Mu Opioid Receptor Splice Variants Research Articles

The Holy Grail of Safer Opioids: Targeting Mu Opioid Receptor Splice Variants

The Holy Grail of Safer Opioids: Targeting Mu Opioid Receptor Splice Variants

Relevance of Mu-Opioid Receptor Splice Variants and Plasticity of Their Signaling Sequelae to Opioid Analgesic Tolerance.

Opioid dose escalation to effectively control pain is often linked to the current prescription opioid abuse epidemic. This creates social as well as medical imperatives to better understand the mechanistic underpinnings of opioid tolerance to develop interventions that minimize it, thereby maximizing the analgesic effectiveness of opioids. Profound opioid analgesic tolerance can be observed in the absence of mu-opioid receptor (MOR) downregulation, aggregate MOR G protein uncoupling, and MOR desensitization, in the absence of impaired G protein coupled receptor kinase phosphorylation, arrestin binding, or endocytosis. Thus, we have explored alternative biochemical sequelae that might better account for opioid analgesic tolerance. Our findings indicate that substantial plasticity among upstream and downstream components of opioid receptor signaling and the emergence of alternative signaling pathways are major contributors to opioid analgesic tolerance. An exemplar of this plasticity is our findings that chronic morphine upregulates the MOR variants MOR-1B2 and MOR-1C1 and phosphorylation of their C-terminal sites not present in MOR-1, events causally associated with the chronic morphine-induced shift in MOR G protein coupling from predominantly Gi/Go inhibitory to Gs-stimulatory adenylyl cyclase signaling. The unique feature(s) of these variants that underlies their susceptibility to adapting to chronic morphine by altering the nature of their G protein coupling reveals the richness and pliability of MOR signaling that is enabled by generating a wide diversity of MOR variants. Furthermore, given differential anatomical expression patterns of MOR variants, MOR splice variant-dependent adaptations to chronic morphine could enable mechanistic underpinnings of tolerance and dependence that are CNS region- and cell-specific.

Pharmacological studies of morphine in a novel humanized mu opioid receptor mouse model

Full‐length 7‐transmembrane (TM) variants of mu opioid receptor (MOR) share similar amino acid sequences of TM domains in rodents and humans; however, interspecies differences in N‐ and C‐terminal amino acid sequences of MOR splice variants dramatically affect the downstream signaling. Thus, it is essential to develop a mouse model that expresses human MOR for opioid pharmacological studies. We generated two lines of fully humanized MOR mice, line #1 and #2. The novel murine model having human OPRM1 genes and human‐specific variants was examined by RT‐PCR. Humanized MOR mice were characterized in vivo using a tail‐flick, charcoal meal, open field, tail suspension, naloxone precipitation tests, and rectal temperature measurement. The data indicated that wild‐type (WT) and humanized MOR mice exhibited different pharmacology of morphine, including antinociception, tolerance, sedation, and withdrawal syndromes, suggesting the presence of species difference between mouse and human MORs. Therefore, humanized MOR mice could serve as a novel mouse model for pharmacogenetic studies of opioids.

Morphine produces potent antinociception, sedation, and hypothermia in humanized mice expressing human mu-opioid receptor splice variants.

Morphine is a strong painkiller acting through mu-opioid receptor (MOR). Full-length 7-transmembrane (TM) variants of MOR share similar amino acid sequences of TM domains in rodents and humans; however, interspecies differences in N- and C-terminal amino acid sequences of MOR splice variants dramatically affect the downstream signaling. Thus, it is essential to develop a mouse model that expresses human MOR splice variants for opioid pharmacological studies. We generated 2 lines of fully humanized MOR mice (hMOR; mMOR mice), line #1 and #2. The novel murine model having human OPRM1 genes and human-specific variants was examined by reverse-transcription polymerase chain reaction and the MinION nanopore sequencing. The differences in the regional distribution of MOR between wild-type and humanized MOR mice brains were detected by RNAscope and radioligand binding assay. hMOR; mMOR mice were characterized in vivo using a tail-flick, charcoal meal, open field, tail suspension, naloxone precipitation tests, and rectal temperature measurement. The data indicated that wild-type and humanized MOR mice exhibited different pharmacology of morphine, including antinociception, tolerance, sedation, and withdrawal syndromes, suggesting the presence of species difference between mouse and human MORs. Therefore, hMOR; mMOR mice could serve as a novel mouse model for pharmacogenetic studies of opioids.

Phosphorylation of unique C-terminal sites of the mu-opioid receptor variants 1B2 and 1C1 influences their Gs association following chronic morphine.

We recently demonstrated in rat spinal cord that a regimen of escalating doses of systemic morphine, analogous to regimens used clinically for chronic pain management, selectively up-regulates the mu-opioid receptor (MOR) splice variants MOR-1B2 and MOR-1C1 mRNA and functional protein. This study investigated the potential relevance of up-regulating MOR-1B2 and MOR-1C1 to the ability of chronic morphine to shift MOR signaling from predominantly Gi /Go inhibitory to Gs stimulatory. Specifically, we tested the hypotheses that chronic morphine induces phosphorylation of carboxyl terminal sites unique to MOR-1B2 and MOR-1C1, and that this phosphorylation is causally related to augmented association of these variants with Gs α. Hypotheses were validated by (i) abolition of the chronic morphine-induced increment in MOR-1C1 and MOR-1B2 association with Gs α by inhibitors of protein kinase A and Casein kinase 2, respectively; (ii) failure of chronic morphine to augment MOR variant Gs α interactions in Chinese hamster ovary cells transiently transfected with either rat MOR-1C1 or MOR-1B2 in which targeted protein kinase A and Casein kinase 2 serine phosphorylation sites, respectively, were mutated to alanine; (iii) abrogation of chronic morphine-induced augmented MOR Gs α association in spinal cord of male rats following intrathecal administration of dicer substrate small interfering RNAs targeting MOR-1B2/MOR-1C1 mRNA. The ability of chronic morphine to not only up-regulate-specific MOR variants but also their carboxyl terminal phosphorylation and consequent augmented association with Gs α may represent a novel component of opioid tolerance mechanisms, suggesting novel potential targets for tolerance abatement.

Characterizing mu Opioid Receptor Splice Variants Using Gene Targeting Rat Models Generated by Easi‐CRISPR

BackgroundA single‐copy mu opioid receptor (OPRM1) gene generates a vast array of mu opioid receptor splice variants through extensive alternative splicing that is conserved from rodent to human. These variants can be divided into two sets: exon 11 (E11) and exon 1 (E1) associated variants. The functional differences between the E1 and E11 sets of variants have been demonstrated using gene targeted mouse models.Rationale/SignificanceWhile mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation.HypothesisE11‐ and E1‐associated splice variants play important and distinct roles In opioid pharmacology.ResultsWe have generated two conditional Oprm1 knockout (KO) models, E11‐KO and E1‐KO, in Sprague Dawley rats using a novel Easi‐CRISPR approach. Cre‐mediated E11 and E1 deletions was confirmed through breeding a CAG‐Cre rat. Initial behavioral studies showed that in both E11 and E1 KO rats, buprenorphine analgesia was lost. Buprenorphine CPP was significantly reduced in E11 KO rats. Morphine analgesia was completely lost in the E1‐KO rat, but intact in the E11‐KO rat.DiscussionOur results suggest that buprenorphine analgesia is dependent on both E11 and E1‐associated variants, a similar scenario seen in the E11‐KO and E1‐KO mouse models. E11‐associated variants also involved in buprenorphine reward in rat. These conditional Oprm1 KO rat models will provide valuable resources for the research community and would expand our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice.Support or Funding InformationSupported by grants from NIH (DA40858, DA06241, DA042888 and CA08748)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced mu opioid receptor-dependent opioidergic modulation of striatal cholinergic transmission in DYT1 dystonia.

Mu opioid receptor activation modulates acetylcholine release in the dorsal striatum, an area deeply involved in motor function, habit formation, and reinforcement learning as well as in the pathophysiology of different movement disorders, such as dystonia. Although the role of opioids in drug reward and addiction is well established, their involvement in motor dysfunction remains largely unexplored. We used a multidisciplinary approach to investigate the responses to mu activation in 2 mouse models of DYT1 dystonia (Tor1a+/Δgag mice, Tor1a+/- torsinA null mice, and their respective wild-types). We performed electrophysiological recordings to characterize the pharmacological effects of receptor activation in cholinergic interneurons as well as the underlying ionic currents. In addition, an analysis of the receptor expression was performed both at the protein and mRNA level. In mutant mice, selective mu receptor activation caused a stronger G-protein-dependent, dose-dependent inhibition of firing activity in cholinergic interneurons when compared with controls. In Tor1a+/- mice, our electrophysiological analysis showed an abnormal involvement of calcium-activated potassium channels. Moreover, in both models we found increased levels of mu receptor protein. In addition, both total mRNA and the mu opioid receptor splice variant 1S (MOR-1S) splice variant of the mu receptor gene transcript, specifically enriched in striatum, were selectively upregulated. Mice with the DYT1 dystonia mutation exhibit an enhanced response to mu receptor activation, dependent on selective receptor gene upregulation. Our data suggest a novel role for striatal opioid signaling in motor control, and more important, identify mu opioid receptors as potential targets for pharmacological intervention in dystonia. © 2017 International Parkinson and Movement Disorder Society.

Mu Opioid Pharmacology: 40 Years to the Promised Land.

Opioids continue to play a major role in medicine, but not without problems. Side effects limit their utility medically, while the potential of addiction has had a major societal impact. Pharmacologists have been trying to develop opioids lacking side effects since the first derivative, heroin, was synthesized in the 1870s. The identification of opioid receptors about 40 years ago opened up new insights into our understanding of opioid action, fueled by the molecular biology revolution of the 1980s and 1990s. A major result of these studies was the discovery that the mu opioid receptor gene, Oprm1, undergoes extensive alternative splicing in mice, rats, and humans. This single gene generates three sets of proteins, each containing many variants. The object of this review is to describe these variants and how they can be targeted to generate safer, effective analgesic drugs. Mu opioid receptor multiplicity was first suggested over 35 years ago based upon a series of selective antagonists and detailed binding assays. The identification of the different classes of mu opioid receptor splice variants enabled us to target one of the classes of splice variants to obtain potent analgesics lacking respiratory depression, physical dependence, and reward behavior. They also displayed no cross tolerance to morphine analgesia and had diminished effects on gastrointestinal transit. Forty years after the identification of opioid-binding sites in brain the promised land of safer, nonaddictive analgesics is in sight.

Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors.

Selective up-regulation of functional mu-opioid receptor splice variants by chronic opioids.

We recently reported (Verzillo, etal. J. Neurochem: 130, 790-796, 2014) that chronic systemic morphine selectively up-regulates mRNA encoding two C-terminal μ-opioid receptor (MOR) splice variants, MOR-1C1 and MOR-1B2 (MOR-1B2/-1C1). Given the known disconnects between changes in levels of mRNA and corresponding protein, it is essential to directly demonstrate that chronic opioid treatment elevates functional MOR-1B2/-1C1 protein prior to inferring relevance of these MOR variants to spinal opioid tolerance mechanisms. Accordingly, we investigated the ability of chronic opioid exposure to up-regulate MOR protein in Chinese hamster ovary cells stably transfected with rat MOR variants MOR-1B2, MOR-1C1, or MOR-1 (considered to be the predominant MOR). Findings revealed that chronic treatment with the clinically relevant opioids morphine, oxycodone and hydrocodone substantially up-regulated membrane MOR-1B2/-1C1 protein. This up-regulation was abolished by the protein synthesis inhibitor cycloheximide, eliminating contributions from receptor redistribution. The increment in MOR-1B2/-1C1 protein was paralleled by a significant increment in opioid agonist-stimulated GTPγS-binding (reflective of increased aggregate MOR G protein coupling) indicating that up-regulated MOR-1B2/-1C1 represented functional receptors. Strikingly, these tolerance-associated adaptations of MOR-1B2/-1C1 differed considerably from those of MOR-1. Antithetical regulation of MOR-1B2/-1C1 and MOR-1 by chronic opioids has significant implications for the design of new therapeutic agents to counteract opioid analgesic tolerance and accompanying addiction. Since chronic opioids induce MOR-1B2/-1C1 up-regulation in spinal cord of males, but not females, elucidating cellular compartments and intracellular pathways mediating MOR-1B2/-1C1 up-regulation and defining their unique signaling attributes would enable a precision medicinal approach to pain management and addiction therapy. In the spinal cord of males, but not females, chronic morphine up-regulates mRNA encoding two mu-opioid receptor (MOR) variants, MOR-1B2 and MOR-1C1 (MOR-1B2/-1C1). We now demonstrate that chronic treatment with the clinically relevant opioids morphine, hydrocodone or oxycodone up-regulates MOR-1B2/-1C1 functional protein, which is dependent on de novo protein synthesis. Findings underscore the importance of unique signaling attributes of MOR variants to sexually dimorphic tolerance mechanisms.

PC-FACS

PC-FACS

Stabilization of morphine tolerance with long-term dosing: Association with selective upregulation of mu-opioid receptor splice variant mRNAs

Chronic morphine administration is associated with the development of tolerance, both clinically and in animal models. Many assume that tolerance is a continually progressive response to chronic opioid dosing. However, clinicians have long appreciated the ability to manage cancer pain in patients for months on stable opioid doses, implying that extended dosing may eventually result in a steady state in which the degree of tolerance remains constant despite the continued administration of a fixed morphine dose. Preclinical animal studies have used short-term paradigms, typically a week or less, whereas the clinical experience is based upon months of treatment. Chronic administration of different fixed morphine doses produced a progressive increase in the ED50 that peaked at 3 wk in mice, consistent with prior results at shorter times. Continued morphine dosing beyond 3 wk revealed stabilization of the level of tolerance for up to 6 wk with no further increase in the ED50. The degree of tolerance at all time points was dependent upon the dose of morphine. The mRNA levels for the various mu opioid receptor splice variants were assessed to determine whether stabilization of morphine tolerance was associated with changes in their levels. After 6 wk of treatment, mRNA levels of the variants increased as much as 300-fold for selected variants in specific brain regions. These findings reconcile preclinical and clinical observations regarding the development of morphine tolerance.

Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine.

The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction.

Buprenorphine analgesia requires exon 11‐associated mu opioid receptor splice variants (659.11)

Buprenorphine is a potent analgesic with an attractive side effect profile. Although it is considered a partial mu opioid receptor (MOR) agonist and a weak partial agonist or antagonist at kappa opioid receptors, it also competes MOR exon 11‐associated IBNtxA binding with high affinity. We therefore sought to examine the roles of exon 11‐ and exon 1‐associated MOR splice variants in buprenorphine analgesia. Exon 11 knockout animals showed a profound loss of analgesia in the tail flick assay at doses over 300 times the analgesic ED50 in wildtype C57 controls, despite the fact that these animals have intact full length MOR‐1 receptors and other opioids such as morphine and methadone retain full analgesic efficacy. Furthermore, administration of a high dose of buprenorphine actually reversed morphine analgesia in these animals. In exon 1 or triple opioid knockout (MOR exon 1, DOR, and KOR) animals, analgesia was also significantly reduced relative to wildtype controls, suggesting that full buprenorphine analgesic efficacy requires both exon 11‐ and exon 1‐associated MOR splice variants, and that the pharmacology of buprenorphine is more complicated than previously believed.Grant Funding Source: Supported by DA02615, DA06241, DA07242 (GWP); DA013997 (YXP); and the PhRMA Foundation (SGG)

Cross‐species and Painless: Targeting truncated mu opioid receptor splice variants in the rat

We recently synthesized a highly potent analgesic, 3′‐Iodobenzoylnaltrexamide (IBNtxA), which exhibited a favorable side effect profile devoid of respiratory depression, dependence, or rewarding behavior in a conditioned place preference assay. Analgesia was retained in a triple knockout mouse lacking traditional mu, delta, and kappa opioid receptors but retaining exon 11‐associated splice variants of MOR‐1; this analgesia was lost in an MOR‐1 exon‐11 knockout animal, implicating these variants in the drug's actions. We sought to determine whether IBNtxA exhibits similar pharmacology in the rat. IBNtxA retained high analgesic potency, and did not show any signs of respiratory depression. Saturation and competition studies in rat brain using 125IBNtxA demonstrated a site possessing a similar pharmacological profile to the site observed in mouse brain. We conclude that this target is not unique to the mouse, and that given the highly conserved splicing between the rat and human MOR‐1 genes, it is likely to have significant clinical relevance. This work was supported by a fellowship from the PhRMA Foundation to SG and grants from the National Institute on Drug Abuse (DA06241, DA02165, and DA07242) to GP.

Expression of mu opioid receptor splice variants mRNA in human blood lymphocytes: A peripheral marker for opioid addiction studies

The expression of the human mu opioid receptor splice variants (hMOR-1A, hMOR-1O, hMOR-1X, and hMOR-1Y) mRNA in peripheral blood lymphocytes (PBLs) was explored in opioid addicted, methadone maintained, long-term abstinent, and control subjects. Real-time PCR showed that hMOR-1X and hMOR-1Y are not expressed in PBLs. The hMOR-1A mRNA was 0.33 fold down-regulated in abstinent and 1.94 fold up-regulated in methadone maintained subjects. The hMOR-1O mRNA was down-regulated 0.39 and 0.53 fold in abstinent and methadone maintained groups, respectively. Expression of both variants in addicted group was not different from controls. It is concluded that expression of hMOR-1A and hMOR-1O variants measured by a suggested peripheral marker can serve to identify people at risk for opioid addiction and also to evaluate the successfulness of methadone therapy.

Coexpression of the mu‐opioid receptor splice variant MOR1C and the vesicular glutamate transporter 2 (VGLUT2) in rat central nervous system

It has been reported that mu-opioid agonists depress glutamate release in some neurons but the specific receptor subtype mediating this effect is unclear. The purpose of the present study was to examine whether a particular mu-opioid receptor (MOR) splice-variant, MOR(1C), is expressed in rat central nervous system (CNS) by terminals expressing the vesicular glutamate transporter2 (VGLUT2), a marker of glutamatergic neurons. Several MOR splice variants have been identified in mice and MOR(1C) appears mainly to be localized to fibers and terminals, from which most neurotransmitter release would be expected. In addition, VGLUT2 has been found in the CNS and antibodies to it are reliable markers for glutamatergic terminals. Using fluorescence immunohistochemistry and confocal microscopy to examine spatial relationships between MOR(1C) and VGLUT2, we found that MOR(1C) and VGLUT2 puncta were widely distributed throughout the rat CNS; moreover, many regions contained terminals that expressed both. Thus, it appears that coexpression of MOR(1C) and VGLUT2 is common in the rat CNS. We hypothesize that activation of MOR(1C) by mu-opioid agonists at some glutamatergic terminals may be a mechanism by which glutamate release is inhibited.

Expression of MOR1C-like mu-opioid receptor mRNA in rats.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone a cDNA fragment of a putative G-protein-coupled receptor from rat brain total RNA. Nucleotide sequencing of this cDNA fragment showed it to be homologous to that of the mu-opioid receptor splice variant MOR(1C) from mice. We used the cDNA to make an RNA probe for a ribonuclease protection assay (RPA). The results from the RPA showed a protected fragment of the size expected for MOR(1C) mRNA, as well as other RNase-protected fragments that may indicate the existence of other MOR1 transcripts. We then used the RNA probe for in situ hybridization (ISH) experiments. We detected strong autoradiographic labeling over much of the rat telencephalon, diencephalon, mesencephalon, cerebellum, spinal cord, and dorsal root ganglia. These findings suggest that MOR(1C), and possibly other MOR1 splice variants, are important components of the system by which the actions of opioids are transduced.

Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1 O and hMOR-1X

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene ( Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1 O and hMOR-1X. The full-length cDNAs of hMOR-1 O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5 kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.

Anatomical and functional correlation of the endomorphins with mu opioid receptor splice variants.

The present study characterizes the relationship between the endogenous mu opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and several splice variants of the cloned mu opioid receptor (MOR-1) encoded by the mu opioid receptor gene (Oprm). Confocal laser microscopy revealed that fibers containing EM-2-like immunoreactivity (-LI) were distributed in close apposition to fibers showing MOR-1-LI (exon 4-LI) and to MOR-1C-LI (exons 7/8/9-LI) in the superficial laminae of the lumbar spinal cord. We also observed colocalization of EM-2-LI and MOR-1-LI in a few fibers of lamina II, and colocalization of EM-2-LI and MOR-1C-LI in laminae I-II, and V-VI. To assess the functional relevance of the MOR-1 variants in endomorphin analgesia, we examined the effects of antisense treatments that targeted individual exons within the Oprm1 gene on EM-1 and EM-2 analgesia in the tail flick test. This antisense mapping study implied mu opioid receptor mechanisms for the endomorphins are distinct from those of morphine or morphine-6beta-glucuronide (M6G).