BACKGROUND/AIMS: 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. CONCLUSION: 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.
BACKGROUND/AIMS: 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. CONCLUSION: 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.