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Abstract
Although pain is important to alert us of potential or actual harm, severe and prolonged pain can be detrimental to our well-being. About half of the adult U.S. population experience some sort of pain within a span of 3 months. Additionally, about 10% of adults in the U.S. suffer from chronic pain, or pain that persists daily for at least 3 months. Individuals with persistent moderate to severe pain are more likely to seek medical services, have disturbances in physical functioning and an overall lower quality of life. Opioids, such as morphine, continue to be standard treatment for moderate to severe pain with approximately 4% of the adult U.S. population prescribed opioids long term. However, adverse effects of opioid use range from relatively minor annoyances such as nausea and vomiting to life threatening respiratory depression. Moreover, the euphoria that occurs with opioids can lead to abuse of prescribed medication and addiction. Many of these adverse effects are due to opioid activation of receptors expressed within the central nervous system. However, these opioid receptors are also expressed on primary afferent pain sensing neurons, referred to as nociceptors, and inhibit pain neurotransmission to the central nervous system. Targeting opioid receptors expressed on nociceptors with analgesic drugs would inhibit pain neurotransmission without central nervous system-mediated adverse effects. Our overall goal is to understand how opioid ligands exert their actions in the periphery for pain relief to lay the foundation for improved pain pharmacotherapy.
The kappa opioid receptor is expressed on nociceptors and like other inhibitory G protein coupled receptors couple to many signaling pathways including inhibition of adenylyl cyclase, inhibition of voltage gated calcium channels, and activation of G protein-coupled inwardly rectifying potassium channels. Although all three of these effectors are known to be involved in analgesic mechanisms, it is not known which of these effector pathways mediate peripheral kappa opioid receptor-mediated antinociception. We have found that peripheral kappa opioid receptormediated antinociception is Gi protein-dependent and involves G protein-coupled inwardly rectifying potassium channel activation but does not necessarily depend on inhibition of adenylyl cyclase and decreased cyclic adenosine monophosphate levels or inhibition of the voltage gated calcium channel CaV2.2. Therefore, my dissertation work focused on the role of G protein-coupled inwardly rectifying potassium channel activation in peripheral kappa opioid receptor-mediated antinociception.
The expression of G protein-coupled inwardly rectifying potassium channels in nociceptors has been controversial, with contradictory findings reported across species. My results from immunofluorescence experiments show that G protein-coupled inwardly rectifying potassium channel subunits and kappa opioid receptor are expressed locally in rat hindpaw tissue of both male and female rats. Importantly, I have demonstrated that kappa opioid receptor-mediated antinociception in both male and female rats is blocked by local administration of the G proteincoupled inwardly rectifying potassium channel blocker, tertiapin Q, as well as by knockdown of GIRK2 subunit following local administration of siRNA. Further, direct activation of G proteincoupled inwardly rectifying potassium channels in the hindpaw elicited antinociceptive effects in both male and female rats. Thus, our data support the overarching hypothesis that G proteincoupled inwardly rectifying potassium channels are the major signaling effectors that mediate peripheral kappa opioid receptor-mediated antinociception. Interestingly, although we originally focused on kappa opioid receptor expressed on nociceptor terminals that innervate the skin, we found that both G protein-coupled inwardly rectifying potassium channels and kappa opioid receptor were abundantly expressed in keratinocytes. In addition to performing barrier functions, recently keratinocytes have been implicated in pain neurotransmission, thus we explored the role of G protein-coupled inwardly rectifying potassium channels expressed on keratinocytes for kappa opioid receptor-mediated antinociception. Following administration of a modified transactivator of transcription protein-containing plasmid complex to selectively knock down GIRK2 subunits in keratinocytes, we found that kappa opioid receptor-mediated antinociception was lost completely in both males and female rats. These exciting findings suggest that kappa opioid receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels in keratinocytes plays a major role in peripherally mediated antinociception.
In summary, the major finding of my thesis work is that G protein-coupled inwardly rectifying potassium channels expressed in the periphery are essential for peripheral kappa opioid receptor-mediated antinociceptive responses. Importantly, I have also found that activation of G protein-coupled inwardly rectifying potassium channels in keratinocytes is involved in the production of peripheral antinociception. The development of peripherally restricted kappa opioid receptor agonists with increased coupling to G protein-coupled inwardly rectifying potassium channel activation may result in more efficacious analgesics for the treatment of pain, devoid of debilitating central nervous system-mediated adverse effects.
