Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain

Abstract

BackgroundA growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons.ResultsThe effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons). Thereafter, the action of these stable, synthetic Ap4A analogs on inflammatory pain and thermal hyperalgesia is studied through the measurement of antinociceptive effects in formalin and Hargreaves plantar tests in rats in vivo. In vitro inhibition of rat P2X3Rs (not P2X2Rs, P2X4Rs nor P2X7Rs) is shown to take place mediated by high-affinity desensitization (at low concentrations; IC50 values 100–250 nM) giving way to only weak partial agonism at much higher concentrations (EC50 values ≥ 10 µM). Similar inhibitory activity is observed with human recombinant P2X3Rs. The inhibitory effects of AppNHppA on nodose, dorsal root, and trigeminal neuron whole cell currents suggest that stable, synthetic Ap4A analogs inhibit homomeric P2X3Rs in preference to heteromeric P2X2/3Rs. Both Ap4A analogs mediate clear inhibition of pain responses in both in vivo inflammation models.ConclusionsStable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) being weak partial agonist provoke potent high-affinity desensitization-mediated inhibition of homomeric P2X3Rs at low concentrations. Therefore, both analogs demonstrate clear potential as potent analgesic agents for use in the management of chronic pain associated with heightened P2X3R activation.

Selective Calcium-Dependent Inhibition of ATP-Gated P2X3 Receptors by Bisphosphonate-Induced Endogenous ATP Analog ApppI

“…Human embryonic kidney HEK293 and HEK293T cell cultures were maintained as reported previously (Fabbretti et al, 2004;Viatchenko-Karpinski et al, 2016) in Dulbecco's modified Eagle's medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. Cells were passaged at 80% confluence.…”

“…We then evaluated the ability of ApppI to induce a highaffinity desensitization of P2X3 receptors leading to inhibitory and thus antinociceptive effects (Giniatullin et al, 2008;Viatchenko-Karpinski et al, 2016). Therefore, we tested the different concentrations of ApppI to determine its inhibitory potency on rP2X3 receptors.…”

“…We compared currents activated by a,b-meATP in trigeminal and nodose ganglia because the nodose neurons, responsible for the visceral pain, express preferentially heteromeric P2X2/3 subunits that produce slow currents with little desensitization (Viatchenko-Karpinski et al, 2016). Trigeminal ganglion nociceptors take part in bone innervations; in particular, they supply the cranial periosteum (Zhao and Levy, 2014).…”

“…However, ATP is a full P2X3 agonist with limited stability, whereas the stability of ApppI appears to be much higher, a property suggesting that it is able to induce prolonged inhibitory antinociceptive action. In a previous study, we characterized synthetic Ap n A-analogs with comparable inhibitory action on hP2X3 receptors and antinociceptive action in vivo (Viatchenko-Karpinski et al, 2016); however, ApppI presents even better characteristics as a stronger inhibitory antinociceptive agent.…”

“…However, the interaction of ApppI with the nociceptive system has not yet been characterized. Since ApppI structurally resembles the antinociceptive diadenosine polyphosphates (Viatchenko-Karpinski et al, 2016), we investigated the effects of the NBP-induced derivative ApppI on the activity of nociceptive receptors P2X2, P2X3, and P2X7.…”