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Differential agonist regulation of the human kappa-opioid receptor
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, U.S.A.
Abstract:
Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.
Insights
Opioid tolerance is a major challenge in pain management. This study shows that selective kappa-opioid agonists, unlike nonselective ones, can desensitize kappa-opioid receptors, offering insights into tolerance mechanisms.
Area of Science:
- Pharmacology
- Neuroscience
- Molecular Biology
Background:
- Opioid analgesics are crucial for pain management but chronic use leads to tolerance, limiting their effectiveness.
- Understanding the cellular mechanisms of opioid tolerance is essential for developing safer and more effective pain therapies.
Purpose of the Study:
- To investigate the cellular mechanisms underlying opioid tolerance by examining the regulation of the human kappa-opioid receptor.
- To determine how selective and nonselective opioid agonists differentially affect kappa-opioid receptor function and signaling.
Main Methods:
- Stable expression of the human kappa-opioid receptor in human embryonic kidney (HEK 293) cells.
- Assessment of receptor binding, G protein coupling, and adenylyl cyclase inhibition.
- Evaluation of receptor down-regulation and desensitization following pretreatment with various opioid agonists.
Main Results:
- Human kappa-opioid receptors in HEK 293 cells couple to Gi/Go proteins and inhibit adenylyl cyclase.
- Selective kappa-opioid agonists (e.g., U50,488) induced receptor down-regulation and desensitization.
- Nonselective opioid agonists (e.g., levorphanol, etorphine) did not cause significant receptor desensitization.
Conclusions:
- The human kappa-opioid receptor is differentially regulated by selective and nonselective opioid agonists.
- Selective kappa-opioid agonists possess the ability to desensitize the receptor, contributing to the development of opioid tolerance.