In neuronal cell lines, activation of opioid receptors has been shown to mobilize intracellular Ca2+ stores. In this report, we describe the excitatory actions of opioid agonists on murine neuroblastoma neuro(2a) cells stably expressing either δ, μ, or κ opioid receptors. Fura-2-based digital imaging was used to record opioid-induced increases in intracellular Ca2+ concentration ([Ca2+](i)). Repeated challenges δ, μ, or κ opioid receptor expressing cells with 100 nM [D-Ala2,D-Leu5]-enkephalin (DADLE), [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO), or trans-(±)-3,4 dichloro N-methyl-N-(2-[1-pyrollidinyl] cyclohexyl) benzene acetamide (U-50488H), respectively, elicited reproducible Ca2+ responses. Non-transfected neuro(2a) cells did not respond to opioid agonists. Removal of extracellular Ca2+ from the bath prior to and during agonist challenge did nor affect significantly the agonist-evoked increase in [Ca2+](i), indicating that the response resulted from the release of Ca2+ from intracellular stores. Naloxone reversibly inhibited responses in all three cell lines, confirming that they were mediated by opioid receptors. Expression of cloned opioid receptors in neuro(2a) cells, coupled with digital [Ca2+](i) imaging, provides a model system for the study of opioid receptors and opioid activated signaling processes. The fact that all three receptors coupled to the same intracellular signaling mechanism suggests that the primary functional difference between opioid responses in vivo results from their selective localization.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institute on Drug Abuse (DAO9293, DA07304, DA01.583, DAO5695, and DA07339) and the National Science Foundation (IBN9412654).
- Ca stores
- Cloned receptors
- Intracellular Ca