Activation of μ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice

In this study, we quantified the ability of opioids present in biological samples to activate the mu-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its. receptor-active metabolite morphine-6 glucuronide (M6G) and its mu receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited mu 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 mu receptor activation, reversed by 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-kappa B activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNF alpha. Circulating TNF alpha was increased after three days of morphine administration, and TNF alpha mRNA elevated in the spleen of morphine-treated mice. (C) 2016 Elsevier Inc. All rights reserved.