Abstract
While humans and most animals respond to µ-opioid receptor (MOR) agonists with analgesia and decreased aggression, in the naked mole rat (NMR) opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1) can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3'-5'-cyclic adenosine monophosphate (cAMP) enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR's extreme reaction to opioids.
Highlights
The μ-opioid receptor (MOR) mediates the analgesic properties of most animals respond to μ-opioid receptor (MOR) ligands, the oldest and most powerful analgesics [1]
Three nucleotide substitution sites were identified in highly conserved regions of the 5’-end of the oprm1 resulting in three unique aa at the N-terminal naked-mole rat (NMR) MOR compared to all other species examined (Figure 1B)
Sequencing of the plasmid verified that pCMVNMRoprm1eGFP was intact. These findings indicate internalization of the NMR MOReGFP predominantly after DAMGO and to a lesser degree after morphine stimulation, which is similar to the findings of others [33,36,37] and supports our data obtained in the whole cell ligand binding assay
Summary
The μ-opioid receptor (MOR) mediates the analgesic properties of MOR ligands (e.g. morphine), the oldest and most powerful analgesics [1]. The behavioral response to MOR agonists is highly conserved and includes analgesia, sedation, and decreased aggression [3]. One exception is the naked-mole rat (NMR) which displays hyperactivity, motor dysfunction and, most notably, extreme aggression in response to MOR agonists [4,5]. These behaviors are reversible by naloxone (NLX), a non-selective opioid receptor antagonist, demonstrating that these effects are mediated by opioid receptor activation. Morphine induced hyperalgesia in the hot-plate test (4) and much higher doses of opioids were required to produce analgesia in the formalin test compared to mice (5). The molecular basis for these unique reactions has not been examined so far
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