Mediators of the inhibitory effect of FTY720 phosphate, an analogue of sphingosine one phosphate, on hepatic Na⁺-K⁺ ATPase -

Abstract

An inhibitory effect of sphingosine -1- phosphate (S1P) on hepatic Na⁺-K⁺ ATPase was previously demonstrated. This work is an attempt to delineate the signaling pathway involved using its agonist FTY720P (FTY720P). HepG2 cells were used as a model and the activity of the Na⁺-K⁺ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the ATPase. FTY720P (7.5 nM, 15min) significantly inhibited the Na⁺-K⁺ATPase. This effect disappeared completely in presence of JTE-013, a specific blocker of S1P2 receptors. The inhibition was similarly abrogated by calphostin, indomethacin, RpcAMP, PTIO, an NF-κB inhibitor, and PD98059, respective inhibitors of protein kinase C (PKC), cyclooxygenases, protein kinase A (PKA), nitric oxide synthase (NOS), NF-κB, and extracellular signal-regulated kinase (ERK1-2). Treatment with prostaglandin E₂ (PGE₂) or selective activators of these enzymes mimicked the effect of FTY720P which was shown by western blot analysis to increase IκB degradation. PGE2 was found to act via its EP2 receptors, and its effect was still manifested in the presence of calphostin, and the NF-κB inhibitor but disappeared completely in presence of inhibitors of PKA, NOS,and ERK, suggesting that PGE2 is downstream of PKC and NF-κB and upstream of PKA, NO, and ERK. The effect of PMA, a PKC activator, was completely abolished by NF-κB inhibitor indicating that NF-κB is downstream of PKC and upstream of PGE₂. Furthermore, western blot analysis showed phosphorylation of ERK by dibutyrylcyclic adenosine monophosphate, a PKA activator. This phosphorylation did not appear in presence of PTIO indicating that PKA is upstream of NO. It was concluded that FTY 720-P activates PKC via S1P2 receptors leading to NF-κB activation and PGE2 generation. PGE₂ acts through its EP2 receptor and activates PKA which in turn activates NOS leading to NO