The effect of FTY720P on NA+-K+ ATPase in CACO2 cells, and the signaling pathway invovled -

Abstract

The colon plays an important role in sodium absorption which is driven by the sodium electro-chemical gradient established by the Na+-K+ ATPase present on the basolateral side. Water follows then by osmosis. Any change in the activity of the ATPase is expected to alter sodium and potassium colonic transport and consequently water, leading to diarrhea or constipation. Diarrhea is the main symptom of Inflammatory Bowel Disease (IBD). IBD patients have lower Na+-K+ ATPase activity and higher levels of sphingosine-1-phosphate (S1P), a lipid mediator essential for cell proliferation, migration, and differentiation. S1P acts as an intracellular and an extracellular messenger. Its extracellular action is mediated via 5 different G-protein coupled receptors (S1PR 1-5). Lately, FTY720-P has been recognized as a structural analogue of S1P and has been approved for the treatment of multiple sclerosis. Since in IBD the high S1P levels are accompanied with low Na+-K+ ATPase activity, this work was undertaken to see if a cause effect relationship exists between S1P and the ATPase, and in case it does to determine the signaling pathway involved using Caco-2 cells as a model and FTY720P, a S1P analogue. 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 inhibitory of the ATPase. The type of S1P receptors expressed was studied by western blot analysis and showed that S1PR2 is the most abundantly expressed receptor in Caco-2 cells. FTY720P (7.5 nM, 15 min) exerted a significant inhibitory effect on the ATPase which disappeared in presence of JTE-013(a specific blocker of S1PR2) and indomethacin (an inhibitor of cyclooxygenase enzymes), and mimicked by CYM5520, (a S1PR2 agonist). Sulprostone (an EP3 agonist) and RpcAMP (a PKA inhibitor) reduced individually the activity of the Na+-K+ ATPase. The effect of FTY720P was abolished in the presence of calphostin (PKC inhibitor), wortmanin (PI3K inhibitor) and PTIO (nitric oxide scavenger