Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2

Abstract

Background and purpose: Prostaglandin E₂ is considered a major mediator of inflammatory pain, by acting on neuronal G_s protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is G_i protein-coupled and antagonizes the pronociceptive prostaglandin E₂ effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons.

Experimental approach: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)^-/- , and PI3Kγ^{kinase dead (KD)/KD} mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry.

Key results: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding.

Conclusion and implications: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E₂ . These new insights hold the potential for a novel approach in pain therapy.

Keywords: EP3 receptor; GRK2; PGE2; PI3Kγ; antinociception; pain; sensory neuron.