Functional Downregulation of P2X₃Receptor Subunit in Rat Sensory Neurons Reveals a Significant Role in Chronic Neuropathic and Inflammatory Pain

Abstract

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.

Pathophysiology and Therapeutic Potential of Purinergic Signaling

“…A-317491 is a potent and selective non-nucleotide antagonist of P2X 3 and P2X 2/3 receptors, and it reduces chronic inflammatory and neuropathic pain in the rat (Jarvis et al, , 2004McGaraughty et al, 2003). Antisense oligonucleotides have been used to down-regulate the P2X 3 receptor, and in models of neuropathic (partial sciatic nerve ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia, were observed within 2 days of treatment (Barclay et al, 2002;Honore et al, 2002;Stone and Vulchanova, 2003). P2X 3 antisense oligonucleotides or antagonists appear to be less effective for treating discogenic (lumbar intervertebral disc) than cutaneous tissue pain (Aoki et al, 2003).…”

Effects of A‐317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration

“…Neuronal expression of the P2X 3 receptor is elevated in both the dorsal root ganglion and the ipsilateral spinal cord following chronic constriction injury (CCI) of the sciatic nerve (Novakovic et al, 1999). Furthermore, P2X 3 (À/À) gene-ablated mice show a significant attenuation in spontaneous pain behaviors following administration of ATP or formalin (Cockayne et al, 2000;Souslova et al, 2000), and animals treated with P2X 3 antisense oligonucleotides exhibit reduced thermal hyperalgesia and mechanical allodynia (Barclay et al, 2002;Honore et al, 2002).…”

Expression of P2X3and its colocalization with trpv1 in the human dental pulp

“…Studies with antagonists selective for P2X3-containing receptor showed marked reduction in chronic inflammation induced by thermal 30) and mechanical 31) hyperalgesia, and spinal nerve ligation-induced mechanical allodynia 30) . Moreover, it has been reported that nociceptive action of ATP are markedly augmented in the presence of inflammation or inflammatory mediators.…”