Opioid Receptor Splice Variants Research Articles

Understanding Opioid Actions, Pain and Analgesia: A Tribute to Dr. Gavril Pasternak.

This special issue is a tribute to our mentor, colleague and friend, Gavril W. Pasternak, MD, PhD. Homage to the breadth and depth of his work (~ 450 publications) over a 40 career in pharmacology and medicine cannot be captured fully in one special issue, but the 22 papers collected herein represent seven of the topics near and dear to Gav's heart, and the colleagues, friends and mentees who held him near to theirs. The seven themes include: (1) sites and mechanisms of opioid actions in vivo; (2) development of novel analgesic agents; (3) opioid tolerance, withdrawal and addiction: mechanisms and treatment; (4) opioid receptor splice variants; (5) novel research tools and approaches; (6) receptor signaling and crosstalk in vitro; and (7) mentorship. This introduction to the issue summarizes contributions and includes formal and personal remembrances of Gav that illustrate his personality, warmth, and dedication to making a difference in patient care and people's lives.

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

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

Biased signaling at mu opioid receptor splice variants

Biased signaling at mu opioid receptor splice variants

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.

Pharmacological Characterization of Levorphanol, a G-Protein Biased Opioid Analgesic.

Levorphanol is a potent analgesic that has been used for decades. Most commonly used for acute and cancer pain, it also is effective against neuropathic pain. The recent appreciation of the importance of functional bias and the uncovering of multiple µ opioid receptor splice variants may help explain the variability of patient responses to different opioid drugs. Here, we evaluate levorphanol in a variety of traditional in vitro receptor binding and functional assays. In vivo analgesia studies using the radiant heat tail flick assay explored the receptor selectivity of the responses through the use of knockout (KO) mice, selective antagonists, and viral rescue approaches. Receptor binding studies revealed high levorphanol affinity for all the μ, δ, and κ opioid receptors. In S-GTPγS binding assays, it was a full agonist at most µ receptor subtypes, with the exception of MOR-1O, but displayed little activity in β-arrestin2 recruitment assays, indicating a preference for G-protein transduction mechanisms. A KO mouse and selective antagonists confirmed that levorphanol analgesia was mediated through classical µ receptors, but there was a contribution from 6 transmembrane targets, as illustrated by a lower response in an exon 11 KO mouse and its rescue with a virally transfected 6 transmembrane receptor splice variant. Compared to morphine, levorphanol had less respiratory depression at equianalgesic doses. While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.

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.

Opioidergic effects on enteric and sensory nerves in the lower GI tract: basic mechanisms and clinical implications.

Opioids are one of the most prescribed drug classes for treating acute pain. However, chronic use is often associated with tolerance as well as debilitating side effects, including nausea and dependence, which are mediated by the central nervous system, as well as constipation emerging from effects on the enteric nervous system. These gastrointestinal (GI) side effects limit the usefulness of opioids in treating pain in many patients. Understanding the mechanism(s) of action of opioids on the nervous system that shows clinical benefit as well as those that have unwanted effects is critical for the improvement of opioid drugs. The opioidergic system comprises three classical receptors (μ, δ, κ) and a nonclassical receptor (nociceptin), and each of these receptors is expressed to varying extents by the enteric and intestinal extrinsic sensory afferent nerves. The purpose of this review is to discuss the role that the opioidergic system has on enteric and extrinsic afferent nerves in the lower GI tract in health and diseases of the lower GI tract, particularly inflammatory bowel disease and irritable bowel syndrome, and the implications of opioid treatment on clinical outcomes. Consideration is also given to emerging developments in our understanding of the immune system as a novel source of endogenous opioids and the mechanisms underlying opioid tolerance, including the potential influence of opioid receptor splice variants and heteromeric complexes.

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.

Expression and physiology of opioid receptors in the gastrointestinal tract.

Stimulation of opioid receptors elicits analgesic effect not only in the central nervous system, but also in the gastrointestinal tract, where a high concentration of opioid receptors can be found within the enteric nervous system as well as muscular and immune cells. Along with antinociception, opioid receptors in the stomach and intestine relay signals crucial for secretory and motor gastrointestinal function. The review focuses on the utility of opioid receptor antagonists, which is generally contributing to the management of postoperative ileus and opioid bowel dysfunction in chronic pain patients nonetheless, opioid receptor antagonists can also be useful in the treatment of irritable bowel syndrome and chronic idiopathic constipation. The study also discusses several antidiarrheal opioid agonists, as well as opioids and opioid mimetics encompassing the concept of ligand-biased agonism and truncated opioid receptor splice variants. Good understanding of the localization and the role of opioid receptors is vital for regulation of various pathophysiological processes in the gastrointestinal tract and may simultaneously provide a tempting approach in eliminating adverse effects related to centrally acting opioids.

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.

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.

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).

Presynaptic localization of the carboxy-terminus epitopes of the μ opioid receptor splice variants MOR-1C and MOR-1D in the superficial laminae of the rat spinal cord

Opioids inhibit nociceptive transmission at the level of the spinal cord, possibly through inhibition of neurotransmitter release by presynaptic μ opioid receptors (MORs) thus preventing the activation of ascending pathways and the perception of pain. Most nociceptive primary afferents are unmyelinated fibers containing peptides such as substance P and/or calcitonin gene-related peptide. However, few terminals contain both substance P and MOR. Recently, we identified new carboxy-terminal MOR splice variants that are localized in the superficial laminae of the dorsal horn. We now report the precise cellular distribution of two of these MOR-1 variants, MOR-1C (exon 7/8/9 epitope) and MOR-1D (exon 8/9 epitope), at the ultrastructural level. In the superficial laminae of the dorsal horn, the majority of the labeling of MOR-1C and MOR-1D was found in unmyelinated axons. This distribution contrasts with that of MOR-1 (exon 4 epitope), in which labeling is equally found in dendrites and soma, as well as in axons. The presence of dense core vesicles in many of the MOR-1C-like immunoreactive terminals implies that this splice variant might be involved in presynaptic inhibition of transmitter release from peptide-containing afferents to the dorsal horn. Consistent with this finding, confocal microscopy analyses showed that many MOR-1C profiles in laminae I–II also contained calcitonin gene-related peptide, whereas fewer MOR-1 profiles contained either substance P or calcitonin gene-related peptide in this same region. From these findings we suggest that there are differential distributions of MOR-1 splice variants as well as distinct peptide colocalizations in the dorsal horn.

Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1F

The MOR-1 gene is large, with a recent study reporting nine exons spanning 250 kb which combine to yield six different mu opioid receptor splice variants. We now report the isolation of exon 10, which is contained within yet another splice variant, MOR-1F, which is composed of exons 1, 2, 3, 10, 6, 7, 8 and 9. Exon 10 comprises 186 bp which predict a unique 58 amino acid sequence extending beyond exon 3. It has been mapped between exons 4 and 6 and has flanking consensus splice sequences. On Northern blot analysis, the MOR-1F mRNA is smaller than the other MOR-1 mRNAs. When expressed in CHO cells, MOR-1F binds the mu opioid radioligand [ 3H]DAMGO with high affinity ( K D=1.04±0.03 nM). Competition studies demonstrated the selectivity of the variant for mu opioid ligands, supporting its classification within the mu opioid receptor family.