Department of Biotechnology, School of Arts and Sciences, American University of Ras Al Khaimah, Ras Al Khaimah, United Arab Emirates.
Department of Biology, Faculty of Arts & Sciences, American University of Beirut, Beirut, Lebanon.
PLoS One. 2021 Jan 14;16(1):e0245400. doi: 10.1371/journal.pone.0245400. eCollection 2021.
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.
钠钾 ATP 酶是调节肝细胞离子稳态的关键酶，其功能改变可导致多种肝脏疾病。我们最近发现，S1P 类似物 FTY720P 处理 HepG2 细胞可显著刺激钠钾 ATP 酶，该作用是通过 PGE2 介导的。然而，目前尚不清楚前列腺素发挥作用的机制，这也是本研究的重点。通过使用药理学抑制剂来确定涉及的受体类型，同时使用 Western blot 分析、GFP 标记的钠钾 ATP 酶荧光成像和活细胞延时成像来检测钠钾 ATP 酶在膜上丰度的变化。通过测量有无哇巴因存在时释放的无机磷酸盐的量来测定 ATP 酶的活性。当阻断 EP4 受体时，未观察到 ATP 酶活性增强，但在 EP1、EP2 和 EP3 受体抑制剂存在的情况下仍观察到该现象。通过观察 EP4 激动剂引起的刺激，证实了 EP4 的参与。在 PKA 抑制剂 Rp-cAMP 的存在下，PGE2 的刺激作用不明显，而 db-cAMP（PKA 激活剂）可模拟该作用。用 BAPTA-AM 螯合细胞内钙可消除 db-cAMP 以及 PGE2 的作用，但在无钙 PBS 培养基中用 PGE2 处理则不会，这表明细胞内钙的参与，这一结果得到了 2-APB 处理结果的证实。活细胞成像显示，PGE2 处理后 GFP-钠钾 ATP 酶阳性囊泡向膜运动，以及膜上 ATP 酶丰度增加。综上所述，PGE2 通过 EP4、PKA 和细胞内钙发挥作用。
