Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K+ channels pathway in the peripheral antinociception of melatonin

European Journal of Pharmacology 596 (2008) 70–76

Contents lists available at ScienceDirect

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

Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K+ channels pathway in the peripheral antinociception of melatonin Alfonso Hernández-Pacheco a, Claudia Ivonne Araiza-Saldaña b, Vinicio Granados-Soto b, Teresa Mixcoatl-Zecuatl c,⁎ a b c

Facultad de Ciencias, Universidad Nacional Autónoma de México, México, D.F., Mexico Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico Centro de Investigación y de Estudios Avanzados, Unidad Monterrey, Apodaca, Nuevo León, Mexico

a r t i c l e

i n f o

Article history: Received 25 February 2008 Received in revised form 2 July 2008 Accepted 31 July 2008 Available online 13 August 2008 Keywords: Melatonin Nitric oxide Guanylyl cyclase Protein kinase G K+ channels Formalin test Peripheral antinociception

a b s t r a c t The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG)-K+ channel pathway on melatonin-induced local antinociception was assessed during the second phase of the formalin test. The local peripheral ipsilateral, but not contralateral, administration of melatonin (150—600 μg/paw) produced a dose-related antinociception during both phases of the formalin test in rats. Moreover, local pretreatment with NG-L-nitro-arginine methyl ester (L-NAME, NO synthesis inhibitor, 10–100 μg/paw), 1H-(1,2,4)-oxadiazolo (4,2-a)quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor, 5–50 μg/paw), (9S, 10R, 12R)-2,3,9,10,11,12hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo [1,2,3-fg:3′,2′,1′-kl]pyrrolo [3,4-i][1,6] benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor, 50–500 ng/paw), glibenclamide (ATP-sensitive K+ channel blocker, 5–50 μg/paw), apamin (small-conductance Ca2+activated K+ channel blocker, 0.1–1 μg/paw) or charybdotoxin (large- and intermediate-conductance Ca2+activated K+ channel blocker, 0.03–0.3 μg/paw), but not NG-D-nitro-arginine methyl ester (D-NAME, inactive isomer of L-NAME, 100 μg/paw) or vehicle, significantly prevented melatonin (300 μg/paw)-induced antinociception. Data suggest that melatonin-induced local peripheral antinociception during the second phase of the test could be due to activation of the NO-cyclic GMP-PKG-ATP-sensitive and Ca2+-activated K+ channels pathway. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Melatonin, N-acetyl-5-methoxytryptamine, is an important neurohormone synthesized primarily in the pineal gland and released into the bloodstream (Vanecek, 1998). This hormone is involved in many physiological processes in mammals such as chronobiotic, anxiolytic, antioxidant, anticonvulsant, and antinociceptive activities (Golombek et al., 1996; Vanecek, 1998; Tomás-Zapico and Coto-Montes, 2005). In addition, melatonin has been widely studied in pain models in animals, as there is evidence that systemic (Golombek et al., 1991; Raghavendra et al., 2000; Yu et al., 2000) or central (Yu et al., 2000; Noseda et al., 2004; Onal et al., 2004; Tu et al., 2004) administration of the hormone produces dose-dependent antinociception in acute (Lakin et al., 1981; Golombek et al., 1991; Yu et al., 2000; El-Shenawy et al., 2002), inflammatory (Cuzzocrea et al., 1997; Raghavendra et al., 2000; Pang et al., 2001; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004;

⁎ Corresponding author. Cinvestav, Unidad Monterrey, Vía del Conocimiento 201, Parque de Investigación e Innovación Tecnológica, Autopista Monterrey-Aeropuerto Km 9.5, 66600 Apodaca, Nuevo León, Mexico. Tel.: +52 81 1156 1740x4512; fax: +52 81 1156 1741. E-mail address: [email protected] (T. Mixcoatl-Zecuatl). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.07.068

Wang et al., 2006), and neuropathic (Ulugol et al., 2006; Ambriz-Tututi and Granados-Soto, 2007) pain. The exact mechanism of action of melatonin to reduce nociception is unclear. Besides activation of melatonin MT2 receptors, melatonin reduces excitatory neurotransmission by several mechanisms such as activation of opioid receptors (Wang et al., 2006; Arreola-Espino et al., 2007) and inhibition of voltage-activated calcium currents (Ayar et al., 2001). Moreover, other studies have shown that melatonin increases a delayed rectifier K+ current in cerebellar granule cells (Huan et al., 2001) and it generates an outward potassium current in the suprachiasmatic nucleus neurons (Van den Top et al., 2001). Since there is evidence that opening of K+ channels is able to produce peripheral antinociception, we hypothesized that local peripheral melatonin could open K+ channels in order to produce its antinociceptive effect. If this were the case, we also hypothesized that melatonin could activate K+ channels as a result of the nitric oxide (NO)-cyclic GMP-cyclic GMP dependent protein kinase (PKG) pathway activation, as previously shown (Soares et al., 2000; Soares and Duarte, 2001; Sachs et al., 2004; Ambriz-Tututi et al., 2005; Ortiz et al., 2006). Therefore, the purpose of the present work was to determine the role of the NO-cyclic GMP-PKG pathway as well as the activation of K+ channels on the local peripheral antinociception induced by melatonin in the 1% rat formalin test.

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

2. Material and methods 2.1. Animals Experiments were performed on adult female Wistar rats (body weight range, 180–220 g) of 10–12 weeks of age. The animals were obtained from our own breeding facilities and had free access to food and drinking water. Experiments were done in normal light/dark cycle and they were started at the same time (10:00 AM). Efforts were made to minimize animal suffering and to reduce the number of animals used. All experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983) and were approved by our local Ethics Committee. 2.2. Measurement of nociceptive activity Nociception was assessed using the formalin test (Dubuisson and Dennis, 1977; Wheeler-Aceto and Cowan, 1991). Rats were placed in open Plexiglas observation chambers for 30 min to allow them to acclimate to their surroundings; then they were removed for formalin administration. Rats were gently restrained while the dorsum of the hind paw was injected with 50 μl of diluted formalin (1%) into the dorsal surface of the right hind paw with a 30-gauge needle. The animals were returned to the chambers and nociceptive behavior was observed immediately after formalin injection. Mirrors were placed in each chamber to enable unhindered observation. Nociceptive behavior was quantified as the number of flinches of the injected paw during 1-min periods every 5 min, up to 60 min after injection (Wheeler-Aceto and Cowan, 1991). Flinching was readily discriminated and was characterized as rapid and brief withdrawal, or as flexing of the injected paw. We decided to evaluate flinching because it is a simple and reliable parameter of pain behavior and one producing high scores (Wheeler-Aceto and Cowan, 1991). Formalininduced flinching behavior was biphasic. The initial acute phase (phase 1, 0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (phase 2, 15–60 min). At the end of the experiment the rats were sacrificed in a CO2 chamber. 2.3. Drugs Melatonin, NG-L-nitro-arginine methyl ester (L-NAME), NG-D-nitroarginine methyl ester (D-NAME), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ), (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo [1,2,3-fg:3′,2′,1′-kl] pyrrolo [3,4-i] [1,6] benzodiazocine-10-carboxylic acid methyl ester (KT-5823), glibenclamide, charybdotoxin and apamin were obtained from Sigma (St. Louis, MO, USA). L-NAME, D-NAME, charybdotoxin and apamin were dissolved in saline. Melatonin and KT-5823 were dissolved in 20% dimethylsulfoxide (DMSO). ODQ and glibenclamide were dissolved in 50% DMSO. 2.4. Study design Rats were randomized to receive a subcutaneous injection (50 μl/paw) into the dorsal surface of the right hind paw of vehicle (20% DMSO) or increasing doses of melatonin (150–600 μg) 30 min before formalin injection into the same paw (ipsilateral). To determine if melatonin (600 μg/paw) acted locally, it was administered to the left (contralateral) paw 30 min before formalin was injected into the right paw, and the corresponding effect on nociceptive behavior was assessed. In these groups, animals received 2 injections, one of vehicle or melatonin and one of formalin. We decided to use melatonin 300 μg/paw in the next experiments based on the following facts. 1) Melatonin 600 μg/paw was difficult to dissolve; 2) melatonin was active during phase 1 only at the

71

highest dose used (600 μg/paw); 3) both melatonin doses (300 and 600 μg/paw) had the same magnitude of effect during phase 2; and 4) we were interested in melatonin's activity during the inflammatory phase of the test. In order to determine whether melatonin (300 μg/paw)-induced peripheral antinociception was mediated by the NO-cyclic GMP-PKG-K+ channel pathway activation, effect of treatment (40 min before) with the appropriate vehicle or L-NAME (10 and 100 μg/paw), D-NAME (100 μg/paw), ODQ (5 and 50 μg/paw), KT-5823 (50 and 500 ng/paw), glibenclamide (5 and 50 μg/paw), apamin (0.1 and 1 μg/paw) or charybdotoxin (0.03 and 0.3 μg/paw) on the antinociceptive effect induced by local peripheral melatonin (−30 min) was assessed. Control groups received 20% DMSO + saline for L-NAME/D-NAME, apamin and charybdotoxin; 20% DMSO + 20% DMSO for KT5823; 20% DMSO + 50% DMSO for ODQ and glibenclamide. Treatment groups received melatonin + 20% DMSO for KT5823; melatonin + 50% DMSO for ODQ and glibenclamide; melatonin + saline for L-NAME/D-NAME, apamin and charybdotoxin. In these groups, rats received 3 injections, one of melatonin, one of vehicle (saline and 20 or 50% DMSO) or inhibitor or blocker and one of formalin. Again, animals were randomized to receive one the above treatments. Drugs were injected in a volume of 50 μl. Appropriate controls for multiple injections and vehicles were performed before starting the formal study. Doses and drug administration schedule of NO synthase, guanylyl cyclase and PKG inhibitors, K+ channel blockers were selected based on previous reports (Ambriz-Tututi et al., 2005; Ortiz et al., 2006) and on pilot experiments in our laboratory. The observer was unaware of the treatment each animal received. 2.5. Data analysis and statistics All results are presented as mean ± S.E.M. for at least 6 animals per group. Curves were constructed by plotting the mean number of flinches as a function of time. The area under the number of flinches against time curves (AUC) for phase 1 and 2, an expression of the duration and intensity of the effect, were calculated by the trapezoidal rule. Analysis of variance (ANOVA), followed by Tukey's test was used to compare differences between treatments. Differences were considered to reach statistical significance when P b 0.05. 3. Results 3.1. Antinociceptive effect of melatonin after local peripheral administration Formalin 1% injection produced a typical pattern of flinching behavior. The first phase started immediately after administration of formalin and then diminished gradually in about 10 min. The second phase started at 15 min and lasted until 1 h. Local peripheral administration of melatonin (Figs.1 and 2), but not vehicle, dose-dependently reduced formalin-induced nociceptive behavior during phase 2. In addition, only the highest dose of melatonin partially reduced the nociceptive behavior during phase 1. In contrast, contralateral administration of melatonin (600 μg/paw) was not able to inhibit flinching behavior (Figs. 1 and 2). 3.2. Effect of L-NAME, D-NAME, ODQ and KT-5823 on the local peripheral antinociceptive activity of melatonin Local peripheral pretreatment with the non-selective NO synthesis inhibitor L-NAME (100 μg/paw), inactive isomer of L-NAME, D-NAME (100 μg/paw), soluble guanylyl cyclase inhibitor ODQ (50 μg/paw) and PKG inhibitor KT-5823 (500 ng/paw) did not produce any effect by themselves on formalin-induced flinching behavior. However, L-NAME (10–100 μg/paw, Fig. 3), ODQ (5–50 μg/paw, Fig. 4) or KT-5823 (50– 500 ng/paw, Fig. 5), but not D-NAME (100 μg/paw, Fig. 3), significantly

72

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

Fig. 1. Time course of the local peripheral antinociceptive effect of melatonin in the formalin test. Data are the mean ± S.E.M. of six animals.

(P b 0.05) reversed the local peripheral antinociception induced by melatonin (300 μg/paw) during phase 2. No changes in nociceptive behavior were observed during the phase 1 (data not shown).

Fig. 3. Effect of NG-L-nitro-arginine methyl ester (L-NAME) and D-NAME (inactive isomer of L-NAME) on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with a local injection of L-NAME or D-NAME (− 40 min) and then melatonin (− 30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from the melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

3.3. Effect of glibenclamide, apamin and charybdotoxin on the local peripheral antinociceptive activity of melatonin Local peripheral pretreatment with the ATP-sensitive K+ channel blocker glibenclamide (5-50 μg/paw, Fig. 6), small-conductance Ca2+activated K+ channel blocker apamin (0.1–1 μg/paw, Fig. 7) and largeconductance Ca 2+-activated K + channel blocker charybdotoxin (0.03–0.3 μg/paw, Fig. 8) significantly (P b 0.05) blocked the antinociception produced by local peripheral melatonin (300 μg/paw) during phase 2. No changes in nociceptive behavior were observed during the phase 1 (data not shown). Given alone, local peripheral administration of K+ channel blockers did not modify formalin-induced nociceptive behavior.

Fig. 2. Local peripheral antinociceptive effect of melatonin on the 1% formalin test. Rats were pretreated with a local injection of vehicle (Veh) or melatonin into the right (ipsilateral, IL) or left paw (contralateral, CL) 30 min before formalin injection. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

Fig. 4. Effect of 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ) on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with a local injection of ODQ (−40 min) and then melatonin (− 30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from the melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

73

4. Discussion 4.1. Local peripheral antinociceptive effect of melatonin In our study, the local peripheral ipsilateral, but not contralateral, administration of melatonin dose-dependently reduced formalininduced nociceptive behavior during phase 2. In addition, melatonin, at the highest dose, reduced flinching behavior during phase 1. These data are similar to that reported by Ray et al. (2004) after systemic administration of melatonin in mice. It is interesting to note that melatonin-induced antinociception was ∼ 50% in the phase 2. The efficacy of this drug is similar to that produced by sildenafil (a phosphodiesterase 5 inhibitor, Mixcoatl-Zecuatl et al., 2000) and gabapentin (an anticonvulsant, Ortiz et al., 2006), and considerably lower than that produced by morphine (Mixcoatl-Zecuatl et al., 2000). The difference in efficacy could be due to the different mechanisms activated by melatonin. However, whatever the case, it seems that melatonin plays a role as modulator of the nociceptive behavior on the formalin test. It has been suggested that phase 1 of the formalin test is associated with direct activation of primary afferent fibers as formalin injection results in an immediate and intense increase in the spontaneous activity of C fibers (Heapy et al., 1987), which results from activation of TRPA1 channels (McNamara et al., 2007). In the case of the second phase, it is associated with peripheral as well as central sensitization (Woolf, 1983; Hunskaar and Hole, 1987; Dickenson and Sullivan, 1987; Coderre et al., 1990; Tjølsen et al., 1992; Puig and Sorkin, 1996). In this sense, our results suggest that local peripheral melatonin inhibits the mechanisms of nociception related to the peripheral inflammatory response (peripheral sensitization), but not the central mechanisms. This could be a reason by which only a partial reduction of nociceptive behavior is observed in the formalin test. To our knowledge, this is the first report about the antinociceptive effect of peripheral melatonin in the formalin test (an inflammatory pain model). Our results agree with those of Mantovani et al. (2006) showing that antinociceptive activity of local peripheral melatonin on glutamate-induced nociceptive behaviors. Therefore, these data support a significant participation of peripheral mechanisms on the antinociceptive effect of melatonin in glutamate and formalin tests (our results). Moreover, our results agree with previous reports showing that melatonin reduces nociception in several inflammatory pain models (Cuzzocrea

Fig. 5. Effect of KT-5823 on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with a local injection of KT-5823 (−40 min) and then melatonin (−30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from the melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

Fig. 6. Effect of glibenclamide on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with glibenclamide (−40 min) and then a local injection of melatonin (−30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

et al., 1997; Raghavendra et al., 2000; Pang et al., 2001; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004; Arreola-Espino et al., 2007). 4.2. Possible mechanisms of action of melatonin The antinociceptive effect of melatonin has been attributed to activation of melatonin MT2 and opioid receptors (Noseda et al., 2004; Shavali et al., 2005; Mantovani et al., 2006; Arreola-Espino et al., 2007). There is controversy concerning the effect of opioid receptor antagonist on melatonin-induced antinociception in the formalin test. On one hand, naltrexone significantly reduced melatonin-induced antinociceptive activity in diabetic rats. On the other hand, naloxone was not able to reduce the antinociceptive effect of melatonin in mice (Ray et al., 2004). Differences could be due to the different species used in each case. However, there is evidence from other models that

Fig. 7. Effect of apamin on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with apamin (−40 min) and then a local injection of melatonin (− 30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

74

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

Fig. 8. Effect of charybdotoxin on the local peripheral antinociception produced by melatonin during the second phase of the formalin test. Rats were pretreated with charybdotoxin (− 40 min) and then a local injection of melatonin (−30 min) into the right paw. Data are expressed as the area under the number of flinches against time curve (AUC). Bars are the mean ± S.E.M. of six animals. ⁎Significantly different from the vehicle (Veh) group (P b 0.05) and #significantly different from melatonin group (P b 0.05), as determined by analysis of variance followed by Tukey's test.

either naloxone or naltrexone indeed block melatonin-induced antinociception (Lakin et al., 1981; Mantovani et al., 2006; AmbrizTututi and Granados-Soto, 2007). Thus, it seems that opioid receptors participate in the antinociceptive effect of systemic melatonin. Peripheral mechanisms of action of melatonin are unknown. Previous data have shown that melatonin increases K+ currents (Huan et al., 2001; Van den Top et al., 2001). Since there is evidence that opening of K+ channels is able to produce peripheral antinociception, we hypothesized that local peripheral melatonin could open K+ channels in order to produce its antinociceptive effect. If this were the case, it was also hypothesized that melatonin could activate K+ channels as a result of the NO-cyclic GMP-PKG pathway activation (Bolotina et al., 1994; Archer et al., 1994; Han et al., 2002). Accordingly, the local peripheral antinociceptive effect of melatonin during phase 2 was blocked by the NO synthesis inhibitor L-NAME (Gibson et al., 1990) and the soluble guanylyl cyclase inhibitor ODQ (Moro et al., 1996), but not by the inactive isomer of L-NAME, D-NAME. These results suggest that the NO-cyclic GMP pathway is involved in the local peripheral melatonin-induced antinociceptive activity. Our data agree with evidence showing that the anticonvulsive effect produced by systemic melatonin is blocked by L-NAME and 7-nitroindazole (preferential neuronal NO synthase inhibitor). Our results are also in line with studies showing that local peripheral NO and cyclic GMP are important for the antinociceptive action of several drugs (Granados-Soto et al., 1997; Lorenzetti and Ferreira, 1996; Sachs et al., 2004; Brito et al., 2006). The participation of the NO-cyclic GMP pathway in melatonin's local peripheral antinociception is consistent with the fact that local administration of NO donors or membrane permeable analogues of cyclic GMP produce antinociception (Duarte et al., 1992; Cunha et al., 1999; Soares et al., 2000; Soares and Duarte, 2001). In support of this, an analogue of cyclic GMP produces inhibition of spontaneous activity and mechanical responses of nociceptive afferents (Levy and Strassman, 2004; Liu et al., 2004). Other researchers have found that NO can either decrease or increase mechanical responsiveness of nociceptors. This action might depend on the baseline level of neuronal excitability (Levy and Strassman, 2004) and may explain the results showing that low doses of NO or cyclic GMP are associated with antinociception, whereas that medium or high doses produce nociception (Prado et al., 2002; Tegeder et al., 2002). The mechanism by which cyclic GMP modulates the neuronal function has not been entirely described. In fact, it has been suggested in three families of cyclic GMP targets: cyclic nucleotide-gated ion

channels, cyclic GMP-regulated phosphodiesterases and PKG (Wang and Robinson, 1997). Activation of PKG would lead to phosphorylation and regulation of ion channels, protein phosphatase and cytoskeleton proteins (Wang and Robinson, 1997). Local peripheral treatment with the inhibitor of PKG KT-5823 (Jin et al., 1993) was able to reduce melatonin-induced antinociception suggesting that PKG activation as well as phosphorylation of K+ channels could be an important step in the peripheral effect produced by melatonin. Our results are in agreement with previous observations in rat models of inflammatory pain (Sachs et al., 2004; Brito et al., 2006; Ortiz et al., 2006) showing that the local peripheral administration of KT-5823 was able to decrease the peripheral antinociceptive actions of several drugs. Thus, data support the participation of the NO-cyclic GMP-PKG pathway in the peripheral antinociception produced by melatonin. When applied alone, the NO-cyclic GMP-PKG pathway inhibitors did not affect formalin-induced nociceptive behavior during phase 2. The lack of effect of these inhibitors is consistent with results of studies in which these drugs were not able to modify the nociceptive activity of thermal noxious stimulus, formalin-induced nociception and mechanical hyperalgesia (Sachs et al., 2004; Brito et al., 2006; Ortiz et al., 2006) thus excluding the possibility that the inhibition of melatonin antinociception could be due to a hyperalgesic or nociceptive effect of the inhibitors used. The results reported here suggest that the modulation of K+ channels may represent an important step in the peripheral mechanism of antinociceptive action of melatonin. Local peripheral administration of glibenclamide (ATP-sensitive K+ channel blocker; Edwards and Weston, 1993) significantly reduced the antinociceptive action of melatonin, suggesting that melatonin activates these channels on peripheral sites. In line with this evidence, it has been reported that melatonin may generate an outward potassium current in rat suprachiasmatic nucleus neurons (Van den Top et al., 2001). Data agree with several reports showing that local peripheral administration of glibenclamide or tolbutamide (ATP-sensitive K+ channel blocker) significantly diminished the antinociceptive effect of morphine as well as ATP-sensitive K+ channel openers (pinacidil and diazoxide) and others antinociceptive drugs (Rodrigues and Duarte, 2000; Ortiz et al., 2003; Sachs et al., 2004; Brito et al., 2006; Ortiz et al., 2006). Considering this, it is likely that local peripheral administration of melatonin could have a synergistic interaction with ATP-sensitive as well as other K+ channel openers. Furthermore, it was found that melatonin-induced antinociception was prevented by apamin and charybdotoxin (small- and large-conductance Ca2+-activated K+ channel blockers, respectively; Romey et al., 1984; Stretton et al., 1992), suggesting that melatonin could activate the small- and largeconductance Ca2+-activated K+ channel in order to produce their peripheral antinociceptive effect during the second phase of the formalin test. The fact that local peripheral resveratrol, an antioxidant like melatonin, behaves as Ca2+-activated K+ channel opener on the formalin test (Granados-Soto et al., 2002) is in line with our suggestion. Contrary to our results, the antinociceptive effect of morphine was unaffected by apamin and charybdotoxin (Rodrigues and Duarte, 2000), suggesting that activation of K+ channels by melatonin is different to that produced by opioids. However, with the present results we cannot discard the possible interaction between local peripheral melatonin and the opioid system. In summary, local melatonin reduced formalin-induced nociceptive behavior, effect being more marked on the second phase. The antinociceptive effect of melatonin during the second phase of the test was blocked by L-NAME, ODQ or KT-5823, but not by D-NAME. Moreover, melatonin-induced antinociception was blocked by glibenclamide, apamin and charybdotoxin. These results suggest that peripheral local melatonin activates the NO-cyclic GMP-PKG-ATPsensitive and small- and large-conductance Ca2+-activated K+ channels pathway in order to produce its local peripheral antinociceptive effect during the second phase of the formalin test.

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

Acknowledgments This work was done at the Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico. Authors greatly appreciate the technical and bibliographic assistance of M.Sc. Guadalupe C. Vidal-Cantú and B.Sc. Héctor Vázquez, respectively. Teresa Mixcoatl-Zecuatl is a post-doctoral fellow from Cinvestav, Unidad Monterrey. This work is part of the B.Sc. dissertation of Alfonso Hernández-Pacheco. References Ambriz-Tututi, M., Granados-Soto, V., 2007. Oral and spinal melatonin reduces tactile allodynia in rats via activation of MT2 and opioid receptors. Pain 132, 273–280. Ambriz-Tututi, M., Velázquez-Zamora, D.A., Urquiza-Marín, H., Granados-Soto, V., 2005. Analysis of the mechanism underlying the peripheral antinociceptive action of sildenafil in the formalin test. Eur. J. Pharmacol. 512, 121–127. Archer, S.L., Huang, J.M., Hampl, V., Nelson, D.P., Shultz, P.J., Weir, E.K., 1994. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K+ channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 91, 7583–7587. Arreola-Espino, R., Urquiza-Marín, H., Ambriz-Tututi, M., Araiza-Saldaña, C.I., CaramSalas, N.L., Rocha-González, H.I., Mixcoatl-Zecuatl, T., Granados-Soto, V., 2007. Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats. Eur. J. Pharmacol. 577, 203–210. Ayar, A., Martin, D.J., Ozcan, M., Kelestimur, H., 2001. Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones. Neurosci. Lett. 313, 73–77. Bilici, D., Akpinar, E., Kiziltunc, A., 2002. Protective effect of melatonin in carrageenaninduced acute local inflammation. Pharmacol. Res. 46, 133–139. Bolotina, V.M., Najibi, S., Palacino, J.J., Pagano, P.J., Cohen, R.A., 1994. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368, 850–853. Brito, G.A.C., Sachs, D., Cunha, F.Q., Vale, M.L., Lotufo, C.M.C., Ferreira, S.H., Ribeiro, R.A., 2006. Peripheral antinociceptive effect of pertussis toxin: activation of the arginine⁄NO⁄cGMP⁄PKG⁄ATP-sensitive K+ channel pathway. Eur. J. Neurosci. 24, 1175–1181. Coderre, T.J., Vaccarino, A.L., Melzack, R., 1990. Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res. 535, 155–158. Cunha, F.Q., Teixeira, M.M., Ferreira, S.H., 1999. Pharmacological modulation of secondary mediator systems-cyclic AMP and cyclic GMP-on inflammatory hyperalgesia. Br. J. Pharmacol. 127, 671–678. Cuzzocrea, S., Zingarelli, B., Gilad, E., Hake, P., Salzman, A.L., Szabo, C., 1997. Protective effect of melatonin in carrageenan-induced models of local inflammation: relationship to its inhibitory effect on nitric oxide production and its peroxynitrite scavenging activity. J. Pineal Res. 23, 106–116. Dickenson, A.H., Sullivan, A.F., 1987. Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones. Neurosci. Lett. 83, 207–211. Duarte, I.D., dos Santos, I.R., Lorenzetti, B.B., Ferreira, S.H., 1992. Analgesia by direct antagonism of nociceptor sensitization involves the arginine-nitric oxide-cGMP pathway. Eur. J. Pharmacol. 217, 225–227. Dubuisson, D., Dennis, S.G., 1977. The formalin test: a quantitative study of the analgesic effect of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4, 161–174. Edwards, G., Weston, A.H., 1993. The pharmacology of ATP-sensitive K+ channels. Annu. Rev. Pharmacol. Toxicol. 33, 597–637. El-Shenawy, S.M., Abdel-Salam, O.M., Baiuomy, A.R., El-Batran, S., Arbid, M.S., 2002. Studies on the anti-inflammatory and anti-nociceptive effects of melatonin in the rat. Pharmacol. Res. 46, 235–243. Gibson, A., Mirzazadeh, S., Hobbs, A.J., Moore, P.K., 1990. L-NG-monomethyl arginine and L-NG-nitro arginine inhibit non-adrenergic, non-cholinergic relaxation of the mouse anococcygeus muscle. Br. J. Pharmacol. 99, 602–606. Golombek, D.A., Escolar, E., Burin, L.J., De Brito Sanchez, M.G., Cardinali, D.P., 1991. Timedependent melatonin analgesia in mice: inhibition by opiate or benzodiazepine antagonism. Eur. J. Pharmacol. 194, 25–30. Golombek, D.A., Pevet, P., Cardinali, D.P., 1996. Melatonin effects on behavior: possible mediation by the central GABAergic system. Neurosci. Biobehav. Rev. 20, 403–412. Granados-Soto, V., Rufino, M.O., Gomes Lopes, L.D., Ferreira, S.H., 1997. Evidence for the involvement of the nitric oxide-cGMP pathway in the antinociception of morphine in the formalin test. Eur. J. Pharmacol. 340, 177–180. Granados-Soto, V., Arguelles, C.F., Ortiz, M.I., 2002. The peripheral antinociceptive effect of resveratrol is associated with activation of potassium channels. Neuropharmacology 43, 917–923. Han, J., Kim, N., Joo, H., Kim, E., Earm, Y.E., 2002. ATP-sensitive K+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes. Am. J. Physiol. Heart Circ. Physiol. 283, H1545–H1554. Heapy, C.G., Jamieson, A., Russell, N.J.W., 1987. Afferent C-fiber and A-delta activity in models of inflammation. Br. J. Pharmacol. 90, 164P. Huan, C., Zhou, M., Wu, M., Zhang, Z., Mei, Y., 2001. Activation of melatonin receptor increases a delayed rectifier K+ current in rat cerebellar granule cells. Brain Res. 917, 182–190.

75

Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Jin, J.G., Murthy, K.S., Grider, J.R., Makhlouf, G.M., 1993. Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells. Am. J. Physiol. 264, G470–G477. Lakin, M.L., Miller, C.H., Stott, M.L., Winters, W.D., 1981. Involvement of the pineal gland and melatonin in murine analgesia. Life Sci. 29, 2543–2551. Levy, D., Strassman, A.M., 2004. Modulation of dural nociceptor mechanosensitivity by the nitric oxide-cyclic GMP signaling cascade. J. Neurophysiol. 92, 766–772. Liu, L., Yang, T., Bruno, M.J., Andersen, O.S., Simon, S.A., 2004. Voltage gated ion channels in nociceptors; Modulation by cGMP. J. Neurophysiol. 92, 2323–2332. Lorenzetti, B.B., Ferreira, S.H., 1996. Activation of the arginine-nitric oxide pathway in primary sensory neurons contributes to dipyrone-induced intrathecal and peripheral analgesia. Inflamm. Res. 45, 308–311. Mantovani, M., Kaster, M.P., Pertile, R., Calixto, J.B., Rodrigues, A.L., Santos, A.R., 2006. Mechanisms involved in the antinociception caused by melatonin in mice. J. Pineal Res. 41, 382–389. McNamara, C.R., Mandel-Brehm, J., Bautista, D.M., Siemens, J., Deranian, K.L., Zhao, M., Hayward, N.J., Chong, J.A., Julius, D., Moran, M.M., Fanger, C.M., 2007. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. U. S. A. 104, 13525–13530. Mixcoatl-Zecuatl, T., Aguirre-Bañuelos, P., Granados-Soto, V., 2000. Sildenafil produces antinociception and increases morphine antinociception in the formalin test. Eur. J. Pharmacol. 400, 81–87. Moro, M.A., Russel, R.J., Cellek, S., Lizasoain, I., Su, Y., Darley-Usmar, V.M., Radomski, M.W., Moncada, S., 1996. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc. Natl. Acad. Sci. U. S. A. 93, 1480–1485. Noseda, R., Hernández, A., Valladares, L., Mondaca, M., Laurido, C., Soto-Moyano, R., 2004. Melatonin-induced inhibition of spinal cord synaptic potentiation in rats is MT2 receptor-dependent. Neurosci. Lett. 360, 41–44. Onal, S.A., Inalkac, S., Kutlu, S., Kelestimur, H., 2004. Intrathecal melatonin increases the mechanical nociceptive threshold in the rat. Agri. 16, 35–40. Ortiz, M.I., Granados-Soto, V., Castañeda-Hernández, G., 2003. The NO-cGMP-K+ channel pathway participates in the antinociceptive effect of diclofenac, but not of indomethacin. Pharmacol. Biochem. Behav. 76, 187–195. Ortiz, M.I., Medina-Tato, D.A., Sarmiento-Heredia, D., Palma-Martínez, J., Granados-Soto, V., 2006. Possible activation of the NO-cyclic GMP-protein kinase G-K+ channels pathway by gabapentin on the formalin test. Pharmacol. Biochem. Behav. 83, 420–427. Pang, C.S., Tsang, S.F., Yang, J.C., 2001. Effects of melatonin, morphine and diazepam on formalin-induced nociception in mice. Life Sci. 68, 943–951. Prado, W.A., Schiavon, V.F., Cunha, F.Q., 2002. Dual effect of local application of nitric oxide donors in a model of incision pain in rats. Eur. J. Pharmacol. 441, 57–65. Puig, S., Sorkin, L.S., 1996. Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 64, 345–355. Raghavendra, V., Agrewala, J.N., Kulkarni, S.K., 2000. Melatonin reversal of lipopolysacharides-induced thermal and behavioral hyperalgesia in mice. Eur. J. Pharmacol. 395, 15–21. Ray, M., Mediratta, P.K., Mahajan, P., Sharma, K.K., 2004. Evaluation of the role of melatonin in formalin-induced pain response in mice. Indian J. Med. Sci. 58, 122–130. Rodrigues, A.R., Duarte, I.D., 2000. The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels. Br. J. Pharmacol. 129, 110–114. Romey, G., Hughes, M., Schmid-Antomarchi, H., LazdunsKi, M., 1984. Apamin: a specific toxin to study a class of Ca2+-dependent K+ channels. J. Physiol. 79, 259–264. Sachs, D., Cunha, F.Q., Ferreira, S.H., 2004. Peripheral analgesic blockade of hypernociception: activation of arginine/NO/cGMP/protein kinase G/ATP-sensitive K+ channel pathway. Proc. Natl. Acad. Sci. U. S. A. 101, 3680–3685. Shavali, S., Ho, B., Govitrapong, P., Sawlom, S., Ajjimaporn, A., Klongpanichapak, S., Ebadi, M., 2005. Melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by increasing the release of beta-endorphin an endogenous opioid. Brain Res. Bull. 64, 471–479. Soares, A.C., Duarte, I.D.G., 2001. Dibutyryl-cyclic GMP induces peripheral antinociception via activation of ATP-sensitive K+ channels in the rat PGE2-induced hyperalgesic paw. Br. J. Pharmacol. 134, 127–131. Soares, A.C., Leite, R., Tatsuo, M.A.K.F., Duarte, I.D.G., 2000. Activation of ATP-sensitive K+ channels: mechanism of peripheral antinociceptive action of the nitric oxide donor, sodium nitroprusside. Eur. J. Pharmacol. 400, 67–71. Stretton, D., Miura, M., Bevisi, M.G., Barnes, P.J., 1992. Calcium-activated potassium channels mediate prejunctional inhibition of peripheral sensory nerves. Proc. Natl. Acad. Sci. U. S. A. 9, 1325–1329. Tegeder, I., Schmidtko, A., Niederberger, E., Ruth, P., Geisslinger, G., 2002. Dual effects of spinally delivered 8-bromo-cyclic guanosinemono-phosphate (8-bromo-cGMP) in formalin-induced nociception in rats. Neurosci. Lett. 332, 146–150. Tjølsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., Hole, K., 1992. The formalin test: an evaluation of the method. Pain 51, 5–17. Tomás-Zapico, C., Coto-Montes, A., 2005. A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. J. Pineal Res. 39, 99–104. Tu, Y., Sun, R.Q., Willis, W.D., 2004. Effects of intrathecal injections of melatonin analogs on capsaicin-induced secondary mechanical allodynia and hyperalgesia in rats. Pain 109, 340–350. Ulugol, A., Dokmeci, D., Guray, G., Sapolyo, N., Ozyigit, F., Tamer, M., 2006. Antihyperalgesic, but not antiallodynic, effect of melatonin in nerve-injured neuropathic mice: Possible involvements of the L-arginine-NO pathway and opioid system. Life Sci. 78, 1592–1597.

76

A. Hernández-Pacheco et al. / European Journal of Pharmacology 596 (2008) 70–76

Van den Top, M., Buijs, R.M., Ruijter, J.M., Delagrange, P., Spanswick, D., Hermes, M.L., 2001. Melatonin generates an outward potassium current in rat suprachiasmatic nucleus neurones in vitro independent of their circadian rhythm. Neuroscience 107, 99–108. Vanecek, J., 1998. Cellular mechanisms of melatonin action. Physiol. Rev. 78, 687–721. Wang, X., Robinson, P.J., 1997. Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system. J. Neurochem. 68, 443–456. Wang, T., Li, S.R., Dai, X., Peng, Y.L., Chen, Q., Wang, R., 2006. Effects of melatonin on orphanin FQ/nociceptin-induced hyperalgesia in mice. Brain Res. 1085, 43–48.

Wheeler-Aceto, H., Cowan, A., 1991. Standardization of the rat paw formalin test for the evaluation of analgesics. Psychopharmacol. 104, 35–44. Woolf, C.J., 1983. Evidence for a central component of post-injury pain hypersensitivity. Nature 306, 686–688. Yu, C.X., Zhu, B., Xu, S.F., Cao, X.D., Wu, G.C., 2000. The analgesic effects of peripheral and central administration of melatonin in rats. Eur. J. Pharmacol. 403, 49–53. Zimmermann, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109–110.