Selective loss of δ opioid analgesia and binding by antisense oligodeoxynucleotides to a δ opioid receptor

Standifer, Kelly M.; Chien, Chih Cheng; Wahlested, Claes; Brown, George P.; Pasternak, Gavril W.

doi:10.1016/0896-6273(94)90333-6

Cited by 185 publications

(64 citation statements)

References 18 publications

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“…S2). The importance of the 6TM variants in kappa opioid analgesia was confirmed in an antisense paradigm (15,16). Antisense approaches offer the ability to down-regulate expression levels in adults without impacting development and potential compensatory systems.…”

Section: Significancementioning

confidence: 94%

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS

Marrone

Grinnell

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3′-iodobenzoyl-6β-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α 2 -adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50,488H, or α 2 -mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa 1 , and α 2 actions from analgesia.GPCR | truncation | analgesia | mu opioid receptor | morphine M odulation of pain is complex, with contributions from a variety of neurotransmitter systems. The opioid systems are particularly prominent clinically because of the availability of many potent and effective drugs, particularly those acting through mu opioid receptors. The mu opioid receptor gene, Oprm1, is complex, containing two independent promoters that generate dozens of variants through both 5′ and 3′ alternative splicing patterns that are highly conserved among multiple species (SI Appendix, Fig. S1) (1). Like the first mu opioid receptor (Oprm1) clone, MOR-1, most of the variants are traditional seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs) generated from the exon 1 (E1) promoter. Each contains exons 1, 2, and 3, differing only at the intracellular C terminus due to 3′ splicing. The exon 11 (E11) promoter, located ∼30 kb upstream of exon 1, generates a set of truncated proteins lacking the first transmembrane domain because of the absence of exon 1, resulting in only six transmembrane domains (6TM).Classically, opioid mechanisms were defined pharmacologically by using selective agonists and antagonists, but genetic approaches offer a more precise approach toward assessing the pharmacological contributions of the various Oprm1 splice variants. Mice have been developed that have the selective loss of only 6TM variants (E11 KO) (2), only the exon 1-containing 7TM and 1TM variants (E1 KO) (3) or the loss of all Oprm1 variants (E1/E11 KO) (4) (SI Appendix, Fig. S1). These models provide insights into the differing pharmacology of the full-length 7TM and the truncated 6TM variants. Full-length 7TM variants are essential for morphine actions (3, 5-7). However, m...

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Section: Significancementioning

confidence: 94%

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS

Marrone

Grinnell

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The fact that the response is reversible assures one that the effect is not due to nonspecific antisense toxicity and provides a degree of specificity which is lacking in "knockout" or "transgenic" experiments unless complemented by subsequent breeding studies to reacquire the original phenotype. There are now several examples in which antisense ODNs have been successfully used in vivo to interfere with specific protein synthesis and its physiologic function (17)(18)(19)(20)(21).…”

Section: Discussionmentioning

confidence: 99%

Antisense technology reveals the alpha2A adrenoceptor to be the subtype mediating the hypnotic response to the highly selective agonist, dexmedetomidine, in the locus coeruleus of the rat.

Mizobe

Maghsoudi²,

Sitwala³

et al. 1996

J. Clin. Invest.

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Abstract␣ 2 adrenergic agonists are used in the anesthetic management of the surgical patient for their sedative/hypnotic properties although the ␣ 2 adrenoceptor subtype responsible for these anesthetic effects is not known. Using a gene-targeting strategy, it is possible to specifically reduce the expression of the individual adrenoceptors expressed in the central nervous system and to thereby determine their role in hypnotic action.Stably transfected cell lines (PC 124D for rat ␣ 2A ; NIH3T3 for rat ␣ 2C adrenoceptors) were exposed to 5 M antisense oligodeoxynucleotides (ODNs) for ␣ 2A and ␣ 2C adrenergic receptor subtypes for 3 d. Individual receptor subtype expression, as determined by radiolabeled ligand binding, was selectively decreased only by the appropriate antisense ODNs and not by the "scrambled" ODNs. These antisense ODNs were then administered three times, on alternate days, into the locus coeruleus of chronically cannulated rats and their hypnotic response to dexmedetomidine (an ␣ 2 agonist) was determined.Only the ␣ 2A antisense ODNs significantly change the hypnotic response causing both an increase in latency to, and a decrease in duration of, the loss of righting reflex following dexmedetomidine; hypnotic response had normalized 8 d after stopping the ODNs. Therefore, the ␣ 2A adrenoceptor subtype is responsible for the hypnotic response to dexmedetomidine in the locus coeruleus of the rat. ( J. Clin. Invest. 1996. 98:1076-1080.)

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“…Conversely, the antinociception produced by intracerebroventricular injection of DPDPE 1 (␦ agonist) was antagonized by ICI-174864 but not by ␤-funaltrexamine and naloxonazine. Moreover, studies have shown that an antisense oligodeoxynucleotide to the cloned ␦-opioid receptor given intrathecally lowers ␦ but not or spinal (20) and central (21) analgesia. These studies confirm, at the molecular level, traditional pharmacological studies implying distinct receptor mechanisms for ␦, , and analgesia.…”

mentioning

confidence: 99%

Novel “Restoration of Function” Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding

Pepin

Yue

Roberts

et al. 1997

Journal of Biological Chemistry

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A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind ␦-selective ligands to an inactive ␦/ receptor chimera in which 10 amino acids of the third extracellular loop of the ␦ receptor were replaced by the corresponding amino acids from the receptor (␦/ 291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for ␦-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the sequence of ␦/ 291-300 were randomly reverted to the corresponding ␦ amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for ␦-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human ␦-opioid receptor were present in all revertants. Single and double point mutations were then introduced in ␦/ 291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of ␦-selective ligands. An increased affinity for ␦-selective ligands was observed when the tryptophan 300 ( residue) of ␦/ 291-300 was reverted to a leucine (␦ residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of ␦-selective ligands to their docking site.The opioid receptors are widely distributed throughout the central nervous system and mediate the diverse effects of endogenous opioid peptides and opiate drugs (1). Pharmacological studies have defined at least three classes of opioid receptors, named ␦, , and , that differ in their affinity for ligands and in their distribution in the nervous system (1-6).The antinociception mediated by supraspinal opioid receptors is thought to act via the -opioid receptor subtype (7-10). The numerous side effects accompanying opioid treatment, including respiratory depression and addiction (11), are generally thought to be mediated by the stimulation of receptors. However, there is growing evidence suggesting that selective stimulation of the ␦ receptor may also mediate antinociception (12-19). The strongest indication of an involvement of ␦-opioid receptors in supraspinal antinociception follows from studies with selective antagonists (14 -16). These studies demonstrated that the antinociception produced by intracerebroventricular injection of morphine and [D-Ala 2 ,MePhe 4 ,Gly-ol 5 ]-enkephalin ( agonists) was antagonized by ␤-funaltrexamine and naloxonazine ( antagonists) but not by ICI-174864 (␦ antagonists). Conversely, the antinociception produced by intracerebroventricular injection of DPDPE 1 (␦ agonist) was antagonized by ICI-174864 but not by ␤-funaltrexamine and naloxonazine. Moreover, studies have shown that an antisense oligodeoxynucleotide to the cloned ␦-opioid receptor given intrathecally lowers ␦ but not or spinal (20) and central (21) analgesia. These studies confirm, at the m...

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Selective loss of δ opioid analgesia and binding by antisense oligodeoxynucleotides to a δ opioid receptor

Cited by 185 publications

References 18 publications

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS

Antisense technology reveals the alpha2A adrenoceptor to be the subtype mediating the hypnotic response to the highly selective agonist, dexmedetomidine, in the locus coeruleus of the rat.

Novel “Restoration of Function” Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding

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