Abstract
Naloxone benzoylhydrazone (NalBzoH) is a potent muantagonist 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 muantagonist properties, whereas the agonist DAMGO showed more than a 3-fold loss of affinity. The dissociation of3H-diprenorphine was monophasic. However, kinetic studies revealed biphasic dissociations for both 3H-NalBzoH and3H-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.
Footnotes
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Send reprint requests to: Dr. Gavril W. Pasternak, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
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↵1 This work was supported, in part, by Grant DA06241 and Research Scientist Award K05 DA00220 from the National Institute on Drug Abuse to G.W.P. and core Grant CA08748 from the National Cancer Institute to MSKCC. G.P.B. was supported by Training Grant T32 DA07274 from the National Institute on Drug Abuse.
- Abbreviations:
- NalBzoH
- naloxone benzoylhydrazone
- PTX
- pertussis toxin
- CTX
- cholera toxin
- Gpp(NH)p
- 5′-guanylylimidophosphate
- ANOVA
- analysis of variance
- CHO
- Chinese hamster ovary
- 3H-DAMGO
- [d-Ala2, MePhe4, Gly(ol)5]enkephalin
- Received October 15, 1997.
- Accepted March 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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