Abstract
A C6 glioma cell line stably transfected with the ratdelta opioid receptor (C6δ) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[d-Pen2,d-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [d-Ser2,l-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPγS binding by 640, 654 and 576%, respectively, over basal. The peptide agonists, DPDPE and deltorphin II, both stimulated [35S]GTPγS binding by 375%. Etorphine, diprenorphine, oxymorphindole and 7-spiroindanyloxymorphone were also partial agonists in this assay, although they were less efficacious than deltorphin II. Stimulation of [35S]GTPγS binding by agonists was blocked completely by pertussis toxin pretreatment. Both delta-1 anddelta-2 selective antagonists 7-benzylidenenaltrexone and a benzofuran analog of naltrindole displayed high affinity for the cloned receptor (0.04 and 0.08 nM) and antagonized the stimulation of [35S]GTPγS binding by BW373U86 and DPDPE with similar potencies. Other evidence suggesting the lack of receptor subtypes includes the finding that stimulation of [35S]GTPγS binding by receptor subtype selective ligands DPDPE and deltorphin II was not additive. BW373U86, SNC80 and DPDPE maximally inhibited forskolin-stimulated adenylyl cyclase. These cells highly express a homogeneous population of delta opioid receptor that couple to inhibitory Go/Gi proteins. Ligand affinity for the delta opioid receptor correlates with ligand EC50 values for stimulation of [35S]GTPγS binding.
Footnotes
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Send reprint requests to: Fedor Medzihradsky, Department of Pharmacology, 1303 MSRB III, 1150 W. Medical Center Drive, University of Michigan, Ann Arbor, MI 48109-0632.
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↵1 This work was supported by grants from the United States Public Health Service to F.M. (RO1 DA04087), J.H.W. (DA 00254) and H.A. (NIDA R01 DA02265 and RO1 DA08920).
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↵2 Present address: Department of Pharmacological and Physiological Sciences the University of Chicago, 947 East 58th Street, Chicago, IL 60637.
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↵3 The International Union of Pharmacology (IUPHAR) subcommittee on Opioid Receptors (Dhawan et al., 1996) recommended the name of OP1 receptor for thedelta opioid receptor (OP for opioids, and the chronological order of the first formal demonstration of the existence of the receptors).
- Abbreviations:
- BW373U86
- (±)-4-((α-R*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, methyl ether of (+)BW373U86 (SNC80)
- DSLET
- [d-Ser2,l-Leu5]enkephalyl-Thr
- DPDPE
- [d-Pen2,d-Pen5]enkephalin
- pCl-DPDPE
- [d-Pen2,pCl-Phe4,d-Pen5]enkephalin
- DAMGO
- Tyr-d-Ala-Gly-(Me)Phe-Gly-ol)
- BNTX
- 7-benzylidenenaltrexone
- NTB
- naltriben, benzofuran derivative of naltrindole
- SIOM
- 7-spiroindinooxymorphone
- ICI 174864
- N,N-diallyl-Tyr-Aib-Phe-Leu-OH (Aib, α-aminoisobutyric acid)
- U69
- 593, 5α,7α,8β(−)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzene acetamide
- G protein
- GTP binding protein
- Go
- pertussis toxin-sensitive G protein highly expressed in brain
- Gi
- pertussis toxin-sensitive G protein that mediates adenylyl cyclase inhibition
- GTP
- guanosine triphosphate
- GDP
- guanosine diphosphate
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- PTX
- pertussis toxin
- CTX
- cholera toxin
- A2 buffer
- 128 mM NaCl, 2.4 mM KCl, 1.3 mM CaCl2, 2.0 mM NaHCO3, 3.0 mM MgSO4, 10 mM Na2HPO4, 10 mM glucose, 8 mM theophylline, pH 7.4
- CHO
- Chinese hamster ovary
- DMEM
- Dulbecco’s modified Eagle’s medium
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- SINTX
- 7-spiroindanylnaltrexone
- Received March 28, 1997.
- Accepted July 1, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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