadol in the FST had no effect on the locomotor activity of mouse in the OFT. Therefore, it excludes the possibility that the antidepressant-like effects of tramadol in the FST might be due to its effect on the locomotor activity in mice. In order to exclude the possibility that the synergistic effect of tramadol and the K+ channel inhibitors in the FST is a reflection of generalized
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increased locomotor activity, mice were also observed in an OFT for ambulation. However, the ability of the K+ channel inhibitors to augment the behavioral response to tramadol in the FST is not due to a nonspecific locomotor stimulant effect of the drug combination. Therefore, the synergistic antidepressant-like effect of tramadol combined with the K+ channel inhibitors observed in this study could not be attributed to general hyperactivity. Tramadol has an antidepressant-like effect predictive of antidepressant activity in some behavioural models, such as the FST in mice (Rojas-Corrales et al., 1998) and the learned helplessness model of depression in rats (Rojas-Corrales et al., 2002). Reports and cases have illustrated that tramadol has a putative antidepressant clinical effect in various depressive states (Spencer, 2000), including resistant depression (Shapira et al., 1997, 2001), these clinical results suggest that tramadol has a putative antidepressant clinical effect. Moreover, the localization of opioid receptors in large concentrations in cortical and limbic brain regions involved in mood and stress response suggests that opioid agonists could regulate mood and induce antidepressant effects in preclinical and clinical conditions (TejedorReal et al., 1995). In addition, the Rojas-Corrales study has shown that, indeed, tramadol displays an antidepressant-like effect in mice, mediated by the noradrenergic system rather than serotonergic or opioidergic pathways (Rojas-Corrales et al., 1998). Other studies have showed a reduction of tramadol antidepressant-like effect on unpredictable chronic mild stress model by 5-HT or NA neurotransmission reduction in the reserpine model (Rojas-Corrales et al., 2004). Berrocoso and collaborators (2006) demonstrated that 5-HT1A receptors modulate the analgesic and the antidepressant-like effects of tramadol in the hot-plate and forced swimming tests. These authors suggested the involvement of the 5-HT1A autoreceptors from the raphe nuclei and spinal 5-HT1A receptors in this antinociceptive effect. In contrast, the 5-HT1A receptors located in the forebrain may be responsible for the blockade of the antidepressant-like effect of tramadol. In addition, 5-HT1B receptors seem not to modify these effects in the models investigated. In mammalian central neurons, 5-HT1A receptors are usually involved in the 5-HT activation of inwardly rectifying K+ currents (Andrade et al., 1986; Jeong et al., 2001a,b). Furthermore, inwardly rectifying K+ channels activated by agonists or neurotransmitters in central neurons are usually mediated by pertussis toxin sensitive G proteins (Andrade et al., 1986; Jeong et al., 2001a,b). Lee and collaborators (2008) reported that 5-HT hyperpolarized the medial preoptic area neurons by the activation of the G-protein-coupled inwardly rectifying K+ currents via 5-HT1A receptors. The pretreatment with tetraethylammonium, a blocker of voltagegated K+ channels, glibenclamide, an ATP-sensitive K+ channel blocker and charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K+ channels was able to produce a synergistic action of subeffective dose of tramadol. However, pretreatment with apamin, a blocker of small (or low)-conductance calcium-gated K+ channels did not modify the action of subeffective doses of tramadol. Therefore, tramadol seems to exert specific action upon K+ channels responsive to voltage- large conductance calcium-gated and to ATP-sensitive K+ channel. The difference between small, large-conductance calciumgated K+ channels and KATP channels resides in the architecture of channels. The small-conductance calcium- gated K+ channels have a calmodulin-binding domain that allows the channel to interact with calmodulin and to be regulated by Ca2+. The KATP channels are constituted by two subunits and the so-called SUR (Sulfonylurea Receptor) subunit, a 17-transmembrane-domain protein that contains two nucleotide binding folds (NBF) while the large-conductance calcium-gated K+ channels have a region termed the “calcium bowl”, that binds directly to the Ca2+ (Ocaña et al., 2004). This structural difference could explain the different responsiveness of these channels to tramadol. However, molecular studies are necessary to address this hypothesis.
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Several studies suggested that NO donors and exogenous cGMP are also capable to activate K+ channels (Fujino et al., 1991; Kaster et al., 2005). Thus, the inhibition of the L-arginine-NO pathway decreases the levels of NO and cGMP preventing the activation of these channels. Besides that, the antidepressant-like effects of several K+ channel inhibitors in the FST were prevented by pretreatment of mice with Larginine or sildenafil, drugs that enhance the levels of NO and cGMP, respectively (Kaster et al., 2005). We have previously demonstrated that tramadol produces an antidepressant-like effect in the FST by a mechanism that involves the inhibition of the L-arginine-NO pathway (Jesse et al., 2008). Taking into account that the K+ channels represent one of the major downstream targets regulated by the activation of Larginine-NO pathway, we expected that the inhibition of NO production elicited by tramadol might also reflect in an inhibition of these channels. Intracerebroventricular treatment of mice with tetraethylammonium (TEA, a non-specific inhibitor of K+ channels), glibenclamide (an ATP-sensitive K+ channel inhibitor) or charybdotoxin (a large- and intermediate conductance calcium-activated K+ channel inhibitor) was able to potentiate the action of a subeffective dose of tramadol (1 mg/kg, p.o.). Pretreatment of mice with the K+ channel opener, cromakalim, prevented the decrease in the immobility time (antidepressant-like effect) induced by an effective dose of tramadol (40 mg/kg, p.o.) in the FST. This result further reinforces the assumption that the antidepressant-like effect of tramadol involves the inhibition of K+ channels. This is in line with the fact that the administration of K+ channel openers such as minoxidil or cromakalim increases the immobility time in the FST (Galeotti et al., 1999). Besides that, the pretreatment of animals with cromakalim was able to antagonize the anti-immobility effect of several antidepressants such as imipramine, amitriptyline, desipramine and paroxetine (Cryan et al., 2002). The pharmacological modulation of these channels may potentially represent a powerful mean of controlling central nervous system disorders, such as epilepsy, dementia, anxiety, pain, depression, and stroke. The involvement of K+ channels in the modulation of depression has been suggested by a preclinical study (Wickenden, 2002). In conclusion, results indicate that TEA, glibenclamide and charybdotoxin were able to potentiate the action of a subeffective dose of tramadol, that acts via a mechanism dependent on the inhibition of the L-arginine-NO pathway in the FST. Together, the results indicate that the modulatory effects of tramadol on neuronal excitability, via inhibition of K+ channels, may represent the pathway of their antidepressant-like effects in the FST.
Acknowledgements The financial support by UFSM, FAPERGS, CAPES and CNPq is gratefully acknowledged. This study was supported in part by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBNNet)” # 01.06.0842-00.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Involvement of different types of potassium channels in the antidepressant-like effect of tramadol in the mouse forced swimming test Cristiano R. Jesse, Ethel A. Wilhelm, Nilda B.V. Barbosa, Cristina W. Nogueira ⁎ Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil
a r t i c l e
i n f o
Article history: Received 16 February 2009 Received in revised form 13 April 2009 Accepted 20 April 2009 Available online 3 May 2009 Keywords: Tramadol Potassium channels Antidepressant Mice (Mouse) Forced swimming test
a b s t r a c t Administration of tramadol elicited an antidepressant-like effect in the rat forced swimming test (FST) by a mechanism dependent on the inhibition of the L-arginine-nitric oxide (NO)-guanylate cyclase pathway. Since it has been reported that NO can activate different types of potassium (K+) channels in several tissues, the present study investigated the possibility of synergistic interactions between different types of K+ channel inhibitors and tramadol in the mouse FST. Intracerebroventricular pretreatment of mice with tetraethylammonium (TEA, a non-specific inhibitor of K+ channels, 25 pg/site), glibenclamide (an ATP-sensitive K+ channel inhibitor, 0.5 pg/site) or charybdotoxin (a large- and intermediate conductance calcium-activated K+ channel inhibitor, 25 pg/site) was able to produce a synergistic action of a subeffective dose of tramadol (1 mg/ kg, p.o.). Conversely, pretreatment with apamin (a small-conductance calcium-activated K+ channel inhibitor, 10 pg/site) did not modify the action of a subeffective dose of tramadol (1 mg/kg, p.o.). Administration of tramadol and the K+ channel inhibitors, alone or in combination, did not affect the number of crossings and rearings in the open field test (OFT). Reduction in the immobility time elicited by an active dose of tramadol (40 mg/kg, p.o.) in the FST was prevented by pretreatment of mice with cromakalim (a K+ channel opener, 10 µg/site, i.c.v.), without affecting the number of crossings and rearings in the OFT. Thus, our findings clearly suggest that oral acute administration of tramadol produces antidepressant-like effect on the FST in mice by a mechanism that involves the K+ channels. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tramadol is a clinically used, orally active, analgesic drug that is considered to act in the central nervous system (Klotz, 2003). One of the cellular mechanisms for effect of tramadol is the activation of µopioid receptors (Hennies et al., 1988; Raffa et al., 1992). It has been shown that tramadol possesses a non-opioid mechanism that contributes to its pharmacological actions (Rojas-Corrales et al., 2005). In accordance with the recognized implication of noradrenaline and serotonin in pharmacological action, tramadol has been shown to inhibit the re-uptake of noradrenaline and serotonin, thereby increasing the concentration of these two neurotransmitters in selected brain areas, thus raising the pain threshold (Driessen and Reimann, 1992). Conversely, the mechanism of action of tramadol remains unclear, because its binding affinity for opioid receptors appears to be too low to account for the pharmacological effect via this system, and the noradrenergic and serotonergic involvement is still not completely understood (Oliva et al., 2002). However, tramadol is not a single-mechanism analgesic and it may also be a regulator of ion channels (Raffa et al., 1992). For example, tramadol has been reported ⁎ Corresponding author. Tel.: +55 55 32208140; fax: +55 55 32208978. E-mail address: [email protected] (C.W. Nogueira). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.04.041
to inhibit Ca2+-activated Cl− current induced by 5-hydroxytryptamine (Shiraishi et al., 2001; Mert et al., 2003; Ogata et al., 2004). Tramadol has been studied in the forced swimming test (FST) in mice, a test developed to predict the antidepressant action of drugs. Reports have illustrated that tramadol has a putative antidepressant clinical effect in various depressive states (Spencer, 2000), including resistant depression (Shapira et al., 2001), these clinical results suggest that tramadol has a putative antidepressant clinical effect. In addition, the mechanism of action and structure of tramadol is very similar to that of some antidepressants such as venlafaxine. A study (Rojas-Corrales et al., 1998) has shown that, tramadol displays an antidepressant-like effect in mice, associated by the noradrenergic system rather than serotonergic or opioidergic pathway (Tejedor-Real et al., 1995). We have previously demonstrated that oral administration of tramadol produces an antidepressant-like effect in the rat FST by a mechanism that involves the inhibition of L-arginine-nitric oxide (NO) pathway (Jesse et al., 2008). It has been reported that both NO and guanosine cyclic monophosphate (cGMP), produced through the activation of soluble guanylate cyclase (sGC), can activate different types of K+ channels in several tissues (Bolotina et al., 1994; Jeong et al., 2001a,b). Different types of K+ channel inhibitors such as tetraethylammonium (TEA), apamin, charybdotoxin, gliquidone or
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glibenclamide were able to produce an antidepressant-like effect in the mouse FST (Galeotti et al., 1999; Budni et al., 2007; Kaster et al., 2005), whereas the K+ channel openers such as minoxidil or cromakalim increased the immobility time, evidencing the induction of a depressant-like effect (Kobayashi et al., 2004; Kaster et al., 2007). Based on the considerations above, the present study was performed to investigate whether tramadol administered by oral route causes antidepressant-like effect, employing the FST in mice. In addition, the present study attempts to investigate the involvement of different types of K+ channels in the antidepressant-like effect of tramadol in the mouse FST. 2. Materials and methods 2.1. Animals The behavioral experiments were conducted using male adults Swiss mice (25–35 g) maintained at 22–25 °C with free access to water and food, under a 12:12 hour light/dark cycle, with lights on at 6:00 a.m. All manipulations were carried out between 08.00 a.m. and 04.00 p.m. All experiments were performed on separate groups of animals and each animal was used only once in each test. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria, Brazil. All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
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cerebral ventricle, the brains were dissected and examined macroscopically after the test.
2.5. Drugs and treatment Tramadol (1RS,2RS)-2-[(dimethylamine)-methyl]-1-(3-methoxyphe cyclohexanol hydrochloride, was a gift from Cristália (São Paulo, Brazil). The following drugs were used: TEA, (Sigma Chemical Co, USA), charybdotoxin, cromakalim and glibenclamide (Tocris Cookson, Ballwin, MO, USA). Cromakalim was dissolved in saline with 10% Tween 80, whereas all the other drugs were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Appropriate vehicle-treated groups were simultaneously assessed. The drugs were administered by i.c.v. route, in a volume of 5 µl per mouse. To test the hypothesis that the antidepressant-like effect of tramadol is mediated through the inhibition of K+ channels, distinct groups of animals were treated with different classes of drugs. For this purpose animals were pretreated by i.c.v. route with subeffective doses of TEA (a non-specific inhibitor of K+ channels, 25 pg/site),
2.2. Forced swimming test (FST) The test was conducted using the method described by Porsolt et al. (1977). Briefly, mice were individually forced to swim in open cylinders (25 cm height× 10 cm diameter) containing 19 cm of water at 25 ± 1 °C. The duration of immobility was scored during the 6 min test period as described previously (Zomkowski et al., 2006). Each mouse was recorded as immobile when floating motionless or making only those movements necessary to keep its head above water. In order to assess the antidepressant-like effect of tramadol in the FST, this compound was orally administered (dose range: 1–40 mg/ kg, p.o.) 1 h before the FST or OFT. 2.3. Open-field test (OFT) To assess the possible effects of tramadol on the locomotor and exploratory activities, mice were evaluated in the OFT. The open-field was made of polywood and surrounded by walls 30 cm in height. The floor of the open field, 45 cm in length and 45 cm in width, was divided by masking tape markers into 09 squares (3 rows of 3). Each animal was placed individually at the center of the apparatus and observed for 6 min to record the locomotor (number of segments crossed with the four paws) and exploratory activities (expressed by the number of time rearing on the hind limbs) (Savegnago et al., 2008). 2.4. Intracerebroventricular injection technique Intracerebroventricular (i.c.v.) administration was performed under anesthesia. Briefly, a 0.4 mm external diameter hypodermic needle attached to a cannula, which was linked to a 25 µl Hamilton syringe, was inserted perpendicularly through the skull and no more than 2 mm into the brain of the mouse. A volume of 5 µl was then administered. The injection was given over 30 s, and the needle remained in place for another 30 s in order to avoid the reflux of the substances injected. The injection site was 1 mm to the right or left from the mid-point on a line drawn through to the anterior base of the ears. To ascertain that the drugs were administered exactly into the
Fig. 1. Effect of tramadol orally administered in the mouse FST (A) and in the number of crossing (B) and rearing (C) in the OFT in mice. Tramadol (1–40 mg/kg) was orally administered 1 h before the challenge tests. Values are expressed as means ± S.E.M. of 5–7 animals.⁎P b 0.05 and ⁎⁎P b 0.001 when compared to the vehicle treated group.
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site, i.c.v.) (Kaster et al., 2005) 30 min before tramadol (40 mg/kg, an effective dose) administration, and 1 h later the FST or OFT was carried out. 2.6. Statistical analysis All experimental results are given as the mean (s) ± S.E.M. Comparisons between experimental and control groups were performed by one-way (tramadol) or two-way ANOVA (inhibitors of K+ channels and/or a K+ channel opener X tramadol) followed by Newman– Keuls' test for post hoc comparison when appropriate. A value of P b 0.05 was considered to be significant. Main effects are presented only when interaction was not significant. 3. Results 3.1. Effect of tramadol orally administered in the mouse FST and OFT One-way ANOVA revealed a significant effect of tramadol in the FST (F(4,35) = 12.16, P b 0.001) (Fig. 1A). It can be seen that tramadol, given (1 h earlier) by oral route at the doses of 20 and 40 mg/kg, decreased immobility time in the FST. Tramadol given by p.o. route, at all doses tested, did not cause any change in the number of crossings (F(4,35) = 1.09, P b 0.127) (Fig. 1B) and rearings (F (4,35) = 1.00, P b 0.422) (Fig. 1C) in the mouse OFT. 3.2. Effect of K+ channel inhibitor on the antidepressant-like action of tramadol in mice evaluated in the FST The results depicted in Fig. 2A show the effect of TEA (25 pg/site, i.c.v.) in producing a synergistic effect with a subeffective dose of tramadol
Fig. 2. Effect of pretreatment of mice with TEA (25 pg/site, i.c.v.) (A), glibenclamide (0.5 pg/site, i.c.v.) (B) and charybdotoxin (25 pg/site, i.c.v.) (C) in the action of a subeffective dose of tramadol (1 mg/kg, p.o.) in the FST in mice. Values are expressed as means ± S.E.M. (n = 5–7 mice/group). Data were analyzed by Two Way Analysis of Variance (ANOVA) followed by Newman-Keuls test. ⁎⁎P b 0.01 compared to the vehicle group pretreated with TEA (A) and with charybdotoxin (C).⁎⁎⁎P b 0.001 compared to the vehicle group pretreated with glibenclamide.
glibenclamide (an ATP-sensitive K+ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calciumactivated K+ channel inhibitor, 25 pg/site) and apamin (a smallconductance calcium-activated K+ channel inhibitor, 10 pg/site) (Kaster et al., 2005). Fifteen minutes later the animals received a single oral administration of tramadol (1 mg/kg, p.o., a subeffective dose) and a further 1 h was allowed to elapse before the animals were tested in the FST. In order to rule out any psychostimulant effect of the interaction of K+ channel inhibitors and tramadol, mice were pretreated by i.c.v. route with TEA (25 pg/site), glibenclamide (0.5 pg/site), charybdotoxin (25 pg/site) or apamin (10 pg/site) 15 min before the administration of tramadol (1 mg/kg, p.o., a subeffective dose). After 1 h the OFT was carried out. To address the role played by the K+ channels in the antidepressant-like effect caused by tramadol in the FST, distinct groups of animals were treated with cromakalim (a K+ channel opener, 10 µg/
Fig. 3. Effect of pretreatment of mice with apamin (10 pg/site, i.c.v.) (A) in the action of a subeffective dose of tramadol (1 mg/kg, p.o.) and with cromakalim (10 µg/site, i.c.v.) (B) in the action of an effective dose of tramadol (40 mg/kg, p.o.) in the FST in mice. Values are expressed as means ± S.E.M. (n = 5–7 mice/group). Data were analyzed by Two Way Analysis of Variance (ANOVA) followed by Newman-Keuls test. #P b 0.01 compared to the vehicle group pretreated with cromakalim.
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(1 mg/kg, p.o.) in the FST (F(1,28)= 10.68, P b 0.002). No significant effects were observed in the number of crossings (F(1,28) = 0.66, Pb 0.424) and rearings (F(1,28) = 1.14, P b 0.293) in the OFT (data not shown). Fig. 2B shows that glibenclamide (0.5 pg/site, i.c.v.) produced a synergistic action with tramadol (1 mg/kg, p.o.) in the FST (F(1,28) = 11.10, P b 0.002). The number of crossings (F(1,28) = 0.55, P b 0.465) and rearings (F(1,28) = 0.84, P b 0.367) was unmodified by the administration of both drugs (data not shown). The results demonstrated in Fig. 2C show that a subeffective dose of tramadol (1 mg/kg, p.o.) produced a synergistic effect with charybdotoxin (25 pg/site, i.c.v.) (F(1,28) = 10.27, P b 0.003). The administration of tramadol with charybdotoxin did not modify the number of crossings (F(1,28) = 0.48, P b 0.496) and rearings (F(1,28) = 1.43, P b 0.245) in the OFT (data not shown). Fig. 3A demonstrates that apamin (10 pg/site, i.c.v.) was not able to produce a synergistic action with a subeffective dose of tramadol (1 mg/kg, p.o.) in the FST in mice (F(1,28) = 0.08, P b 0.77). The combined administration of tramadol with the K+ channel inhibitor, apamin (10 pg/site, i.c.v.), did not produce any effect on the number of crossings (F(1,28) = 0.05, P b 0.951) and rearings (F(1,28) = 1.03, P b 0.318) in the OFT (data not shown). 3.3. Effects of K+ channel opener on the antidepressant-like effect of tramadol in mice evaluated in the FST Fig. 3B shows the effect of pretreatment with cromakalim (10 µg/ site, i.c.v.) in the antidepressant-like effect of tramadol (40 mg/kg, p.o.) in the mouse FST. Cromakalim was able to reverse the reduction of the immobility time produced by tramadol (40 mg/kg, p.o.) (F(1,28) = 21.34, P b 0.001). No significant differences were observed in the number of crossings (F(1,28) = 0.02, P b 0.931) and rearings (F(1,28) = 0.14, P b 0.708) in the OFT (data not shown). 4. Discussion The present study, for the first time, demonstrates that the inhibition of different types of K+ channels augments the antidepressant-like effect of oral administration of tramadol, a synthetic opioid, in the mouse FST, a widely used animal model of antidepressant-like activity (Cryan et al., 2002). Treatment of mice with pharmacological compounds able to block different types of K+ channels, such as TEA, glibenclamide and charybdotoxin was able to cause a synergistic effect with a subeffective dose of tramadol in the FST. In addition, pretreatment of mice with the K+ channel opener, cromakalim, prevented the decrease in the immobility time (antidepressant-like effect) induced by an effective dose of tramadol in the FST. The FST is the most widely employed screening test for antidepressants in rodents (Cryan et al., 2002; Porsolt et al., 1977). Rodents when forced to swim in a cylinder from which they cannot escape will, after an initial period of vigorous activity, display a characteristic immobile posture which can be readily identified and is said to reflect a state of ‘behavioural despair’. Moreover, it has some drawbacks represented by the possibility of obtaining some false positives or negatives (Borsini and Meli, 1988). Drugs enhancing motor activity may give a “false” positive effect in the forced swimming test. Therefore, this test would not be as a good test for antidepressants such as bupropion, nomifensine, and amineptine, since these agents increase motor activity (Borsini and Meli, 1988). In the present study, active doses of tramadol in the FST had no effect on the locomotor activity of mouse in the OFT. Therefore, it excludes the possibility that the antidepressant-like effects of tramadol in the FST might be due to its effect on the locomotor activity in mice. In order to exclude the possibility that the synergistic effect of tramadol and the K+ channel inhibitors in the FST is a reflection of generalized
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increased locomotor activity, mice were also observed in an OFT for ambulation. However, the ability of the K+ channel inhibitors to augment the behavioral response to tramadol in the FST is not due to a nonspecific locomotor stimulant effect of the drug combination. Therefore, the synergistic antidepressant-like effect of tramadol combined with the K+ channel inhibitors observed in this study could not be attributed to general hyperactivity. Tramadol has an antidepressant-like effect predictive of antidepressant activity in some behavioural models, such as the FST in mice (Rojas-Corrales et al., 1998) and the learned helplessness model of depression in rats (Rojas-Corrales et al., 2002). Reports and cases have illustrated that tramadol has a putative antidepressant clinical effect in various depressive states (Spencer, 2000), including resistant depression (Shapira et al., 1997, 2001), these clinical results suggest that tramadol has a putative antidepressant clinical effect. Moreover, the localization of opioid receptors in large concentrations in cortical and limbic brain regions involved in mood and stress response suggests that opioid agonists could regulate mood and induce antidepressant effects in preclinical and clinical conditions (TejedorReal et al., 1995). In addition, the Rojas-Corrales study has shown that, indeed, tramadol displays an antidepressant-like effect in mice, mediated by the noradrenergic system rather than serotonergic or opioidergic pathways (Rojas-Corrales et al., 1998). Other studies have showed a reduction of tramadol antidepressant-like effect on unpredictable chronic mild stress model by 5-HT or NA neurotransmission reduction in the reserpine model (Rojas-Corrales et al., 2004). Berrocoso and collaborators (2006) demonstrated that 5-HT1A receptors modulate the analgesic and the antidepressant-like effects of tramadol in the hot-plate and forced swimming tests. These authors suggested the involvement of the 5-HT1A autoreceptors from the raphe nuclei and spinal 5-HT1A receptors in this antinociceptive effect. In contrast, the 5-HT1A receptors located in the forebrain may be responsible for the blockade of the antidepressant-like effect of tramadol. In addition, 5-HT1B receptors seem not to modify these effects in the models investigated. In mammalian central neurons, 5-HT1A receptors are usually involved in the 5-HT activation of inwardly rectifying K+ currents (Andrade et al., 1986; Jeong et al., 2001a,b). Furthermore, inwardly rectifying K+ channels activated by agonists or neurotransmitters in central neurons are usually mediated by pertussis toxin sensitive G proteins (Andrade et al., 1986; Jeong et al., 2001a,b). Lee and collaborators (2008) reported that 5-HT hyperpolarized the medial preoptic area neurons by the activation of the G-protein-coupled inwardly rectifying K+ currents via 5-HT1A receptors. The pretreatment with tetraethylammonium, a blocker of voltagegated K+ channels, glibenclamide, an ATP-sensitive K+ channel blocker and charybdotoxin, a blocker of large (or fast)-conductance calcium-gated K+ channels was able to produce a synergistic action of subeffective dose of tramadol. However, pretreatment with apamin, a blocker of small (or low)-conductance calcium-gated K+ channels did not modify the action of subeffective doses of tramadol. Therefore, tramadol seems to exert specific action upon K+ channels responsive to voltage- large conductance calcium-gated and to ATP-sensitive K+ channel. The difference between small, large-conductance calciumgated K+ channels and KATP channels resides in the architecture of channels. The small-conductance calcium- gated K+ channels have a calmodulin-binding domain that allows the channel to interact with calmodulin and to be regulated by Ca2+. The KATP channels are constituted by two subunits and the so-called SUR (Sulfonylurea Receptor) subunit, a 17-transmembrane-domain protein that contains two nucleotide binding folds (NBF) while the large-conductance calcium-gated K+ channels have a region termed the “calcium bowl”, that binds directly to the Ca2+ (Ocaña et al., 2004). This structural difference could explain the different responsiveness of these channels to tramadol. However, molecular studies are necessary to address this hypothesis.
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Several studies suggested that NO donors and exogenous cGMP are also capable to activate K+ channels (Fujino et al., 1991; Kaster et al., 2005). Thus, the inhibition of the L-arginine-NO pathway decreases the levels of NO and cGMP preventing the activation of these channels. Besides that, the antidepressant-like effects of several K+ channel inhibitors in the FST were prevented by pretreatment of mice with Larginine or sildenafil, drugs that enhance the levels of NO and cGMP, respectively (Kaster et al., 2005). We have previously demonstrated that tramadol produces an antidepressant-like effect in the FST by a mechanism that involves the inhibition of the L-arginine-NO pathway (Jesse et al., 2008). Taking into account that the K+ channels represent one of the major downstream targets regulated by the activation of Larginine-NO pathway, we expected that the inhibition of NO production elicited by tramadol might also reflect in an inhibition of these channels. Intracerebroventricular treatment of mice with tetraethylammonium (TEA, a non-specific inhibitor of K+ channels), glibenclamide (an ATP-sensitive K+ channel inhibitor) or charybdotoxin (a large- and intermediate conductance calcium-activated K+ channel inhibitor) was able to potentiate the action of a subeffective dose of tramadol (1 mg/kg, p.o.). Pretreatment of mice with the K+ channel opener, cromakalim, prevented the decrease in the immobility time (antidepressant-like effect) induced by an effective dose of tramadol (40 mg/kg, p.o.) in the FST. This result further reinforces the assumption that the antidepressant-like effect of tramadol involves the inhibition of K+ channels. This is in line with the fact that the administration of K+ channel openers such as minoxidil or cromakalim increases the immobility time in the FST (Galeotti et al., 1999). Besides that, the pretreatment of animals with cromakalim was able to antagonize the anti-immobility effect of several antidepressants such as imipramine, amitriptyline, desipramine and paroxetine (Cryan et al., 2002). The pharmacological modulation of these channels may potentially represent a powerful mean of controlling central nervous system disorders, such as epilepsy, dementia, anxiety, pain, depression, and stroke. The involvement of K+ channels in the modulation of depression has been suggested by a preclinical study (Wickenden, 2002). In conclusion, results indicate that TEA, glibenclamide and charybdotoxin were able to potentiate the action of a subeffective dose of tramadol, that acts via a mechanism dependent on the inhibition of the L-arginine-NO pathway in the FST. Together, the results indicate that the modulatory effects of tramadol on neuronal excitability, via inhibition of K+ channels, may represent the pathway of their antidepressant-like effects in the FST.
Acknowledgements The financial support by UFSM, FAPERGS, CAPES and CNPq is gratefully acknowledged. This study was supported in part by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBNNet)” # 01.06.0842-00.
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