α2-Adrenoceptor agonist induces peripheral antinociception via the endocannabinoid system

  • Thiago Roberto Lima RomeroEmail author
  • Marina Gomes Miranda e Castor
  • Cosimo Parrella
  • Fabiana Piscitelli
  • Vincenzo Di Marzo
  • Igor Dimitri Gama Duarte



Xylazine is an α2 adrenoceptor agonist that is extensively used in veterinary medicine and animal experimentation procedures to produce analgesia, sedation and muscle relaxation without causing general anesthesia. Considering the lack of knowledge of the mechanisms involved in peripheral antinociception induced by xylazine and the potential interactions between the adrenergic and endocannabinoid systems, the present study investigated the contribution of the latter system in the mechanism of xylazine.


The rat paw pressure test, in which hyperalgesia was induced by the intraplantar injection of prostaglandin E2, was performed.


Xylazine administered via an intraplantar injection (25, 50 and 100 μg) induced a peripheral antinociceptive effect against prostaglandin E2 (2 μg)-induced hyperalgesia. This effect was blocked by treatment with the selective CB1 cannabinoid antagonist AM251 (20, 40 and 80 μg) but not by the selective CB2 cannabinoid antagonist AM630 (100 μg). The anandamide reuptake inhibitor VDM11 (2.5 μg) intensified the peripheral antinociceptive effect of a submaximal dose of xylazine (25 μg), and the inhibitor of endocannabinoid enzymatic hydrolysis, MAFP (0.5 μg), showed a tendency towards this same effect. In addition, liquid-chromatography mass spectrometric analysis indicated that xylazine (100 μg) treatment was associated with an increase in anandamide levels in the rat paws treated with PGE2.


The present results provides evidence that the peripheral antinociceptive effect of the α2 adrenoceptor agonist xylazine probably results from anandamide release and subsequent CB1 cannabinoid receptor activation.


Xylazine α2-Adrenoceptor Anandamide CB1 cannabinoid Peripheral antinociception 



TRLR would like to thank the financial support from Fundação de Amparo a Pesquisa de Minas Gerais—FAPEMIG—PPMFAPEMIG 2015 Process no 00474-15. MGMC would like to thank the financial support from Fundação de Amparo a Pesquisa de Minas Gerais—FAPEMIG—UNIVERSAL Process no 01307-14. TRLR would like to thank the Fellowships by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES.

Compliance with ethical standards

Conflict of interest

The authors inform non conflicts of interest.


  1. 1.
    Willian WMIII, John AEH, Roman TS, Richard MB. Fármacos usados na medicação pré-anestésica. In: Willian WMIII Manual de anestesia veterinária. Proc Artmed, 3ª ed, Porto Alegre; 2001. pp. 31–44.Google Scholar
  2. 2.
    Kroneberg G, Oberdorf A, Hoffmeiter F, Wirth W. On the pharmacology of 2-(2,6-dimethylphenylamino)-4H-5,6-dihydro-1,3-thiazine (Bayer 1470), a substance inhibitory for adrenergic and cholinergic neurons. Naunyn Schmiedebergs Arch Pharmacol Exp Pathol. 1967;256:257–80.CrossRefGoogle Scholar
  3. 3.
    Hsu WH. Xylazine-induced depression and its antagonism by alpha adrenergic blocking agents. J Pharmacol Exp Ther. 1981;218:188–92.PubMedGoogle Scholar
  4. 4.
    Fürst S. Transmitters involved in antinociception in the spinal cord. Brain Res Bull. 1999;48:129–41.PubMedCrossRefGoogle Scholar
  5. 5.
    Tham SM, Angus JA, Tudor EM, Wright CE. Synergistic and additive interactions of the cannabinoid agonist CP55,940 with mu opioid receptor and alpha2-adrenoceptor agonists in acute pain models in mice. Br J Pharmacol. 2005;144:875–84.PubMedCrossRefGoogle Scholar
  6. 6.
    Yoon MH, Choi JI. Pharmacologic interaction between cannabinoid and either clonidine or neostigmine in the rat formalin test. Anesthesiology. 2003;99:701–7.PubMedCrossRefGoogle Scholar
  7. 7.
    Yaksh TL. Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing. Pharmacol Biochem Behav. 1985;22:845–58.PubMedCrossRefGoogle Scholar
  8. 8.
    Reis GM, Ramos MA, Pacheco DF, Klein A, Perez AC, Duarte ID. Endogenous cannabinoid receptor agonist anandamide induces peripheral antinociception by activation of ATP-sensitive K+ channels. Life Sci. 2001;88:653–7.CrossRefGoogle Scholar
  9. 9.
    Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, et al. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002;54:161–202.PubMedCrossRefGoogle Scholar
  10. 10.
    Khodayar MJ, Shafaghi B, Naderi N, Zarrindast MR. Antinociceptive effect of spinally administered cannabinergic and 2-adrenoceptor drugs on the formalin test in rat: possible interactions. J Psychopharmacol. 2006;20:67–74.PubMedCrossRefGoogle Scholar
  11. 11.
    Ferreira RCM, Castor MGM, Piscitelli F, Di Marzo V, Duarte IDG. Romero TRL the involvement of the endocannabinoid system in the peripheral antinociceptive action of ketamine. J Pain. 2018;19:487–95.PubMedCrossRefGoogle Scholar
  12. 12.
    Zimmermann M. Ethical guidelines for investigation of experimental pain in conscious animals. Pain. 1983;16:109–10.PubMedCrossRefGoogle Scholar
  13. 13.
    Randall LO, Sellito JJ. A method for measurement of analgesic activity on inflamed tissues. Arch Int Pharmacodyn Ther. 1957;111:409–19.PubMedGoogle Scholar
  14. 14.
    Bisogno T, Maurelli S, Melck D, De Petrocellis L, Di Marzo V. Biosynthesis, uptake, and degradation of anandamide and palmitoylethanolamide in leukocytes. J Biol Chem. 1997;272:3315–23.PubMedCrossRefGoogle Scholar
  15. 15.
    Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, et al. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science. 2003;302:84–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Bentley GA, Copeland IW, Starr J. The actions of some alpha-adrenoceptor agonists and antagonists in an antinociceptive test in mice. Clin Exp Pharmacol Physiol. 1977;4:405.PubMedCrossRefGoogle Scholar
  17. 17.
    Naves LA, Freire AC. Xylazine antinociception in mice: evidence for mediation by postsynaptic adrenoceptors. Braz J Med Biol Res. 1989;22:1009–100.PubMedGoogle Scholar
  18. 18.
    Goodchild CS, Guo Z, Davies A, Gent JP. Antinociceptive actions of intratecal xylazine: interations with spinal cord opioid pathways. Br J Anaesth. 1996;76:544–51.PubMedCrossRefGoogle Scholar
  19. 19.
    Schmitt H, Le Douarec JC, Petillot N. The antinociceptive effect of some alpha-sympathomimetic agents. Neuropharmacology. 1974;13:289–94.PubMedCrossRefGoogle Scholar
  20. 20.
    Gold MS, Dastmalchi S, Levine JD. α2-Adrenergic receptor subtypes in rat dorsal root and superior cervical ganglion neurons. Pain. 1997;69:179–90.PubMedCrossRefGoogle Scholar
  21. 21.
    Romero TRL, Perez AC, Francischi JN, Duarte IDG. Probable involvement of α2C-adrenoceptor subtype and endogenous opioid peptides in the peripheral antinociceptive effect induced by Xylazine. Eur J Pharmacol. 2009;608:23–7.PubMedCrossRefGoogle Scholar
  22. 22.
    Ferreira SH. Prostaglandins, aspirin like drugs and analgesia. Nature (New Biol). 1972;240:200–3.CrossRefGoogle Scholar
  23. 23.
    Bevan S. Nociceptive peripheral neurons: cellular properties. In: Melzack R, Wall D 4 Eds.), Textbook of pain, Churchill Livingstone 4ª ed, London; 1999. pp. 85–103.Google Scholar
  24. 24.
    Vinegar R, Truax JF, Selph JL, Johnston PR, Venable AL, McKenzie KK. Pathway to carrageenan-induced inflammation in the hind limb of the rat. Fed Proc. 1987;46:118–26.PubMedGoogle Scholar
  25. 25.
    Soares AC, Leite R, Tatsuo MA, Duarte ID. Activation of ATP-sensitive K(+) channels: mechanism of peripheral antinociceptive action of the nitric oxide donor, sodium nitroprusside. Eur J Pharmacol. 2000;400:67–71.PubMedCrossRefGoogle Scholar
  26. 26.
    Nakamura M, Ferreira SH. Peripheral analgesic action of clonidine: mediation by release of endogenous enkephalin-like substances. Eur J Pharmacol. 1988;146:223–8.PubMedCrossRefGoogle Scholar
  27. 27.
    Khasar SG, Green PG, Chou B, Levine DJ. Peripheral nociceptive effects of α2-adrenergic receptor agonist in the rat. Neuroscience. 1995;66:427–32.PubMedCrossRefGoogle Scholar
  28. 28.
    Bentley GA, Newton SH, Jennifer S. Studies on the antinociceptive action of α-agonist drugs and their interactions with opioid mechanisms. Br J Pharmacol. 1983;79:125–34.PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson SE, Porrino LJ. Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology. 2004;1:345–58.CrossRefGoogle Scholar
  30. 30.
    Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–5.PubMedCrossRefGoogle Scholar
  31. 31.
    Malan TP, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T. CB2 cannabinoid receptor-mediated peripheral antinociception. Pain. 2001;93:239–45.PubMedCrossRefGoogle Scholar
  32. 32.
    Clayton N, Marshall FH, Bountra C, O'Shaughnessy CT. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. Pain. 2002;96:253–60.PubMedCrossRefGoogle Scholar
  33. 33.
    Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F, Makriyannis A, et al. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology. 2003;99:955–60.PubMedCrossRefGoogle Scholar
  34. 34.
    Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A, et al. CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proc Natl Acad Sci USA. 2005;102:3093–8.PubMedCrossRefGoogle Scholar
  35. 35.
    Romero TRL, Resende LC, Guzzo LS, Duarte ID. CB1 and CB2 cannabinoid receptor agonists induce peripheral antinociception by activation of the endogenous noradrenergic system. Anesth Analg. 2013;116:463–72.PubMedCrossRefGoogle Scholar
  36. 36.
    Di Marzo V. Endocannabinoids: synthesis and degradation. Rev Physiol Biochem Pharmacol. 2006;160:1–24.CrossRefGoogle Scholar
  37. 37.
    Di Marzo V. Targeting the endocannabinoid system: to enhance or reduce? Nat Rev Drug Discov. 2008;7:438–55.PubMedCrossRefGoogle Scholar
  38. 38.
    Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D. Functional role of highaffinity anandamide transport, as revealed by selective inhibition. Science. 1997;277:1094–7.PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N. Molecular characterization of a phospholipase D generating anandamide and its congeners. J Biol Chem. 2004;279:5298–305.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Kim J, Isokawa M, Ledent C, Alger BE. Activation of muscarinic acetylcholine receptors enhances the release of endogenous cannabinoids in the hippocampus. J Neurosci. 2002;22:1182–91.Google Scholar
  41. 41.
    Schallreuter KU, Körner C, Pittelkow MR, Swanson NN, Gardner ML. The induction of the alpha-1-adrenoceptor signal transduction system on human melanocytes. Exp Dermatol. 1996;5:20–3.PubMedCrossRefGoogle Scholar
  42. 42.
    McSwigan JD, Hanson DR, Lubiniecki A, Heston LL, Sheppard JR. Down syndrome fibroblasts are hyperresponsive to beta-adrenergic stimulation. Proc Natl Acad Sci USA. 1981;78:7670–3.PubMedCrossRefGoogle Scholar
  43. 43.
    Gillbro JM, Marles LK, Hibberts NA, Schallreuter KU. Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes. J Invest Dermatol. 2004;123:346–53.PubMedCrossRefGoogle Scholar
  44. 44.
    Johnson M. Beta2-adrenoceptors: mechanisms of action of beta2-agonists. Paediatr Respir Rev. 2001;2:57–62.PubMedGoogle Scholar
  45. 45.
    Steenhuis P, Huntley RE, Gurenko Z, Yin L, Dale BA, Fazel N, et al. Adrenergic signaling in human oral keratinocytes and wound repair. J Dent Res. 2011;90:86–192.CrossRefGoogle Scholar
  46. 46.
    Maccarrone M, Di Rienzo M, Battista N, Gasperi V, Guerrieri P, Rossi A, et al. The endocannabinoid system in human keratinocytes. Evidence that anandamide inhibits epidermal differentiation through CB1 receptor-dependent inhibition of protein kinase C, activation protein-1, and transglutaminase. J Biol Chem 2003;278:3896–903.PubMedCrossRefGoogle Scholar
  47. 47.
    Pacheco DF, Klein A, Perez AC, Pacheco FCM, 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.CrossRefGoogle Scholar
  48. 48.
    Binder W, Mousa SA, Sitte NA, Kaiser M, Stein C, Schäfer M. Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue. Eur J Neurosci. 2004;20:92–100.PubMedCrossRefGoogle Scholar
  49. 49.
    Maisel AS, Knowlton KU, Fowler P, Rearden A, Ziegler MG, Motulsky HJ, et al. Adrenergic control of circulating lymphocyte subpopulations. Effects of congestive heart failure, dynamic exercise, and terbutaline treatment. J Clin Invest 1990;85:462–7.PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Kohm AP, Sanders VM. Norepinephrine and beta 2-adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev. 2001;53:487–525.PubMedGoogle Scholar
  51. 51.
    Sanders VM, Straub RH. Norepinephrine, the beta-adrenergic receptor, and immunity. Brain Behav Immun. 2007;16:290–332.CrossRefGoogle Scholar
  52. 52.
    Pestonjamasp VK, Burstein SH. Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system. Biochim Biophys Acta. 1998;1394:249–60.PubMedCrossRefGoogle Scholar
  53. 53.
    Mnich SJ, Hiebsch RR, Huff RM, Muthian S. Anti-inflammatory properties of CB1-receptor antagonist involves beta2 adrenoceptors. J Pharmacol Exp Ther. 2010;333:445–53.PubMedCrossRefGoogle Scholar

Copyright information

© Maj Institute of Pharmacology Polish Academy of Sciences 2020

Authors and Affiliations

  • Thiago Roberto Lima Romero
    • 1
    Email author
  • Marina Gomes Miranda e Castor
    • 1
  • Cosimo Parrella
    • 2
  • Fabiana Piscitelli
    • 2
  • Vincenzo Di Marzo
    • 2
  • Igor Dimitri Gama Duarte
    • 1
  1. 1.Department of Pharmacology, Institute of Biological SciencesICB-UFMGBelo HorizonteBrazil
  2. 2.Endocannabinoid Research Group, Institute of Biomolecular ChemistryNational Research CouncilPozzuoliItaly

Personalised recommendations