AKAP‐dependent sensitization ofCa_v3.2 channels via theEP₄receptor/cAMPpathway mediatesPGE₂‐induced mechanical hyperalgesia

Abstract

BACKGROUND AND PURPOSEThe Cav3.2 isoform of T-type Ca 2+ 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. EXPERIMENTAL APPROACHIn 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. KEY RESULTSIn 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 IMPLICATIONSOur 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. AbbreviationsAKAP, A-kinase anchoring protein; AKAPI, AKAP St-Ht31 inhibitor peptide; AUC, area under the curve;

Protein kinase a regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle

“…Past work conducted in cardiac and neuronal tissues has consistently shown that T-type Ca 2+ channels are a rich target for protein kinase regulation (Li et al, 2012;Sekiguchi et al, 2013). Of particular note is the reported ability of PKA through receptor stimulation to selectively augment Ca V 3.1 or Ca V 3.2 activity in a tissue-dependent manner (Li et al, 2012;Sekiguchi et al, 2013).…”

“…Of particular note is the reported ability of PKA through receptor stimulation to selectively augment Ca V 3.1 or Ca V 3.2 activity in a tissue-dependent manner (Li et al, 2012;Sekiguchi et al, 2013). In vascular smooth muscle, PKA signaling plays a prominent role in enabling vasodilation through mechanisms involving reductions in cytosolic Ca 2+ .…”

“…This control experiment is important in that it: (1) addresses the target specificity of positive modulators such as forskolin; and (2) indicates that T-type channels are not subject to PKA regulatory control under basal conditions. Recent literature has noted that PKA modulation of voltagegated Ca 2+ channels occurs discretely within compartmental signaling domains (Fuller et al, 2010;Sekiguchi et al, 2013). Compartmentalization of PKA signaling is achieved through Akinase anchoring proteins (AKAP), scaffolding elements that bring PKA in close proximity to end targets such as neuronal Ca V 3.2 (Sekiguchi et al, 2013) or cardiac Ca V 1.2 (Fuller et al, 2010) channels.…”

“…For example, vascular studies have yet to separate Ca V 3.1 from Ca V 3.2 and to determine whether either subtype is modulated by signaling pathways that influence arterial tone development. The latter is particularly prescient as T-type Ca 2+ channels have been reported to be a rich regulatory target in neuronal and cardiac tissues (Li et al, 2012;Sekiguchi et al, 2013).…”

T-type Calcium Channels: Functional Regulation and Implication in Pain Signaling

“…2). AKAP-dependent functional upregulation of Ca v 3.2 by PKA following EP 4 receptor activation mediates PGE 2 -induced mechanical hyperalgesia (15), although sensitization of TRPV1 channels by PKC following EP 1 -receptor activation participates in the PGE 2 -induced thermal hyperalgesia (24) (Fig. 2).…”