Functional coupling, desensitization and internalization of virally expressed μ opioid receptors in cultured dorsal root ganglion neurons from μ opioid receptor knockout mice

Abstract

Although μ opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional μ opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged μ opioid receptor is returned to dorsal root ganglia neurons from μ opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged μ opioid receptor in neurons resulted in approximately 1.3×10 6 receptors/cell. Many μ opioid receptor cell lines express a similar density of receptors but this is approximately 7× greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the μ agonist, d-Ala 2, N-MePhe 4, Gly 5-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71±3 vs 29±7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17±1% after 20 min and 55±2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous μ opioid receptor function previously identified in non-neuronal cell lines.