title: Desensitization of the mu-opioid activation of phospholipase C in SH-SY5Y cells: the role of protein kinases C and A and Ca(2+)-activated K+ currents.
abstract: 1. In SH-SY5Y cells, mu-opioids cause a rapidly desensitizing activation of phospholipase C (PLC), that appears secondary to Ca2+ influx via L-type voltage-sensitive Ca2+ channels (VSCCs). The aim of the present study was to characterize the mechanisms of desensitization of the mu-opioid-induced inositol (1,4,5) triphosphate (Ins(1,4,5)P3) response, by use of a stereospecific radioreceptor mass assay. 2. (R+)-Bay K 8644 (1 nM-10 microM) dose-dependently inhibited fentanyl-induced Ins(1,4,5)P3 formation, with an IC50 of 28.5 nM, confirming our earlier observations that mu-opioids open L-type VSCCs, thus allowing Ca2+ influx to activate PLC. 3. Ro 31-8220 (0.1 nM-10 microM), a protein kinase C inhibitor, dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 20.0 nM), whilst acute phorbol 12,13-dibutrate (1 microM) abolished the response. 4. H-89 (1 nM-10 microM), a protein kinase A inhibitor, also dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 93 nM), whilst dibutryl cyclic AMP (0.5 mM) abolished the response. 5. Blockade of Ca(2+)-activated K+ currents with 4-aminopyridine (2 mM) or iberiotoxin (10 nM) had no effect on fentanyl-induced Ins(1,4,5)P3 formation but further increased the Ro 31-8220-enhanced response. 6. All three mechanisms had additive, or even supra-additive, effects, but only at later (120-300 s) time points. In addition, fentanyl-induced Ins(1,4,5)P3 formation, even if enhanced by H-89, Ro 31-8220 and/or 4-aminopyridine, was inhibited by nifedipine (1 nM-10 microM). 7. In conclusion, desensitization of the mu-opioid-induced activation of PLC is multifactorial, involving protein kinases C and A and Ca(2+)-activated K+ efflux, but the L-type VSCC is of critical importance and may be a possible common site of action.
authors.affiliation: University Department of Anaesthesia, Leicester Royal Infirmary.
authors.forename: D
authors.initials: D
authors.lastname: Smart
authors.forename: D G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005283:Fentanyl / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology*; D009447:Neuroblastoma / Q000201:enzymology*; D009543:Nifedipine / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000502:physiology*; D012680:Sensitivity and Specificity; D013449:Sulfonamides; D014407:Tumor Cells, Cultured / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D000701:Analgesics, Opioid; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007211:Indoles; D007546:Isoquinolines; D009294:Narcotics; D015221:Potassium Channels; D017450:Receptors, Opioid, mu; D013449:Sulfonamides; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D015544:Inositol 1,4,5-Trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D009543:Nifedipine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium; D005283:Fentanyl; C064758:Ro 31-8220