The Effect of Two Different Buffers on the High Affinity 3H-Ethylketocyclazocine and 3H-SKF10047 Binding to Guinea Pig Brain Membranes

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Abstract
In vitro .mu. and .delta. opioid receptor binding is influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and may be similar to .mu. opioid receptor binding. The high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a .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 [N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid] 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 2 buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and 3H- .mu. and .delta. opioid ligands by 7 unlabeled ligands is then compared in the 2 buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and 3H .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 inihibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the 7 unlabeled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. 3H-Ethylketocyclazocine 1 nM 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.