REGULATION OF MU-OPIOID RECEPTOR IN NEURAL CELLS BY EXTRACELLULAR-SODIUM

SH-SY5Y neural cells expressing mu- and delta-opioid receptors were ma intained viable in isotonic, sodium-free buffer in vitro. Intracellula r sodium levels were manipulated by various methods, and ligand bindin g to intact cells was studied. In physiological buffer containing 118 mM sodium, [H-3]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([H-3]DAMGO) and [H-3]nal trexone bound to mu receptor with K-D values of 3.1 and 0.32 nM 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 inc reased B-max for [H-3]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 mM) in physiological buffer decreased the intracellular sodium lev els by 67%, but this did not decrease the [H-3]DAMGO binding to the ce lls. Incubation of cells with monensin and ouabain increased the intra cellular sodium levels dramatically (from 78 to 250 and 300 nmol/mg, r espectively), with no changes in agonist binding parameters. Ethylisop ropylamiloride inhibited [H-3]DAMGO and [H-3]naloxone binding to intac t cells with EC(50) values of 24 and 3,600 nM, respectively. Adenylyl cyclase activities measured in intact cells, at different concentratio ns of sodium, showed the physiological significance of this ion in sig nal transduction. Potency of DAMGO in inhibiting the forskolin-stimula ted adenylyl cyclase activity was significantly higher at lower concen trations of sodium. However, inhibition reached the maximal level only at 50 mM sodium, and typical sigmoidal dose-response curves were obta ined only in the presence of 118 mM sodium. Furthermore, even at low o r high intracellular sodium levels, DAMGO inhibition of cyclic AMP lev els 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 mu receptor.