Antisense mapping the MOR-1 opioid receptor: Evidence for alternative splicing and a novel morphine-6β-glucuronide receptor

Cornell University, Итак, New York, United States
FEBS Letters (Impact Factor: 3.17). 09/1995; 369(2-3):192-6. DOI: 10.1016/0014-5793(95)00757-Z
Source: PubMed

ABSTRACT Although MOR-1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes result from alternative splicing of MOR-1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine-6 beta-glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR-1 while morphine-6 beta-glucuronide acts through a novel, previously unidentified receptor which is yet another MOR-1 splice variant.

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Available from: Grace Rossi, Dec 20, 2013
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    • "The greater potency of M6G has been attributed to greater efficacy in activating the MOR (Osborne et al. 2000; Ulens et al. 2001) or to its actions at a unique MOR subtype. The existence of a MOR-1 subtype with greater affinity for M6G than for morphine was first proposed by Rossi et al. (1995a) and was later confirmed by others (Brown et al. 1997; Mantione et al. 2002). Experiments using antisense probes or knockout mice have demonstrated the existence of splice variants of MOR-1 with differential affinity for morphine versus heroin and M6G (Rossi et al. 1995b; Matthes et al. 1996; Sora et al. 1997; Loh et al. 1998; Schuller et al. 1999; Unterwald et al. 1999; Pan et al. 2009). "
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    ABSTRACT: Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronide (M6G), a potent MOR agonist. We have found that rats that had received repeated intraperitoneal injections of heroin exhibit measurable levels of M6G (which is usually undetectable in this species). The goal of the present study was to investigate whether M6G synthesis can be induced by intravenous (i.v.) heroin self-administration (SA). Rats were trained to self-administer either heroin (50 μg/kg per infusion) or saline for 20 consecutive 6-h sessions and then challenged with an intraperitoneal challenge of 10 mg/kg of heroin. Plasma levels of heroin, morphine, 6-mono-acetyl morphine, M3G, and M6G were quantified 2 h after the challenge. In vitro morphine glucuronidation was studied in microsomal preparations obtained from the liver of the same rats. Heroin SA induced the synthesis of M6G, as indicated by detectable plasma levels of M6G (89.7 ± 37.0 ng/ml vs. 7.35 ± 7.35 ng/ml after saline SA). Most important, the in vitro V (max) for M6G synthesis was correlated with plasma levels of M6G (r (2) = 0.78). Microsomal preparations from saline SA rats produced negligible amounts of M6G. Both in vivo and in vitro data indicate that i.v. heroin SA induces the synthesis of M6G. These data are discussed in the light of previous studies conducted in heroin addicts indicating that in humans heroin enhances the synthesis of the active metabolite of heroin and morphine.
    Psychopharmacology 10/2011; 221(2):195-203. DOI:10.1007/s00213-011-2534-7 · 3.88 Impact Factor
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    • "However, to date only a single mu opioid receptor gene has been identified, raising the questions of how a single OPRM1 gene could explain the complex pharmacology of mu opioids in animals and humans. Our early antisense mapping studies suggested different exon combinations for the analgesic actions of the two mu agonists morphine and M6G, raising the possibility of alternative splicing in the OPRM1 gene [17,46]. Since then, much effort has been devoted to identifying these OPRM1variants. "
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    ABSTRACT: The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat. The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding. The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various μ opioids seen in rats.
    Molecular Pain 01/2011; 7(1):9. DOI:10.1186/1744-8069-7-9 · 3.65 Impact Factor
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    • "A mismatched antisense was used as control (Table 1). All antisense olygodeoxynucleotides were administered i.c.v. in dose of 10 μg in 10 μl volume saline [50,52,53]. Treatment with antisenses was performed on day 1, 3 and 5 and the behavioral test was performed at day 6 [51] (Additional file 3). "
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    ABSTRACT: Hydrogen sulphide (H2S) is a gaseous neuro-mediator that exerts analgesic effects in rodent models of visceral pain by activating KATP channels. A body of evidence support the notion that KATP channels interact with endogenous opioids. Whether H2S-induced analgesia involves opioid receptors is unknown. The perception of painful sensation induced by colorectal distension (CRD) in conscious rats was measured by assessing the abdominal withdrawal reflex. The contribution of opioid receptors to H2S-induced analgesia was investigated by administering rats with selective mu, kappa and delta opioid receptor antagonists and antisenses. To investigate whether H2S causes mu opioid receptor (MOR) transactivation, the neuronal like cells SKNMCs were challenged with H2S in the presence of MOR agonist (DAMGO) or antagonist (CTAP). MOR activation and phosphorylation, its association to beta arrestin and internalization were measured. H2S exerted a potent analgesic effects on CRD-induced pain. H2S-induced analgesia required the activation of the opioid system. By pharmacological and molecular analyses, a robust inhibition of H2S-induced analgesia was observed in response to central administration of CTAP and MOR antisense, while kappa and delta receptors were less involved. H2S caused MOR transactivation and internalization in SKNMCs by a mechanism that required AKT phosphorylation. MOR transactivation was inhibited by LY294002, a PI3K inhibitor, and glibenclamide, a KATP channels blocker. This study provides pharmacological and molecular evidence that antinociception exerted by H2S in a rodent model of visceral pain is modulated by the transactivation of MOR. This observation provides support for development of new pharmacological approaches to visceral pain.
    Molecular Pain 06/2010; 6(1):36. DOI:10.1186/1744-8069-6-36 · 3.65 Impact Factor
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