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

1 The rat mu-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous mu-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 mu-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P-3), [Ca2+](i) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned mu-opioid receptor. 2 Opioid receptor binding was assessed with [H-3]-diprenorphine ([H-3]-DPN) as a radiolabel. Ins(1,4,5)P-3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+](i) was measured fluorimetrically with Fura-2. 3 Scatchard analysis of [H-3]-DPN binding revealed that the B-max varied between passages. Fentanyl (10 pM-1 mu M) dose-dependently displaced [H-3]-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 mu M) 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 mu M) had no effect on basal, but dose-dependently inhibited forskolin (1 mu M)-stimulated, cyclic AMP formation (pIC(50)=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 mu M) and [D-Ala(2), MePhe(4), gly(ol)(5)]-enkephalin (DAMGO, 1 mu M) also inhibited forskolin (1 mu M)-stimulated cyclic AMP formation, whilst [D-Pen(2), D-Pen(5)], enkephalin (DPDPE, 1 mu M) did not. 5 Fentanyl (0.1 nM-10 mu M) caused a naloxone (1 mu M)-reversible, dose-dependent stimulation of Ins(1,4,5)P-3 formation, with a pEC(50) 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)P-3 response. Morphine (1 mu M) and DAMGO (1 mu M), but not DPDPE (1 mu M), also stimulated Ins(1,4,5)P-3 formation. Fentanyl (1 mu M) 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 mu M). Pre-incubation with naloxone (1 mu M, 3 min) completely abolished fentanyl-induced increases in [Ca2+](i). 6 In conclusion, the cloned mu-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 mu-opioid receptor.