Expression of μ-, κ- and δ-opioid receptors in P19 mouse embryonal carcinoma cells
Section snippets
Cell culture
P19 cells were purchased from ATCC and cultured in α-minimal essential medium containing 7.5% newborn calf serum and 2.5% fetal calf serum in humid incubators supplied with 5% CO2. The procedures to differentiate P19 cells have been described previously.40 In brief, cells were aggregated in Petri dishes in the presence of RA (5×10−7 M) for four days. After aggregation, cells were trypsinized and plated on tissue culture grade flasks in a chemically defined medium, N2, at a density of 7.5×105
The existence of μ-, κ- and δ-opioid receptors in P19 cells
The μ-opioid receptor was not detectable in undifferentiated P19 cells by RT–PCR, ICC or opioid ligand binding methods. After differentiation, however, μ-opioid receptor could be detected, although only weakly, one day after plating samples. The expression level increased over time and was maximal four days after plating, the last time examined (Fig. 1A, B). The expression of μ-opioid receptor was specific to RA-induced neuronal differentiation, since no μ-specific signal was detected in the
Discussion
P19 EC cells were established in the early 1980s23 and have been used as a model of developmental studies in many laboratories.34 Our demonstration of the presence of μ-, κ- and δ-opioid receptors in these cells thus opens up a new avenue of research for these receptors, particularly as they are known to be involved in developmental processes.2., 15. In our laboratory, P19 cells have recently been used to study the regulation of κ-opioid receptor gene.18
Especially significant is our finding
Conclusions
P19 EC cells can be induced to express μ-, κ- and δ-opioid receptors, suggesting that this cell line can be a useful model system for studying the developmental regulation of these opioid receptors at the cellular and molecular levels. Moreover, the ability to turn the expression of μ-opioid receptor on with RA treatment and cellular aggregation is an especially valuable feature of this system. Finally, co-expression of all three opioid receptors and GAD provides a new model for studying the
Acknowledgements
This research was supported by National Institutes of Health research grants DA00564, DA01583, DA11806, and the F. and A. Stark Fund of the Minnesota Medical Foundation.
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