Probable involvement of Ca2 +-activated Cl− channels (CaCCs) in the activation of CB1 cannabinoid receptors

Life Sciences 92 (2013) 815–820

Contents lists available at SciVerse ScienceDirect

Life Sciences journal homepage: www.elsevier.com/locate/lifescie

Probable involvement of Ca 2 +-activated Cl − channels (CaCCs) in the activation of CB1 cannabinoid receptors Thiago Roberto Lima Romero, Daniela da Fonseca Pacheco, Igor Dimitri Gama Duarte ⁎ Department of Pharmacology, Institute of Biological Sciences, UFMG, Av. Antônio Carlos, 6627, 31.270.100, Belo Horizonte, Brazil

a r t i c l e

i n f o

Article history: Received 24 January 2012 Accepted 11 October 2012 Keywords: Anandamide Palmitoylethanolamine Niflumic acid CaCC Peripheral antinociception

a b s t r a c t Aims: Recently, we demonstrated that peripheral antinociception induced by δ opioid receptor is dependent of Ca2+-activated Cl− channels (CaCCs). Because opioid and cannabinoid receptors share some common mechanisms of action, our objective was to identify a possible relationship between CaCCs and the endocannabinoid system. Main methods: To induce hyperalgesia, rat paws were treated with intraplantar prostaglandin E2 (PGE2, 2 μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3 h following injection. Probabilities were calculated using ANOVA/Bonferroni's test, and values that were less than 5% were considered to be statistically significant. Key findings: Administration of the cannabinoid agonist CB1 anandamide (12.5, 25 and 50 μg/paw) and the cannabinoid agonist CB2 PEA (5, 10 and 20 μg/paw) decreased the PGE2-induced hyperalgesia in a dose-dependent manner. The possibility of the higher doses of anandamide (50 μg) and PEA (20 μg) having a central or systemic effect was excluded because the administration of the drug into the contralateral paw did not elicit antinociception in the right paw. As expected, the antinociceptive effects induced by anandamide and PEA were blocked by the CB1 and CB2 receptor antagonists AM251 and AM630, respectively. The peripheral antinociception was induced by anandamide but not PEA and was dose-dependently inhibited by the CaCC blocker niflumic acid (8, 16 and 32 μg). Significance: These results provide the first evidence for the involvement of CaCCs in the peripheral antinociception induced by activation of the CB1 cannabinoid receptor. © 2012 Elsevier Inc. All rights reserved.

Introduction Cannabinoids are a distinct class of psychoactive compounds that produce a large spectrum of pharmacological effects, including analgesia, sedation, hypothermia and inhibition of motor activity (Manzaneres et al., 1999; Massi et al., 2001; Varvel et al., 2005). Their effects are mediated through the CB1 and CB2 cannabinoid receptors at the peripheral, spinal and supraspinal levels (Pertwee, 2001; Hohmann, 2002). The CB1 receptor is highly expressed in the central nervous system, where cannabinoids act at the presynaptic CB1 receptors to elicit changes in the synaptic efficacy of neuronal circuits (Freud et al., 2003). However, it has been verified that the activation of the CB1 receptors present at the peripheral, spinal and supraspinal sites produces antinociceptive effects (Hohmann, 2002; Hohmann and Suplita, 2006). The CB2 receptor is primarily located on the immune cells in the periphery (Galiègue et al., 1995), though studies have

⁎ Corresponding author at: Departamento de Farmacologia, ICB-UFMG, Av. Antônio Carlos, 6627 — Campus da Pampulha, Belo Horizonte, MG, CEP: 31.270-100, Brazil. Fax: + 55 31 499 2695. E-mail address: [email protected] (I.D.G. Duarte). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.10.006

demonstrated the presence of CB2 receptors in a number of brain regions (Onaivi et al., 2006; Gong et al., 2006). Pharmacological studies that elucidate the role of the endocannabinoid system are dependent on the availability of selective agents that interact specifically and selectively with each of the endocannabinoid proteins. With the identification of endogenous ligands for the different cannabinoid receptors, opportunities and discrepancies have arisen (Pacher et al., 2006). Anandamide, an endocannabinoid, shows preferential affinity for the CB1 receptors (Howlett et al., 2002). It is synthesized on demand instead of being stored in the synaptic vesicles and is hydrolyzed into arachidonic acid and ethanolamine by a membrane bound enzyme named fatty acid amide hydrolase (FAAH) (Hohmann and Suplita, 2006). Furthermore, palmitoylethanolamide (PEA) has been proposed to be a possible endogenous ligand of the CB2 receptor (Facci et al., 1995; Calignano et al., 1998; Helyes et al., 2003) or as a fatty acid with related pharmacology that activates the peroxisome proliferatoractivated receptor-alpha (PPAR-alpha) (LoVerme et al., 2006). There are similarities between this compound and the endogenous cannabinoid, anandamide. Both are structurally similar ethanolamide derivatives of membrane lipids, palmitic acid and arachidonic acid and are thought to be inactivated by the same enzyme (Di Marzo et al., 1998).

816

T.R.L. Romero et al. / Life Sciences 92 (2013) 815–820

The CB1 and CB2 receptors share some common signal transduction pathways, including the inhibition of adenylyl cyclase and the stimulation of the mitogen-activated protein kinase (Goutopoulos and Makriyannis, 2002). However, in contrast to CB1, the CB2 receptor stimulation is believed not to modulate ion channel function (Pertwee, 1997). The CB1 receptors are involved in inhibiting the N- and Q-type calcium channels and in activating potassium channels (Felder and Glass, 1998). Recently, it was demonstrated that the peripheral antinociceptive effect of the cannabinoid receptor agonist, anandamide, is primarily caused by the activation of ATP-sensitive K + channels (Reis et al., 2011). Chloride channels display a variety of important physiological and cellular roles, including the regulation of nerve and muscle excitability. Consistent with other ion channels, chloride channels can perform their functions in the plasma membrane or in the membranes of intracellular organelles (Jentsch et al., 2002). Three distinct channel families have been characterized in detail, according to their molecular structure and tissue distribution. They are voltage-gated Cl− channels, cystic fibrosis transmembrane conductance regulator (CFTR) and ligand-gated Cl− channels that open upon binding of the neurotransmitters GABA. A fourth family of Cl− channels is regulated by the cytosolic Ca2+ concentration (CaCCs), but little is known about its structure and function (Pauli et al., 2000). The CaCCs are expressed in a variety of neurons, including those in the dorsal root ganglion, the spinal cord, and the autonomic system. The functions of CaCCs in neurons, however, remain poorly understood. Studies have suggested that these channels are involved in action potential repolarization, generation of after-polarizations, and membrane oscillatory behavior (Hartzell et al., 2005). Due to the lack of information pertaining to the activation of CaCCs by cannabinoid receptors, our objective was to establish a possible relationship between CaCC activation and the endocannabinoid system.

Materials and methods

Chemicals The following drugs and chemicals were used: PGE2 (Sigma, USA), anandamide (Tocris, EUA), PEA (palmitoylethanolamine; Tocris, USA), AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)4-methyl-1H-pyrazole-3-carboxamide; Tocris, USA), AM630 (6-iodo2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-ethoxyphenyl) methanone; Tocris, USA) and niflumic acid (Sigma, USA), The drugs were dissolved as follows: PGE2 (ethanol 2% in saline); anandamide and PEA (saline); AM251 and AM630 (20% DMSO in saline); niflumic acid (DMSO 16% in saline) and injected in a volume of 50 μl per paw.

Experimental protocol Anandamide and PEA were administered subcutaneously in the right hind paw 2:45 and 2:55 h after local injection of PGE2. Dose– response curves were done to determine effective doses for this study. In the protocol used to determine whether the drugs were acting outside the injected paw, PGE2 was injected into both hind paws, while anandamide and PEA were administered into the left paw. AM251, AM630 and niflumic acid were intraplantarly injected 0:15, 0:15 and 1:10 h prior to the measurement of hyperalgesia (3 h). The nociceptive threshold was always measured in the right hind paw and also in the left hind paw for control experiments (e.g., Fig. 1). The protocol above was assessed in pilot experiments to determine the best moment for the injection of each substance.

Statistical analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) with post-hoc Bonferroni's test for multiple comparisons. Probabilities less than 5% (P b 0.05) were considered to be statistically significant.

Measurement of the hyperalgesia After manual restraint, rats were injected with prostaglandin E2 (PGE2, 2 μg) into the plantar surface (subcutaneous) of its hind paw and measured by the paw pressure test described by Randall and Selitto (1957). An analgesimeter (Ugo-Basile, Italy) with a cone-shaped paw-presser with a rounded tip was used to apply a linearly increasing force to the rat's right hind paw. The weight in grams required to elicit nociceptive response, the paw withdrawal, was determined as the nociceptive threshold. A cut-off value of 300 g was used to prevent damage to the paws. The nociceptive threshold was measured in the right paw and determined by the average of three consecutive trials recorded before (zero time) and 3 h after PGE2 injection (peak of effect). The results were calculated by the difference between these two averages (Δ of nociceptive threshold) and expressed as grams. To reduce stress, the rats were habituated to the apparatus one day prior to the experiments.

125

Δ of Nociceptive threshold (g)

The experiments were performed on 160–200 g male Wistar rats (N= 5 per group) from the CEBIO (The Animal Centre) of the Federal University of Minas Gerais (UFMG). The rats were housed in a temperature-controlled room (23 ± 1 °C) on an automatic 12-h light/ dark cycle (06:00–18:00 h of light phase). All testing was carried out during the light phase (08:00–15:00 h). Food and water were freely available until the onset of the experiments. The local Ethics Committee on Animal Experimentation (CETEA) of UFMG approved all animal experiments.

100

*

75

Δ of Nociceptive threshold (g)

Animals 125

Same Group

Same Group

R Paw L Paw

100 75 50 25

# 0 Veh 1

AEA 50

Veh 1

PGE 2

50

*

25

*

0 12.5 Veh 1

25 AEA PGE2

50 AEA 50 Veh 2

Fig. 1. Effect of anandamide on PGE2-induced hyperalgesia in rats. Anandamide (AEA; 12.5, 25, and 50 μg) was administered 2:45 h after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in the Materials and methods section. Each column represents the mean ± S.E.M. (n = 5). * indicates a significant difference from the PGE2 + veh 1-injected group (P b 0.05, ANOVA + Bonferroni's test). Veh 1 = saline, Veh 2 = ethanol 2% in saline. Insert above. Exclusion of outside paw antinociceptive effect of anandamide. PGE2 (2 μg) was administered in both hind paws, right (R) and left (L). Anandamide (50 μg/paw) was administrated 2 h and 45 min after PGE2 in the right hind paw (AEA 50 R paw). Antinociceptive responses were measured in both hind paws, as described in the Materials and methods section. Each column represents the mean ± S.E.M. (n = 5). # indicates a significant difference from the PGE2 R paw + veh R paw-injected group (P b 0.05, ANOVA + Bonferroni's test).

T.R.L. Romero et al. / Life Sciences 92 (2013) 815–820

817

The peripheral antinociceptive effect of anandamide The administration of anandamide (12.5, 25 and 50 μg) into the right hind paw produced an antinociceptive response against PGE2-induced hyperalgesia (2 μg/paw) in a dose-dependent manner (Fig. 1). Despite the finding that a dose of 50 μg/paw was able to reverse PGE2-induced hyperalgesia almost completely, this dose of anandamide alone did not alter the nociceptive threshold (Fig. 1). When administered into the right paw, anandamide, at a dose of 50 μg, did not produce an antinociceptive effect in the left paw, indicating that, at this dose, it exhibited only a peripheral site of action (Fig. 1, inset).

Δ of Nociceptive threshold (g)

125

Results

75

#*

50

#*

25

# 0

08

16 AN AEA 50 PGE2

Veh 1 Veh 2

The effects of AM251 and AM630 on anandamide-induced antinociception The intraplantar injection of the CB1 receptor antagonist, AM251 (20, 40 and 80 μg), inhibited the anandamide-induced peripheral antinociception (50 μg/paw) in a dose-dependent manner (Fig. 2). The highest dose of AM251, given without PGE2 or without anandamide, did not induce hyperalgesia or anti-hyperalgesic effects. The CB2 receptor antagonist, AM630 (100 μg), did not modify the peripheral antinociception induced by anandamide (50 μg/paw) (Fig. 2).

*

100

32 AN 50

Veh 1

Veh 2 Veh 3 PGE2

Fig. 3. Antagonism induced by intraplantar administration of niflumic acid of the peripheral antinociception produced by anandamide in hyperalgesic paws (PGE2, 2 μg). Niflumic acid (8, 16, 32 μg/paw) was administered 55 min prior anandamide (AEA, 50 μg/paw). Each column represents the mean ± S.E.M. (n = 5). # and * indicate significant differences compared to (PGE2 + veh 1 + veh 2) and (PGE2 + veh 2 + AEA 50)-injected groups, respectively (P b 0.05, ANOVA + Bonferroni's test). Veh 1 = saline, Veh 2 = 16% DMSO in saline.

The antagonism of anandamide-induced antinociception by niflumic acid

The intraplantar injection of PEA (5, 10 and 20 μg) into the right hind paw produced an antinociceptive response against the PGE2-induced hyperalgesia (2 μg/paw) in a dose-dependent manner (Fig. 4). Despite the finding that a dose of 20 μg/paw was able to reverse the hyperalgesia induced by PGE2 almost completely, this dose of PEA alone did not alter the nociceptive threshold (Fig. 4). When administered into the right paw, 20 μg of PEA did not produce

Δ of Nociceptive threshold (g)

125

*

100

75

*#

50

*#

The effects of AM251 and AM630 on PEA-induced antinociception The intraplantar injection of AM630 (25, 50 and 100 μg) inhibited the PEA-induced peripheral antinociception (20 μg/paw) in a dosedependent manner (Fig. 5). The highest dose of AM630, given without PGE2 or without PEA, did not induce hyperalgesia or anti-hyperalgesic effects. AM251 (80 μg) did not modify the peripheral antinociception induced by PEA (20 μg/paw) (Fig. 5, inset).

125

100

#

75

20 Veh 2 Veh 1

40 80 100 AM630 AM251 AEA 50 PGE3

PGE2

Veh 3

Fig. 2. Effect of intraplantar administration of AM251 and AM630 on the peripheral antinociception produced by anandamide in the hyperalgesic paw (PGE2, 2 μg). AM251 (20, 40, and 80 μg) or AM630 (100 μg) was administered 2:45 h after local injection of PGE2, at the same time of anandamide (AEA, 50 μg). Each column represents the mean ± S.E.M. (n = 5). # and * indicate significant differences compared to (PGE2 + veh 1 + veh 2) and (PGE2 + veh 2 + AEA 50)-injected groups, respectively (P b 0.05, ANOVA + Bonferroni's test). Veh 1 = saline, Veh 2 = 20% DMSO in saline.

R Paw L Paw

75 50 25

# 0 Veh 1

PEA 20

Veh 1

# 25

# 05

10 PEA PGE2

Veh 2 AM251 Veh 1

Same Group

2

Veh 1

0

Same Group

100

PGE

25

#

125

50

0

#

Δ of Nociceptive threshold (g)

The peripheral antinociceptive effect of PEA

an antinociceptive effect in the left paw, indicating that it exhibited only a peripheral site of action at this dose (Fig. 4, inset).

Δ of Nociceptive threshold (g)

Intraplantar administration of the CaCC blocker, niflumic acid (8, 16 and 32 μg), inhibited the anandamide-induced peripheral antinociception (50 μg/paw) in a dose-dependent manner (Fig. 3). The highest effective dose of niflumic acid did not induce hyperalgesia or any anti-hyperalgesic effects.

20 PEA 20 Veh 2

Fig. 4. Effect of PEA on PGE2-induced hyperalgesia in rats. PEA (5, 10, 20 μg) was administered 2:55 h after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in the Materials and methods section. Each column represents the mean±S.E.M. (n=5). * indicates a significant difference from the PGE2 +veh 1-injected group (Pb 0.05, ANOVA+Bonferroni's test). Veh 1 = saline, Veh 2 = ethanol 2% in saline. Insert above. Exclusion of outside paw antinociceptive effect of PEA. PGE2 (2 μg) was administered in both hind paws, right (R) and left (L). PEA (20 μg/paw) was administrated 2 h and 55 min after PGE2 in the right hind paw (PEA 20 R paw). Antinociceptive responses were measured in both hind paws, as described in the Materials and methods section. Each column represents the mean±S.E.M. (n=5). # indicates a significant difference from the PGE2 R paw+veh R paw-injected group (Pb 0.05, ANOVA+Bonferroni's test).

818

T.R.L. Romero et al. / Life Sciences 92 (2013) 815–820

Δ of Nociceptive threshold (g)

125

*

100

75

*# 50

*#

25

#

# 0

25 Veh 2 Veh 1

50 100 80 AM251 AM630 PEA 20

Veh 2 AM630 Veh 1

Veh 3

PGE2

PGE2

Fig. 5. Effect of intraplantar administration of AM251 and AM630 on the peripheral antinociception produced by PEA in the hyperalgesic paw (PGE2, 2 μg). AM630 (25, 50 and 100 μg) or AM251 (80 μg) was administered 10 min prior PEA (20 μg). Each column represents the mean ± S.E.M. (n = 5). # and * indicate significant differences compared to (PGE2 + veh 1 + veh 2) and (PGE2 + veh 2 + PEA 20)-injected groups, respectively (P b 0.05, ANOVA + Bonferroni's test). Veh 1 = saline, Veh 2 = 20% DMSO in saline.

The effect of niflumic acid on PEA-induced antinociception As shown in Fig. 6, the administration of the niflumic acid at 32 μg/paw, which was the same effective dose used against anandamide, did not modify the peripheral antinociception induced by PEA (20 μg/paw). Discussion Ca2+-activated Cl− channels (CaCCs) are expressed in a variety of different neurons, including those in the dorsal root ganglion, spinal cord, and autonomic system (Hartzell et al., 2005). Generally, CaCCs are expressed in a subset of neurons rather than an entire group, suggesting that these channels perform a specific function for these neurons. The opening of CaCCs generates membrane potential depolarization or hyperpolarization depending on whether the Cl− equilibrium

Δ of Nociceptive threshold (g)

125

100

75

50

25

#

#

0 AN 32

Veh 1 Veh 2

PEA 20 PGE2

Fig. 6. Effect of intraplantar administration of AM251 and AM630 on the peripheral antinociception produced by PEA in hyperalgesic paws (PGE2, 2 μg). Niflumic acid (32 μg/paw) was administered 1:05 h prior PEA (20 μg/paw). Each column represents the mean ± S.E.M. (n = 5). # indicates significant differences compared to PGE2 + veh 1 + veh 2 (P b 0.05, ANOVA + Bonferroni's test). Veh 1 = saline, Veh 2 = 16% DMSO in saline.

potential is more positive or more negative than the resting potential (Frings et al., 2000). Previous work has demonstrated that CaCCs are, at least in part, involved in the production of the δ-opioid receptor-mediated antinociception in the mouse spinal cord (Yamazaki et al., 2000). This finding indicates that the release of Ca2+ from the IP3-sensitive Ca2+ pool due to δ-opioid receptor stimulation may lead to the opening of Ca2+-activated ion channels, CaCCs and Ca2+-activated K + channels, resulting in neuron hyperpolarization (Narita et al., 2000). Recently, our group demonstrated for the first time that the peripheral and central antinociceptive effects of the δ opioid receptor agonist SNC80 were inhibited, at least in part, by the CaCC blocker niflumic acid (Pacheco et al., 2012a, 2012b). The interaction between the opioid and cannabinoid systems in nociceptive modulation has been the focus of much research in recent years (Welch and Eads, 1999; Finn et al., 2004). Receptors for both drugs are coupled to similar intracellular signaling mechanisms, resulting in a decrease in the cAMP production through the activation of Gi proteins (Bidaut-Russell et al., 1990; Childers, 1991). Additionally, endogenous opioids might be involved in the regulation of pain control by cannabinoids. For example, cannabinoids Δ9-THC and levonantradol appear to enhance the antinociceptive effect of the μ opioid receptor agonist morphine by releasing dynorphin A and dynorphin B, respectively (Welch and Eads, 1999). Our group demonstrated the participation of endocannabinoids in the peripheral and central antinociception of the morphine (Pacheco et al., 2008; Pacheco et al., 2009). Given the lack of information regarding the participation of chloride channels in the analgesic mechanism of endogenous cannabinoids, the present work used niflumic acid to elucidate the mechanism of the peripheral antinociception induced by cannabionids. Initially, we tested the ability of the anandamide and PEA to induce peripheral antinociception. Our data showed that anandamide and PEA produced a dose-dependent peripheral antinociceptive effect in the rat paw by a prostaglandin E2-induced hyperalgesia test. To exclude the possibility that anandamide (50 μg/paw) and PEA (20 μg/paw) produced antinociception by acting at sites outside the paw, a strategy was employed to evaluate the efficacy of ipsi versus contralateral paw administration. PGE2 was injected into both hind paws, thus creating the same tissue conditions and an equal possibility that the agents tested would reach sites outside the injected paw. Anandamide and PEA were then administered into the right paw and the nociceptive threshold was measured in both hind paws; however, anandamide and PEA proved ineffective at producing antinociception in the left paw, indicating that they only exhibited local peripheral action at these doses. Our results demonstrated that AM251 prevented the peripheral antinociception induced by anandamide in a dose-dependent manner. AM251 is a potent CB1 receptor antagonist with a 306-fold selectivity over CB2 receptors (Gatley et al., 1997; Lan et al., 1999), and anandamide shows preferential affinity for CB1 receptors (Howlett et al., 2002). The participation of CB1 receptors in peripheral antinociception has been mentioned in various studies (Rice et al., 2002). For example, the intraplantar administration of the CB1 agonist WIN55212-2 attenuated the development of carrageenan-induced mechanical hyperalgesia ad allodynia (Nackley et al., 2003). Recently, our group demonstrated the participation of these receptors in the peripheral antinociception induced by morphine (Pacheco et al., 2008) and anandamide (Reis et al., 2011). These receptors are expressed on the peripheral axons of primary sensory neurons. Substantial analgesia can be achieved in somatic and visceral pain, as well as in inflammatory and neuropathic pain (Agarwal et al., 2007). Additionally, another study provided strong evidence that the peripheral CB1 receptors, which are presumed to be located on the peripheral endings of the A- and C- fiber primary afferents, are able to modulate the transmission of innocuous and noxious somatosensory information from the periphery to the spinal cord (Kelly et al., 2003).

T.R.L. Romero et al. / Life Sciences 92 (2013) 815–820

CB1 receptors are involved in inhibiting the N- and Q-type calcium channels and activation of potassium channels (Felder and Glass, 1998). Several studies have shown a relationship between drugs that open K + channels and the induction of antinociception (Ocaña et al., 2004; Pacheco and Duarte, 2005). Recently, it was demonstrated that the peripheral antinociceptive effect of anandamide is primarily caused by the activation of ATP-sensitive K + channels (Reis et al., 2011). In mouse sympathetic neurons, there appears to be a selective coupling of different types of voltage-gated Ca 2+ channels to CaCCs (Ca 2+-activated Cl − channels) and K + channels. The entrance of Ca 2+ through L- and P-type channels activates CaCCs; whereas Ca 2+ that enters through the N-type channels activates the Ca 2+-activated K + channels (Martínez-Pinna et al., 2000). Interestingly, the present study showed that the CaCC blocker niflumic acid was able to inhibit the peripheral antinociception induced by anandamide in a dose-dependent manner. Specific high-affinity blockers for CaCCs are currently unavailable, but several studies have demonstrated that niflumic acid effectively inhibits these channels (Kirkup et al., 1996). According to White and Aylwin (1990), the most common blockers for native CaCCs are the flufenamates niflumic and flufenamic acids. Based on voltage–clamp step response measurements, niflumic acid is more potent than flufenamic acid in inhibiting the electrical coupling decreases based on voltage–clamp step response measurements (Harks et al., 2001). Although these data are encouraging, the effects of niflumic acid are not specific to CaCCs. The undesirable effects of niflumic acid include blocking volume-regulated anion channels and K+ channels (Wang et al., 1997; Xu et al., 1997). Niflumic acid can also affect Ca 2+ currents and complicate the interpretation of the effects on ICLCa (Reinsprecht et al., 1995). Studies conducted by Hartzell et al. (2005) considered niflumic acid to be a specific blocker, and they used it to classify certain anion currents through CaCCs in various tissues. Additionally, some researchers consider niflumic acid to be the most potent blocker of the calcium activated Cl− current (ICL(Ca)) in smooth muscle because it can inhibit this current at micromolar concentrations (Hogg et al., 1994). Other commonly used chloride channel blockers, such as NPPB, tamoxifen, DIDS, SITS, A9C, and DPC, are less effective than the flufenamates in inhibiting CaCCs (Jentsch et al., 2002). In contrast, niflumic acid did not modify the peripheral antinociception induced by PEA, at the same effective dose against anandamide. PEA has been proposed as a possible endogenous ligand of the CB2 receptor (Facci et al., 1995) and stimulation of this receptor is not believed to modulate the ion channel function (Pertwee, 1997). Indeed, our results show that AM630 prevented the peripheral antinociception induced by PEA in a dose-dependent manner. AM630 is a CB2 ligand that is 165-fold selective over CB1 receptors (Ross et al., 1999). However, PEA may not be a direct CB2 receptor agonist because it does not bind to the CB2 receptors transfected into host cells (Showalter et al., 1996). Therefore, PEA activates CB2 receptors indirectly by initiating a chain of events resulting in CB2 receptor activation. For example, it has been proposed that PEA may enhance the effects of other endocannabinoids by inhibiting their inactivation (Lambert and Di Marzo, 1999). CB2 receptors have not been found in peripheral neurons. This observation suggests that the activation of CB2 receptors produces antinociception indirectly by causing the release of mediators from non-neuronal cells that alter the responsiveness of the primary afferent neurons to noxious stimuli. One cell type that might mediate the actions of CB2 receptor-selective agonists is the keratinocytes, which have been reported to express CB2 receptors (Casanova et al., 2003). In conclusion, our data demonstrate, for the first time, that the peripheral antinociceptive effect induced by anandamide is inhibited by niflumic acid, suggesting that CaCCs participate in the analgesic effects induced by the activation of CB1 receptors. However, there seems to be no relationship between CB2 receptors and CaCCs. Because this is a new direction in the field, additional research is necessary to further elucidate the interaction between cannabinoids and CaCCs.

819

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments Fellowships by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Pesquisa) are acknowledged. References Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, et al. Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 2007;10:870–9. Bidaut-Russell M, Devane WA, Howlett AC. Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain. J Neurochem 1990;55:21–6. Calignano A, La Rana G, Giuffrida A, Piomelli D. Control of pain initiation by endogenous cannabinoids. Nature 1998;394:277–81. Childers SR. Opioid receptor-coupled second messengers. Life Sci 1991;48:1991–2003. Casanova ML, Blázquez C, Martínez-Palacio J, Villanueva C, Fernández-Aceñero MJ, Huffman JW, et al. Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J Clin Invest 2003;111:43–50. Di Marzo V, Melck D, Bisogno T, De Petrocellis L. Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action. Trends Neurosci 1998;21: 521–8. Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A. Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci U S A 1995;92:3376–80. Felder CC, Glass M. Cannabinoid receptors and their endogenous agonists. Annu Rev Pharmacol Toxicol 1998;38:179–200. Finn DP, Beckett SR, Roe CH, Madjd A, Fone KC, Kendall DA, et al. Effects of coadministration of cannabinoids and morphine on nociceptive behaviour, brain monoamines and HPA axis activity in a rat model of persistent pain. Eur J Neurosci 2004;19:678–86. Freud TF, Katona I, Piomelli D. Role of endogenous cannabinoids in synaptic signaling. Physiol Rev 2003;83:1017–66. Frings S, Reuter D, Kleene SJ. Neuronal Ca2+-activated Cl− channels—homing in on an elusive channel species. Prog Neurobiol 2000;60:247–89. Galiègue S, Mary S, Marchand J, Dussossoy D, Carrière D, Carayon P, et al. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 1995;232:54–61. Gatley SJ, Lan R, Pyatt B, Gifford AN, Volkow ND, Makriyannis A. Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors. Life Sci 1997;61:191–7. Goutopoulos A, Makriyannis A. From cannabis to cannabinergics: new therapeutic opportunities. Pharmacol Ther 2002;95:103–17. Gong JP, Onaivi ES, Ishiguro H, Liu QR, Tagliaferro PA, Brusco A, et al. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res 2006;1071: 10–23. Harks EG, de Roos AD, Peters PH, de Haan LH, Brouwer A, Ypey DL, et al. Fenamates: a novel class of reversible gap junction blockers. J Pharmacol Exp Ther 2001;298: 1033–41. Hartzell C, Putzier I, Arreola J. Calcium-activated chloride channels. Annu Rev Physiol 2005;67:719–58. Helyes Z, Németh J, Thán M, Bölcskei K, Pintér E, Szolcsányi J. Inhibitory effect of anandamide on resiniferatoxin-induced sensory neuropeptide release in vivo and neuropathic hyperalgesia in the rat. Life Sci 2003;73:2345–53. Hogg RC, Wang Q, Large WA. Action of niflumic acid on evoked and spontaneous calcium-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein. Br J Pharmacol 1994;112:977–84. Hohmann AG. Spinal and peripheral mechanisms of cannabinoid antinociception: behavioral, neurophysiological and neuroanatomical perspectives. Chem Phys Lipids 2002;121:173–90. Hohmann AG, Suplita RL. Endocannabinoid mechanisms of pain modulation. AAPS J 2006;8: 693–708. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, et al. International Union of Pharmacology XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002;54:161–202. Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503–68. Kelly S, Jhaveri MD, Sagar DR, Kendall DA, Chapman V. Activation of peripheral cannabinoid CB1 receptors inhibits mechanically evoked responses of spinal neurons in noninflamed rats and rats with hindpaw inflammation. Eur J Neurosci 2003;18: 2239–43. Kirkup AJ, Edwards G, Weston AH. Investigation of the effects of 5-nitro-2(phenylpropylamino)-benzoic acid (NPPB) on membrane currents in rat portal vein. Br J Pharmacol 1996;117:175–83. Lambert DM, Di Marzo V. The palmitoylethanolamide and oleamide enigmas: are these two fatty acid amides cannabimimetic? Curr Med Chem 1999;6:757–73. Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D, et al. Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists. J Med Chem 1999;42: 769–76.

820

T.R.L. Romero et al. / Life Sciences 92 (2013) 815–820

LoVerme J, Russo R, La Rana G, Fu J, Farthing J, Mattace-Raso G, et al. Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-alpha. J Pharmacol Exp Ther 2006;319:1051–61. Manzaneres J, Corchero J, Romero JJ, Fernandez-Ruiz JA, Ramos JÁ, Fuentes JÁ. Pharmacological and biochemical interactions between opioids and cannabionoids. Trends Pharmacol Sci 1999;20:287–94. Martínez-Pinna J, McLachlan EM, Gallego R. Distinct mechanisms for activation of Cl− and K+ currents by Ca2+ from different sources in mouse sympathetic neurones. J Physiol 2000;527:249–64. Massi P, Vaccani A, Romorini S, Parolaro D. Comparative characterization in the rat of the interaction between cannabinoids and opiates for their immunosuppressive and analgesic effects. J Neuroimmunol 2001;117:116–24. Nackley AG, Suplita 2nd RL, Hohmann AG. A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience 2003;117:659–70. Narita M, Ohsawa M, Mizoguchi H, Aoki T, Suzuki T, Tseng LF. Role of the phosphatidylinositol-specific phospholipase C pathway in δ-opioid receptor-mediated antinociception in the mouse spinal cord. Neuroscience 2000;99:327–31. Ocaña M, Cendán CM, Cobos EJ, Entrena JM, Baeyens JM. Potassium channels and pain: present realities and future opportunities. Eur J Pharmacol 2004;500:203–19. Onaivi ES, Ishiguro H, Gong JP, Patel S, Perchuk A, Meozzi PA, et al. Discovery of the presence and functional expression of cannabinoid CB2 receptors in brain. Ann N Y Acad Sci 2006;1074:514–36. Pacheco DF, Duarte IDG. δ-Opioid receptor agonist SNC80 induces peripheral antinociception via activation of ATP-sensitive K+ channels. Eur J Pharmacol 2005;512:23–8. Pacheco DF, Klein A, Perez AC, Pacheco CMF, Francischi JN, Duarte ID. The mu-opioid receptor agonist morphine, but not agonists at delta- or kappa-opioid receptors, induces peripheral antinociception mediated by cannabinoid receptors. Br J Pharmacol 2008;154:1143–9. Pacheco D, Klein A, Perez C, Pacheco CM, Francischi JN, Duarte IDG. The μ-opioid receptor agonist morphine, but not agonists at δ or к-opioid receptors, induces central antinociception mediated by cannabinoid receptors. Br J Pharmacol 2009;158:225–31. Pacheco DF, Pacheco CM, Duarte ID. δ-Opioid receptor agonist SNC80 induces central antinociception mediated by Ca2+-activated Cl– channels. J Pharm Pharmacol 2012a;64:1084–9. Pacheco DF, Pacheco CM, Duarte ID. Peripheral antinociception induced by δ-opioid receptors activation, but not μ- or κ-, is mediated by Ca2+-activated Cl– channels. Eur J Pharmacol 2012b;674:255–9. Pacher P, Bátkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 2006;58:389–462.

Pauli BU, Abdel-Ghany M, Cheng HC, Gruber AD, Archibald HA, Elble RC. Molecular characteristics and functional diversity of CLCA family members. Clin Exp Pharmacol Physiol 2000;27:901–5. Pertwee RG. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 1997;74:129–80. Pertwee RG. Cannabinoid receptors and pain. Prog Neurobiol 2001;63:569–611. Randall LD, Selitto JJ. A method for measurement of analgesic activity on inflamed tissues. Arch Int Pharmacol 1957;113:233–49. Reinsprecht M, Rohn MH, Spadinger RJ, Pecht I, Schindler H, Romanin C. Blockade of capacitive Ca2+ influx by Cl− channel blockers inhibits secretion from rat mucosal-type mast cells. Mol Pharmacol 1995;47:1014–20. Reis GM, Ramos MA, Pacheco Dda F, Klein A, Perez AC, Duarte ID. Endogenous cannabinoid receptor agonist anandamide induces peripheral antinociception by activation of ATP-sensitive K+ channels. Life Sci 2011;88:653–7. Rice AS, Farquhar-Smith WP, Nagy I. Endocannabinoids and pain: spinal and peripheral analgesia in inflammation and neuropathy. Prostaglandins Leukot Essent Fatty Acids 2002;66:243–56. Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A, et al. Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630. Br J Pharmacol 1999;126:665–72. Showalter VM, Compton DR, Martin BR, Abood ME. Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther 1996;278:989–99. Varvel SA, Bridgen DT, Tao Q, Thomas BF, Martin BR, Lichtman AH. Delta9tetrahydrocannbinol accounts for the antinociceptive, hypothermic, and cataleptic effects of marijuana in mice. J Pharmacol Exp Ther 2005;314:329–37. Wang HS, Dixon JE, Mckinnon D. Unexpected and differential effects of Cl− channel blockers on the Kv4.3 and Kv4.2 K+ channels. Implications for the study of the I(to2) current. Circ Res 1997;81:711–8. Welch SP, Eads M. Synergistic interactions of endogenous opioids and cannabinoids systems. Brain Res 1999;848:183–90. White MM, Aylwin M. Niflumic and flufenamic acids are potent reversible blockers of Ca+2-activated Cl− channels in xenopus oocytes. Mol Pharmacol 1990;37:720–4. Xu WX, Kim SJ, So I, Kang TM, Rhee JC, Kim KW. Volume-sensitive chloride current activated by hyposmotic swelling in antral gastric myocytes of the guinea-pig. Pflugers Arch 1997;435:9-19. Yamazaki M, Mizoguchi H, Ohsawa M, Tseng LF, Suzuki T, Narita M. Implications of Ca(2 +)-activated Cl(−) channels in the delta-opioid receptor-mediated antinociception in the mouse spinal cord. Neurosci Lett 2000;295:113–5.