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

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    1. Introduction

    AM1241, administered systemically or locally produces acutethermal antinociception inhibited by the cannabinoid CB2

    inhibits formalin-induced nociception (Malan et al., 2002) and

    hypersensitivity 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 CB / CB agonist, CP55, 940, attenuates acute thermal

    logyreceptor antagonist, AM630, and not the cannabinoid CB1receptor antagonist AM251 (Malan et al., 2001). AM1241 alsoCannabinoids have been shown to produce pain relief in avariety of animal models (Richardson, 2000). Cannabinoid CB1receptors 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,

    suppresses 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 thermalboth cannabinoid CB1 and CB2 receptors in mechanonociception in non-arthritic and arthritic rats. The antinociceptive effect of -tetrahydrocannabinol (9THC) was determined in rats following administration of the cannabinoid CB1 receptor-selective antagonist,SR141716A, the cannabinoid CB2 receptor-selective antagonist, SR144528, or vehicle. Male SpragueDawley rats were rendered arthritic usingFreunds 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 9THC (4mg/kg; i.p) or 1:1:18 vehicle and antinociception determined 30min post 9THC. 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

    9THC in thearthritic rats [AD50 = 3.3 (2.74) mg/kg], but not in the non-arthritic rats at a dose of 10/mg/kg. SR141716A significantly attenuated

    9THC-induced antinociception in both the non-arthritic [AD50 = 1.4 (0.82) mg/kg] and arthritic rat [AD50 = 2.6 (1.83.1) 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; ArthritisCannabinoid CB2 receptors have been implicated in antinociceptionAbstract

    in animal models of both acute and chronic pain. We evaluated the roleThe antinociceptive effect of 9-tetinvolves the CB2 c

    Melinda L. Cox, Victoria L

    Department of Pharmacology and Toxicology, Virginia Comm

    Received 11 August 2006; received in revisAvailable onl

    European Journal of Pharmaco Corresponding author. Tel.: +1 804 828 8446; fax: +1 804 828 2117.E-mail address: vhaller@hsc.vcu.edu (V.L. Haller).

    0014-2999/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.ejphar.2007.05.024hydrocannabinol in the arthritic ratnabinoid receptor

    Haller , Sandra P. Welch

    wealth University, Richmond, VA 23298-0524, United States

    orm 10 May 2007; accepted 15 May 20075 June 2007

    570 (2007) 5056www.elsevier.com/locate/ejphar1 2

    nociceptive responses, as well as neuropathic tactile allodyniafollowing spinal nerve ligation in the rat. The cannabinoidCB1 receptor antagonist, SR141716A, inhibits only the acute

  • l ofantinociception 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 9-tetrahydrocan-nabinol (9THC), a mixed cannabinoid CB1/CB2 receptoragonist alone, or following administration of cannabinoid CB1and 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 9THC-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 SpragueDawley rats (Harlan Laboratories, Indiana-polis, IN), which weighed 350 to 375g were housed in ananimal care facility maintained at 22 2C 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 Freundscomplete adjuvant (heat-killed Mycobacterium butyricum;5mg/ml) was injected intradermally into the base of the tail

    M.L. Cox et al. / European Journa(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. Inflammationproceeds 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). 9-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 doseeffectcurves for 9THC in arthritic and non-arthritic rats and havedetermined the peak time for 9THC activity in the paw-pressure test in the rats (Cox and Welch, 2004).

    2.5. Statistical evaluation

    Doseresponse curves were generated using three or fourdoses of antagonist in combination with the ED80 dose of9THC (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 the

    51Pharmacology 570 (2007) 5056guidelines established by the Institutional Animal Care andUse Committee of Virginia Commonwealth University andadhere to the National Institute of Health Guide for the care

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

    2.6. Tissue preparation for Western immunoassays

    Non-arthritic and arthritic nave 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 80C until prepared. Frozen tissue wasallowed to thaw, then homogenized in 10 ml of cold suspensionbuffer [10mmol EGTA, 20mmol EDTA, 10mmol Tris, 20mmol

    was 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

    52 M.L. Cox et al. / European Journal of-glycerophosphate, 50mmol sodium fluoride, 50mmol sodiumpyrophosphate, 1mmol p-nitrophenylphosphate, 200 molmicrocystin LR (Sigma Chemical Co., St. Louis, MO)] andprotease inhibitor cocktail (Boehringer Mannheim, Indianapo-lis, IN). A P2 membrane preparation was made with a 10min1500g centrifugation, followed by a 30min spin of thesupernatant at 48,000g at 4C. 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 4C. The samples were then centrifuged for 20min at17,000g to remove insoluble material. Protein concentrationswere determined by the Bio-Rad DC protein assay (Hercules,CA). Tissue was aliquoted and stored at 80C.

    2.7. Western immunoassays

    Electrophoresis was performed using a standard Laemmlimethod. Samples were diluted 1:1 with 2 sample buffer to aconcentration of 10g 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 CB2cannabinoid 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 0.3140.06 0.3550.07Arthritic 0.2880.05 0.4550.04

    Spinal cordNon-arthritic 0.3010.08 0.1940.02

    Arthritic 0.3440.05 0.2120.02

    Absorbance readings were obtained using scanning densitometry. The data ispresented as the average absorbance of four trialsS.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 experiment

    Fig. 1. Cannabinoid CB1 receptor antagonist, SR141716A (0.3 to 10 mg/kg, i.p.),administered 1 hour before 9-THC (4 mg/kg, i.p.) significantly attenuated theantinociceptive effect of 9-THC in arthritic rats. Animals (6 per treatment) weretested for antinociception 30 min after 9-THC administration. The %MPE foreach rat was determined. The AD50 for SR141716A was determined as in theMethods.

    Pharmacology 570 (2007) 5056To 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 nave

  • confidence limits) calculated...

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