Opioid signal transduction in intact and fragmented SH-SY5Y neural cells.
Parameters of ligand binding, stimulation of low-Km 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 mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]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 [3H]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 Bmax for [3H]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.(ABSTRACT TRUNCATED AT 250 WORDS)
Carter
B D
BD
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Medzihradsky
F
F
D004723:Endorphins / Q000502:physiology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008024:Ligands; D009423:Nervous System Neoplasms / Q000473:pathology / Q000503:physiopathology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D004723:Endorphins; D004745:Enkephalins; D008024:Ligands; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D006160:Guanosine Triphosphate