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Page 1: The antinociceptive effect of Δ9-tetrahydrocannabinol in the arthritic rat involves the CB2 cannabinoid receptor

logy 570 (2007) 50–56www.elsevier.com/locate/ejphar

European Journal of Pharmaco

The antinociceptive effect of Δ9-tetrahydrocannabinol in the arthritic ratinvolves the CB2 cannabinoid receptor

Melinda L. Cox, Victoria L. Haller ⁎, Sandra P. Welch

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298-0524, United States

Received 11 August 2006; received in revised form 10 May 2007; accepted 15 May 2007Available online 5 June 2007

Abstract

Cannabinoid CB2 receptors have been implicated in antinociception in animal models of both acute and chronic pain. We evaluated the roleboth cannabinoid CB1 and CB2 receptors in mechanonociception in non-arthritic and arthritic rats. The antinociceptive effect of Δ9-tetrahydrocannabinol (Δ9THC) was determined in rats following administration of the cannabinoid CB1 receptor-selective antagonist,SR141716A, the cannabinoid CB2 receptor-selective antagonist, SR144528, or vehicle. Male Sprague–Dawley rats were rendered arthritic usingFreund’s complete adjuvant and tested for mechanical hyperalgesia in the paw-pressure test. Arthritic rats had a baseline paw-pressure of 83 ±3.6g versus a paw-pressure of 177 ± 6.42g in non-arthritic rats. SR144528 or SR141716A (various doses mg/kg; i.p.) or 1:1:18 (ethanol:emulphor:saline) vehicle were injected 1 h prior to Δ9THC (4mg/kg; i.p) or 1:1:18 vehicle and antinociception determined 30min post Δ9THC. AD50's forboth antagonists were calculated with 95% confidence limits. In addition, midbrain and spinal cord were removed for determination ofcannabinoid CB1 and CB2 receptor protein density in the rats. SR144528 significantly attenuated the antinociceptive effect of Δ9THC in thearthritic rats [AD50 = 3.3 (2.7–4) mg/kg], but not in the non-arthritic rats at a dose of 10/mg/kg. SR141716A significantly attenuated Δ9THC-induced antinociception in both the non-arthritic [AD50 = 1.4 (0.8–2) mg/kg] and arthritic rat [AD50 = 2.6 (1.8–3.1) mg/kg]. SR141716A orSR144528 alone did not result in a hyperalgesic effect as compared to vehicle. Our results indicate that the cannabinoid CB2 receptor plays acritical role in cannabinoid-mediated antinociception, particularly in models of chronic inflammatory pain.Published by Elsevier B.V.

Keywords: Cannabinoid; CB1 receptor; CB2 receptor; SR141716A; SR144528; Arthritis

1. Introduction

Cannabinoids have been shown to produce pain relief in avariety of animal models (Richardson, 2000). Cannabinoid CB1

receptors are found primarily in the central nervous system(Matsuda et al., 1990), while CB2 cannabinoid receptors arefound outside the central nervous system, mainly in peripheraltissues with immune function (Munro et al., 1993). Cannabi-noid CB2 receptors have been implicated in the production ofantinociception in animal models of both acute and chronicpain. Administration of the cannabinoid CB2 receptor agonist,AM1241, administered systemically or locally produces acutethermal antinociception inhibited by the cannabinoid CB2

receptor antagonist, AM630, and not the cannabinoid CB1

receptor antagonist AM251 (Malan et al., 2001). AM1241 also

⁎ Corresponding author. Tel.: +1 804 828 8446; fax: +1 804 828 2117.E-mail address: [email protected] (V.L. Haller).

0014-2999/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.ejphar.2007.05.024

inhibits formalin-induced nociception (Malan et al., 2002) andsuppresses development of intradermal capsaicin-evoked ther-mal and mechanical hyperalgesia and allodynia, and nocifen-sive behavior (Hohmann et al., 2004). These effects are blockedby the cannabinoid CB2 receptor antagonist, N-[(1S)-Endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide(SR144528), but not by the cannabinoid CB1 receptor antag-onist, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidine-1-yl)-1H-pyrazole-3-carboxamide (SR141716A).Furthermore, the reversal by AM1241 of tactile and thermalhypersensitivity occurred in cannabinoid CB1 receptor knock-out mice, confirming its actions independent of the cannabinoidCB1 receptor (Ibrahim et al., 2003). A non-selective cannabi-noid CB1/ CB2 agonist, CP55, 940, attenuates acute thermalnociceptive responses, as well as neuropathic tactile allodyniafollowing spinal nerve ligation in the rat. The cannabinoidCB1 receptor antagonist, SR141716A, inhibits only the acute

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antinociception to a thermal stimulus, while the cannabinoidCB2 receptor antagonist, SR144528, inhibits CP55, 940-induced antinociception in both cases. SR141716A andSR144528 produce greater-than-additive inhibition of theantinociceptive effect of CP55, 940 (Scott et al., 2004). Thecannabinoid CB2 receptor agonist, HU-308, inhibits nocicep-tion in the late phase after injection of formalin, an effectblocked by SR144528 (Hanus et al., 1999). Another cannabi-noid CB2 agonist, GW405833, produces mechanical anti-hyperalgesia in arthritic rats and decreases tactile allodynia,effects not observed in cannabinoid CB2 receptor knockoutmice. Calignano et al. (1998) showed that palmitoylethanola-mide inhibition of formalin-evoked nociception to thermalstimuli in the mouse paw is blocked by SR144528. The abovestudies indicate that activation of a cannabinoid CB2 receptormechanism is sufficient to suppress the transmission ofinflammation-evoked neuronal activity. In addition, it hasbeen shown that activation of peripheral cannabinoid CB2receptors attenuates innocuous-and noxious mechanical stimuli-evoked responses of wide dynamic range neurons in rat modelsof acute, neuropathic, and inflammatory pain (Elmes et al.,2004).

In the present study, we evaluated the role of the cannabinoidCB1 and CB2 receptors in mechanociception in non-arthriticand arthritic rats by the administration of Δ9-tetrahydrocan-nabinol (Δ9THC), a mixed cannabinoid CB1/CB2 receptoragonist alone, or following administration of cannabinoid CB1

and CB2 receptor-selective antagonists. In addition, weevaluated the midbrain and spinal cord tissues of both arthriticand non-arthritic rats for the density of cannabinoid CB1 andCB2 receptor proteins using Western immunoblotting. Our dataindicate that SR144528 is selective in blocking Δ9THC-induced antinociception in arthritic rats. However, no differ-ences in receptor protein density for either cannabinoid receptorwere observed in arthritic versus non-arthritic rats.

2. Methods

2.1. Animals

Male Sprague–Dawley rats (Harlan Laboratories, Indiana-polis, IN), which weighed 350 to 375g were housed in ananimal care facility maintained at 22 ± 2°C on a 12-hr light/darkcycle with free access to food and water. All experiments wereconducted according to guidelines established by the Institu-tional Animal Care and Use Committee of Virginia Common-wealth University, and adhered to the European Communityguidelines for the use of experimental animals.

2.2. Freund's adjuvant-induced arthritis treatment

A volume of 0.1ml of vehicle (mineral oil) or Freund’scomplete adjuvant (heat-killed Mycobacterium butyricum;5mg/ml) was injected intradermally into the base of the tail(Cox and Welch, 2004; Millan et al., 1986a). Animals remainedin their cages for 12days and were acclimated to paw-pressuretesting until day 19, on which they were tested. Inflammation

proceeds into a generalized polyarthritis within 19days. Paw-pressure baseline measurements on day 19 indicated that theFreund's adjuvant-induced arthritic rats are more sensitive tomechanical nociception than non-arthritic rats.

2.3. Paw-pressure test

The paw-pressure test consisted of gently holding the bodyof the rat while the hind-paw was exposed to increasingmechanical pressure. The Analgesy-Meter (Ugo-Basile, Varese,Italy) is designed to exert a force on the paw that increases at aconstant rate, similar to the Randall and Sellito (1957) test ofmechanical nociception. Force was applied to the hind-paw thatwas placed under a small plinth under a cone-shaped pusherwith a rounded tip. The operator depressed a pedal-switch tostart the mechanism that exerted force. The force in grams atwhich the rat withdrew its paw was defined as the paw-pressurethreshold. The baseline paw-pressure was measured beforeinjecting vehicle or drug. Non-arthritic rats, with a baselinepaw-pressure greater than 100g (average = 177 ± 6.4g), wereused in further testing. Freund's adjuvant-induced arthritic ratswith a baseline paw-pressure less than 100g (average = 83 ±3.6g) were used in further experimentation. The upper limit of500g was imposed for the experiments to allow the foot to notbecome immobilized due to undue pressure.

2.4. Administration of drugs

Arthritic and non-arthritic rats were injected with SR144528(Research Triangle Institute, Research Triangle Park, NC),SR141716A (Pfizer, Groton, CT) or vehicle (1:1:18, emulphor:ethanol:saline; various doses; i.p.) tested 1 h later formechanical nociception, the time of the peak antinociceptiveantagonism previously shown for SR144528 and SR141716A(Anikwue et al, 2002; Haller et al, 2006). Δ9-THC [4 mg/kg; i.p. National Institute on Drug Abuse (Rockville, MD)] in 1:1:18vehicle was administered and rats were tested again 30min later.We have previously published evaluation of the dose–effectcurves for Δ9THC in arthritic and non-arthritic rats and havedetermined the peak time for Δ9THC activity in the paw-pressure test in the rats (Cox and Welch, 2004).

2.5. Statistical evaluation

Dose–response curves were generated using three or fourdoses of antagonist in combination with the ED80 dose ofΔ9THC (4mg/kg, i.p.). AD50 values and 95% confidence limitswere determined for each antagonist using the methods ofTallarida and Murray (1987). Mechanical nociception wasquantified as the percent maximum possible effect (%MPE),determined for each rat based on the formula %MPE = [test (g) −baseline (g) / 500g − baseline (g)] × 100 (Cox and Welch,2004). At least 6 rats were used for each treatment group.Animal studies were carried out in accordance with theguidelines established by the Institutional Animal Care andUse Committee of Virginia Commonwealth University andadhere to the National Institute of Health Guide for the care

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Fig. 1. Cannabinoid CB1 receptor antagonist, SR141716A (0.3 to 10 mg/kg, i.p.),administered 1 hour before Δ9-THC (4 mg/kg, i.p.) significantly attenuated theantinociceptive effect of Δ9-THC in arthritic rats. Animals (6 per treatment) weretested for antinociception 30 min after Δ9-THC administration. The %MPE foreach rat was determined. The AD50 for SR141716A was determined as in the“Methods”.

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and Use of Laboratory Animals (NIH Publications No. 80-23, 1996).

2.6. Tissue preparation for Western immunoassays

Non-arthritic and arthritic naïve rats were sacrificed bydecapitation. Spinal cords and midbrains (2 animals pooled persample) were removed separately and quickly frozen on dry ice.All tissue was stored at − 80°C until prepared. Frozen tissue wasallowed to thaw, then homogenized in 10 ml of cold suspensionbuffer [10mmol EGTA, 20mmol EDTA, 10mmol Tris, 20mmolβ-glycerophosphate, 50mmol sodium fluoride, 50mmol sodiumpyrophosphate, 1mmol p-nitrophenylphosphate, 200μ molmicrocystin LR (Sigma Chemical Co., St. Louis, MO)] andprotease inhibitor cocktail (Boehringer Mannheim, Indianapo-lis, IN). A P2 membrane preparation was made with a 10min1500×g centrifugation, followed by a 30min spin of thesupernatant at 48,000×g at 4°C. The second pellet wasresuspended in cold suspension buffer plus detergents (1%Triton X-100 and 0.5% Igepal). The pellet was slowlyhomogenized for 30s and placed on a rocking platform for1 h at 4°C. The samples were then centrifuged for 20min at17,000×g to remove insoluble material. Protein concentrationswere determined by the Bio-Rad DC protein assay (Hercules,CA). Tissue was aliquoted and stored at − 80°C.

2.7. Western immunoassays

Electrophoresis was performed using a standard Laemmlimethod. Samples were diluted 1:1 with 2× sample buffer to aconcentration of 10μg and loaded in a 1.5mm 8% sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)gel. Following protein separation, transfer onto Immobilon-Ppolyvinylidene fluoride (PVDF) membrane (Millipore, Bedford,MA) was performed by tank method. Blots were blocked at least1h in casein blocker in TBS (Pierce, Rockford, IL). The blots wereincubated overnight at room temperature with the CB1 cannabi-noid receptor antibody (Invitrogen-BioSource Cytokines &Signaling, Camarillo, CA) at a 1:100 dilution, and the CB2

cannabinoid receptor antibody (Cayman Chemical, Ann Arbor,MI) at a 1:250 dilution in casein blocker. After washing 3 times inTris-buffered saline plus 0.5% Tween 20 (TBST) for 5min, blotswere incubated in horseradish peroxidase-conjugated goat anti-

Table 1Absorbance readings for cannabinoid receptor proteins

Treatment Absorbance

CB1 CB2

MidbrainNon-arthritic 0.314±0.06 0.355±0.07Arthritic 0.288±0.05 0.455±0.04

Spinal cordNon-arthritic 0.301±0.08 0.194±0.02Arthritic 0.344±0.05 0.212±0.02

Absorbance readings were obtained using scanning densitometry. The data ispresented as the average absorbance of four trials±S.E.M.

rabbit IgG antiserum (Sigma Chemical Co.) at a 1:50,000 dilutionin casein blocker for 2h at room temperature. Blots were againwashed 3 times in TBST and then incubated 5min in SuperSignalWest Pico CL-HRP chemiluminescence (Pierce, Rockford, IL).Blots were then exposed on XAR-2 film (Eastman Kodak,Rochester, NY). Bands were quantified using scanning densitom-etry, and comparisons of absorbance values were done byStudent's unpaired t-test. As a control for gel loading accuracy,some blots were stripped and reprobedwith anti-GAPDHantibody(Abcam, Inc, Cambridge, MA). A peptide blocking experimentwas conducted by pre-incubating the antibody with the blockingpeptide 1:1 in casein blocker 1h prior to incubation of the blot.

2.8. Antibodies

The anti-cannabinoid CB1 receptor antibody was obtainedfrom Invitrogen-BioSource Cytokines & Signaling (Camarillo,CA), and was produced against a synthetic peptide from the N-terminal region of the human central cannabinoid receptor witha molecular weight of approximately 64kDa. The anti-cannabinoid CB2 receptor antibody was obtained from CaymanChemical (Ann Arbor, MI), and was produced against asynthetic peptide sequence between the N-terminus and the firsttransmembrane domain of the human CB2 cannabinoid receptorwith an approximate molecular weight of 45kDa. The anti-GAPDH antibody used for the loading control was obtainedfrom Abcam, Inc. (Cambridge, MA), and was produced againstrabbit muscle glyceraldehydes-3-phosphate dehydrogenase(GAPDH) with an approximate molecular weight of 36kDa.

2.9. Western immunoassays

To further examine any differences between the non-arthriticand arthritic rats, levels of receptor protein in both midbrain andspinal cord were measured in non-arthritic and arthritic naïve

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Fig. 2. Cannabinoid CB2 receptor antagonist, SR144528 (1 to 10 mg/kg, i.p.),administered 1 hour before Δ9-THC (4 mg/kg, i.p.) significantly attenuated theantinociceptive effect of Δ9-THC in arthritic rats. Animals (6 per treatment)were tested for antinociception 30 min after Δ9-THC administration. The %MPE for each rat was determined. The AD50 for SR141716Awas determined asin the “Methods”.

Fig. 4. Cannabinoid CB2 receptor antagonist, SR144528 (1, 3 and 10 mg/kg, i.p.),administered 1 hour beforeΔ9-THC (4mg/kg, i.p.) did not significantly attenuatedthe antinociceptive effect of Δ9-THC in non-arthritic rats. Animals (6 pertreatment) were tested for antinociception 30 min after Δ9-THC administration.The %MPE for each rat was determined.

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rats (N = 2 rats) using Western immunoassays. Table 1 lists theabsorbance readings of bands from the Western immunoblots.No differences in receptor protein were detected.

3. Results

3.1. In vivo studies

We have previously shown that the ED50's for Δ9THC innon-arthritic and arthritic rats did not differ in the paw-pressuretest [2.1 (1.8–2.5) versus 2.5 (2.2–3.0) mg/kg, respectively](Cox and Welch, 2004). The ED80 for Δ9THC in both non-arthritic and arthritic rats was 4mg/kg, when administered i.p.

Fig. 3. Cannabinoid CB1 receptor antagonist, SR141716A (0.3 to 10 mg/kg, i.p.),administered 1 hour before Δ9-THC (4 mg/kg, i.p.) significantly attenuated theantinociceptive effect of Δ9-THC in non-arthritic rats. Animals (6 per treatment)were tested for antinociception 30 min after Δ9-THC administration. The %MPEfor each rat was determined. The AD50 for SR141716Awas determined as in the“Methods”.

30min prior to testing. Thus, we chose a similar dose ofΔ9THCand time point for the studies of antagonists.

Both the cannabinoid CB1 and the CB2 receptor-selectiveantagonists significantly attenuated the antinociceptive effect ofΔ9THC in the arthritic rat (Figs. 1 and 2). Administration ofΔ9THC (Fig. 1) resulted in a %MPE of 89 ± 5.5% and theantinociceptive effect was reduced in a dose-related manner bySR141716A, doses 0.3mg/kg–10mg/kg. The AD50 (+95%confidence limits) calculated for SR141716Awas 2.6 (1.8–3.1)mg/kg. Administration of Δ9THC (Fig. 2) resulted in a %MPE

Fig. 5. Representative gel indicating the quantity of cannabinoid CB2 receptorproteins in the spinal cord (Panel A) or midbrain (Panel B) of either non-arthritic(non-arth) or arthritic (arth) rats. The rats were “naïve” in that they did notreceive any drug treatments other than those to induce arthritis. N1–N4 indicatethat the gel reflects the results of four trials, either non-arthritic or arthritic.Statistical analyses of the gel indicated no significant differences between thearthritic and non-arthritic rats as shown in Table 1.

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of 90 ± 6% and the antinociceptive effect was reduced in a dose-related manner by SR144528, doses 1mg/kg–10mg/kg. TheAD50 (+ 95% confidence limits) calculated for SR144528 was3.3 (2.7–4) mg/kg and did not differ significantly from thatproduced by SR141716A due to overlap of confidence limits.Administration of either antagonist alone did not result in ahyperalgesic effect as compared to vehicle in arthritic rats (datanot shown).

Only the cannabinoid CB1 receptor-selective antagonistsignificantly attenuated the antinociceptive effect of Δ9THC inthe non-arthritic rat (Fig. 3). Administration of SR144528 atdoses up to 10mg/kg produced no attenuation of theantinociception produced by Δ9THC in non-arthritic rats(Fig. 4). Administration of Δ9THC (Fig. 3) resulted in a %MPE of 76 ± 3% and the antinociceptive effect was reduced in adose-related manner by SR141716A doses 0.3mg/kg–10mg/kg.The AD50 (+ 95% confidence limits) calculated forSR141716Awas 1.4 (0.8–2.0) mg/kg. Administration of eitherantagonist alone did not result in a hyperalgesic effect ascompared to vehicle in non-arthritic rats (data not shown).

3.2. In vitro studies

The midbrain and spinal cord from both non-arthritic andarthritic rats were removed and processed for evaluation ofcannabinoid CB1 and CB2 receptor proteins using Westernimmunoblotting as described in the Methods. Figs. 5 and 6indicate representative gels from naïve (no drug treatment) ratsthat are either non-arthritic or arthritic and from which themidbrain or spinal cord had been removed. Fig. 5—panel A

Fig. 6. Representative gel indicating the quantity of CB1 receptor proteins in thespinal cord (Panel A) or midbrain (Panel B) of either non-arthritic (non-arth) orarthritic (arth) rats. The rats were “naïve” in that they did not receive any drugtreatments other than those to induce arthritis. N1–N4 indicate that the gelreflects the results of four trials, either non-arthritic or arthritic. Statisticalanalyses of the gel indicated no significant differences between the arthritic andnon-arthritic rats as shown inTable 1.

indicates the cannabinoid CB2 protein density from the spinalcord of non-arthritic and arthritic rats. Fig. 5—panel B indicatesthe protein levels of the cannabinoid CB2 receptor from themidbrain. The densities of all proteins are listed in Table 1 andindicate no changes in any protein.

Fig. 6—panel A indicates the cannabinoid CB1 proteindensity from the spinal cord of non-arthritic and arthritic rats.Fig. 6—panel B indicates the protein levels of the cannabinoidCB1 receptor from the midbrain. “N” indicates a pooled sampleof two rats per trial that were evaluated in the experiments. InFigs. 5 and 6 four trials were evaluated for midbrain regions andspinal cord in the arthritic state and non-arthritic state. Table 1indicates the average absorbance readings of the four trials. Nosignificant differences in either receptor protein in either thenon-arthritic or the arthritic rats were observed.

4. Discussion

In this study, we determined if differences in efficacy andpotency of cannabinoid receptor-selective antagonists versusthe antinociceptive effects of Δ9THC exist in non-arthritic ratsversus those with Freund's adjuvant-induced chronic arthriticpain. Although Δ9THC is equipotent and equiefficacious in thenon-arthritic and arthritic rats (Cox and Welch, 2004), theantinociceptive effects of Δ9THC in arthritic rats are producedvia activation of both cannabinoid CB1 and CB2 receptors. Innon-arthritic rats, Δ9THC appears to produce antinociceptionvia cannabinoid CB1 receptor activation only. The differencesbetween the non-arthritic and arthritic rats appear not to be dueto an increase in cannabinoid receptor proteins in eithermidbrain or spinal cord tissue. However, one cannot rule outa more region-specific effect on cannabinoid CB1 or CB2

receptor proteins. We also cannot rule out a change in thenumber or affinity of the receptors in the arthritic rats given thatΔ9THC is a partial agonist for CB1 and CB2 receptors (Sugiuraet al, 1999; Sugiura et al, 2000).

Several studies have shown that cannabinoids produceantinociception in mice and rats through both supraspinal andspinal mechanisms (Lichtman and Martin, 1991). The preva-lence of cannabinoid CB2 receptors in immune cells hasdirected attention to an array of immunological disorders,including inflammation and pain. Early studies failed to detectcannabinoid CB2 receptors in the central nervous systemthereby suggesting cannabinoid CB2 selective agonists wouldbe devoid of cannabinoid CB1-like behavioral effects. However,cannabinoid CB2 receptors were recently identified in brainstemneurons which could be a possible site of action (Van Sickleet al., 2005). Our data clearly indicate the presence ofcannabinoid CB2 receptor protein in brain and cord homo-genates, although it is possible that such proteins are due to non-neuronal contaminants. Sciatic nerve section or spinal nerveligation causes an upregulation of cannabinoid CB2 receptors inthe dorsal horn of the spinal cord (Wotherspoon et al., 2005).However, we did not observe such an increase due to arthritis.These data are consistent with the results of Zhang et al. (2003),who showed no change in CB2 mRNA in arthritic rats. It isreasonable to speculate that the analgesic and anti-inflammatory

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effects of cannabinoid CB2 selective agonists and non-selectiveagonists such asΔ9THC result from a combination of actions atboth neuronal and immune sites. A large number of cannabinoidCB2 selective analogs have been developed and shown to beactive in wide range of pain models including neuropathic pain(Malan et al., 2002) and capsaicin-induced thermal andmechanical hyperalgesia (Hohmann et al., 2005; Quartilhoet al., 2003). Cannabinoid CB2 selective agonists are effectivein acute pain and inflammatory models at doses that do notproduce behavioral effects (Valenzano et al., 2005).

Our results are consistent with findings that the cannabinoidCB2 receptor plays a role in cannabinoid-mediated antinocicep-tion, particularly in models of chronic inflammatory pain. Thedistribution of the cannabinoid CB2 receptor makes it a perfectcandidate to play a role in nociception in an inflammatory paincondition. Cannabinoid CB2 receptors are found primarily inperipheral tissues and inhibit the release of inflammatorymediators that excite nociceptors (Mazzari et al., 1996; Munroet al., 1993). It is not known exactly how activation of peripheralcannabinoid CB2 receptors produces antinociception. There isevidence that cannabinoid CB2-like receptors are present onperipheral neurons (Griffin et al., 1997). In addition, cannabinoidCB2 receptors are found on cultured neonatal dorsal root ganglionneurons (Ross et al., 2001). The existence of cannabinoid CB2

receptors on primary afferent terminals has not been demonstrat-ed. Cannabinoid CB2 receptor agonists may act indirectly viamodulation of immune cell activity at the site of injury resulting indecreased local levels of agents that sensitize the peripheralnociceptor (Facci et al., 1995; Galiegue et al., 1995; Munro et al.,1993). Such a mechanism may explain cannabinoid CB2-mediated antinociception in inflammation, as in this study.

It has also been shown that in arthritic animals, a model ofchronic pain with high levels of dynorphin expression, Δ9THCreduces dynorphin levels (Cox and Welch, 2004) via bothcannabinoid CB1-and CB2-mediated mechanisms. Thus, can-nabinoid/opioid interactions in chronic pain states may differfrom those in normal animals. A recent review addresses severalanimal models in order to evaluate the complex cannabinoid/opioid interactions and the neurochemical substrates involvedin such interactions (Tanda and Goldberg, 2003). Theinteraction between Δ9THC and the dynorphin A systemcontributes to Δ9THC-induced antinociception in acute painmodels, such as the tail-flick and hot-plate tests. Less is knownabout antinociceptive effects of Δ9THC in mechanicalnociception and in chronic pain models. An early study bySofia et al. (1973) demonstrated that Δ9THC (p.o.) is effectivein the paw-pressure test for mechanical nociception in rats. Inrats with chronic inflammatory arthritis induced by Freund’scomplete adjuvant, Δ9THC-elicited antinociceptive efficacywas no different from that in non-arthritic rats (Smith et al.,1998). Freund’s adjuvant treatment produces chronic inflam-mation, edema, and hyperalgesia in rats (Millan et al., 1986b).The inflammation and hyperalgesia produced by Freund'sadjuvant is associated with alterations in several neuropeptidesystems, including opioids. Tissue levels of dynorphins andenkephalins, as well as the mRNAs encoding their precursors,are up-regulated in the spinal dorsal horn of rats with chronic

inflammation (Millan et al., 1986a; Pohl et al., 1997). Anincreased spinal release of dynorphin-like material has beenreported in polyarthritic rats, and a decreased spinal release ofmet-enkephalin-like material has been reported in these animals(Pohl et al., 1997). The modulation of endogenous opioidsystems by opioid receptor agonists and antagonists is altered inpolyarthritic rats as compared to non-arthritic rats (Ballet et al.,1998, Ballet et al., 2000). Therefore, although not directlyquantified in this study, the modulation of endogenous opioidsbyΔ9THC is modified in arthritic rats as shown in our previousstudies (Cox and Welch, 2004).

The endogenous cannabinoid system appears to play a role inthe suppression of chronic pain, an effect that is not opioid-mediated. It has been shown that in chronic neuropathic pain, anendocannabinoid analog retains the ability to modulatenociception while opioids lose the ability to reduce nociception(Kawasaki et al., 2006). Thus, the endocannabinoid system doesnot appear to require the opioid system for antinociception in achronic pain state. Similar results using Δ9THC and morphinein chronic intractable pain have led to the conclusion thatcannabinoid and opioid pathways are independent in such typesof pain and further, that the cannabinoid system may be superiorto the opioid system in terms of pain relief (Mao et al., 2000).Thus, it is possible that modulation of endocannabinoid tone bythe cannabinoid antagonists in combination withΔ9THC differsin non-arthritic and arthritic rats.

In summary, we have shown that Δ9THC-induced antinoci-ception in a model of inflammatory pain is both cannabinoid CB1

and CB2 receptor-mediated. It is unclear as to why both receptorsappear to play equiefficacious roles in modulation of Δ9THC-induced antinociception in that the AD50's for both antagonistsdid not differ in the arthritic rats and both were totally efficacious.One might hypothesize from such data that chronic pain as that inarthritics has two distinct, but interacting components involvingan interaction of cannabinoid CB1 and CB2 receptor-mediatedmechanisms. One could envision in such a scenario such thatblock of either the cannabinoid CB1 or CB2 component mightcompletely block the effects of Δ9THC. We have demonstratedthat Δ9THC-induced antinociception in non-arthritic rats iscannabinoid CB1 receptor-mediated. Future studies will beimportant to determine if there is some physical interaction ofcannabinoid CB1 andCB2 receptors in chronic pain states, such asa receptor dimerization event precipitated by the chronic pain.

Acknowledgement

This work was supported by NIDA Grants DA-09789, DA-05274 and KO2-DA-00186.

Send reprint requests to: Sandra P. Welch Dept. ofPharmacology/Toxocology P.O. Box 980524 Virginia Com-monwealth University Richmond, VA 23298-0524.

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