Regulation of Μ-Opioid Receptor in Neural Cells by Extracellular Sodium

Abstract

SH-SY5Y neural cells expressing Μ- and Δ-opioid receptors were maintained viable in isotonic, sodium-free buffer in vitro. Intracellular sodium levels were manipulated by various methods, and ligand binding to intact cells was studied. In physiological buffer containing 118 m M sodium, [ 3 H]Tyr-d-Ala-Gly-(Me)Phe-Gly-ol ([ 3 H]-DAMGO) and [ 3 H]naltrexone bound to Μ receptor with K D values of 3.1 and 0.32 n M and B max values of 94 and 264 fmol/mg of protein, respectively. Replacement of sodium by choline decreased the affinity of the antagonist and increased B max for [ 3 H]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 m M ) in physiological buffer decreased the intracellular sodium levels by 67%, but this did not decrease the [ 3 H]DAMGO binding to the cells. Incubation of cells with monensin and ouabain increased the intracellular sodium levels dramatically (from 78 to 250 and 300 nmol/mg, respectively), with no changes in agonist binding parameters. Ethylisopropylamiloride inhibited [ 3 H]DAMGO and [ 3 H]naloxone binding to intact cells with EC 50 values of 24 and 3,600 n M , respectively. Adenylyl cyclase activities measured in intact cells, at different concentrations of sodium, showed the physiological significance of this ion in signal transduction. Potency of DAMGO in inhibiting the forskolin-stimulated adenylyl cyclase activity was significantly higher at lower concentrations of sodium. However, inhibition reached the maximal level only at 50 m M sodium, and typical sigmoidal dose-response curves were obtained only in the presence of 118 m M sodium. Furthermore, even at low or high intracellular sodium levels, DAMGO inhibition of cyclic AMP levels was normal. These results support a role for extracellular sodium in regulating not only the ligand interactions with the receptor, but also the signal transduction through the Μ receptor.