title: Novel indomethacin action: selective and direct activation of protein kinase C-ε.
abstract: OBJECTIVE
  In our earlier study, indomethacin potentiated α7 acetylcholine (ACh) receptor responses by activating protein kinase C (PKC). The present study was conducted to gain further insight into the indomethacin action on PKC.
  
  
  METHODS
  PKC activity was assayed in PC-12 cells or under the cell-free conditions. PKC-ε was knocked-down using the siRNA to silence the PKC-ε-targeted gene. A fluorescein-conjugated indomethacin was synthesized to examine the interaction of indomethacin with PKC-ε.
  
  
  RESULTS
  In the in situ PKC assay, indomethacin activated PKC in PC-12 cells in a concentration (1-100 µM)-dependent manner, and the activation was suppressed by knocking-down PKC-ε. In the cell-free PKC assay, indomethacin (100 µM) activated PKC-ε in the absence of diacylglycerol, phosphatidylserine, and calcium, but other PKC isozymes such as α, βΙ, βΙΙ, γ, δ, ι, and ζ were not activated. In the indomethacin binding assay using a fluorescent-conjugated indomethacin on blue native-polyacrylamide gel electrophoresis (blue native-PAGE), a fluorescent signal was detected at the site consistent with PKC-ε protein and the signal was attenuated by adding non-conjugated indomethacin or eliminated by pretreatment with non-conjugated indomethacin.
  
  
  CONCLUSIONS
  The results of the present study show that indomethacin has the potential to selectively activate PKC-ε through its direct binding, independently of cyclooxygenase (COX) inhibition.
authors.affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kanno
authors.forename: Ayako
authors.initials: A
authors.lastname: Tsuchiya
authors.forename: Tadashi
authors.initials: T
authors.lastname: Shimizu
authors.forename: Akito
authors.initials: A
authors.lastname: Tanaka
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nishizaki
mesh_terms: D000818:Animals; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects*; D019793:Fluorescein / Q000737:chemistry; D007213:Indomethacin / Q000494:pharmacology*; D016716:PC12 Cells; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D016861:Cyclooxygenase Inhibitors; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011451:Prostaglandin-Endoperoxide Synthases; D051744:Protein Kinase C-epsilon; D019793:Fluorescein; D007213:Indomethacin