Title : Spinal opioid tolerance depends upon platelet-derived growth factor receptor-β signaling , not mu-opioid receptor internalization

Opioids are some of the most potent analgesics available. However, their effectiveness is limited by the development of analgesic tolerance. Traditionally, tolerance was thought to occur by termination of mu-opioid receptor (MOR) receptor signaling via desensitization and internalization. Contradictory findings led to a more recent proposal that sustained MOR signaling caused analgesic tolerance. However, this view has also been called into question. We recently discovered that the platelet-derived growth factor receptor beta (PDGFR-β) signaling system is both necessary and sufficient to cause opioid tolerance. We therefore propose a completely new hypothesis, that opioid tolerance is mediated by selective cellular signals and is independent of MOR internalization. To test this hypothesis, we developed an automated software-based method to perform unbiased analyses of opioid-induced MOR internalization in the rat substantia gelatinosa. We induced tolerance with either morphine, which did not cause MOR internalization, or fentanyl, which did. We also blocked tolerance by administering morphine or fentanyl with the PDGFR-β inhibitor imatinib. We found that imatinib blocked tolerance without altering receptor internalization induced by either morphine or fentanyl. We also showed that imatinib blocked tolerance to other clinically used opioids. Our findings indicate that opioid tolerance is not dependent upon MOR internalization and support the novel hypothesis that opioid tolerance is mediated by intracellular signaling that can be selectively targeted. This suggests the exciting possibility that undesirable opioid side effects can be selectively eliminated, dramatically improving the safety and efficacy of opioids. This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on July 28, 2020 as DOI: 10.1124/mol.120. 119552 at A PE T Jornals on N ovem er 4, 2020 m oharm . aspeurnals.org D ow nladed from Significance Statement: Classically, it was thought that analgesic tolerance to opioids was caused by desensitization and internalization of mu opioid receptors (MORs). More recently, it was proposed that sustained, rather than reduced MOR signaling caused tolerance. Here we present conclusive evidence that opioid tolerance occurs independently of MOR internalization, and is selectively mediated by platelet-derived growth factor receptor signaling. This novel hypothesis suggests that dangerous opioid side effects can be selectively targeted and blocked, improving the safety and efficacy of opioids. This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on July 28, 2020 as DOI: 10.1124/mol.120. 119552 at A PE T Jornals on N ovem er 4, 2020 m oharm . aspeurnals.org D ow nladed from Introduction: For centuries, opioids have been first line drugs for the treatment of severe pain. However, their effectiveness is limited by the development of opioid analgesic tolerance. Analgesic tolerance develops more rapidly than tolerance to other severe and potentially life-threatening opioid effects such as itching, constipation, and respiratory depression (Gutstein and Akil, 2006). Thus, the severity of side effects and risk of lethal overdose increases as opioid doses escalate, limiting their prolonged use. Better understanding the mechanisms underlying tolerance is critical for the development of safer and more effective treatments for chronic pain. Opioids induce cellular signaling through the mu-opioid receptor (MOR), a Gi/o-protein coupled protein receptor (GPCR) (Pasternak, 2010). MOR is expressed in pain-modulating neuronal pathways in peripheral sensory neurons, spinal cord and brain (Mansour et al., 1994, 1995). Initially it was postulated that opioid tolerance was caused by termination of opioid signaling by opioid-induced receptor desensitization and downregulation (internalization) (Cox, 1991). However, it was observed that chronic morphine administration caused tolerance without inducing receptor internalization (the ‘morphine paradox’) (Keith et al., 1996; Gutstein and Akil, 2006). It was subsequently proposed that sustained receptor signaling, rather than termination of signaling by internalization mediated analgesic tolerance (Kieffer and Evans, 2002; He and Whistler, 2005). This hypothesis has also been called into question by conflicting data (Bailey et al., 2003; Koch et al., 2005). Contradicting both theories, all opioids, regardless of whether they induce MOR internalization or not, induce analgesic tolerance when administered at equianalgesic doses (Duttaroy and Yoburn, 1995; Raehal et al., 2011). We recently discovered that the platelet-derived growth factor receptor beta (PDGFR-β) signaling system is both necessary and sufficient to cause opioid tolerance. We also demonstrated that the analgesic properties of opioids could be completely dissociated from opioid tolerance (Wang et al., 2012). This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on July 28, 2020 as DOI: 10.1124/mol.120. 119552 at A PE T Jornals on N ovem er 4, 2020 m oharm . aspeurnals.org D ow nladed from Based these findings, we propose a completely new hypothesis for the mechanisms underlying opioid tolerance; namely, that opioid tolerance is caused by selective intracellular signaling and occurs independently of MOR internalization. To test this hypothesis, we developed an automated software-based method that allowed us to perform an unbiased analysis of the amount of MOR internalization induced by opioids in the dorsal horn of the spinal cord of rats. We induced opioid tolerance in rats with morphine, which did not cause MOR internalization, and with fentanyl, which did cause receptor internalization. We also blocked the development of tolerance by treating rats with morphine or fentanyl in combination with the PDGFR-β inhibitor imatinib. We then analyzed the degree of MOR internalization induced in each treatment group. We also treated rats with several clinically used opioids to determine whether PDGFR-β antagonism would block analgesic tolerance to a wider range of clinically used opioids. This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on July 28, 2020 as DOI: 10.1124/mol.120. 119552 at A PE T Jornals on N ovem er 4, 2020 m oharm . aspeurnals.org D ow nladed from