Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Abstract

Parameters of ligand binding, stimulation of low- K m GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the Μ-selective opioid agonist [ 3 H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([ 3 H]DAMGO) bound to two populations of sites with K D values of 3.9 and 160 n M , with <10% of the sites in the high-affinity state. Both sites were also detected at 4°C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [ 3 H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [ 3 H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [ 3 H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the B max for [ 3 H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP. Opioid receptor coupling to adenylate cyclase in intact and fragmented cells occurred with similar efficiency: DAMGO inhibited adenylate cyclase with K i , values of 11 n M in cells and 26 n M in lysates, with 30% maximal inhibition in both preparations. Receptor coupling to G protein in membranes occurred with similar parameters: DAMGO stimulated low- K m GTPase with a K s of 31 n M and an S max of 48%. Both effector responses were blocked by naloxone and were strongly impaired by rigorous cell homogenization. These results indicate that opioid signal transduction in intact SH-SY5Y cells and their appropriately isolated membranes functions with similar efficiencies involving a large reserve of uncoupled receptors.