Vol. 286, Issue 1, 376-381, July 1998

³H-Naloxone Benzoylhydrazone Binding in MOR-1-Transfected Chinese Hamster Ovary Cells: Evidence for G-Protein-Dependent Antagonist Binding¹

George P. Brown and Gavril W. Pasternak

The Cotzias Laboratory of Neuro-Oncology (G.P.B., G.W.P.), Memorial Sloan-Kettering Cancer Center and Departments of Neurology and Neuroscience (G.P.B., G.W.P.) and Pharmacology (G.W.P.), Cornell U. Medical College, New York, New York

Naloxone benzoylhydrazone (NalBzoH) is a potent mu antagonist in vivo. In a cell line stably transfected with MOR-1 (CHO/MOR-1), NalBzoH also was an antagonist when examined in adenylyl cyclase studies. In binding studies, it displayed high affinity for the mu receptor, confirming its earlier characterization in brain membranes. In competition studies under equilibrium conditions, NalBzoH and diprenorphine both retained their potency in the presence of the stable GTP analog 5'-guanylylimidophosphate, consistent with their mu antagonist properties, whereas the agonist DAMGO showed more than a 3-fold loss of affinity. The dissociation of H-diprenorphine was monophasic. However, kinetic studies revealed biphasic dissociations for both ³H-NalBzoH and ³H-DAMGO. The slow component of ³H-NalBzoH dissociation, corresponding to the higher affinity state, was dependent on coupling to G-proteins. It is selectively abolished by guanine nucleotides, leaving only the rapid dissociation phase. Furthermore, the slow dissociation component is eliminated by treatment of the cells with pertussis toxin, but not cholera toxin. In conclusion, NalBzoH is an unusual opioid. Functionally it is an antagonist, a classification consistent with its equilibrium binding in the presence of guanine nucleotides. Yet, kinetic studies reveal that it labels a G-protein coupled state of the receptor with high affinity.