Novel Indomethacin Action: Selective and Direct Activation of Protein Kinase C-ε

Background/Aims: In our earlier study, indomethacin potentiated alpha 7 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-epsilon was knocked-down using the siRNA to silence the PKC-epsilon-targeted gene. A fluorescein-conjugated indomethacin was synthesized to examine the interaction of indomethacin with PKC-epsilon. Results: In the in situ PKC assay, indomethacin activated PKC in PC-12 cells in a concentration (1-100 mu M)-dependent manner, and the activation was suppressed by knocking-down PKC-epsilon. In the cell-free PKC assay, indomethacin (100 mu M) activated PKC-epsilon in the absence of diacylglycerol, phosphatidylserine, and calcium, but other PKC isozymes such as alpha, beta I, beta II, gamma, delta, iota, and zeta 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-epsilon 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-epsilon through its direct binding, independently of cyclooxygenase (COX) inhibition. Copyright (C) 2012 S. Karger AG, Basel