Prostaglandin E(2) (PGE(2)) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na(+),K(+)-ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE(2) decreases Na(+),K(+)-ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE(2). Na(+),K(+)-ATPase activity was determined by assessing ouabain-sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE(2) (0.1-10 microM) for 30 min decreased Na(+),K(+)-ATPase activity in a concentration-dependent manner. However, PGE(2) did not alter Na(+),K(+)-ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE(2) on Na(+),K(+)-ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic alpha subunit of Na(+),K(+)-ATPase. We found that the inhibitory effect of PGE(2) (1 microM) on Na(+),K(+)-ATPase activity was receptor-mediated, as incubation with selective antagonists for EP1 (SC-19220, 10 microM), EP3 (L-826266, 1 microM) or EP4 (L-161982, 1 microM) receptors prevented the PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1-10 microM) increased enzyme activity per se in a concentration-dependent manner, but did not prevent the inhibitory effect of PGE(2). Incubation with a protein kinase A (PKA) inhibitor (H-89, 1 microM) and a protein kinase C (PKC) inhibitor (GF-109203X, 300 nM) also prevented PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. Accordingly, PGE(2) increased phosphorylation of Ser943 at the alpha subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE(2) decreases Na(+),K(+)-ATPase activity in vivo. The results presented here imply Na(+),K(+)-ATPase as a target for PGE(2)-mediated signaling, which may underlie PGE(2)-induced increase of brain excitability.