Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons

Christopher P Bailey, S Oldfield, J Llorente, Christopher J Caunt, AG Teschemacher, L Roberts, CA McArdle, FL Smith, WL Dewey, E Kelly, G Henderson

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Abstract

BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.
LanguageEnglish
Pages157-164
Number of pages8
JournalBritish Journal of Pharmacology
Volume158
Issue number1
Early online date20 Mar 2009
DOIs
StatusPublished - Sep 2009

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G-Protein-Coupled Receptor Kinase 2
mu Opioid Receptor
Opioid Receptors
Morphine
Ala(2)-MePhe(4)-Gly(5)-enkephalin
Neurons
Protein Kinase C
Brain
G-Protein-Coupled Receptor Kinases
Locus Coeruleus
Knockout Mice
Protein Isoforms
Viral Genes
Electrophysiology

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Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons. / Bailey, Christopher P; Oldfield, S; Llorente, J; Caunt, Christopher J; Teschemacher, AG; Roberts, L; McArdle, CA; Smith, FL; Dewey, WL; Kelly, E; Henderson, G.

In: British Journal of Pharmacology, Vol. 158, No. 1, 09.2009, p. 157-164.

Research output: Contribution to journalArticle

Bailey, CP, Oldfield, S, Llorente, J, Caunt, CJ, Teschemacher, AG, Roberts, L, McArdle, CA, Smith, FL, Dewey, WL, Kelly, E & Henderson, G 2009, 'Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons' British Journal of Pharmacology, vol. 158, no. 1, pp. 157-164. DOI: 10.1111/j.1476-5381.2009.00140.x
Bailey, Christopher P ; Oldfield, S ; Llorente, J ; Caunt, Christopher J ; Teschemacher, AG ; Roberts, L ; McArdle, CA ; Smith, FL ; Dewey, WL ; Kelly, E ; Henderson, G. / Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons. In: British Journal of Pharmacology. 2009 ; Vol. 158, No. 1. pp. 157-164
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abstract = "BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.",
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T1 - Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons

AU - Bailey,Christopher P

AU - Oldfield,S

AU - Llorente,J

AU - Caunt,Christopher J

AU - Teschemacher,AG

AU - Roberts,L

AU - McArdle,CA

AU - Smith,FL

AU - Dewey,WL

AU - Kelly,E

AU - Henderson,G

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N2 - BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

AB - BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

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