The effects of recombinant rat μ‐opioid receptor activation in CHO cells on phospholipase C, [Ca 2+ ] i and adenylyl cyclase

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 [Ca 2+ ] 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)P 3 ), [Ca 2+ ] 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 [ 3 H]‐diprenorphine ([ 3 H]‐DPN) as a radiolabel. Ins(1,4,5)P 3 and cyclic AMP were measured by specific radioreceptor assays. [Ca 2+ ] i was measured fluorimetrically with Fura‐2. Scatchard analysis of [ 3 H]‐DPN binding revealed that the B max varied between passages. Fentanyl (10 p m –1 μ m ) dose‐dependently displaced [ 3 H]‐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 p K 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 μ m ) and Na + (100 m m ), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a p K i value of 6.76±0.04. Fentanyl (0.1 n m –1 μ m ) had no effect on basal, but dose‐dependently inhibited forskolin (1 μ 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 n m ). Morphine (1 μ m ) and [ d ‐Ala 2 , MePhe 4 , gly(ol) 5 ]‐enkephalin (DAMGO, 1 μ m ) also inhibited forskolin (1 μ m )‐stimulated cyclic AMP formation, whilst [ d ‐Pen 2 , d ‐Pen 5 ], enkephalin (DPDPE, 1 μ m ) did not. Fentanyl (0.1 n m –10 μ m ) caused a naloxone (1 μ 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 Ni 2+ (2.5 m m ) inhibited (by 52%), the fentanyl‐induced Ins(1,4,5)P 3 response. Morphine (1 μ m ) and DAMGO (1 μ m ), but not DPDPE (1 μ m ), also stimulated Ins(1,4,5)P 3 formation. Fentanyl (1 μ m ) also caused an increase in [Ca 2+ ] i (80±16.4 n m , n =6), reaching a maximum at 26.8±2.5 s. The increase in [Ca 2+ ] i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μ m ). Pre‐incubation with naloxone (1 μ m , 3 min) completely abolished fentanyl‐induced increases in [Ca 2+ ] 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 [Ca 2+ ] i . These findings are consistent with the previously described effector‐second messenger coupling of the endogenous μ‐opioid receptor.British Journal of Pharmacology (1997) 120 , 1165–1171; doi: 10.1038/sj.bjp.0701012