Background
The mu opioid receptor (MuOR) is a member of the superfamily of G protein-coupled receptors that mediates the analgesic actions of endogenous opioid peptides and the narcotic alkaloid derivatives of morphine. Activation and translocation of protein kinase C (PKC) by N-methyl-D-aspartate receptor stimulation correlates with resistance to opioid drugs in experimental states of neuropathic pain, but the cellular mechanisms of resistance have not been identified. One possibility is that PKC activation regulates MuOR mRNA expression and thus the ability to generate functional receptors. Using a human neuroblastoma cell line, the authors tested the hypothesis that phorbol ester activation of PKC regulates MuOR mRNA levels.
Methods
SH-SY5Y cells were maintained in a continuous monolayer culture and treated with phorbol esters or other agents before extraction of total cellular RNA. Slot-blot hybridization was used to measure the level of MuOR mRNA using 32P-labeled MuOR cDNA probes under high-stringency conditions. Autoradiograms were analyzed by scanning and densitometry.
Results
MuOR mRNA levels decreased in a dose- and time-dependent manner after tetradecanoyl phorbol acetate (TPA) was administered to activate PKC. The nadir, a level of approximately 50% of control, was at 2-8 h, followed by gradual recovery. The actions of TPA were blocked by pretreatment with the selective PKC inhibitor bisindolylmaleimide, but not by inhibition of protein synthesis with cycloheximide or anisomycin. The combination of TPA treatment and transcription inhibition with actinomycin D was associated with a transient increase in MuOR mRNA.
Conclusions
Mu opioid receptor mRNA levels are regulated by activation of PKC in a neuronal model. Protein kinase C effects which decrease MuOR mRNA levels appear largely independent of new protein synthesis, and cytotoxicity does not account for the findings. Plasticity of MuOR gene expression may contribute to variations in clinical responses to opioid analgesics in clinical states such as neuropathic pain.
Stabilization of morphine tolerance with long-term dosing: Association with selective upregulation of mu-opioid receptor splice variant mRNAs
