The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant μ-opioid receptors and SH-SY5Y cells

C. Harrison, S. McNulty, D. Smart, D. J. Rowbotham, David Grandy, L. A. Devi, D. G. Lambert

    Research output: Contribution to journalArticle

    31 Citations (Scopus)

    Abstract

    1. Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the μ-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+](i) levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant μ-opioid receptors. 2. E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHOμ and SH-SY5Y membranes with pK(i) values of 8.02 ± 0.09 and 8.54 ± 0.13 respectively. E-2 displaced [3H]-DPN binding in CHOμ and SH-SY5Y cells with pK(i) values of 7.82 ± 0.11 and 8.43 ± 0.13 respectively. E-1/E-2 bound weakly to CHOδ and CHOκ membranes, with IC50 values of greater than 10 μM. 3. In CHOμ cells, E-1/E-2 inhibited forskolin (1 μM) stimulated cyclic AMP formation with pIC50 values of 8.03 ± 0.16 (I(max) = 53.0 ± 9.3%) and 8.15 ± 0.24 (I(max) = 56.3 ± 3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72 ± 0.13 (I(max) = 46.9 ± 5.6%) and 8.11 ± 0.31 (I(max) = 40.2 ± 2.8%) respectively. 4. E-1/E-2 (1 μM) increased [Ca2+](i) in fura-2 loaded CHOμ cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106 ± 28 and 69 ± 6.7 nM respectively. In single adherent cells E-1/E-2 (1 μM) increased [Ca2+](i) with a mean 340/380 ratio change of 0.81 ± 0.09 and 0.40 ± 0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOδ, CHOκ and SH-SY5Y cells. 5. These data show that E-1/E-2 bind with high affinity and selectivity to μ-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the μ-opioid receptor.

    Original languageEnglish (US)
    Pages (from-to)472-478
    Number of pages7
    JournalBritish Journal of Pharmacology
    Volume128
    Issue number2
    DOIs
    StatePublished - 1999

    Fingerprint

    Opioid Receptors
    Cricetulus
    Ovary
    Cyclic AMP
    Colforsin
    Diprenorphine
    Ligands
    endomorphin 2
    endomorphin 1
    Thapsigargin
    Membranes
    Fura-2
    Naloxone
    NAD
    Opioid Analgesics
    Inhibitory Concentration 50
    Signal Transduction
    Suspensions
    Calcium
    Peptides

    Keywords

    • Cyclic AMP
    • Endomorphin
    • Intracellular calcium
    • Opioid receptor

    ASJC Scopus subject areas

    • Pharmacology

    Cite this

    Harrison, C., McNulty, S., Smart, D., Rowbotham, D. J., Grandy, D., Devi, L. A., & Lambert, D. G. (1999). The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant μ-opioid receptors and SH-SY5Y cells. British Journal of Pharmacology, 128(2), 472-478. https://doi.org/10.1038/sj.bjp.0702798

    The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant μ-opioid receptors and SH-SY5Y cells. / Harrison, C.; McNulty, S.; Smart, D.; Rowbotham, D. J.; Grandy, David; Devi, L. A.; Lambert, D. G.

    In: British Journal of Pharmacology, Vol. 128, No. 2, 1999, p. 472-478.

    Research output: Contribution to journalArticle

    Harrison, C, McNulty, S, Smart, D, Rowbotham, DJ, Grandy, D, Devi, LA & Lambert, DG 1999, 'The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant μ-opioid receptors and SH-SY5Y cells', British Journal of Pharmacology, vol. 128, no. 2, pp. 472-478. https://doi.org/10.1038/sj.bjp.0702798
    Harrison, C. ; McNulty, S. ; Smart, D. ; Rowbotham, D. J. ; Grandy, David ; Devi, L. A. ; Lambert, D. G. / The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant μ-opioid receptors and SH-SY5Y cells. In: British Journal of Pharmacology. 1999 ; Vol. 128, No. 2. pp. 472-478.
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    AU - McNulty, S.

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    AU - Rowbotham, D. J.

    AU - Grandy, David

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    N2 - 1. Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the μ-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+](i) levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant μ-opioid receptors. 2. E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHOμ and SH-SY5Y membranes with pK(i) values of 8.02 ± 0.09 and 8.54 ± 0.13 respectively. E-2 displaced [3H]-DPN binding in CHOμ and SH-SY5Y cells with pK(i) values of 7.82 ± 0.11 and 8.43 ± 0.13 respectively. E-1/E-2 bound weakly to CHOδ and CHOκ membranes, with IC50 values of greater than 10 μM. 3. In CHOμ cells, E-1/E-2 inhibited forskolin (1 μM) stimulated cyclic AMP formation with pIC50 values of 8.03 ± 0.16 (I(max) = 53.0 ± 9.3%) and 8.15 ± 0.24 (I(max) = 56.3 ± 3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72 ± 0.13 (I(max) = 46.9 ± 5.6%) and 8.11 ± 0.31 (I(max) = 40.2 ± 2.8%) respectively. 4. E-1/E-2 (1 μM) increased [Ca2+](i) in fura-2 loaded CHOμ cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106 ± 28 and 69 ± 6.7 nM respectively. In single adherent cells E-1/E-2 (1 μM) increased [Ca2+](i) with a mean 340/380 ratio change of 0.81 ± 0.09 and 0.40 ± 0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOδ, CHOκ and SH-SY5Y cells. 5. These data show that E-1/E-2 bind with high affinity and selectivity to μ-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the μ-opioid receptor.

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