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, D. K. Grandy, D. G. Lambert

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42 Scopus citations


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
Issue number6
StatePublished - 1997
Externally publishedYes


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

ASJC Scopus subject areas

  • Pharmacology


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