Inhibition of Spinal Cord Hsp90 Enhances Src Kinase Signaling to Increase Opioid Anti‐Nociception

Abstract

Chronic pain remains a huge social and economic burden on the United States and around the world. With few safe treatment options for these patients, many physicians turn to opioid analgesics such as morphine. These medications are not without serious side effects including constipation, respiratory depression, and addiction, which has led to an opioid addiction crisis affecting over 10 million Americans each year. This crisis emphasizes the need to find new ways to improve opioid therapy and decrease side effects like addiction. One such research area is the signal transduction cascades evoked by opioid receptor activation, which could be modulated to enhance their effects on pain management while decreasing negative side effects. Previously, our lab discovered that the molecular chaperone protein Heat shock protein 90 (Hsp90) plays a key role in mu opioid receptor (MOR) signal transduction. When inhibiting Hsp90 in the spinal cord through intrathecal administration of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) in mice we observed a significant increase in morphine anti-nociception, which could enable an opioid dose-reduction strategy. A preliminary proteomic evaluation in 17-AAG treated mouse spinal cord suggested that the inhibitory protein Src-kinase inhibitor 1 was downregulated by 17-AAG, thus suggesting that Src kinase signaling might be upregulated. To test this hypothesis, male and female CD-1 mice were treated with 0.5 nmol 17-AAG or vehicle intrathecally. After 24 hours, mice were treated with 10 nmol of the Src kinase inhibitor Src-I1 or vehicle intrathecally, then after 10 minutes, mice were administered 3.2 mg/kg morphine and a tail flick assay was performed. The enhanced anti-nociceptive effects seen with 17-AAG were completely abolished in groups that received the Src inhibitor, suggesting that Hsp90 inhibition enhances Src kinase signaling to lead to enhanced opioid pain relief. Interestingly, the group that did not receive 17-AAG but did receive Src-I1 also showed a decrease in the anti-nociceptive effects of morphine, thus suggesting a role for Src in the opioid receptor pathway at baseline without Hsp90 inhibitor treatment. Next, mice were treated with only Src-I1 or vehicle and then given morphine or vehicle, showing that Src-I1 administration had no effect on pain by itself. We also tested a post-surgical paw incision pain model to test if Src regulates opioid anti-nociception in other pain types. Similar results were seen with a complete loss of the enhanced anti-nociception of 17-AAG as well as a decrease in the anti-nociceptive effects of morphine with Src inhibitor treatment. Lastly, initial analysis by Western Blotting confirmed what we observed behaviorally. Groups treated with 17-AAG and the opioid agonist DAMGO had a significant increase in phosphorylated Src compared to groups treated with vehicle alone, thus supporting our hypothesis that Hsp90 inhibition in the spinal cord enhances opioid pain relief by upregulation of Src signaling. This has helped elucidate a key molecular mechanism by which Hsp90 regulates opioid signaling and leads to enhanced opioid antinociception, creating a potential target for future efforts to improve opioid treatment.