Opioid Efficacy in a C6 Glioma Cell Line Stably Expressing the Human Kappa Opioid Receptor
- Ann E. Remmers1,
- Mary J. Clark1,
- Alfred Mansour4,
- Huda Akil4,
- James H. Woods1,3 and
- Fedor Medzihradsky1,2
- Departments of 1Pharmacology (A.E.R., M.J.C., J.H.W., F.M.),2Biological Chemistry (F.M.), 3Psychology (J.H.W.), and 4Mental Health Research Institute (A.M., H.A.) University of Michigan, Ann Arbor, Michigan
Abstract
A C6 glioma cell line stably transfected with the humankappa opioid receptor (κOR) was used to characterize receptor binding and G protein activation via the κOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [3H]5α,7α,8β(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [35S]GTPγS binding. The stimulation of [35S]GTPγS binding was blocked by pretreatment of cells with pertussis toxin. Partial stimulation of [35S]GTPγS binding via the κOR was observed for several ligands that are antagonists at the mu opioid receptor, suggesting an additional mechanism of drug action. The ability of isomers of tifluadom and levallorphan to stimulate [35S]GTPγS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition, (−)tifluadom, the less potent isomer of tifluadom, which is also a γ-aminobutyric acidA receptor agonist, stimulated [35S]GTPγS binding. In contrast,d-pentazocine, (+)SKF10047, (+)cyclazocine, andd-ethylketocyclazocine displayed no agonist activity. κOR-selective antagonist norbinaltorphimine competitively inhibited the stimulation of [35S]GTPγS binding by the active isomers of ethylketocyclazocine, cyclazocine, and nalorphine to the same degree, indicating that all three ligands are eliciting an effect via the κOR. The results suggest that these cells express a homogeneous population of κOR, and that their [35S]GTPγS-binding properties make them an excellent means to assess κOR efficacy.
Footnotes
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Send reprint requests to: Ann E. Remmers. Ph.D., Department of Pharmacology, 1303 MSRB III, 1150 W. Medical Center Drive, University of Michigan, Ann Arbor, Michigan 48109-0632. Email:aremmers{at}umich.edu
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1 This work was supported by grants from the U.S. Public Health Service to F.M (DA04087), J.H.W. (DA00254), and H.A. (National Institute on Drug Abuse DA02265 and DA08920).
- Abbreviations:
- κOR
- kappa opioid receptor
- C6κ
- C6 glioma cells stably expressing the human kappaopioid receptor
- E2078
- [N-methyl-Tyr1,N-α-methyl-Arg7-d-Leu8]dynorphin A-(1–8) ethylamide
- EKC
- ethylketocyclazocine
- U69593
- 5α,7α,8β(−)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzene acetamide
- U50488H
- (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide
- β-CNA
- β-chlornaltrexamine
- norBNI
- nor-binaltorphimine
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- DMEM
- Dulbecco’s modified Eagle’s medium
- CHO
- Chinese hamster ovary
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- Received April 14, 1998.
- Accepted September 9, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
