Elsevier

Neuroscience

Volume 92, Issue 3, June 1999, Pages 1143-1155
Neuroscience

Expression of μ-, κ- and δ-opioid receptors in P19 mouse embryonal carcinoma cells

https://doi.org/10.1016/S0306-4522(99)00030-5Get rights and content

Abstract

P19 embryonal carcinoma cells are pluripotential and able to differentiate into a variety of cell types, including neurons, glia and fibroblast-like cells, upon retinoic acid treatment and cellular aggregation. Using reverse transcription–polymerase chain reaction, ligand binding and immunocytochemical methods, κ- and δ-opioid receptors were detected in undifferentiated P19 cells. The μ-opioid receptor was not observed until one day after plating, following one essential step of differentiation, but increased in number in the four days after plating. Several different expression patterns were detected in these differentiated cells. Some cells exhibited μ- and δ-opioid receptors co-expressed, with or without κ-opioid receptor; whereas some of the cells expressed only κ-opioid receptor. All three opioid receptors are detected on aggregated cells which are postmitotic and also expressing neurofilaments, indicating neuronal characteristics. Furthermore, those cells expressing μ- and δ-opioid receptors also expressed glutamate decarboxylase, characteristic of the GABAergic phenotype.

Based on these findings, we propose that P19 cells may serve as a model system to study the developmental regulation of opioid receptors, and in particular their relationship with GABA.

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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