The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+](i) and adenylyl cyclase

D. Smart, R. A. Hirst, K. Hirota, David Grandy, D. G. Lambert

    Research output: Contribution to journalArticle

    40 Citations (Scopus)

    Abstract

    1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+](i) and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+](i) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+](i) was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the B(max) varied between passages. Fentanyl (10 pM-1 μM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 ± 0.1), and was best fit to a two site model, with pK(i) values (% of sites) of 9.97 ± 0.4 (27 ± 4.8%) and 7.68 ± 0.07 (73 ± 4.8%). In the presence of GppNHp (100 μM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 ± 0.1) with a pK(i) value of 6.76 ± 0.04. 4. Fentanyl (0.1 nM-1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC50 = 7.42 ± 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K(i) = 1.7 nM). Morphine (1 μM) and [D-Ala2, MePhe4, gly(ol)5]enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 μM) did not. 5. Fentanyl (0.1 nM-10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 ± 0.15 (n = 5). PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2+ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μM) also caused an increase in [Ca2+](i) (80 ± 16.4 nM, n = 6), reaching a maximum at 26.8 ± 2.5 s. The increase in [Ca2+](i) remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca2+](i). 6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+](i). These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

    Original languageEnglish (US)
    Pages (from-to)1165-1171
    Number of pages7
    JournalBritish Journal of Pharmacology
    Volume120
    Issue number6
    DOIs
    StatePublished - 1997

    Fingerprint

    Opioid Receptors
    Type C Phospholipases
    Fentanyl
    Cricetulus
    Adenylyl Cyclases
    D-Penicillamine (2,5)-Enkephalin
    Naloxone
    Cyclic AMP
    Ala(2)-MePhe(4)-Gly(5)-enkephalin
    Second Messenger Systems
    Colforsin
    NAD
    Morphine
    Diprenorphine
    Radioligand Assay
    Inositol 1,4,5-Trisphosphate
    Fura-2
    Enkephalins
    Pertussis Toxin
    GTP-Binding Proteins

    Keywords

    • μ-opioid receptor
    • [Ca](i)
    • Adenylyl cyclase
    • Cyclic AMP
    • Inositol(1,4,5)trisphosphate
    • Phospholipase C
    • Transfected CHO cells

    ASJC Scopus subject areas

    • Pharmacology

    Cite this

    The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+](i) and adenylyl cyclase. / Smart, D.; Hirst, R. A.; Hirota, K.; Grandy, David; Lambert, D. G.

    In: British Journal of Pharmacology, Vol. 120, No. 6, 1997, p. 1165-1171.

    Research output: Contribution to journalArticle

    Smart, D. ; Hirst, R. A. ; Hirota, K. ; Grandy, David ; Lambert, D. G. / The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+](i) and adenylyl cyclase. In: British Journal of Pharmacology. 1997 ; Vol. 120, No. 6. pp. 1165-1171.
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    abstract = "1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+](i) and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+](i) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+](i) was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the B(max) varied between passages. Fentanyl (10 pM-1 μM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 ± 0.1), and was best fit to a two site model, with pK(i) values ({\%} of sites) of 9.97 ± 0.4 (27 ± 4.8{\%}) and 7.68 ± 0.07 (73 ± 4.8{\%}). In the presence of GppNHp (100 μM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 ± 0.1) with a pK(i) value of 6.76 ± 0.04. 4. Fentanyl (0.1 nM-1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC50 = 7.42 ± 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K(i) = 1.7 nM). Morphine (1 μM) and [D-Ala2, MePhe4, gly(ol)5]enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 μM) did not. 5. Fentanyl (0.1 nM-10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 ± 0.15 (n = 5). PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2+ (2.5 mM) inhibited (by 52{\%}), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μM) also caused an increase in [Ca2+](i) (80 ± 16.4 nM, n = 6), reaching a maximum at 26.8 ± 2.5 s. The increase in [Ca2+](i) remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca2+](i). 6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+](i). These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.",
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    TY - JOUR

    T1 - The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+](i) and adenylyl cyclase

    AU - Smart, D.

    AU - Hirst, R. A.

    AU - Hirota, K.

    AU - Grandy, David

    AU - Lambert, D. G.

    PY - 1997

    Y1 - 1997

    N2 - 1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+](i) and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+](i) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+](i) was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the B(max) varied between passages. Fentanyl (10 pM-1 μM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 ± 0.1), and was best fit to a two site model, with pK(i) values (% of sites) of 9.97 ± 0.4 (27 ± 4.8%) and 7.68 ± 0.07 (73 ± 4.8%). In the presence of GppNHp (100 μM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 ± 0.1) with a pK(i) value of 6.76 ± 0.04. 4. Fentanyl (0.1 nM-1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC50 = 7.42 ± 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K(i) = 1.7 nM). Morphine (1 μM) and [D-Ala2, MePhe4, gly(ol)5]enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 μM) did not. 5. Fentanyl (0.1 nM-10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 ± 0.15 (n = 5). PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2+ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μM) also caused an increase in [Ca2+](i) (80 ± 16.4 nM, n = 6), reaching a maximum at 26.8 ± 2.5 s. The increase in [Ca2+](i) remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca2+](i). 6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+](i). These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

    AB - 1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+](i) and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+](i) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+](i) was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the B(max) varied between passages. Fentanyl (10 pM-1 μM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 ± 0.1), and was best fit to a two site model, with pK(i) values (% of sites) of 9.97 ± 0.4 (27 ± 4.8%) and 7.68 ± 0.07 (73 ± 4.8%). In the presence of GppNHp (100 μM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 ± 0.1) with a pK(i) value of 6.76 ± 0.04. 4. Fentanyl (0.1 nM-1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC50 = 7.42 ± 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K(i) = 1.7 nM). Morphine (1 μM) and [D-Ala2, MePhe4, gly(ol)5]enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 μM) did not. 5. Fentanyl (0.1 nM-10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 ± 0.15 (n = 5). PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2+ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μM) also caused an increase in [Ca2+](i) (80 ± 16.4 nM, n = 6), reaching a maximum at 26.8 ± 2.5 s. The increase in [Ca2+](i) remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca2+](i). 6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+](i). These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

    KW - μ-opioid receptor

    KW - [Ca](i)

    KW - Adenylyl cyclase

    KW - Cyclic AMP

    KW - Inositol(1,4,5)trisphosphate

    KW - Phospholipase C

    KW - Transfected CHO cells

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    U2 - 10.1038/sj.bjp.0701012

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