Friday, November 22, 2024
11/22/2024
PROJECT SUMMARY Spinal administration of morphine, a potent opioid, is commonly used to treat post-operative pain. However, the desired opioid-induced analgesia consistently has a negative side effect of opioid-induced pruritus, causing debilitating itch in the already distressed patient. The first line treatment is opioid antagonists, but this may compete with the initial opioid treatment and cause the patient to choose between unwanted pain or itch. Both sensations are closely related, but the mechanism of how opioids can cause both pruritus and analgesia remains unclear. Morphine acts on mu-opioid receptors (MOR) to inhibit the activity of pain-sensing neurons. MOR is a G-protein coupled receptor (GPCR) coupled to the inhibitory Gi-heterotrimeric proteins (Gαi and Gβγ) which cause downstream actions to inhibit depolarization of the neuron. One mechanism by which this inhibition occurs is by direct inhibition by Gβγ on the Transient Receptor Potential melastatin 3 (TRPM3) ion channel, a thermosensitive and nociceptive cation channel expressed on neurons. Recently, a naturally occurring splice variant of TRPM3 that does not bind to Gβγ was identified. With this knowledge, a novel mouse line was generated (TRPM3DEx17) in which all the TRPM3 channels express this isoform. Utilizing the TRPM3DEx17 mouse line we performed preliminary behavioral studies on morphine induced pruritus and analgesia. Data showed that the TRPM3DEx17 mice experienced significantly less pruritus upon intrathecal injection of morphine compared to WT. In the plantar incision model, TRPM3DEx17 mice had no change in morphine analgesia when injected intrathecally in heat and mechanical nociception. Analgesia by morphine occurs through inhibition of excitatory (Vglu2+) neurons, while pruritus occurs by inhibition of inhibitory (Vgat+) neurons, or disinhibition, in the spinal cord. Interestingly, TRPM3 and MOR are more frequently coexpressed on the itch-inhibitory Vgat+ neurons compared to the pain-excitatory Vglut2+ neurons, supporting our behavioral findings. Thus, Aim 1 of the proposal will explore via in-situ hybridization, the co-expression of TRPM3 and MOR in Vglu2+ and Vgat+ neurons in the TRPM3DEx17 mouse spinal cord, and use two reporter mouse lines with GFP tagged excitatory and inhibitory neurons to perform whole-cell patch clamping of spinal cord slices to compare morphine inhibition in each neuronal population. Aim 2 will attempt to overcome the MOR inhibition of TRPM3 by co-injecting a TRPM3 agonist, pregnenolone sulfate, in the WT mice intrathecally with morphine, and observe if opioid-induced pruritus can be alleviated while analgesia is maintained. These results may suggest that TRPM3 is the main depolarizing ion channel responsible for the disinhibition of pruritus and can be a potential target for alleviating morphine induced pruritus in patients undergoing opioid therapy without affecting the desired analgesia.
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