AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cAMP pathway mediates PGE2-induced mechanical hyperalgesia
Background and Purpose
The Cav3.2 isoform of T-type Ca2+ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE2-induced hyperalgesia. Here we examined and analysed Cav3.2 sensitization via the PGE2/cAMP pathway in NG108-15 cells that express Cav3.2 and produce cAMP in response to PGE2, and its impact on mechanical nociceptive processing in rats.
In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Cav3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats.
In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE2 increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE2 was abolished by RQ-00015986-00, an EP4 receptor antagonist. AKAP150 was co-immunoprecipitated with Cav3.2, regardless of stimulation with db-cAMP, and Cav3.2 was phosphorylated by db-cAMP or PGE2. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE2 caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl2, known to inhibit Cav3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE2-induced mechanical hyperalgesia.
Conclusion and Implications
Our findings suggest that PGE2 causes AKAP-dependent phosphorylation and sensitization of Cav3.2 through the EP4 receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.