CHARACTERIZATION OF OPIOID AGONIST EFFICACY IN A C-6 GLIOMA CELL-LINEEXPRESSING THE MU-OPIOID RECEPTOR

In C-6 glioma cells stably expressing a homogeneous population of the cloned rat mu opioid receptor, the binding affinities of opioid agonis ts and subsequent activation of G protein were examined. Opioid recept or number in membranes of these cells was high (10-30 pmol/mg protein [H-3]diprenorphine binding sites). Opioids were found to bind to the r eceptor with high affinity [Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) 0.23 nM; sufentanil 0.034 nM; morphine 0.16 nM]. Activation of G protein by opioid agonists was examined by measuring the stimulation of guanosin e-5'-0-(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding. Sufenta nil increased [S-35]GTP gamma S binding by 326% with an EC(50) value o f 2.39 nM. Agonist stimulation of [S-35]GTP gamma S binding was stereo selective, naltrexone-reversible, and pertussis toxin-sensitive. The ' 'intrinsic activity'' of opioids at the mu receptor was reflected by t he magnitude of agonist-mediated activation of G protein. The rank ord er of the stimulation of [S-35]GTP gamma S binding was etonitazene = s ufentanil = DAMGO = PL017 = fentanyl > morphine > profadol > meperidin e > butorphanol = nalbuphine = pentazocine > cyclazocine = nalorphine > levallorphan > naltrexone. High affinity binding of ligands to the m u opioid receptor was reduced by the addition of sodium and guanosine diphosphate at concentrations used in the [S-35]GTP gamma S binding as say. Ligand affinity was reduced in a manner correlating with ''intrin sic activity'': DAMGO, 1229-fold, nalbuphine 35-fold, naltrexone, 3-fo ld. The results presented show that the stable expression of the rat m u opioid receptor in C-6 cells provides an effective tool to examine o pioid receptor signal transduction mechanisms and evaluate the activit y of novel opioids at the mu receptor.