Traditional avenues for therapeutic pain intervention primarily target opioid receptors. Opioid compounds activate mu (μ), delta (δ), and kappa (κ) opioid receptors to produce analgesia. Systemic therapies targeting mu opioid receptors remain the gold standard for treating acute, severe pain, but can lead to debilitating side effects that contraindicate long-term use. Targeting delta opioid receptors produces comparable analgesia with reduced side effect profiles in rodents and primates. Activation of competent opioid receptors in the periphery relieve pain to the same extent as systemic administration, but require inflammatory priming. Peripherally-restricted delta opioid receptor agonists would be expected to have reduced side effects, but only be efficacious for individuals with severe inflammatory pain. Understanding mechanisms that underlie inflammatory priming would increase the application of peripherally-restrictive delta opioid receptor agonists to treat other pain modalities with a reduced side effect profile.
The first aim identified the mechanism that underlies delta opioid receptor priming in the periphery by the potent inflammatory mediator bradykinin. Agonist-induced desensitization of delta opioid receptors depends on hierarchical phosphorylation by G protein-coupled receptor kinase 2 to mediate receptor desensitization through G protein-uncoupling followed by internalization that further attenuates signaling. Biochemical, molecular, imaging, functional techniques, and behavioral experiments collectively revealed that G protein-coupled receptor kinase 2 chronically down-regulates delta opioid receptor competence in peripheral pain-sensing neurons, as well as peripheral delta opioid receptor-mediated anti-nociception in vivo. G protein-coupled receptor kinase 2–dependent delta opioid receptor incompetence was found to be dependent on a constitutive interaction between G protein-coupled receptor kinase 2 and the receptor, not kinase activity. This hinders delta opioid receptor coupling with G-proteins following priming by bradykinin and, thus, delta opioid receptor competence. The mechanism elucidated within this aim found that bradykinin stimulates G protein-coupled receptor kinase 2 movement away from delta opioid receptor and onto rraf kkinase iinhibitory pprotein. Protein kinase C–dependent phosphorylation of raf kinase inhibitory proteind induces its self-dimerization and subsequent G protein-coupled receptor kinase 2 sequestration, restoring G protein-coupling and delta opioid receptor functionality in sensory neurons.
The second aim characterized the mechanism that maintains peripheral delta opioid receptor incompetence. Protein kinase A phosphorylation of G protein-coupled receptor kinase 2 at serine 685 plays an important role in its plasma membrane targeting. A-kinase anchoring protein 79/150, a scaffolding protein that resides at the plasma membrane in sensory neurons, scaffolds protein kinase A and mediates downstream signal transduction. Imaging, biochemical, mutagenesis, and functional techniques collectively demonstrated that this also occurs in peripheral sensory neurons and determines delta opioid receptor responsiveness to agonist stimulation. Results from these experiments suggest that A-kinase anchoring protein scaffolds protein kinase A to increase plasma membrane targeting and phosphorylation of G protein-coupled receptor kinase 2 to maintain delta opioid receptor analgesic incompetence in peripheral sensory neurons.
The third aim identifies a novel analgesic approach that repurposes the well-tolerated Food and Drug Administration-approved selective serotonin reuptake inhibitor paroxetine (Paxil®) for opioid co-therapy. Paroxetine potently binds G protein-coupled receptor kinase 2 and has opioid-mediated analgesic properties. Importantly, paroxetine’s analgesic effect can be effectively antagonized by the delta opioid receptor-selective antagonist naltrindole. The potential role of paroxetine on delta opioid receptor-mediated analgesic competence in the periphery was assessed using biochemical, molecular, functional, and behavioral techniques. Paroxetine dose-dependently reduces G protein-coupled receptor kinase 2 association with plasma membrane delta opioid receptor in peripheral sensory neurons and reduces protein kinase A–dependent phosphorylation of G protein-coupled receptor kinase 2, thereby enhancing receptor activity. Paroxetine’s effect on delta opioid receptor functional competence is notably antagonized by G protein-coupled receptor kinase 2 overexpression in sensory neurons. Paroxetine also enhances peripheral delta opioid receptor analgesic competence in vivo without the requirement for inflammatory priming by targeting G protein-coupled receptor kinase 2 in the periphery. Collectively, these results suggest that the Food and Drug Administration-approved drug paroxetine targets G protein-coupled receptor kinase 2 to enhance peripheral delta opioid receptor competence in sensory neurons, which may improve analgesic efficacy in non-inflammatory pain conditions.
The results from the studies presented in this thesis provide a clearer understanding of the mechanisms that underlie delta opioid receptor incompetence and govern functional delta opioid receptor competence in peripheral sensory neurons. Novel pharmacological strategies that target and/or influence proteins identified within this thesis warrants further investigation and research for the treatment of inflammatory and non-inflammatory pain conditions.
|Advisor:||Jeske, Nathaniel A.|
|Commitee:||Akopian, Armen N., Hargreaves, Kenneth M., Kavelaars, Annemieke M., Mooberry, Susan L.|
|School:||The University of Texas Health Science Center at San Antonio|
|School Location:||United States -- Texas|
|Source:||DAI-B 78/10(E), Dissertation Abstracts International|
|Subjects:||Biology, Neurosciences, Pharmacology|
|Keywords:||GRK2, Opioid, Opioids, Pain, Paroxetine, Sensory neurons|
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