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Tolerance to morphine at the mu-opioid receptor differentially induced by cAMP-dependent protein kinase activation
1Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA. zwang@u.arizona.edu
Abstract:
Human neuroblastoma SH-SY5Y cells express endogenous mu-opioid receptor and develop cellular tolerance to morphine after prolonged (>/=4 h) treatment with morphine. Treatment with forskolin (25 microM, 12 h), an adenylyl cyclase activator, also desensitized mu-opioid receptor response to morphine (10 microM) by 38% (P<0. 001), which was reversed by the cyclic AMP (cAMP) dependent kinase inhibitor N-(2-aminoethyl)-5-isoquinolinesulfonamide (H8) (100 microM). Treatment with both morphine and forskolin appeared to cause an additive effect in desensitizing mu-opioid receptor. In mu-opioid receptor stably transfected human embryonic kidney 293 (HEK-mu) cells, morphine treatment produced cAMP upregulation, yet failed to induce mu-opioid receptor tolerance. However, treatment with forskolin (25 microM) or 8-bromo-cAMP (1mM) led to profound mu-opioid receptor tolerance, which was reversed by H8. These results demonstrate that cAMP-dependent kinase activation causes mu-opioid receptor tolerance. However, morphine-induced mu-opioid receptor tolerance in SH-SY5Y cells is not mediated by cAMP-dependent kinase activation. In addition, our results indicate that cAMP-upregulation does not necessarily lead to mu-opioid receptor tolerance.
Insights
Cellular tolerance to morphine involves cyclic AMP (cAMP)-dependent kinase activation. However, morphine-induced tolerance in neuroblastoma cells does not rely on this pathway, suggesting alternative mechanisms.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Human neuroblastoma SH-SY5Y cells exhibit mu-opioid receptor (MOR) tolerance after prolonged morphine exposure.
- Understanding the molecular mechanisms underlying MOR tolerance is crucial for pain management and addiction treatment.
Purpose of the Study:
- To investigate the role of cyclic AMP (cAMP) and cAMP-dependent kinase in mediating cellular tolerance to morphine.
- To differentiate between cAMP-dependent and independent pathways in MOR tolerance.
Main Methods:
- Utilized human neuroblastoma SH-SY5Y cells and stably transfected HEK-293 cells expressing MOR.
- Administered morphine, forskolin (adenylyl cyclase activator), and 8-bromo-cAMP, with or without the cAMP-dependent kinase inhibitor H8.
- Measured MOR response desensitization and cAMP levels.
Main Results:
- Forskolin and 8-bromo-cAMP induced significant MOR tolerance in HEK-293 cells, which was reversed by H8, indicating cAMP-dependent kinase activation causes tolerance.
- Morphine treatment in SH-SY5Y cells led to tolerance but not through cAMP-dependent kinase activation.
- Morphine treatment in HEK-293 cells caused cAMP upregulation but failed to induce tolerance.
Conclusions:
- cAMP-dependent kinase activation is sufficient to induce MOR tolerance.
- Morphine-induced MOR tolerance in SH-SY5Y cells is mediated by a pathway independent of cAMP-dependent kinase.
- cAMP upregulation alone does not necessarily result in MOR tolerance.