Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures.

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.