title: Delta opioid modulation of the binding of guanosine-5'-O-(3-[35S]thio)triphosphate to NG108-15 cell membranes: characterization of agonist and inverse agonist effects.
abstract: The ability of the delta opioid agonist DPDPE ([D-Pen2, D-Pen4]enkephalin) to stimulate binding of the GTP analog guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) to pertussis toxin-sensitive G proteins has been characterized in membranes from NG108-15 mouse neuroblastoma X rat glioma cells. The presence of GDP, or its hydrolysis-resistant analog GDPbetaS, and Mg++ ions was essential to observe agonist-mediated stimulation of [35S]GTPgammaS binding, although the guanine dinucleotides alone had complex inhibitory and stimulatory effects on [35S]GTPgammaS binding. The relative ability of the delta antagonists benzylidenenaltrexone and naltriben to inhibit DPDPE-stimulated [35S]GTPgammaS binding suggested the opioid receptor involved was of the delta-2 subtype. Ligand binding assays demonstrated biphasic binding of these antagonists to this single receptor type. [35S]GTPgammaS binding was also stimulated by [D-Ser2,Leu5,Thr6]enkephalin > deltorphin II = DPDPE = etorphine > levallorphan = diprenorphine = nalorphine = naltrindole. The delta antagonists benzylidenenaltrexone, TIPP (Tyr-Tic-Phe-Phe) and naltriben had no effect, but ICI 174864 (N, N-diallyl-Tyr-Aib-Phe-Leu-OH) acted as an inverse agonist and inhibited [35S]GTPgammaS binding. Pertussis toxin pretreatment blocked agonist stimulation of [35S]GTPgammaS binding and also reduced basal binding, thus confirming the presence of constitutively active delta receptors. Replacement of Na+ in the assay buffer with K+ afforded an increased level of basal [35S]GTPgammaS binding and an apparent increase in both the inverse agonist activity of ICI 174864 and the agonist activity of the partial agonist diprenorphine relative to the full agonist [D-Ser2, Leu5,Thr6]enkephalin. The stimulation of [35S]GTPgammaS binding to NG108-15 cell membranes allows a functional measure of delta opioid activity that can provide systems of differing relative efficacy.
authors.affiliation: Department of Chemistry, Loughborough University, Leicester, LE11 3TU, UK.
authors.forename: P G
authors.initials: PG
authors.lastname: Szekeres
authors.forename: J R
authors.initials: JR
authors.lastname: Traynor
mesh_terms: D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D020881:Enkephalin, D-Penicillamine (2,5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D051379:Mice; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017465:Receptors, Opioid, delta / Q000819:agonists*; D012964:Sodium / Q000494:pharmacology
chemical_list: D001597:Benzylidene Compounds; D004745:Enkephalins; D017465:Receptors, Opioid, delta; C077358:7-benzylidenenaltrexone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004743:Enkephalin, Leucine; D009271:Naltrexone; D020881:Enkephalin, D-Penicillamine (2,5)-; C040643:N,N-diallyl-tyrosyl-alpha-aminoisobutyric acid-phenylalanyl-leucine; D012964:Sodium