Abstract
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.
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.
Scatchard analysis of [3H]-DPN binding revealed that the Bmax 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 pKi 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 pKi value of 6.76±0.04.
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 (Ki=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.
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.
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.
Keywords: μ-Opioid receptor; phospholipase C; adenylyl cyclase; transfected CHO cells; [Ca2+]i; inositol(1,4,5)trisphosphate; cyclic AMP
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