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CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

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Abstract

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

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References

  1. Woodhams SG, Sagar DR, Burston JJ, Chapman V (2015) The role of the endocannabinoid system in pain. Handb Exp Pharmacol 227:119–143

    Article  CAS  PubMed  Google Scholar 

  2. Ulugöl A (2014) The endocannabinoid system as a potential therapeutic target for pain modulation. Balkan Med J 31:115–120

    Article  PubMed Central  PubMed  Google Scholar 

  3. Farquhar-Smith WP, Egertová M, Bradbury EJ, McMahon SB, Rice AS, Elphick MR (2000) Cannabinoid CB(1) receptor expression in rat spinal cord. Mol Cell Neurosci 15:510–521

    Article  CAS  PubMed  Google Scholar 

  4. Hegyi Z, Kis G, Holló K, Ledent C, Antal M (2009) Neuronal and glial localization of the cannabinoid-1 receptor in the superficial spinal dorsal horn of the rodent spinal cord. Eur J Neurosci 30: 251–262

    Article  PubMed  Google Scholar 

  5. Yang F, Xu Q, Shu B, Tiwari V, He SQ, Vera-Portocarrero LP, Dong X, Linderoth B, Raja SN, Wang Y, Guan Y (2016) Activation of cannabinoid CB1 receptor contributes to suppression of spinal nociceptive transmission and inhibition of mechanical hypersensitivity by Aβ-fiber stimulation. Pain 157(11):2582–2593

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Bardoni R, Goldstein PA, Lee CJ, Gu JG, MacDermott AB (1997) ATP P2Xreceptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord. J Neurosci 17:5297–5304

    CAS  PubMed  Google Scholar 

  7. Gu JG, MacDermott AB (1997) Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses. Nature 389:749–753

    Article  CAS  PubMed  Google Scholar 

  8. Nakatsuka T, Gu JG (2001) ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord. J Neurosci 21(17):6522–6531

    CAS  PubMed  Google Scholar 

  9. Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A, Buell G (1996) Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16:2495–2507

    CAS  PubMed  Google Scholar 

  10. Aoyama R, Okada Y, Yokota S, Yasui Y, Fukuda K, Shinozaki Y, Yoshida H, Nakamura M, Chiba K, Yasui Y, Kato F, Toyama Y (2011) Spatiotemporal and anatomical analyses of P2X receptor-mediated neuronal and glial processing of sensory signals in the rat dorsal horn. Pain 152:2085–2097

    Article  CAS  PubMed  Google Scholar 

  11. Jo YH, Schlichter R (1999) Synaptic corelease of ATP and GABA in cultured spinal neurons. Nat Neurosci 2:241–245

    Article  CAS  PubMed  Google Scholar 

  12. Paukert M, Osteroth R, Geisler HS, Brandle U, Glowatzki E, Ruppersberg JP, Gründer S (2001) Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit. J Biol Chem 276(24):21077–21082

    Article  CAS  PubMed  Google Scholar 

  13. Vial C, Tobin AB, Evans RJ (2004) G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation. Biochem J 382(Pt 1):101–110

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Brown DA, Yule DI (2007) Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation. Biochim Biophys Acta 1773(2):166–175

    Article  CAS  PubMed  Google Scholar 

  15. Wang C, Gu Y, Li GW, Huang LY (2007) A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2-induced potentiation of P2X3 receptor currents in inflamed rats. J Physiol 584(Pt 1):191–203

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Liu XH, Zeng JW, Zhao YD, Chen PH, Xiao Z, Ruan HZ (2008) Rapid inhibition of ATP-induced currents by corticosterone in rat dorsal root ganglion neurons. Pharmacology 82(2):164–170

    Article  CAS  PubMed  Google Scholar 

  17. Liu XH, Zeng JW, Zhao YD, Xiao Z, Fang CQ, Ruan HZ (2010) Inhibition of ATP-induced Ca2+ influx by corticosterone in dorsal root ganglion neurons. Neurochem Res 35(5):804–810

    Article  CAS  PubMed  Google Scholar 

  18. Han JZ, Lin W, Chen YZ (2005) Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids: involvement of protein kinase A. Acta Pharmacol Sin 26(2):199–204

    Article  PubMed  Google Scholar 

  19. Dalton GD, Bass CE, Van Horn C, Howlett AC (2009) Signal transduction via cannabinoid receptors. CNS Neurol Disord Drug Targets 8:422–431

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Jo YH, Stoeckel ME, Freund-Mercier MJ, Schlichter R (1998) Oxytocin modulates glutamatergic synaptic transmission between cultured neonatal spinal cord dorsal horn neurons. J Neurosci 18:2377–2386

    CAS  PubMed  Google Scholar 

  21. Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I (2000) Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27:97–106

    Article  CAS  PubMed  Google Scholar 

  22. Lai KM, Wong PC (1991) Metabolism of extracellular adenine nucleotides by cultured rat brain astrocytes. J Neurochem 57(5):1510–1515

    Article  CAS  PubMed  Google Scholar 

  23. Prendergast J, Umanah GK, Yoo SW, Lagerlöf O, Motari MG, Cole RN, Huganir RL, Dawson TM, Dawson VL, Schnaar RL (2014) Ganglioside regulation of AMPA receptor trafficking. J Neurosci 34(39):13246–13258

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Zeng JW, Liu XH, Zhao YD, Xiao Z, He WJ, Hu ZA, Ruan HZ (2009) Role of P2Y1 receptor in neuron-to-glia signaling at dorsal spinal cord. J Neurosci Res 87(12):2667–2676

    Article  CAS  PubMed  Google Scholar 

  25. Solymosi K, Köfalvi A (2017) Cannabis: a treasure trove or pandora’s box? Mini Rev Med Chem 17(13):1223–1291

    CAS  PubMed  Google Scholar 

  26. Tomiyama K, Funada M (2014) Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement ofcannabinoid CB1 receptors and apoptotic cell death. Toxicol Appl Pharmacol 274(1):17–23

    Article  CAS  PubMed  Google Scholar 

  27. Grabiec U, Koch M, Kallendrusch S, Kraft R, Hill K, Merkwitz C, Ghadban C, Lutz B, Straiker A, Dehghani F (2012) The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB(1)). Neuropharmacology 62(4):1797–1807

    Article  CAS  PubMed  Google Scholar 

  28. Felder CC, Joyce KE, Briley EM, Glass M, Mackie KP, Fahey KJ, Cullinan GJ, Hunden DC, Johnson DW, Chaney MO, Koppel GA, Brownstein M (1998) LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation. J Pharmacol Exp Ther 284:291–297

    CAS  PubMed  Google Scholar 

  29. Glass M, Felder C (1997) Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a G s linkage to the CB1 receptor. J Neurosci 17:5327–5333

    CAS  PubMed  Google Scholar 

  30. McAllister SD, Glass M (2002) CB(1) and CB(2) receptor-mediated signalling: a focus on endocannabinoids. Prostaglandins Leukot Essent Fat Acids 66:161–171

    Article  CAS  Google Scholar 

  31. Burnstock G (2006) Purinergic P2 receptors as targets for novel analgesics. Pharmacol Ther 110(3):433–454

    Article  CAS  PubMed  Google Scholar 

  32. Donnelly-Roberts DL, Jarvis MF (2007) Discovery of P2X7 receptor selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states. Br J Pharmacol 151(5):571–579

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Andó RD, Méhész B, Gyires K, Illes P, Sperlágh B (2010) A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain. Br J Pharmacol 159(5):1106–1117

    Article  PubMed Central  PubMed  Google Scholar 

  34. Dunn PM, Zhong Y, Burnstock G (2001) P2X receptors in peripheral neurons. Prog Neurobiol 65:107–134

    Article  CAS  PubMed  Google Scholar 

  35. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492

    CAS  PubMed  Google Scholar 

  36. Zeng JW, Liu XH, Zhang JH, Wu XG, Ruan HZ (2008) P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes. J Neurochem 106(5):2106–2118

    Article  CAS  PubMed  Google Scholar 

  37. Zeng J, Wang G, Liu X, Wang C, Tian H, Liu A, Jin H, Luo X, Chen Y (2014) P2Y13 receptor-mediated rapid increase in intracellular calcium induced by ADP in cultured dorsal spinal cord microglia. Neurochem Res 39(11):2240–2250

    Article  CAS  PubMed  Google Scholar 

  38. Jahr CE, Jessell TM (1983) ATP excites a subpopulation of rat dorsal horn neurones. Nature 304(5928):730–733

    Article  CAS  PubMed  Google Scholar 

  39. Li J, Perl ER (1995) ATP modulation of synaptic transmission in the spinal substantia gelatinosa. J Neurosci 15:3357–3365

    CAS  PubMed  Google Scholar 

  40. Burston JJ, Sagar DR, Shao P, Bai M, King E, Brailsford L, Turner JM, Hathway GJ, Bennett AJ, Walsh DA, Kendall DA, Lichtman A, Chapman V (2013) Cannabinoid CB2 receptors regulate central sensitization and pain responses associated with osteoarthritis of the knee joint. PLoS ONE 8(11):e80440

    Article  PubMed Central  PubMed  Google Scholar 

  41. Romero-Sandoval A, Nutile-McMenemy N, DeLeo JA (2008) Spinal microglial and perivascular cell cannabinoid receptor type 2 activation reduces behavioralhypersensitivity without tolerance after peripheral nerve injury. Anesthesiology 108(4):722–734

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Krishtal O, Lozovaya N, Fedorenko A, Savelyev I, Chizhmakov I (2006) The agonists for nociceptors are ubiquitous, but the modulators are specific: P2X receptors in the sensory neurons are modulated by cannabinoids. Pflugers Arch 453:353–360

    Article  CAS  PubMed  Google Scholar 

  43. Sagar DR, Kelly S, Millns PJ, O’Shaughnessey CT, Kendall DA, Chapman V (2005) Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats. Eur J Neurosci 22(2):371–379

    Article  PubMed  Google Scholar 

  44. Xiong W, Wu X, Li F, Cheng K, Rice KC, Lovinger DM, Zhang L (2012) A common molecular basis for exogenous and endogenous cannabinoid potentiation of glycinereceptors. J Neurosci 32(15):5200–5208

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Liu C, Walker JM (2006) Effects of a cannabinoid agonist on spinal nociceptive neurons in a rodent model of neuropathic pain. J Neurophysiol 96(6):2984–2994

    Article  CAS  PubMed  Google Scholar 

  46. Chen XP, Yang W, Fan Y, Luo JS, Hong K, Wang Z, Yan JF, Chen X, Lu JX, Benovic JL, Zhou NM (2010) Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to G(s) and G(i). Br J Pharmacol 161:1817–1834

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Sántha P, Jenes A, Somogyi C, Nagy I (2010) The endogenous cannabinoid anandamide inhibits transient receptor potential vanilloid type 1 receptor-mediated currents in rat cultured primary sensory neurons. Acta Physiol Hung 97:149–158

    Article  PubMed  Google Scholar 

  48. Shen JJ, Liu CJ, Li A, Hu XW, Lu YL, Chen L, Zhou Y, Liu LJ (2007) Cannabinoids inhibit ATP-activated currents in rat trigeminal ganglionic neurons. Sheng Li Xue Bao 59(6):745–752

    CAS  PubMed  Google Scholar 

  49. Brown David A., David I (2010) Yule protein kinase A regulation of P2X4 receptors: requirement for a specific motif in the C-terminus. Biochim Biophys Acta 1803(2):275–287

    Article  CAS  PubMed  Google Scholar 

  50. Chow YW, Wang HL (1998) Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase. J Neurochem 70(6):2606–2612

    Article  CAS  PubMed  Google Scholar 

  51. Serpa A, Correia S, Ribeiro JA, Sebastião AM, Cascalheira JF (2015) The combined inhibitory effect of the adenosine A1 and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus is additive and independent of A1 receptor desensitization. Biomed Res Int. doi:10.1155/2015/872684

    PubMed Central  PubMed  Google Scholar 

  52. Veeraraghavan P, Nistri A (2015) Modulatory effects by CB1 receptors on rat spinal locomotor networks after sustained application of agonists or antagonists. Neuroscience 303:16–33

    Article  CAS  PubMed  Google Scholar 

  53. Roscioni SS, Kistemaker LE, Menzen MH, Elzinga CR, Gosens R, Halayko AJ, Meurs H, Schmidt M (2009) PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells. Respir Res 10:88

    Article  PubMed Central  PubMed  Google Scholar 

  54. Oliveira-Fusaro MC, Zanoni CI, Dos Santos GG, Manzo LP, Araldi D, Bonet IJ, Tambeli CH, Dias EV, Parada CA (2017) Antihyperalgesic effect of CB1 receptor activation involves the modulation of P2X3 receptor in the primary afferent neuron. Eur J Pharmacol 798:113–121

    Article  CAS  PubMed  Google Scholar 

  55. Walter L, Dinh T, Stella N (2004) ATP induces a rapid and pronounced increase in 2-arachidonoylglycerol production by astrocytes, a response limited by monoacylglycerol lipase. J Neurosci 24(37):8068–8074

    Article  CAS  PubMed  Google Scholar 

  56. Witting A, Walter L, Wacker J, Möller T, Stella N (2004) P2X7 receptors control 2-arachidonoylglycerol production by microglial cells. Proc Natl Acad Sci USA 101(9):3214–3219

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 31360253). We are very grateful to the other staff of Department of Physiology. We also thank Guo Luo (Central Laboratory, Zunyi Medical College) for his technique assistance in laser scanning confocal microscopy.

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  1. Jingdong Long and Xiaolu Lei contributed equally to this study.

Authors and Affiliations

  1. Department of Physiology, Zunyi Medical College, Zunyi, 563000, People’s Republic of China

    Jingdong Long, Xiaolu Lei, Meiyun Chen, Shulei Yang, Tao Sun, Junwei Zeng, Deqian Yu, Hong Tian & Xiaohong Liu

  2. Department of Physiology, Zunyi Medical College, No. 6 West Xuefu Road, Xinpu District, Zunyi, 563006, Guizhou Province, People’s Republic of China

    Xiaohong Liu

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  1. Jingdong Long
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Long, J., Lei, X., Chen, M. et al. CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons. Neurochem Res 43, 267–275 (2018). https://doi.org/10.1007/s11064-017-2414-6

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