Indomethacin serves as a potential inhibitor of protein phosphatases

We have shown that indomethacin has the potential to activate Ca2+/ calmodulin-dependent protein kinase II (CaMKII), regardless of cyclooxygenase (COX) inhibition. To understand the underlying mechanism, the present study investigated the effect of indomethacin on protein phosphatases such as protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein tyrosine phosphatase 1B (PTP1B).

Activity of CaMKII was assayed in cultured rat hippocampal neurons and under the cell-free conditions. Activities of protein phosphatases were monitored under the cell-free conditions. Indomethacin binding assay was carried out using a fluorescein-conjugated indomethacin.

Indomethacin enhanced CaMKII activity in cultured rat hippocampal neurons, that is abolished the CaMKII inhibitor KN-93. In the cell-free assay, no CaMKII activation was obtained with indomethacin, but indomethacin otherwise inhibited PP1 in a concentration (10 µM-1 mM)-dependent manner, the maximum reaching 70% of basal levels. This indicates that indomethacin indirectly activates CaMKII due to PP1 inhibition. Likewise, indomethacin still inhibited PP2A and PTP1B in a concentration (10 µM-1 mM)-dependent manner, reaching 80 and 10% of basal levels at 1 mM, respectively. In the indomethacin binding assay, indomethacin bound to all the investigated protein phosphatases.

The results of the present study indicate that indomethacin inhibits PP1, PP2A, and PTP1B, possibly through its direct binding and that the inhibitory effect of indomethacin on PP1 could cause indirect CaMKII activation. This may represent the novel indomethacin action.