Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171 S
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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2019/07/15/jpet.119.259242.DC1 1521-0103/371/1/56–62$35.00 https://doi.org/10.1124/jpet.119.259242 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 371:56–62, October 2019 Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171 s Max V. McDermott, Leela Afrose, Ivone Gomes, Lakshmi A. Devi, and Erin N. Bobeck Department of Biology, Utah State University, Logan, Utah (M.V.M., L.A., E.N.B.) and Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York (I.G., L.A.D.) Received April 19, 2019; accepted July 10, 2019 ABSTRACT Downloaded from ProSAAS is one of the most widely expressed proteins through- the agonist enhancing and antagonist reducing antinociception. out the brain and was recently found to be upregulated in chronic Lastly, we showed that the GPR171 antagonist or receptor fibromyalgia patients. BigLEN is a neuropeptide that is derived knockdown decreases signaling by the mu-opioid receptor, but from ProSAAS and was recently discovered to be the endoge- not the delta-opioid receptor. Taken together, these results nous ligand for the orphan G protein-coupled receptor GPR171. suggest that antagonism of the GPR171 receptor reduces mu Although BigLEN-GPR171 has been found to play a role in opioid receptor signaling and morphine-induced antinocicep- feeding and anxiety behaviors, it has not yet been explored in tion, whereas the GPR171 agonist enhances morphine antino- jpet.aspetjournals.org pain and opioid modulation. The purpose of this study was to ciception, suggesting that GPR171 may be a novel target toward evaluate this novel neuropeptide-receptor system in opioid- the development of pain therapeutics. induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is SIGNIFICANCE STATEMENT a key brain area involved in pain modulation and opioid GPR171 is a recently deorphanized receptor that is expressed functions. We also found that, although the GPR171 agonist within the periaqueductal gray and can regulate mu opioid and antagonist do not have nociceptive effects on their own, receptor signaling and antinociception. This research may they oppositely regulate morphine-induced antinociception with contribute to the development of new therapeutics to treat pain. at ASPET Journals on September 30, 2021 Introduction Studies from our laboratory showed that this system regu- lates anxiety (Bobeck et al., 2017), and feeding behaviors Despite many years of research, opioids remain among the in mice (Wardman et al., 2016). Our studies used homology best therapeutics for the treatment of chronic pain, but side modeling to identify and characterize a small molecule effects with acute and long-term use limit their effectiveness. agonist and antagonist. We found that administration of The development of novel therapeutics that increase opioid the GPR171 antagonist within the basolateral amygdala potency would allow for smaller prescribed doses of opioids decreased anxiety behaviors, while in the hypothalamus the to treat pain, and would thus decrease the detrimental agonist increased feeding and body weight (Wardman et al., side effects. A new and promising avenue of study toward 2016; Bobeck et al., 2017). developing novel pain therapeutics is targeting novel Recently, ProSAAS was shown to be upregulated in the neuropeptide-receptor systems, given that many neuropep- cerebrospinal fluid of fibromyalgia patients (Khoonsari et al., tides play important roles in many sensory modalities, 2019); however, alterations in specific brain regions have not including pain. ProSAAS is one of the most widely expressed been assessed. Possible sites of action are within the descend- proteins in the brain and has been implicated in a wide range ing pain modulatory pathway, which includes the periaque- of functions (Wei et al., 2004; Morgan et al., 2010; Hoshino ductal gray (PAG), rostral ventromedial medulla (RVM), and et al., 2014; Berezniuk et al., 2017). It was recently found that the dorsal horn of the spinal cord. Opioids produce their the ProSAAS derived peptide, BigLEN, is the endogenous antinociceptive effects in part by excitation of neurons projec- ligand for the previously orphan G protein-coupled receptor, ting from the PAG to the RVM, which in turn inhibits GPR171 (Gomes et al., 2013), however, little is known about incoming pain signals at the dorsal horn of the spinal cord the physiologic functions of this peptide-receptor system. (Heinricher et al., 2009; Lau and Vaughan, 2014). Many studies have highlighted the importance of the PAG in opioid This work was supported by National Institutes of Health [Grants DA0886 antinociception (Depaulis et al., 1985; Morgan et al., 1991, and NS026880 to L.A.D. and by a Young Investigator Grant from Brain and 2009; Bobeck et al., 2009). Behavior Research Foundation to E.N.B. https://doi.org/10.1124/jpet.119.259242. Given that the PAG is an important structure in pain s This article has supplemental material available at jpet.aspetjournals.org. modulation, the goal of this current research was to ABBREVIATIONS: CHO, Chinese hamster ovary cells; DMEM, Dulbecco’s modified Eagle’s medium; DOPr, delta opioid receptor; GTPgS, 59-O-(3- [35S]thio)triphosphate; KD, knockdown; MOPr, mu opioid receptor; N2A, neuro 2A cells; PAG, periaqueductal gray; RVM, rostral ventromedial medulla. 56 GPR171 Regulates Opioid Signaling and Antinociception 57 investigate GPR171 expression in PAG, the antinociceptive Immunohistochemistry. Immunohistochemistry was conducted properties of GPR171 ligands, and GPR171 interactions as described previously (Bobeck et al., 2017). Briefly, five mice were with the opioid system. To do this, we used GPR171 small deeply anesthetized with isoflurane and perfused transcardially molecule ligands that were characterized previously (Ward- through the ascending aorta with 4% paraformaldehyde. Brains were man et al., 2016; Bobeck et al., 2017). We first sought to postfixed for 1 hour and stored in PBS. Immunohistochemistry was performed on free-floating coronal cut brain tissue sections (50 mm) investigate GPR171 localization in neuronal subtypes containing PAG. Sections were incubated in 1% sodium borohydride in within the PAG utilizing immunohistochemistry. We found PBS for 30 minutes followed by blocking buffer (5% normal goat GPR171 primarily in the GABAergic neurons. Next, we serum and 0.3% Triton X-100 in PBS) at room temperature for 1 hour. examined the behavioral effects of the agonist and antag- Tissues were incubated overnight at 4°C in primary antibodies onist on antinociception and morphine-induced antinoci- against GPR171 (rabbit, 1:400; GeneTex, Irvine, CA), GAD67 (mouse, ception in vivo using the hot plate and tail flick behavioral 1:500; Millipore, Temecula, CA), or vGlut2 (guinea pig, 1:500; Milli- assays and found that the agonist and antagonist oppositely pore, Burlington, MA) in 1% bovine serum albumin (Sigma-Aldrich) regulate morphine antinociception. Lastly, we examined and 0.1% Triton X-100 (Sigma-Aldrich). The GPR171 antibody was the effect of GPR171 ligands on opioid receptor signaling previously validated in mouse brain tissue using knockdown or in vitro utilizing a radioligand guanosine 59-O-(3-[35S]thio) knockout approaches (Wardman et al., 2016). Primary antibodies triphosphate (GTPgS) signaling assay. We found that were visualized with goat anti-rabbit 594, goat anti-mouse 488, goat GPR171 blockade decreased MOPr-mediated G protein anti-guinea pig 647, and goat anti-rabbit 488 (1:1000; Invitrogen, activity. Taken together, these experiments give us a better Carlsbad, CA) followed by 5 minutes of incubation with the nuclear Downloaded from stain DAPI (100 ng/ml; Invitrogen). Sections were mounted with understanding of how GPR171 is interacting with the opioid ProLong Diamond Antifade (Invitrogen). Microscopy was performed system to regulate signaling and behavior. at the Utah State University Microscopy CORE using Zeiss 710 confocal microscope. Cell Culture. Chinese Hamster Ovary (CHO) cells stably express- Materials and Methods ing N-terminally Flag-epitope tagged mu opioid receptors (CHO- MOPr) or N-terminally myc-epitope tagged GPR171 (CHO-GPR171) jpet.aspetjournals.org Subjects. Eighty-seven male C57BL/6 mice (Charles River Labo- were grown in F12 media containing 10% FBS, 1X streptomycin- – – ratories, Wilmington, VA), 6 8 weeks old, weighing 18 26 g at the penicillin and 500 mg/ml geneticin (G418). Neuro 2A (N2A) cells were start of the experiment, were used for behavior experiments. Food grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1Â and water were available ad libitum, except during testing. Mice were streptomycin-penicillin. N2A cells were chosen given the vast housed (four to five per cage) in a humidity- and temperature- expression of many GPCRs including MOPr, DOPr, and GPR171 – controlled room with a 12:12-hour light/dark cycle (on 0700 1900). (Supplemental Fig. 1; Gomes et al., 2013). N2A cells stably express- Testing took place between 0800 and 1600. All procedures were ing GPR171 (N2A-GPR171) were generated as described (Trapaidze conducted in accordance with the Guide for the Care and Use of at ASPET Journals on September 30, 2021 et al., 2000; Gomes et al., 2013) and maintained in DMEM containing Laboratory Animals adopted by the National Institutes of Health and 1Â streptomycin-penicillin and 500 mg/ml geneticin (G418). Neuro were approved by the Utah State University Institutional Care and 2A cells stably expressing GPR171 lentiviral shRNA (N2A GPR171 Use Committee (Protocol #2775). KD) were generated as described (Gomes et al., 2013) and main- Drug Treatment. Morphine sulfate (2 or 5 mg/kg, s.c.; Hikma, tained in DMEM containing 1Â streptomycin-penicillin and 8 mg/ml London, UK) was suspended in 0.9% saline. The GPR171 antagonist, puromycin. MS21570 (5 mg/kg, i.p.; Santa Cruz Biotechnology, Inc., Santa Cruz, Membrane Preparation.