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Mu and delta opioid receptor desensitization in undifferentiated human neuroblastoma SHSY5Y cells
P L Prather1, A W Tsai, P Y Law
1Department of Pharmacology, School of Medicine, University of Minnesota, Minneapolis.
Abstract:
Both mu and delta opioid receptors are expressed in undifferentiated human neuroblastoma SHSY5Y cells and are negatively coupled to adenylate cyclase. The ability of various mu opioid, delta opioid and alpha-2 adrenergic agonists to inhibit acutely forskolin-stimulated adenylate cyclase activity in undifferentiated SHSY5Y cells after chronic administration with the selective mu opioid agonist [N-MePhe3,D-Pro4]morphiceptin (PLO17) or delta opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE) was assessed. In control cells, both PLO17 and DPDPE inhibited cyclic AMP (cAMP) formation with equal maximal inhibition, i.e., 60 +/- 3 and 66 +/- 2%, having IC50 values of 51.1 +/- 1.3 and 3.7 +/- 1.0 nM, respectively. The inhibition of intracellular cAMP formation by both agonists could be blocked by pertussis toxin pretreatment. After 24 hr of chronic administration of PLO17 (50 nM to 10 microM), a concentration-dependent loss of the ability of mu opioid agonists PLO17 and DAMGO, but not the delta opioid agonists DPDPE, nor alpha-2 adrenergic agonist UK-14304 (5-Bromo-N-(4,5,-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) to inhibit adenylate cyclase activity was observed. In contrast, chronic administration of DPDPE (0.1 nM to 0.3 microM) resulted in a concentration-dependent reduction in the inhibition of cAMP formation produced by delta opioid agonists DPDPE and DSLET, but not mu opioid, nor alpha-2 adrenergic agonists tested. The observed homologous desensitization was also time-dependent. In addition, antagonist-induced increases in adenylate cyclase activity were observed only after chronic PLO17 administration.2+ Finally, chronic pretreatment of cells with PLO17 (10 microM) resulted in a significant decrease in mu opioid, but not delta opioid receptor, binding, whereas treatment with DPDPE (0.3 microM) resulted in a significant decrease in delta opioid, but not mu opioid receptor binding. Therefore, undifferentiated SHSY5Y cells may provide an excellent model system to study not only the signal transduction mechanisms of mu and/or delta opioid receptors, but also the cellular adaptations of specific opioid receptors.
Insights
This study shows that chronic exposure to specific opioid agonists causes homologous desensitization of mu and delta opioid receptors in SHSY5Y cells. This desensitization is receptor-specific and time-dependent, offering a model for studying opioid receptor adaptations.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Mu and delta opioid receptors are expressed in human neuroblastoma SHSY5Y cells.
- These receptors are negatively coupled to adenylate cyclase, regulating cyclic AMP (cAMP) formation.
- Understanding opioid receptor desensitization is crucial for pain management and addiction research.
Purpose of the Study:
- To investigate the effects of chronic administration of selective mu and delta opioid agonists on adenylate cyclase activity in SHSY5Y cells.
- To assess the receptor specificity and time-dependency of opioid receptor desensitization.
- To evaluate SHSY5Y cells as a model system for studying opioid receptor signal transduction and adaptation.
Main Methods:
- SHSY5Y cells were chronically treated with selective mu opioid agonist PLO17 or delta opioid agonist DPDPE.
- Adenylate cyclase activity was measured by forskolin-stimulated cAMP formation.
- Receptor binding assays were performed to quantify changes in mu and delta opioid receptor expression.
Main Results:
- Chronic PLO17 treatment led to homologous desensitization of mu opioid receptors, reducing their ability to inhibit cAMP formation.
- Chronic DPDPE treatment resulted in homologous desensitization of delta opioid receptors.
- Desensitization was concentration- and time-dependent, and receptor-specific, confirmed by decreased receptor binding.
- Antagonist-induced increases in adenylate cyclase activity were observed after chronic PLO17 treatment.
Conclusions:
- Undifferentiated SHSY5Y cells exhibit homologous desensitization of mu and delta opioid receptors upon chronic agonist exposure.
- This cellular model allows for the specific study of opioid receptor signal transduction and adaptive mechanisms.
- Findings support the utility of SHSY5Y cells for investigating opioid receptor pharmacology and cellular responses.