Koshimizu Taka-Aki, Kashiwazaki Aki, Taniguchi Junichi
Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, Tochigi 329-0498, Japan.
Sci Rep. 2016 May 3;6:25327. doi: 10.1038/srep25327.
Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.
降低细胞外环境中的Na⁺可能会对增加激动剂与细胞表面G蛋白偶联受体（GPCRs）的结合产生两种有益效果：减少Na⁺介导的结合阻滞以及减少受体内化。然而，尚未对这种联合效应进行探索。我们使用表达血管加压素V1b受体的中国仓鼠卵巢细胞作为模型，来探究激动剂结合和受体内化过程中的Na⁺敏感性。在基础条件下，大部分V1b受体位于细胞内，小部分位于质膜中。细胞外Na⁺的减少增加了细胞表面[³H]AVP的结合，并减少了受体内化。用Cs⁺或NH₄⁺替代Na⁺会抑制激动剂结合。为了抑制受体内化，由于内化机制对Na⁺的敏感性高于Cs⁺，NaCl的浓度必须低于50 mM，而CsCl则不必。即使在低NaCl环境中，等渗补充葡萄糖或NH₄Cl也能维持V1b受体的内化。此外，作为抗衡阴离子的碘离子会抑制V1b激动剂的结合。总之，我们发现了外部离子条件，这些条件可以在激动剂刺激期间以最小的内化增加细胞表面高亲和力状态受体的存在。
