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Pain and Analgesic Mechanisms

CB1 and CB2 Cannabinoid Receptor Agonists Induce Peripheral Antinociception by Activation of the Endogenous Noradrenergic System

Romero, Thiago R. L. PhD; Resende, Livia C. MD; Guzzo, Luciana S. PhD; Duarte, Igor D. G. PhD

Author Information
doi: 10.1213/ANE.0b013e3182707859
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Abstract

Peripheral adrenergic mechanisms contribute to ongoing maintenance of pain,1 and noradrenergic transmission is a component of nociception.2 Briefly, nociception seems to involve protein kinase C3 and Gq protein via α1 adrenergic receptors and Gs protein and protein kinase A activation to β adrenoceptors.4 Data obtained by our group demonstrated that the peripheral antinociceptive effect of the adrenoceptor agonist xylazine involves the activation of α2C adrenoreceptor5 thus inducing nitric oxide synthase to produce nitric oxide with a subsequent increase of cyclic guanosine monophosphate in peripheral nociceptors6 and opening of adenosine triphosphate–sensitive K+ channels.7 Activation of the β adrenoceptor is responsible for analgesia in patients with rheumatoid arthritis8 and in rats with adjuvant-induced arthritis.9,10 In addition, norepinephrine injected directly into inflamed rat hindpaw, via immune cells participation, produced dose-dependent antinociception, blocked by α1, α2, and β2 adrenoceptor antagonists.11

The literature suggests an interaction between the adrenergic and cannabinoidergic systems. Kurihara et al.12 suggested that the endocannabinoid arachidonoylglycerol, but not anandamida, participates in the CB1 cannabinoid receptor-mediated sympathetic stimulation resulting in norepinephrine release. On the other hand, activation of presynaptic CB1 cannabinoid receptors mediating the inhibition of adrenergic sensory transmission has been frequently observed13 in vitro and in vivo in peripheral sympathetic nerve terminals,14 for example, in cardiovascular sympathetic regulation15 and in the rat perfused mesenteric bed.16 Direct application of the selective CB1 cannabinoid agonist WIN 55,212-2 into the rat frontal cortex elicited a significant increase in extracellular norepinephrine efflux, suggesting that the activation of cortical cannabinoid receptors contributes to changes in norepinephrine levels in this region of the brain.17 When systemically administered, cannabinoid agonists stimulate norepinephrine release in the frontal cortex by activation of noradrenergic neurons in the coeruleofrontal cortex pathway.18

Three lines of evidence suggesting an antinociceptive interaction between the adrenergic and the cannabinoid systems have also been proposed.19–21 First, both α2 adrenoceptor and cannabinoid receptors induce antinociception through inhibition of postsynaptic membrane excitation, Ca2+-inducing neurotransmitter release,22–24 and the opening of adenosine triphosphate–sensitive K+ channels.7,25 Second, some cannabinoid receptor agonists, such as N-palmitoyl-ethanolamine (PEA), release norepinephrine in the central nervous system, inducing antinociception.19 Third, there is synergy in the antinociceptive effect between the cannabinoid agonist WIN 55,212-2 and the α2 adrenoceptor agonist clonidine at the spinal level.21

The aim of this study was to determine whether the CB1 and CB2 cannabinoid agonists anandamide and PEA, respectively, induce peripheral antinociception through an interaction with the endogenous noradrenergic system.

METHODS

Animals

All experiments were performed on 160 g to 200 g male Wistar rats (from CEBIO-UFMG). The rats were housed in a temperature-controlled room (23 ± 1°C) on an automatic 12-hour light/dark cycle (06:00–18:00 hours). All tests were conducted during the light phase (08:00–15:00 hours). Food and water were freely available until the onset of the experiments. The animals were killed after experiments. All animal procedures and protocols were approved by the Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais.

Measurement of Hyperalgesia

Hyperalgesia was induced by subcutaneous injection of prostaglandin E2 (PGE2, 2 μg) into the plantar surface of the hindpaw. Hyperalgesia was measured according to the paw pressure test described by Green and Young26 and Randall and Sellito.27 An analgesimeter was used (Ugo-Basile, Comerio, Italy) with a cone-shaped paw-presser with a rounded tip, which applies a linearly increasing force to the hindpaw. The weight in grams (g) required to elicit the nociceptive response of paw flexion was determined as the nociceptive threshold. A cutoff value of 300 g was used to reduce the possibility of damage to the paws. The nociceptive threshold was measured in the right paw and determined as the average of the 3 consecutive trials recorded before and 3 hours after PGE2 injection. Hyperalgesia was calculated as the difference between these 2 averages (Δ of nociceptive threshold) and expressed in grams.

Drug Administration

All drugs were administered using an injected volume of 50 μL/paw, with the exception of PGE2, 100 μL/paw. The CB1 cannabinoid receptor agonist anandamide (Tocris Bioscience, Minneapolis, MN) was dissolved in Tocrisolve™ 100 10%; the CB2 cannabinoid receptor agonist PEA (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide 6% in saline; the α2 adrenoceptor antagonist yohimbine (Sigma), the α2A adrenoceptor antagonist BRL 44 480 (2-[{4,5-Dihydro-1H-imidazol-2-yl}methyl]-2,3-dihydro-1-methyl-1H-isoindole maleato; BRL; Tocris), the α2B adrenoceptor antagonist imiloxan (Tocris), the α2C adrenoceptor antagonist rauwolscine (Tocris), the α2D adrenoceptor antagonist RX 821002 (2-[2,3-Dihydro-2-methoxy-1,4-benzodioxin-2-yl]-4,5-dihydro-1H-imidazole hydrochloride; RX; Tocris), the α1 adrenoceptor antagonist prazosin (Sigma), the β adrenoceptor antagonist propranolol (Sigma), the norepinephrine depletory guanethidine (Sigma), and the norepinephrine reuptake inhibitor reboxetine (Pfizer, New York, NY) were dissolved in saline. The CB1 cannabinoid antagonist AM251 (1-[2,4-dichlorophenyl]-5-[4-iodophenyl]-4-methyl-N-[1-piperidyl] pyrazole-3-carboxamide; Tocris) and the CB2 cannabinoid antagonist AM630 (6-Iodo-2-methyl-1-[2-{4-morpholinyl}ethyl]-1H-indol-3-yl) [4-ethoxyphenyl] methanone; Tocris) were dissolved in dimethyl sulfoxide 10%, whereas PGE2 (Sigma) was dissolved in ethanol 2% in isotonic saline.

Experimental Protocol

Anandamide or PEA was administered in the right hindpaw 2:55 hours after local injection of PGE2. In the protocol used to determine whether anandamide or PEA was acting in the central nervous system or the contralateral paw, PGE2 was injected into both hindpaws, whereas anandamide or PEA was administered just into the right paw. After 5 minutes, the nociceptive threshold was measured in both hindpaws. Yohimbine was injected 40 minutes before anandamide or PEA. BRL 44 480, IMI, rauwolscine, RX 821002, prazosin, propranolol, and reboxetine were administered 30 minutes before anandamide or PEA. AM251 and AM630 were administered 15 minutes before cannabinoid agonists. Guanethidine, 30 mg/kg, was injected to deplete peripheral norepinephrine once a day for 3 days before the threshold measures as described by Nakamura and Ferreira.28 The protocols concerning dose and time of administration of each drug used in this study were obtained through literature data and pilot experiments.

Statistical Analysis

The data were statistically analyzed by 1-way analysis of variance with post hoc Bonferroni test for multiple comparisons. All data are expressed as the mean (SD) in n = 4 animals. P < 0.05 was considered to be statistically significant.

RESULTS

Participation of the CB1 and CB2 Cannabinoid Receptors in the Peripheral Antinociceptive Effect Induced by Anandamide and PEA

The administration of anandamide, 12.5 ng/paw, 25 ng/paw, and 50 ng/paw, or PEA, 5 μg/paw, 10 μg/paw, and 20 μg/paw into the right hindpaw produced an antinociceptive response against PGE2-induced hyperalgesia, 2 μg/paw, in a dose-dependent manner (Fig. 1A and B). Despite the fact that high doses of anandamide or PEA were able to almost completely reverse the hyperalgesia induced by PGE2, it was found that these doses alone did not alter the nociceptive threshold. When administered into the right hindpaw, anandamide, 50 ng/paw, or PEA, 20 μg/paw, did not produce an antinociceptive effect in the left hindpaw, indicating that, at this dose, they are only locally effective (Fig. 1A and B insert).

Figure 1
Figure 1:
Effect of anandamide (AEA) on prostaglandin (PG)E2–induced hyperalgesia in rats (A). AEA (ng/paw) was administrated 2:55 hours after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in Materials and Methods. # indicates a significant difference from the PGE2 + vehicle (veh) 1-injected group (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve™ 100 10% in saline; Veh 2 = Ethanol 2% in saline. Exclusion of outside paw antinociceptive effect of AEA (Insert A). PGE2 (2 μg) was administered in both hindpaws, right (R) and left (L). AEA (50 ng/paw) was administrated 2:55 hours after PGE2 in the right hindpaw. Antinociceptive responses were measured in both hindpaws. # indicates a significant difference from the PGE2 R paw + veh 1 R paw-injected group (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = Tocrisolve 100 10% in saline. Effect of N-palmitoyl-ethanolamine (PEA) on PGE2–induced hyperalgesia in rats (B). PEA (μg/paw) was administrated 2:55 hours after local administration of PGE2 (2 μg). Antinociceptive response was measured by the paw pressure test, as described in Materials and Methods. # indicates a significant difference from the PGE2 + veh 1-injected group (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide (DMSO) 6% in saline; Veh 2 = ethanol 2% in saline. Exclusion of outside paw antinociceptive effect of PEA (Insert B). PGE2 (2 μg) was administered in both hindpaws, right (R) and left (L). PEA (20 μg/paw) was administrated 2:55 hours after PGE2 in the right hindpaw. Antinociceptive responses were measured in both hindpaws. # indicates a significant difference from the PGE2 R paw + veh 1 R paw-injected group (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = DMSO 6% in saline. Effect induced by AM251 or AM630 of the peripheral AEA-induced antinociception (C). AM251 (μg/paw) and AM630 (μg/paw) were administered 15 minutes before AEA (50 μg/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + AEA 50 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = Tocrisolve 100 10% in saline; Veh 2 = DMSO 10%; Veh 3 = ethanol 2% in saline. Effect induced by AM251 or AM630 of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (D). AM251 (μg/paw) and AM630 (μg/paw) were administered 15 minutes before PEA (20 μg/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + PEA 20 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = DMSO 6% in saline; Veh 2 = DMSO 10%; Veh 3 = ethanol 2% in saline.

The selective CB1 cannabinoid receptor antagonist AM251, 20 μg/paw, 40 μg/paw, and 80 μg/paw, was able to block the peripheral antinociceptive effect of anandamide, 50 ng/paw (Fig. 1C), but not the effect of PEA, 20 μg/paw, (Fig. 1D). Conversely, the selective CB2 cannabinoid receptor antagonist AM630, 25 μg/paw, 50 μg/paw, and 100 μg/paw, antagonized the peripheral antinociceptive effect of PEA, 20 μg/paw, (Fig. 1D), but not the effect of anandamide, 50 ng/paw, (Fig. 1C). AM251, 80 μg/paw, or AM630, 100 μg/paw, when injected alone, did not induce any effect in hyperalgesic or normal paws.

Participation of Adrenoceptors in the Peripheral Antinociceptive Effect Induced by Anandamide and PEA

Participation of the α2 adrenoceptor in the peripheral antinociceptive effect induced by anandamide, 50 ng/paw, or PEA, 20 μg/paw, was verified using the classic, nonselective α2 adrenoceptor antagonist yohimbine, 5 μg/paw, 10 μg/paw, and 20 μg/paw, which dose-dependently antagonized the peripheral effect of the drugs tested (Fig. 2A and B). Yohimbine did not induce hyperalgesia or antinociception when administered alone.

Figure 2
Figure 2:
Antagonism induced by yohimbine of the peripheral anandamide (AEA)-induced antinociception (A). Yohimbine (YOH; μg/paw) was administered 40 minutes before AEA (50 ng/paw). # and * indicate a significant difference compared with (PGE2 + vehicle [veh] 1 + veh 2) and (PGE2 + AEA 50 + veh 2)-injected controls, respectively (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve 100 10% in saline; Veh 2 = saline, Veh 3 = ethanol 2% in saline. Antagonism induced by yohimbine of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). Yohimbine (YOH; μg/paw) was administered 40 minutes before PEA (20 μg/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + PEA 20 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide [DMSO] 6% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline.

Evidence that anandamide or PEA induce peripheral antinociception by a specific α2 adrenoceptor is shown in Figure 3A and B. In these figures, it is possible to observe that the α2C adrenoceptor antagonist rauwolscine, 10 μg/paw, 15 μg/paw, and 20 μg/paw, dose-dependently blocked the peripheral antinociceptive effect of anandamide, 50 ng/paw, or PEA, 20 μg/paw. No effect induced by rauwolscine was seen when it was injected alone into normal or hyperalgesic paws.

Figure 3
Figure 3:
Antagonism induced by rauwolscine (RAU) of the peripheral anandamide (AEA)-induced antinociception (A). Rauwolscine (RAU; μg/paw) was administered 30 minutes before AEA (50 ng/paw). # and * indicate a significant difference compared with (prostaglandin [PG]E2 + vehicle [veh] 1 + veh 2) and (PGE2 + AEA 50 + veh 2)-injected controls, respectively (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve™ 100 10% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline. Antagonism induced by rauwolscine of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). RAU (μg/paw) was administered 30 minutes before PEA (20 μg/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + PEA 20 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide (DMSO) 6% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline.

The involvement of other α2 adrenoceptor subtypes in peripheral antinociception induced by anandamide or PEA was rejected in the present experiments because the α2 adrenoceptor antagonists for subtypes A, B, and D (BRL 44 480, IMI, and RX 82100, respectively, 20 μg/paw) did not significantly reduce the peripheral effect of the drugs tested (Fig. 4A and B).

Figure 4
Figure 4:
Effect induced by BRL 44 480 (BRL), imiloxan (IMI) and RX 821002 (RX; μg/paw) of the peripheral anandamide (AEA)-induced antinociception (A). All antagonists were administered 30 minutes prior AEA (50 ng/paw). # indicates a significant difference from the prostaglandin (PG)E2 + vehicle (veh)1 + veh 2-injected group (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve 100 10%; Veh 2 = saline. Effect induced by BRL, IMI, and RX (μg/paw) of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). All antagonists were administered 30 minutes prior PEA (20 μg/paw). # indicates a significant difference from the prostaglandin (PG)E2 + veh 1 + veh 2-injected group (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide (DMSO) 6%; Veh 2 = saline.

Administration of the nonselective α1 and β adrenoceptor antagonists prazosin, 0.5 μg/paw, 1 μg/paw, and 2 μg/paw, and propranolol, 150 ng/paw, 300 ng/paw, and 600 ng/paw dose-dependently antagonized the peripheral antinociceptive effect induced by anandamide, 50 ng/paw, or PEA, 20 μg/paw, indicating the participation of these adrenoceptors in the antinociceptive effect (Figs. 5A, B and A, B). No antagonist tested induced hyperalgesia or antinociception when administered alone.

Figure 5
Figure 5:
Antagonism induced by prazosin (PRA) of the peripheral anandamide (AEA)-induced antinociception (A). PRA (μg/paw) was administered 30 minutes before AEA (50 ng/paw). # and * indicate a significant difference compared with (prostaglandin [PG]E2 + vehicle [veh] 1 + veh 2) and (PGE2 + AEA 50 + veh 2)-injected controls, respectively (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve 100 10% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline. Antagonism induced by PRA of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). PRA (μg/paw) was administered 30 minutes before PEA (20 μg/paw). # and * indicate a significant difference compared with (prostaglandin [PG]E2 + veh 1 + veh 2) and (PGE2 + PEA 20 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide (DMSO) 6% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline.
Figure 6
Figure 6:
Antagonism induced by propranolol (PROP) of the peripheral anandamide (AEA)-induced antinociception (A). PROP (μg/paw) was administered 30 minutes before AEA (50 ng/paw). # and * indicate a significant difference compared with (prostaglandin [PG]E2 + vehicle [veh] 1 + veh 2) and (PGE2 + AEA 50 + veh 2)-injected controls, respectively (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test). Veh 1 = Tocrisolve 100 10% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline. Antagonism induced by PROP of the peripheral N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). PROP (μg/paw) was administered 30 minutes before PEA (20 μg/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + PEA 20 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = dimethyl sulfoxide (DMSO) 6% in saline; Veh 2 = saline; Veh 3 = ethanol 2% in saline.

Involvement of Endogenous Norepinephrine in the Peripheral Antinociceptive Effect Induced by Anandamide and PEA

The involvement of endogenous norepinephrine in anandamide and PEA-induced peripheral antinociception was confirmed using the norepinephrine depletor guanethidine, and the norepinephrine reuptake inhibitor reboxetine. Guanethidine (30 mg/kg/animal, once a day for 3 days) restored approximately 70% of the peripheral antinociceptive effect induced by anandamide, 50 ng/paw, or PEA, 20 μg/paw, (Fig. 7A and B). An injection of reboxetine alone, 30 μg/paw, intensified the peripheral antinociceptive effect of low-dose anandamide, 12.5 ng/paw, and PEA, 5 μg/paw (Fig. 7C and D). Guanethidine or reboxetine injection did not induce any effect in PGE2-induced hyperalgesia, 2 μg/paw.

Figure 7
Figure 7:
Effect induced by guanethidine (GUA) of the anandamide (AEA)-induced antinociception (A). GUA was administered 30 mg/kg/animal once a day for 3 days before PGE2. #, *, and Δ indicate a significant difference from the (vehicle [veh] 1 + PGE2 + veh 2), (veh 1 + prostaglandin [PG]E2 + AEA 50), and (GUA + PGE2 + veh 2)-injected group (P < 0.05, analysis of variance [ANOVA] + the Bonferroni test).Veh 1 = saline; Veh 2 = Tocrisolve 100 10% in saline. Effect induced by guanethidine of the N-palmitoyl-ethanolamine (PEA)-induced antinociception (B). GUA was administered 30 mg/kg/animal once a day for 3 days before PGE2. #, *, and Δ indicate a significant difference from the (veh 1 + PGE2 + veh 2), (veh 1 + PGE2 + PEA 20), and (GUA + PGE2 + veh 2)-injected group (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = saline; Veh 2 = dimethyl sulfoxide (DMSO) 6% in saline. Effect induced by reboxetine (REB) of the peripheral anandamide (AEA)-induced antinociception (C). REB (30 μg/paw) was administered 30 minutes before AEA (12.5 ng/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + AEA 12.5 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = saline; Veh 2 = Tocrisolve 100 10% in saline; Veh 3 = ethanol 2% in saline. Effect induced by REB of the peripheral PEA-induced antinociception (D). REB (30 μg/paw) was administered 30 minutes before PEA (5 ng/paw). # and * indicate a significant difference compared with (PGE2 + veh 1 + veh 2) and (PGE2 + PEA 5 + veh 2)-injected controls, respectively (P < 0.05, ANOVA + the Bonferroni test). Veh 1 = saline; Veh 2 = dimethyl sulfoxide (DMSO) 6% in saline; Veh 3 = ethanol 2% in saline.

DISCUSSION

It is well established that the endocannabinoids anandamide and PEA exert inhibitory effects on mechanical hyperalgesia.29 This study demonstrated that anandamide and PEA elicited a peripheral antinociceptive effect in rat paw PE2-induced hyperalgesia, as observed in the paw mechanical pressure test.

Besides the central cannabinoid antinociceptive effect,30 a possible peripheral antinociceptive action has also been proposed for CB1 cannabinoid receptor agonists in the carrageenan,31 formalin,32 neuropathic,33 cancer,34 and capsaicin35 models. The activation of the CB2 cannabinoid receptor inhibited thermal, mechanical,36,37 and inflammatory nociception.37,38 We previously showed the peripheral antinociceptive effect of anandamide in the same experimental model39 and that PEA could reduce inflammatory pain elicited by intraplantar injection of formalin, complete Freund’s adjuvant and carrageenan.40

There are conflicting data on the selectivity of the cannabinoid agonists for antinociception. Anandamide activated CB1 or CB2 cannabinoid receptors.41 Peripherally administered, anandamide attenuated inflammatory31,32 and noninflammatory hyperalgesia by a selective CB1 receptor-mediated mechanism.39 PEA has been classified as both a CB2 cannabinoid agonist29,42–44 and a nuclear receptor peroxisome proliferator-activated receptor-α agonist.40 On the other hand, it was shown that PEA induces antiallodynic and antihyperalgesic effects in a murine model of neuropathic pain, activating the CB1, transient receptor potential vanilloid 1 and peroxisome proliferator-activated receptor-α receptors.45

In this present study, using the CB1 and CB2 selective cannabinoid antagonists AM251 and AM630, respectively, we observed the selectivity of anandamide for the CB1 cannabinoid receptor and PEA for the CB2 cannabinoid receptor in the experimental model tested. These results are in agreement with the consistent anatomic location of CB146,47 and CB248 cannabinoid receptor subtypes demonstrated in dorsal root ganglion (DRG).

The present investigation of the role of the noradrenergic system in the peripheral antinociceptive effect of anandamide and PEA verified that the classic nonselective α2 adrenoceptor antagonist yohimbine antagonized the antinociceptive effect of the drugs tested. The α2 adrenoceptor is classified in α2A, α2B, α2C, and α2D subtypes based on radioligand-binding studies in the DRG49,50 and pharmacologic classification.51,52 The antinociceptive effect of α2 adrenoceptor agonists seems to be mediated by specific receptor subtypes.23,53 In this context, and in contrast with in vitro binding data,54 a preferential requirement for α2A or α2C subtypes in vivo assays of the antinociceptive effect was demonstrated,55,56 underscoring the importance of whole animal studies. In the present investigation of the selective α2A, α2B, α2C, and α2D adrenoceptor antagonists, it was verified that only the α2C subtype could be implicated in the peripheral antinociceptive effect of anandamide and PEA.

Despite the extensive involvement in spinal23,53 and supraspinal23,57 antinociception, the nonparticipation of α2A adrenoceptors in the present study might be because they are not associated with analgesic effects in peripheral events.5,51 The nonparticipation of the α2D subtype is controversial because it is uncertain whether it exists as an actual separate subtype or as a more commonly accepted variant of subtype α2A. In addition, the α2B adrenergic receptor has been associated with peripheral hyperalgesia.51 Finally, the α2 adrenoceptor agonists, xylazine5 and clonidine51 induce peripheral antinociceptive effects mediated by only α2C adrenoceptors.

Matsuoka et al.58 demonstrated that the central antinociception induced by PEA was enhanced by intracisternal injection of epinephrine or clonidine and was attenuated by intracisternal injection of phentolamine or yohimbine. However, it was not affected by intracisternal injection of prazosin in the mouse hotplate method. The α159,60 and β261 subtypes were characterized in the DRG. They have traditionally been associated with peripheral hyperalgesia,1,28,62–64 but previous studies also describe their participation in peripheral antinociceptive events.8–11 The present study found evidence for the participation of α1 and β adrenoceptors in the peripheral antinociceptive mechanism of anandamide and PEA. There are reports indicating that this hypothesis is possible. Using cell cultures from the DRG of rats, Pluteanu et al.65 verified that epinephrine depolarized 46% of neurons observed and hyperpolarized 18% of neurons. Despite these data indicating that epinephrine has a more pronounced pronociceptive effect in DRG neurons, the data have led researchers to conclude that epinephrine or isoproterenol acting in the α1 and β adrenoceptors could induce a hyperpolarization in the DRG with a resultant decrease in neuronal excitability.

The participation of adrenoceptors in the antinociception mediated by cannabinoid agonists suggests that these drugs might be releasing endogenous norepinephrine. The possible release of norepinephrine by cannabinoid agonists has been reported in the literature.12,17,18

The CB1 agonist arachidonoylglycerol increased perivascular nerve stimulation–evoked norepinephrine release, but this effect was not observed with anandamide or with the synthetic cannabinoid HU210.12 Another CB1 cannabinoid receptor agonist, CP 55,940, intrathecally induces antinociception that was not blocked by yohimbine in the formalin test.66 Systemically administered cannabinoid agonists could stimulate norepinephrine release in the frontal cortex by activating noradrenergic neurons in the coeruleofrontal cortex pathway.18 Intracisternal administration of norepinephrine increased the antinociception induced by PEA. PEA decreased norepinephrine in the brain and tended to increase the norepinephrine metabolite normetanephrine 15 minutes after administration, indicating a release of norepinephrine in the central nervous system, inducing antinociception.19

To evaluate the hypothesis that norepinephrine is involved in mediating peripheral cannabinoid antinociception in our model, we studied this effect in rats treated with guanethidine, a depletor of peripheral sympathomimetic amines,28 and the norepinephrine reuptake inhibitor, reboxetine.67 Without the presence of norepinephrine, guanethidine induced an approximately 70% reversal of the peripheral effect of anandamide and PEA. Furthermore, norepinephrine reuptake blockade by reboxetine resulted in an intensification of the peripheral antinociceptive effect induced by low-dose anandamide and PEA, indicating that CB1 and CB2 cannabinoid agonists induce peripheral antinociception and are partially dependent on the interaction with the noradrenergic system.

The cannabinoid receptors were characterized in cells of the immune system68 and in keratinocytes.68–70 Immune system cells71 or other resident cells, such as keratinocytes or melanocytes, can synthesize and release endogenous catecholamines.72,73 In addition to this cellular context, a CB12 receptor complex possibly exists in human kidney embryonic cells, indicating a physical and functional interaction between cannabinoid receptor and adrenoceptor.74

Thus, knowing that α2C adrenoceptor activation inhibits the excitation of the DRG5 and that the activation of α1 and β adrenoceptors can induce hyperpolarization with consequent desensitization of the DRG,65 the present research provides evidence showing that besides direct activation of the CB1 and CB2 cannabinoid receptor in nociception, the cannabinoid agonists can activate their respective receptors on cells. This initializes an indirectly dependent mechanism of endogenous norepinephrine release to induce antinociception via adrenergic receptors. Moreover, one cannot exclude the possibility of a direct activation of a “cannabinoid-adrenoceptor complex” in nociception.

In conclusion, our results contribute to a greater understanding about norepinephrine as an important monoamine involved in endogenous pain modulation. It functions not only as a peripheral sympathetic component of inflammatory hyperalgesia28 or in an analgesic-dependent pathway,75 but also functions as an important endogenous local mediator of peripheral antinociception via α2C, α1 and β adrenoceptor activation.

DISCLOSURES

Name: Thiago R. L. Romero, PhD.

Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.

Attestation: Thiago R. L. Romero has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Livia C. Resende, MD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Livia C. Resende has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Luciana S. Guzzo, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Luciana S. Guzzo has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Igor D. G. Duarte, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Igor D. G. Duarte has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Steven L. Shafer, MD.

REFERENCES

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