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The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

The Australian redclaw crayfish Cherax quadricarinatus is a freshwater crustacean aquacultured in many countries. Redclaw crayfish are often exposed to saline waters in attempts to improve flavor, to relieve stress during transport, and to prevent or treat parasitic infestations. However, the effect of salinity on the crayfish osmoregulatory process is not well studied. In the present work, we assessed the effect of various salinities on survival, growth, and osmoregulation in redclaw crayfish. Adult crayfish were maintained at seven salinities (0, 2, 4, 6, 8, 10, and 12 ppt) for 4 weeks, and hemolymph osmolality, gill sodium potassium ATPase activity, and sodium potassium chloride cotransporter expression were assessed. In another experiment, juvenile crayfish were size sorted to similar weights and stocked at four salinities (0, 2, 4, and 6 ppt) for 8 weeks. The animals were group weighed every 2 weeks and individually weighed at the end of the eighth week. Hemolymph osmolality was constant as salinity increased from 0 to 10 ppt then a significant increase was observed at 12 ppt. Redclaw growth rate decreased with an increase in salinity from 0 to 6 ppt. Sodium potassium ATPase activity and sodium potassium chloride cotransporter expression increased with salinity increase. Although salinity might help treat ectoparasites, improve taste, and reduce transportation stress, long‐term exposure increases osmoregulatory metabolic costs and affects growth and energy consumption.

FTY720P, an analogue of sphingosine 1‐phosphate, has emerged lately as a potential causative agent of inflammatory bowel disease, in which electrolytes movements driven by the sodium gradient established by the Na+/K+ ATPase are altered. We showed previously in Caco‐2 cells, a 50% FTY720P‐induced decrease in the ATPase activity, mediated via S1PR2 and PGE2. This work aims at delineating the mechanism underlying PGE2 release and at investigating if the ATPase inhibition is due to changes in its abundance. The activity of the ATPase and the localization of a GFP‐tagged Na+/K+‐ATPase α1‐subunit were assessed in cells treated with 7.5 nM FTY720P. The involvement of ERK, p38 MAPK, PKC, and PI3K was studied in cells treated with 7.5 nM FTY720P or 1 nM PGE2 in presence of their inhibitors, or by determining changes in the protein expression of their activated phosphorylated forms. Imaging data showed ∼30% reduction in the GFP‐tagged Na+/K+ ATPase at the plasma membrane. Both FTY720P and PGE2 showed, respectively, 50% and 60% reduction in ATPase activity that disappeared when p38 MAPK, PKC, and PI3K were inhibited individually but not with ERK inhibition. The effect of FTY720P was imitated by PMA, an activator of PKC. Western blotting revealed inhibition of ERK by FTY720P. It was concluded that FTY720P, through activation of S1PR2, downregulates the Na+/K+ ATPase by inhibiting ERK, which in turn activates p38 MAPK leading to the sequential activation of PKC and PI3K, PGE2 release, and a decrease in the Na+/K+ ATPase activity and membrane abundance.

Inflammatory Bowel Disease was shown to be accompaniedwith higher levels of sphingosine‐1‐ phosphate (S1P) and lower 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 main mediators involved, using Caco‐2 cells as a model and fingolimod phosphate (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 inhibitor of the Na+/K+ATPase. Western blot analysis showed that sphingosine‐1‐phosphate receptor 2 (S1PR2) is the mostly expressed receptor in Caco‐2 cells. FTY720P, applied for 15 min, reduced significantly the activity of the Na+/K+ ATPase, with a maximal inhibition observed at a concentration of 7.5nM. The effect of FTY720P disappeared completely in presence of JTE‐013 (a specific blocker of S1PR2) and indomethacin (an inhibitor of cyclooxygenase enzymes), and was mimicked by CYM5520 (a S1PR2 agonist), prostaglandin E2 (PGE2), and sulprostone (an agonist of E prostanoid receptor 3 (EP3)). Carboxy‐PTIO a nitric oxide scavenger, abrogated the effect of FTY720P as well as that of PGE2. Treating the cells with Glyco‐SNAP (nitric oxide donor) reduced significantly the ATPase activity. It was concluded that FTY720‐P inhibits the ATPase by binding to S1PR2 leading to PGE2 production. PGE2 binds to EP3 receptors and increases nitric oxide levels resulting in an inhibitory effect on the Na+/K+ ATPase.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.