title: AKAP-dependent sensitization of Ca(v) 3.2 channels via the EP(4)  receptor/cAMP pathway mediates PGE(2) -induced mechanical hyperalgesia.
abstract: OBJECTIVE
  The Ca(v) 3.2 isoform of T-type Ca(2+)  channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE(2) -induced hyperalgesia. Here we examined and analysed Ca(v) 3.2 sensitization via the PGE(2) /cAMP pathway in NG108-15 cells that express Ca(v) 3.2 and produce cAMP in response to PGE(2) , and its impact on mechanical nociceptive processing in rats.
  
  
  METHODS
  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 Ca(v) 3.2 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.
  
  
  RESULTS
  In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE(2)  increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE(2)  was abolished by RQ-00015986-00, an EP(4)  receptor antagonist. AKAP150 was co-immunoprecipitated with Ca(v) 3.2, regardless of stimulation with db-cAMP, and Ca(v) 3.2 was phosphorylated by db-cAMP or PGE(2) . In rats, intraplantar (i.pl.) administration of db-cAMP or PGE(2)  caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl(2) , known to inhibit Ca(v) 3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE(2) -induced mechanical hyperalgesia.
  
  
  CONCLUSIONS
  Our findings suggest that PGE(2)  causes AKAP-dependent phosphorylation and sensitization of Ca(v) 3.2 through the EP(4)  receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.
authors.affiliation: Division of Pharmacology & Pathophysiology, Kinki University School of Pharmacy, Higashi-Osaka, Japan.
authors.forename: Fumiko
authors.initials: F
authors.lastname: Sekiguchi
authors.forename: Yuka
authors.initials: Y
authors.lastname: Aoki
authors.forename: Maiko
authors.initials: M
authors.lastname: Nakagawa
authors.forename: Daiki
authors.initials: D
authors.lastname: Kanaoka
authors.forename: Yuta
authors.initials: Y
authors.lastname: Nishimoto
authors.forename: Maho
authors.initials: M
authors.lastname: Tsubota-Matsunami
authors.forename: Rumi
authors.initials: R
authors.lastname: Yamanaka
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Atsufumi
authors.initials: A
authors.lastname: Kawabata
mesh_terms: D054758:A Kinase Anchor Proteins / Q000502:physiology*; D000818:Animals; D020747:Calcium Channels, T-Type / Q000502:physiology*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D015232:Dinoprostone / Q000502:physiology*; D005727:Ganglia, Spinal / Q000166:cytology; D006930:Hyperalgesia / Q000139:chemically induced / Q000503:physiopathology; D008297:Male; D051379:Mice; D009474:Neurons; D051381:Rats; D017208:Rats, Wistar; D058309:Receptors, Prostaglandin E, EP4 Subtype / Q000502:physiology*
chemical_list: D054758:A Kinase Anchor Proteins; C504883:Cacna1h protein, mouse; C509958:Cacna1h protein, rat; D020747:Calcium Channels, T-Type; D058309:Receptors, Prostaglandin E, EP4 Subtype; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone