Full-length ArticleActivation of μ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice

- Both μ-opioid receptor and TLR4 can be activated by opioids or their metabolites.
- Cell based assays can be used to quantify receptor agonism in biological samples.
- Morphine administration results in circulating MOR and TLR4 agonism.
- IP injections of morphine to mice every 12 h lead to intermittent MOR activation.
- This mode of morphine administration results in TLR4-independent NF-κB activation.

In this study, we quantified the ability of opioids present in biological samples to activate the μ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its μ receptor-active metabolite morphine-6 glucuronide (M6G) and its μ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited μ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10 mg/kg every 12 h for 3 days) or saline. Morphine administration led to intermittent μ receptor activation, reversed by μ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.