Research Paper Activation of m-d opioid receptor heteromers inhibits neuropathic pain behavior in rodents Vinod Tiwaria, Shao-Qiu Hea, Qian Huanga, Lingli Lianga, Fei Yanga, Zhiyong Chena, Vineeta Tiwaria, Wakako Fujitab, Lakshmi A. Devib, Xinzhong Dongc,d, Yun Guana,e, Srinivasa N. Rajaa,* 10/27/2020 on Lc+LFovvA8kwLlXep+KVmBVRPo9c/w+jP3jM+VLppt6EIGY6AvlsMLr0U4h2Wl4ClQhoxQwnxSxh7PCm3iIk+ncF1wL2pK61fHxkxqEG1VREkSDLiG2Pu36FVfirG5EBAMD/bDeF43o= by http://journals.lww.com/pain from Downloaded Downloaded from Abstract http://journals.lww.com/pain Several reports support the idea that m- and d-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of m-d heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on m-d heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a m-d by heteromer–targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats Lc+LFovvA8kwLlXep+KVmBVRPo9c/w+jP3jM+VLppt6EIGY6AvlsMLr0U4h2Wl4ClQhoxQwnxSxh7PCm3iIk+ncF1wL2pK61fHxkxqEG1VREkSDLiG2Pu36FVfirG5EBAMD/bDeF43o= significantly decreased m-d heteromer expression in L5 DRG but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC50: 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, subcutaneously). HEK-293T cell surface-labeled with m- and d-ORs internalized both receptors after exposure to CYM51010. By contrast, in cells transfected with m-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats but was markedly attenuated in m-OR knockout mice. Our studies show that spinal nerve injury may increase m-d heterodimerization in uninjured DRG neurons, and that m-d heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance. Keywords: Opioid receptor, Heteromers, Windup, Neuropathic pain, Dorsal root ganglion 1. Introduction Whether m-andd-ORs colocalize in primary sensory neurons d Opioid receptors (ORs) are G-protein–coupled receptors (GPCRs) has been a subject of debate. Using -OR tagged with enhanced 50 d that are widely distributed along the pain pathways. In sensory green fluorescent protein, Scherrer et al. found that -OR and m neurons, m-, d-, and k-ORs play an important role in the modulation -OR were expressed in distinct subsets of mouse DRG neurons. of pain signals. The analgesic effect of morphine is mediated However, evidence from the early immunohistochemical studies d 50 primarily by m-OR activation.41 In the peripheral nervous system, m- was challenged because the -OR antibody lacked specificity. and d-ORs are present in dorsal root ganglion (DRG) neurons.25,34 More recent studies that have used in situ hybridization, single-cell Although m-ORs are expressed primarily on small-diameter PCR, electrophysiologic, and immunostaining techniques provide peptidergic neurons in DRG,60 d-OR mRNA has been found in evidence for the coexistence of functional d-ORs and m-ORs in both small-diameter peptidergic and nonpeptidergic (IB41) cells and a subpopulation of small-diameter peptidergic DRG neurons.25,59 large-diameter neurons, in large dense-core vesicles.43,58 Noxious Importantly, both d-OR– and m-OR–specific agonists inhibited 1 stimulus-induced Ca21 influx causes exocytosis of large dense-core depolarization-induced Ca2 currents in the same DRG neurons.59 vesicles and translocation of d-ORs to the plasma membrane.17 A pharmacologic study provided further evidence that the 2 Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. V. Tiwari, S.-Q. He, and Q. Huang contributed equally to this work. on a Department of Anesthesiology and Critical Care Medicine, the Johns Hopkins University, School of Medicine, Baltimore, MD, United States. Dr. Tiwari is now with the 10/27/2020 Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (IIT-BHU), Varanasi, India. Dr. Liang is now with the Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, China. Dr. Yang is now with the Department of Neurobiology, School of Basic Medical Sciences, Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, China, b Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States. Dr. Fujita is now with the Department of Frontier Life Science, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, c The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, School of Medicine, The Johns Hopkins University, Baltimore, MD, United States, d Howard Hughes Medical Institute, School of Medicine, the Johns Hopkins University, Baltimore, MD, United States, e Department of Neurological Surgery, School of Medicine, The Johns Hopkins University, Baltimore, MD, United States *Corresponding author. Address: Division of Pain Medicine, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University, Phipps 460, 600 N. Wolfe St, Baltimore, MD 21287, United States. Tel.: 410-955-1822. E-mail address: [email protected] (S.N. Raja). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.painjournalonline.com). PAIN 161 (2020) 842–855 © 2019 International Association for the Study of Pain http://dx.doi.org/10.1097/j.pain.0000000000001768 842 V. Tiwari et al.·161 (2020) 842–855 PAIN® Copyright © 2019 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited. April 2020· Volume 161· Number 4 www.painjournalonline.com 843 receptors colocalize on a functionally important population of Biologicals (Salem, MA) and CYM51010, a m-d heteromer– IB4-/TrkA1 nociceptors.35 Intriguingly, d-OR agonists can en- targeting agonist, was purchased from ChemBridge (San Diego, hance m-OR–mediated analgesia in opioid-naıve¨ animals. How- CA). m-d heteromer antibody was purchased from Kerafast ever, antagonists of d-OR decrease tolerance to the analgesic (Boston, MA). Morphine sulfate (Hospira, Inc, Lake Forest, IL) was effects of morphine,1,51 and animals lacking d-ORs or the gene- procured from the Johns Hopkins Hospital pharmacy. Other encoding preprotachykinin do not exhibit morphine tolerance.22,61 drugs were purchased from Sigma-Aldrich (St. Louis, MO) or G-protein–coupled receptors can form homomers or hetero- Tocris Bioscience (Bristol, United Kingdom). Stock solutions were mers. Heteromers exhibit novel pharmacology, such as altered freshly prepared as instructed by the manufacturer. ligand-binding properties, G-protein coupling, and trafficking, that differs from that of homomers.5,11,18 Studies suggest that m-d 2.3. Neuropathic pain model in rodents heteromers have distinct pharmacologic and functional proper- ties.14,15,44,49 For example, whereas activation of the m-ORs L5 spinal nerve ligation (L5-SNL) surgery was used for the induces G-protein–mediated signaling, the same agonist acting on induction of neuropathic pain-related behavior in rats. The the m-d heteromer may evoke b-arrestin–mediated signaling.15,49 procedure was a modification of that described in our previous Chronic morphine exposure upregulates m-d heteromers in brain studies.24,55 Briefly, rats were anesthetized with isoflurane (2%; regions involved in pain signaling (eg, rostroventral medulla and Abbott Laboratories, Chicago, IL) delivered through a nose cone. nucleus accumbens), suggesting that the central m-d heteromer Using aseptic conditions, we incised the skin at the midline over may play a role in opioid tolerance.27 The analgesic effects of the lumbar spine to expose the L5, L6, and upper sacral opioids in inflammatory and neuropathic pain are mediated, in part, vertebrae. The left transverse process of the L6 vertebra was by m-ORs expressed on primary sensory neurons. However, the removed, and the left L5 spinal nerve was exposed and dissected role of m-d heteromers in pain and peripheral opioid tolerance has from the underlying tissue. The left L5 spinal nerve was tightly not been well examined, in part, because specific histologic and ligated with a 6-0 silk suture and cut distal to the ligature. The pharmacologic tools have been lacking. The development of overlying muscle layer was approximated with 4-0 silk suture and a heteromer-selective antibody has helped researchers to the skin closed with metal clips. The rats were returned to their characterize the m-d heteromer distribution in DRG neurons and cages after surgery, kept warm under a heat lamp, and monitored explore heteromer-mediated signaling.27 Using this antibody, we during recovery. In control rats, the spinal nerve was exposed but examined the effects of