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Abstract
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.
Highlights
The liver plays an important role in metabolic homeostasis that is highly dependent on cellular ionic balance
We have shown previously a significant increase in the activity of the Na+/K+ ATPase in HepG2 cells, treated with FTY720P for 2 hrs [10], that was mediated via S1PR3 and sequential activation of PKC, ERK, and NF-κB leading to a higher expression level of COX-2 enzyme and Prostaglandin E2 (PGE2) release
PGE2 exerted a significant increase in the activity of the Na+/K+ ATPase that was still manifested in presence of blockers of EP1(SC-19220), EP2 (PF-04418948) and EP3 (L-798106), but disappeared completely in presence of BGC 20–1531, an antagonist of EP4 receptors (Fig 1), suggesting that PGE2 acts via EP4 receptors
Summary
The liver plays an important role in metabolic homeostasis that is highly dependent on cellular ionic balance. The latter is accomplished in hepatocytes, by the synchronized activity of various carriers and transporters [1]. PGE2 upregulates Na+/K+ ATPase in HepG2 metabolism, proliferation, differentiation and most importantly cell volume. It establishes the Na+ gradient needed for the transport of bile salts [2] and drives the activity of many other transporters like the Na+ /H+ exchanger and the Na+ -K + 2Cl cotransporter involved respectively in the regulation of intracellular pH and cell volume. An alteration in the Na+ -K + ATPase activity is expected to result in a derangement of many of the liver functions
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