Endomorphin-1 induced desensitization and down-regulation of the recombinant μ-opioid receptor

C. Harrison, D. J. Rowbotham, David Grandy, D. G. Lambert

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    1. Endomorphin-1 (E1) is a peptide with high affinity and selectivity for the μ-opioid receptor. The aim of this study was to determine if endomorphin-1 caused desensitization and down-regulation of the μ-opioid receptor expressed in Chinese hamster ovary cells. 2. Following 10 μM E1 pre-treatment, desensitization was assessed by measuring cyclic AMP inhibition, down-regulation was assessed by [ 3H]-diprenorphine ([ 3H]-DPN) binding and immunoblotting. 3. Pre-treatment of CHOμ cells with 10 μM E1 for 11 and 18 h caused significant reduction in cyclic AMP inhibition. (11 h=39.0±16.7%, 18 h 47.0±11.1% reduction). 4. At 18 h E1 pre-treatment there was an enhancement (4.5 fold) of cyclic AMP production under forskolin stimulated conditions accompanied by a small rightward shift in the concentration-response curve (pEC 50 control = 7.8±0.3, pEC 50 E1 = 7.3±0.2) when cells were re-challenged with E1. 5. In membranes prepared from untreated and 0.5 h E1 pre-treated cells, addition of GTPγS produced a significant rightward shift in the concentration response curves for E1 displacement of [ 3H]-DPN (0 h K(i) control=7.86±0.11, GTPγS=7.37±0.15; 0.5 h K(i) control=7.92±0.12, GTPγS=7.36±0.08) This was not observed in membranes prepared from cells that had been treated with E1 for 18 h (18 h K(i) control=7.69±0.11, GTPγS=7.75±0.08). 6. In whole cells E1 treatment caused a rapid loss of cell surface receptors such that at 0.5 h there was a 30.5±1.5 reduction (this was unchanged for 18 h). In crude membranes a loss of receptors was also observed using radioligand binding or immuno-blotting protocols. 7. These data show that E1 causes desensitization and down-regulation of the rat μ-opioid receptor expressed in CHO cells. However, these two responses appear temporally distinct.

    Original languageEnglish (US)
    Pages (from-to)1220-1226
    Number of pages7
    JournalBritish Journal of Pharmacology
    Volume131
    Issue number6
    StatePublished - 2000

    Fingerprint

    Opioid Receptors
    Down-Regulation
    Cyclic AMP
    CHO Cells
    endomorphin 1
    Diprenorphine
    Membranes
    Cell Surface Receptors
    Colforsin
    Cricetulus
    Immunoblotting
    NAD
    Ovary
    Cell Membrane

    Keywords

    • Desensitization
    • Down-regulation
    • Endomorphin
    • Opioid receptor

    ASJC Scopus subject areas

    • Pharmacology

    Cite this

    Harrison, C., Rowbotham, D. J., Grandy, D., & Lambert, D. G. (2000). Endomorphin-1 induced desensitization and down-regulation of the recombinant μ-opioid receptor. British Journal of Pharmacology, 131(6), 1220-1226.

    Endomorphin-1 induced desensitization and down-regulation of the recombinant μ-opioid receptor. / Harrison, C.; Rowbotham, D. J.; Grandy, David; Lambert, D. G.

    In: British Journal of Pharmacology, Vol. 131, No. 6, 2000, p. 1220-1226.

    Research output: Contribution to journalArticle

    Harrison, C, Rowbotham, DJ, Grandy, D & Lambert, DG 2000, 'Endomorphin-1 induced desensitization and down-regulation of the recombinant μ-opioid receptor', British Journal of Pharmacology, vol. 131, no. 6, pp. 1220-1226.
    Harrison, C. ; Rowbotham, D. J. ; Grandy, David ; Lambert, D. G. / Endomorphin-1 induced desensitization and down-regulation of the recombinant μ-opioid receptor. In: British Journal of Pharmacology. 2000 ; Vol. 131, No. 6. pp. 1220-1226.
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    AB - 1. Endomorphin-1 (E1) is a peptide with high affinity and selectivity for the μ-opioid receptor. The aim of this study was to determine if endomorphin-1 caused desensitization and down-regulation of the μ-opioid receptor expressed in Chinese hamster ovary cells. 2. Following 10 μM E1 pre-treatment, desensitization was assessed by measuring cyclic AMP inhibition, down-regulation was assessed by [ 3H]-diprenorphine ([ 3H]-DPN) binding and immunoblotting. 3. Pre-treatment of CHOμ cells with 10 μM E1 for 11 and 18 h caused significant reduction in cyclic AMP inhibition. (11 h=39.0±16.7%, 18 h 47.0±11.1% reduction). 4. At 18 h E1 pre-treatment there was an enhancement (4.5 fold) of cyclic AMP production under forskolin stimulated conditions accompanied by a small rightward shift in the concentration-response curve (pEC 50 control = 7.8±0.3, pEC 50 E1 = 7.3±0.2) when cells were re-challenged with E1. 5. In membranes prepared from untreated and 0.5 h E1 pre-treated cells, addition of GTPγS produced a significant rightward shift in the concentration response curves for E1 displacement of [ 3H]-DPN (0 h K(i) control=7.86±0.11, GTPγS=7.37±0.15; 0.5 h K(i) control=7.92±0.12, GTPγS=7.36±0.08) This was not observed in membranes prepared from cells that had been treated with E1 for 18 h (18 h K(i) control=7.69±0.11, GTPγS=7.75±0.08). 6. In whole cells E1 treatment caused a rapid loss of cell surface receptors such that at 0.5 h there was a 30.5±1.5 reduction (this was unchanged for 18 h). In crude membranes a loss of receptors was also observed using radioligand binding or immuno-blotting protocols. 7. These data show that E1 causes desensitization and down-regulation of the rat μ-opioid receptor expressed in CHO cells. However, these two responses appear temporally distinct.

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    KW - Down-regulation

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