INVESTIGATING THE EFFECTS OF PROLONGED MU OPIOID RECEPTOR ACTIVATION UPON OPIOID RECEPTOR HETEROMERIZATION
Opioid receptors are the sites of action for morphine and most other clinically-used opioid drugs. Abundant evidence now demonstrates that different opioid receptor types can physically associate to form heteromers. Owing to their constituent monomers’ involvement in analgesia, mu/delta opioid receptor (M/DOR) heteromers have been a particular focus of attention. Understandings of the physiological relevance of M/DOR formation remain limited in large part due to the reliance of existing M/DOR findings upon contrived heterologous systems. This thesis investigated the physiological relevance of M/DOR generation following prolonged MOR activation. To address M/DOR in endogenous tissues, suitable model systems and experimental tools were established. This included a viable dorsal root ganglion (DRG) neuron primary culture model, antisera specifically directed against M/DOR, a quantitative immunofluorescence colocalizational analysis method, and a floxed-Stop, FLAG-tagged DOR conditional knock-in mouse model. The development and implementation of such techniques make it possible to conduct experiments addressing the nature of M/DOR heteromers in systems with compelling physiological relevance. Seeking to both reinforce and extend existing findings from heterologous systems, it was first necessary to demonstrate the existence of M/DOR heteromers. Using antibodies directed against M/DOR itself as well as constituent monomers, M/DOR heteromers were identified in endogenous tissues and demonstrated to increase in abundance following prolonged mu opioid receptor (MOR) activation by morphine. The next experiments addressed an aspect of the functional consequences of M/DOR formation in endogenous tissues by investigating the post-internalization trafficking of delta opioid receptor (DOR) in conditions of augmented M/DOR formation. These experiments identified perturbations to DOR trafficking consistent with incorporation into a M/DOR species with probabilistic post-internalisation trafficking behaviour intermediate to that of either constituent and responsive to both MOR and DOR agonists in a manner subject to blockade by DOR antagonist. These studies provide concurrent evidence of the existence and functionality of physiologically-relevant M/DOR heteromers in endogenous tissues.
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