Abstract:
Na+-K+ ATPase is ubiquitously expressed in all animal cells serving a myriad of fundamental functions. In the intestines, it regulates sodium, water, and nutrient absorption; hence, any significant alteration in its activity can trigger the onset of diarrhea or constipation. Patients with Inflammatory Bowel Disease (IBD) suffer from diarrhea with their colonic mucosal tissues showing elevated sphingosine-1-phosphate (S1P) levels. Previous studies in our lab have shown a modulatory effect of S1P on the activity of Na+-K+ ATPase in HepG2 cells. This work was undertaken to examine whether S1P exerts a similar modulatory effect in colonocytes, and in case it does, to determine the signaling pathway. FTY720P was used as an analogue of S1P and Caco-2 cells as a model. Na+-K+ ATPase activity was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain, a specific inhibitor of the ATPase. A time-response study, conducted between 75 and 180 min, revealed a significant stimulatory effect of FTY720P on Na+-K+ ATPase, reaching maximal effects at 90 min and beyond. Two hours were considered as the optimal period for FTY720P action and were adopted in all subsequent experiments. The stimulatory effect of FTY720P was completely abolished in presence of JTE-013, a S1P receptor 2 (S1PR2) antagonist but not in the presence of CAY10444, a blocker of S1P receptor 3 (S1PR3). Furthermore, the S1PR2 agonist, CYM5520, stimulated the Na+-K+ ATPase in a similar fashion to that of FTY720P while CYM5541, a S1PR3 agonist, had no effect. The effect of FTY720P completely disappeared also in presence of calphostin (PKC inhibitor), BAPT-AM (calcium chelator), NFκB inhibitor, indomethacin (COX inhibitor), RpcAMP (PKA inhibitor), and Wortmannin (PI3K inhibitor) and was mimicked by PMA (PKC activator), exogenous PGE2 (prostaglandin E2), and dbcAMP (PKA activator). The stimulatory effect of PMA disappeared completely when Caco-2 cells were pre-treated with NFκB inhibitor, Indomethacin,
Description:
Thesis. M.S. American University of Beirut. Department of Biology, 2018. Advisor : Dr. Sawsan Kreydiyyeh, Professor, Biology ; Members of Committee : Dr. Khouzama Knio, Professor, Biology ; Dr. Zakaria Kambris, Associate Professor, Biology.
Includes bibliographical references (leaves 67-89)