Abstract
Chronic treatment of C6 glioma cells stably expressing the ratdelta opioid receptor (C6δ) with full agonists resulted in receptor down-regulation. Chronic [d-Ser2,l-Leu5]enkephalyl-Thr treatment caused a decrease in cell surface as well as a decrease in agonist-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding. Treatment with full agonists for 12 hr resulted in a 90% decrease in receptor number that was paralleled by a decrease in the ability of agonist to stimulate [35S]guanosine-5′-O-(3-thio)triphosphate binding and inhibit forskolin-stimulated adenylyl cyclase. Of the remaining receptors, a smaller fraction of receptors (41 ± 4 vs.56 ± 4% in control) exhibited high affinity for agonist as compared to receptors in control membranes. Elimination of functional guanosine triphosphate binding protein (G protein) by Pertussis toxin pretreatment did not alter the ability of agonist to down regulate receptor. We hypothesized that agonist affinity (not efficacy) would be a predictor of an agonist’s ability to down-regulate receptor. However, we found that only full agonists were able to down-regulate receptor number, G protein activation and adenylyl cyclase inhibition. Chronic exposure to partial agonist 7-spiroindinooxymorphone, which has a very high affinity for the receptor, as well as morphine, did not cause receptor down-regulation. Taken together, these results suggest that full agonists alter receptor conformation such that the altered conformation is recognized by G protein as well as proteins involved in receptor down-regulation. In addition, down-regulation is independent of agonist-mediated G protein activation and subsequent down-stream signaling.
Footnotes
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Send reprint requests to: Dr. Ann E. Remmers, Research Investigator, Department of Pharmacology, University of Michigan, 1303 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0632.
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1 This work was supported by National Institutes of Health Grant DA 04087.
- Abbreviations:
- C6δ
- C6 glioma cells stably expressing the rat delta opioid receptor
- DSLET
- [d-Ser2,l-Leu5]enkephalyl-Thr
- SIOM
- 7-spiroindinooxymorphone
- DPDPE
- [d-Pen2,d-Pen5]enkephalin
- SNC80
- methyl ether of (+)-4-((α-R*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide (BW373U86)
- G protein
- GTP binding protein
- GTP
- guanosine triphosphate
- GDP
- guanosine diphosphate
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- PTX
- Pertussis 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. PBS, phosphate-buffered saline
- HEPES
- (N-[2-Hydroxyethyl]piperazine-N’-[2-ethane sulfonic acid])
- Received April 7, 1998.
- Accepted June 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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