title: Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures.
abstract: Primary cultures of dissociated cerebral cortex cells were used to characterize the muscarinic acetylcholinergic receptors (mAChR) present and to study receptor down-regulation and receptor mediated 2nd messenger responses induced by muscarinic agonists. Binding of the hydrophilic antagonist [3H]N-methyl scopolamine ([3H]NMS) to the cultured cells was saturated after one hour at 4 degrees C with a Kd of 93 pM and a Bmax of 958 fmol/mg protein. Competition binding studies with several antagonists and agonists indicated that the mAChR present in the culture were of a mixed M1/M3 subtype. The number of muscarinic receptors at the cell surface decreased by 60% after one hour pre-incubation of the cultures with 10 microM carbachol or oxotremorine. After down-regulation with carbachol affinity for pirenzepine was decreased, while low affinity sites for 4-DAMP were lost, indicating that especially M1 subtypes are sensitive to this type of regulation. Carbachol and oxotremorine-M induced a 2-3 fold increase in phosphatidyl inositide (PI) turnover, which was blocked with high affinity by both pirenzepine and 4-DAMP. Down-regulation of the mAChR and stimulation of PI-turnover by agonists with different potency and intrinsic activity appeared highly correlated. These data suggest that activation of the PI second-messenger system is involved in the desensitization and down-regulation of the muscarinic acetylcholine receptor.
authors.affiliation: Organon Intl. B.V., Dept. of Neuropharmacology, Oss, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: van Huizen
authors.forename: J A
authors.initials: JA
authors.lastname: Tonnaer
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000196:embryology; D015536:Down-Regulation; D010095:Oxotremorine / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects*; D015290:Second Messenger Systems / Q000187:drug effects*
chemical_list: D010276:Parasympatholytics; D010880:Piperidines; D011976:Receptors, Muscarinic; D010890:Pirenzepine; D010095:Oxotremorine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002217:Carbachol