FTY720‐P Activates Hepatic Na+/K+ ATPase via S1PR3, PKC and PGE2

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BackgroundAlterations in hepatocytes' volume contribute to the pathophysiology of several hepatic disorders including liver insufficiency, diabetic ketoacidosis, hyper‐catabolism and infection. A key regulator of ionic homeostasis and hepatocellular volume is the Na+/K+ ATPase which establishes a sodium electro‐chemical gradient involved in a wide array of cellular activities. Emerging evidence ascribes a role for sphingosine 1‐phosphate (S1P) in the development and progression of liver diseases. The sphingolipid signal via 5 different types of G protein coupled sphingosine 1‐phosphate receptors (S1PR1‐S1PR5), and induces many cellular responses. Previous studies in our lab showed that S1P promotes at 2hrs, an increase in the Na+/K+ ATPase activity in HepG2 (Hepatic carcinoma cell line) cells, but its signaling pathways was not determined. FTY720‐P, a S1P analogue, has been approved recently by US‐FDA as an oral treatment for relapsing form of multiple sclerosis. The drug is a S1P receptor agonist and has been shown to exert some side effects on different organs like the heart. Its side effects on the liver have not been however sufficiently studied.AimThe aim of this work is investigate the effect of FTY720P on hepatic Na+/K+ ATPase and identify the signaling pathway involved, using HepG2 cells as a model. An effect of the drug on liver Na+/K+ ATPase may alter ionic homeostasis and exert adverse effects leading to liver disease. Unraveling the mode of action of the drug would help in reducing the undesirable effects, by inhibiting any of the specific mediators involved.Methods and resultsHepG2 cells were incubated with FTY720P for 2 hrs and the activity of the pump was assayed by measuring the amount of inorganic‐phosphate in the presence and absence of ouabain, a specific inhibitor of the Na+/K+ ATPase. FTY720P induced a 2.5 fold increase in the activity of the Na+/K+ ATPase which was maintained in the presence of JTE‐013, a specific blocker of S1PR2, but disappeared completely in presence of CAY 10444, a specific S1PR3 antagonist. Inhibiting protein kinase C (PKC) and the cyclooxygenase (COX) enzymes with respectively calphostin and indomethacin abolished also the effect of FTY720P. Western blot analysis showed an increase in the expression of COX‐2 enzyme. The PKC activator Phorbol 12‐ myristate 13‐acetate (PMA), induced as FTY720P, a significant increase in the activity of the ATPase and a significant increase in the expression of COX‐2 enzyme. Exogenous prostaglandin E2 (PGE2), exerted a stimulatory effect on the ATPase which was maintained in presence of calphostin.ConclusionsIt was concluded that FTY720P, when applied for 2 hours, binds to S1PR3 and activates PKC, which increases directly or indirectly the expression of COX‐2 enzyme leading to PGE2 release. The latter exerts a stimulatory effect on the Na+/K+ ATPase through a pathway that still needs to be determined.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.