Molecular assays for characterization of alternatively spliced isoforms of the u opioid receptor (MOR).
- Authors
- Gris, Pavel; Cheng, Philip; Pierson, John; Maixner, William; Diatchenko, Luda
- Year
- 2010
- Journal
- Methods in molecular biology (Clifton, N.J.)
- PMID
- 20336438
- DOI
- 10.1007/978-1-60327-323-7_30
- PMCID
- PMC3991784
Mu-opioid receptor (MOR) belongs to a family of heptahelical G-protein-coupled receptors (GPCRs). Studies in humans and rodents demonstrated that the OPRM1 gene coding for MOR undergoes extensive alternative splicing afforded by the genetic complexity of OPRM1. Evidence from rodent studies also demonstrates an important role of these alternatively spliced forms in mediating opiate analgesia via their differential signaling properties. MOR signaling is predominantly G(ia) coupled. Release of the alpha subunit from G-protein complex results in the inhibition of adenylyl cyclase/cAMP pathway, whereas release of the betagamma subunits activates G-protein-activated inwardly rectifying potassium channels and inhibits voltage-dependent calcium channels. These molecular events result in the suppression of cellular activities that diminish pain sensations. Recently, a new isoform of OPRM1, MOR3, has been identified that shows an increase in the production of nitric oxide (NO) upon stimulation with morphine. Hence, there is a need to describe molecular techniques that enable the functional characterization of MOR isoforms. In this review, we describe the methodologies used to assay key mediators of MOR activation including cellular assays for cAMP, free Ca(2+), and NO, all of which have been implicated in the pharmacological effects of MOR agonists.
Inhibition of FSK-stimulated cAMP accumulation by morphine (1 μM). cAMP levels in FLAG-MOP stable colonies were measured using an enzyme-immunoassay kit either in unstimulated cells (no FSK), or in cells treated with FSK (50 μM) and IBMX (100 μM). Morphine (1 μM) significantly reduced cAMP levels in FSK-stimulated cells (morphine) compared to stimulated cells treated with water (vehicle). Morphine inhibited cAMP production by 67.0 ± 4.6% for FLAG-MOP/TRPV1 colony 13 (dotted bars) and 79.2 ± 7.4% for colony 21 (cross-hatched bars). Data are presented as mean ± SEM from n = 3 samples read in triplicate. **p < 0.01 compared to vehicle. (Vetter et al. Molecular Pain 2006 2:22)
Capsaicin dose-response curves from two independent FLAG-MOP/TRPV1 expressants. Ca2+ responses of Fluo-3-loaded cells to injection of capsaicin were measured using a fluorescent Microplate reader and maximum change in fluorescence, expressed as ΔF/F, was plotted as a function of capsaicin concentration. A 4-parameter Hill function was fitted to the data using GraphPad Prism. (a) Capsaicin dose-response from FLAG-MOP/TRPV1 double expressant colony 13. (b) Capsaicin dose–response from FLAG-MOP/TRPV1 double expressant colony 21. (Data are expressed as mean ± SEM with n = 8.) Vetter et al. Molecular Pain 2006 2:22
Effect of the different doses of morphine on the Ca2+ levels in BE(2)C cells. The low doses (L) of morphine (10−3 μM) and high (H) doses (102 μM) result in increase in Ca2+ levels in BE2C cells. The 1 μM dose (M) of morphine caused a small decrease in Ca2+ levels. Data are presented as mean ± SEM from n = 8 *p < 0.01 compared to vehicle
(a) Real-time NO production measurements in μ3-transfected COS-1 cells after addition of the opiate alkaloid, morphine, or the opioid peptides, Met- and Leu-enkephalin. The control represents the addition of PBS to the cells. Each experiment was replicated four times ± SEM. (b), Peak concentration-dependent morphine-stimulated NO release from COS-1 cells and μ3-transfected COS-1 cells. Each experiment was replicated four times; results shown are the mean ± SEM. (Copyright 2003 The American Association of Immunologists, Inc.)
| Name | Type |
|---|---|
| adenylyl cyclase | drug |
| alternatively-spliced isoforms of MOR local | drug |
| Analgesic tolerance local | phenotype |
| Ca2+ | drug |
| cAMP | drug |
| capsaicin | drug |
| cGMP | drug |
| dependence | phenotype |
| forskolin | drug |
| GNAI | gene |
| guanylyl cyclase local | drug |
| Gβγ dimer local | drug |
| HEK293 cells | cohort |
| hyperalgesia | phenotype |
| Intracellular calcium local | drug |
| Kcnj6 | gene |
| MOR | drug |
| MOR-3 isoform local | variant |
| MOR-3 six transmembrane variant local | variant |
| MOR agonist local | drug |
| morphine | drug |
| naloxone | drug |
| neuronal activity | phenotype |
| nitric oxide synthase | drug |
| NO | drug |
| opioid | drug |
| OPRM1 | cohort |
| PGE2 | drug |
| PKA | drug |
| PKG local | drug |
| pronociceptive phenotype local | phenotype |
| tolerance | phenotype |
| TrpV1 | gene |
| VDCC | drug |
| μ-opioid receptor agonists local | drug |
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In this knowledge base
| Title | Year | PMID |
|---|---|---|
| KAT2B polymorphism identified for drug abuse in African Americans with regulatory links to drug abuse pathways in human prefrontal cortex. | 2016 | 26202629 |
External
| Title | Authors | Journal | Year | Link |
|---|---|---|---|---|
| Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model. | Ross CL et al. | — | 2016 | → |
| KAT2B polymorphism identified for drug abuse in African Americans with regulatory links to drug abuse pathways in human prefrontal cortex. | Johnson EO et al. | — | 2016 | → |
| Schizophrenia is primed for an increased expression of depression through activation of immuno-inflammatory, oxidative and nitrosative stress, and tryptophan catabolite pathways. | Anderson G et al. | — | 2013 | → |
| Endogenous opiates and behavior: 2011. | Bodnar RJ | — | 2012 | → |