Real-time RT-PCR measurement of the modulation of Mu opiate receptor expression by nitric oxide in human mononuclear cells

Background: In previous studies, we have attributed opiate alkaloid selectivity to a subtype of the neuronal μ receptor known as μ3, expressed on human blood cells. Opiate alkaloid activation of this receptor subtype leads to the release of constitutively derived nitric oxide. In this report, we show by real-time RT-PCR that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) initiates the down regulation of μ receptor gene expression in human mononuclear cells after 30 minutes. Superoxide dismutase, a free radical scavenger, blocks the effect of SNAP. Material and methods: Human mononuclear cells isolated from whole blood were treated with SNAP (100 μM), and also with SNAP plus superoxide dismutase (100 U/ml) at different time points. Real-time RT-PCR with total RNA extracted from the cells was used to analyze expression of the μ opiate receptor. Results: Mu opiate receptor gene expression was significantly down regulated in cells treated with SNAP at 30 min, and superoxide dismutase blocked the effect of SNAP. At 2 and 6 hours, a rebound effect was observed as noted by an increase in μ receptor expression, and at 24 hours μ receptor expression returned to control levels in the SNAP-treated cells. Conclusion: This study confirms that human mononuclear cells express the μ opiate receptor transcript and demonstrates that nitric oxide is involved in regulation of its expression.