Spontaneous firing and evoked responses of spinal nociceptive neurons are attenuated by blockade of P2X3 and P2X2/3 receptors in inflamed rats.
ABSTRACT P2X3 and P2X2/3 receptors are selectively expressed on primary afferent nociceptors and have been implicated in modulating nociception in different models of pathological pain, including inflammatory pain. In an effort to delineate further the role of P2X3 receptors (homomeric and heteromeric) in the modulation of nociceptive transmission after a chronic inflammation injury, A-317491, a potent and selective P2X3-P2X2/3 antagonist, was administered to CFA-inflamed rats in order to examine its effects on responses of spinal dorsal horn neurons to mechanical and thermal stimulation. Systemic injection of A-317491 (30 μmol/kg, i.v.) reduced the responses of wide-dynamic-range (WDR) and nociceptive specific (NS) neurons to both high-intensity mechanical (pinch) and heat (49°C) stimulation. A-317491 also decreased low-intensity (10 g von Frey hair) mechanically evoked activity of WDR neurons but did not alter WDR neuronal responses to cold stimulation (5°C). Spontaneous firing of WDR neurons in CFA-inflamed rats was also significantly attenuated by A-317491 injection. By using immunohistochemistry, P2X3 receptors were demonstrated to be enhanced in lamina II of the spinal dorsal horn after inflammation. In summary, blockade of P2X3 and P2X2/3 receptors dampens mechanical- and heat-related signaling, as well as nonevoked activity of key classes of spinal nociceptive neurons in inflamed animals. These data suggest that P2X3 and/or P2X2/3 receptors have a broad contribution to somatosensory/nociceptive transmission in rats with a chronic inflammatory injury and are consistent with previous behavioral data demonstrating antiallodynic and antihyperalgesic effects of receptor antagonists.
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ABSTRACT: P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22-92 nM) and was highly selective (IC50 >10 microM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 micromolkg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10-15 micromolkg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 micromolkg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.Proceedings of the National Academy of Sciences 12/2002; 99(26):17179-84. · 9.74 Impact Factor
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ABSTRACT: The mechanism of mechanical hyperalgesia in inflammation might involve a 'mechanochemical' process whereby stretch evokes the release of adenosine 5'-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (alphabetame-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after alphabetame-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or 2'- (or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.Pain 05/2004; 108(3):258-66. · 5.64 Impact Factor
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ABSTRACT: The effect of A-317491 (5-([(3-Phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid), a recently described selective P2X3 and P2X(2/3) receptor antagonist, on inflammatory mechanical hyperalgesia was examined. In the rat Freund's complete adjuvant model of inflammatory pain, s.c. administration of A-317491 dose-dependently reversed mechanical hyperalgesia. Maximum percent reversal (72%) was seen 3 h after administration at 10 mg/kg. Substantial plasma concentrations were measured for A-317491 after s.c. dosing 3, 10 and 30 mg/kg. However, the brain-to-plasma concentration ratio, determined 1 h after a 10 mg/kg s.c. dose, indicated limited penetration of A-317491 into the central nervous system. As revealed by neural activity recorded from single C-fiber nociceptive afferent in a Freund's complete adjuvant-inflamed rat skin-nerve preparation, topical application of A-317491 completely blocked afferent activation and mechanical sensitization induced by alpha,beta-methylene ATP, a P2X agonist. These results suggest that A-317491 is a peripherally acting P2X blocker. Its efficacy demonstrates the importance of peripheral P2X3/P2X(2/3) receptors in mediating ATP-associated mechanical hyperalgesia following inflammation, confirming previous suggestions of a significant role for P2X(2/3).European Journal of Pharmacology 12/2004; 504(1-2):45-53. · 2.59 Impact Factor