The effect of two different buffers on the high affinity 3H-ethylketocyclazocine and 3H-SKF10047 binding to guinea pig brain membranes.
In vitro mu and delta opioid receptor binding is known to be influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and postulated to be similar to mu opioid receptor binding. To investigate this postulate, we have studied how the high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a tritiated mu agonist, mu antagonist and delta agonist is altered when the radioreceptor binding assay incubation buffer is changed. The binding of 3H-ethylketocyclazocine and the mu antagonist (3H-naloxone) is highest in isotonic HEPES buffer, while the binding of the mu (3H-dihydromorphine) and delta (3H-D-ala-D-leu-enkephalin) agonist is highest in hypotonic Tris-HCl buffer. 3H-SKF10047 binding is similar in the two buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and tritiated mu and delta opioid ligands by seven unlabeled ligands is then compared in the two buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and tritiated mu ligand in hypotonic Tris-HCl buffer than in isotonic HEPES buffer. The potency of naloxone, nalorphine, SKF10047, D-ala-D-leu-enkephalin, cyclazocine and phencyclidine in inhibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the seven unlabelled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. In sum our results show that 1 nM 3H-ethylketocyclazocine binding is influenced by buffer change in a manner very similar to mu ligand binding, while the 1.2 nM 3H-SKF10047 binding is only slightly influenced by buffer change and therefore different from mu ligand binding.
D000701:Analgesics, Opioid / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D003496:Cyclazocine / Q000031:analogs & derivatives* / Q000378:metabolism; D016314:Ethylketocyclazocine; D006168:Guinea Pigs; D006531:HEPES / Q000494:pharmacology*; D066298:In Vitro Techniques; D009020:Morphine / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D010620:Phenazocine / Q000031:analogs & derivatives* / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D011957:Receptors, Opioid / Q000378:metabolism*; D014316:Tritium; D014325:Tromethamine / Q000494:pharmacology*
D000701:Analgesics, Opioid; D010879:Piperazines; D011957:Receptors, Opioid; D014325:Tromethamine; D014316:Tritium; D009270:Naloxone; D016314:Ethylketocyclazocine; C012134:SK&F 10047; D009020:Morphine; D010620:Phenazocine; D003496:Cyclazocine; D006531:HEPES