Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X₃Receptors of Nociceptive Trigeminal Ganglion Neurons

Abstract

The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X 3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X 3 receptor-mediated currents and Ca 2ϩ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X 3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X 2/3 receptors, consistent with enhanced membrane location of P2X 2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X 2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X 3 subunits and their membrane assembly with P2X 2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X 3 receptor activity, indicating separate NGF-or CGRP-mediated mechanisms to upregulate P2X 3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X 3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.

P2Y purinergic receptors: New targets for analgesic and antimigraine drugs

“…As a final outcome, recovery from P2X3 receptor desensitization is accelerated, and P2X3 activity is consequently increased [15,16,17]. Moreover, an increased expression of P2X3 receptors in TG neurons was observed upon algogen application in vitro [15,18], or following induction of trigeminalassociated pain in vivo ( Fig.…”

Mechanisms Mediating the Enhanced Gene Transcription of P2X3 Receptor by Calcitonin Gene-related Peptide in Trigeminal Sensory Neurons

“…Real Time Reverse Transcription-PCR-Total mRNA extraction from TG cultures or ganglia and reverse transcription reactions was performed as previously reported (22), using the specific primers listed in Table 1. Data were normalized with respect to GAPDH and ␤-tubulin III housekeeping mRNA contents.…”

Activation and Regulation of Purinergic P2X Receptor Channels

“…For the C-terminal SRC kinase-dependent regulation, the C-terminal Tyr 393 residue has been proposed as a target for phosphorylation (D'Arco et al, 2009;Nair et al, 2010). In mouse trigeminal sensory neurons, the role of this kinase is controlled by nerve growth factors (D'Arco et al, 2007).…”