title: The effects of recombinant rat mu-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase.
abstract: 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)P3), [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 [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 Bmax varied between passages. Fentanyl (10 pM 1 microM) 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 pK1 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 microM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 +/- 0.1) with a pK1 value of 6.76 +/- 0.04. 4. Fentanyl (0.1 nM-1 microM) had no effect on basal, but dose-dependently inhibited forskolin (1 microM)-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 (K1 = 1.7 nM). Morphine (1 microM) and [D-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 microM) also inhibited forskolin (1 microM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 microM) did not. 5. Fentanyl (0.1 nM-10 microM) caused a naloxone (1 microM)-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 microM) and DAMGO (1 microM), but not DPDPE (1 microM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 microM) 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 microM). Pre-incubation with naloxone (1 microM, 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.
authors.affiliation: University Department of Anaesthesia, Leicester Royal Infirmary.
authors.forename: D
authors.initials: D
authors.lastname: Smart
authors.forename: R A
authors.initials: RA
authors.lastname: Hirst
authors.forename: K
authors.initials: K
authors.lastname: Hirota
authors.forename: D K
authors.initials: DK
authors.lastname: Grandy
authors.forename: D G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004174:Diprenorphine / Q000378:metabolism / Q000494:pharmacology; D005283:Fentanyl / Q000378:metabolism / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D051381:Rats; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D017450:Receptors, Opioid, mu; D004174:Diprenorphine; D005576:Colforsin; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases; D002118:Calcium; D005283:Fentanyl