Abstract
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 [3H]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.
Highlights
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
Using the vasopressin V1b receptor expressed in Chinese hamster ovary (CHO) cells as a model, we quantitatively investigated the interaction between the agonist and cell surface receptors and subsequent receptor
Using [3H]AVP, agonist binding was evaluated by the radioactivity of [3H] AVP bound to cell surface receptors, whereas receptor internalization was evaluated by the radioactivity of [3H]AVP taken up by CHO cells[20]
Summary
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. Such combined effects have not been explored. 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. Hypotonic shock and the subsequent reduction in extracellular potassium ion levels effectively depletes intracellular potassium ions and blocks internalization of the receptor for low density lipoprotein (LDL) and epidermal growth factors (EGF), which may be a result of the inhibition of new clathrin pit formation[8,9]. The V1b receptor is located in the colonic epithelia, where the ionic composition around the cells can be widely altered in physiological and pathophysiological conditions[18,19]
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