We have studied the mechanisms of regulating the m2- and m3-muscarinic and serotonergic 5-HT2 receptors in cultured cerebellar granule cells. The selectivity of muscarinic antagonists to bind to specific receptor subtypes is greater in intact cells than in membrane preparations. Two m2 antagonists, AF-DX 116 and methoctramine, and two m3-selective antagonists, 4-DAMP and pFHHSiD, showed a decrease in affinity for the binding of the receptor subtype for which they had the higher affinity to start with. When the intact cell binding assays were decreased from 37 degrees C to 4 degrees C the selectivity of the muscarinic antagonists was similar to that of membranes. The difference between whole cell and membrane binding assays may therefore be due to a temperature-sensitive cellular component which is lost when the cells are disrupted. Northern blot analysis of granule cell RNA were examined for the effect of dibutyryl phorbol ester on muscarinic agonist-induced down-regulation of the m3-receptor mRNA. Although a short-term phorbol ester pretreatment to inhibit inositol phosphate second messenger generation failed to block agonist-induced down-regulation, overnight pretreatment which would down- regulate protein kinase C (PKC) completely inhibited the agonist-induced decrease in m3-mRNA, suggesting a role of PKC in the regulation of muscarinic receptor mRNA levels. We have also extended our studies on the agonist-induced up-regulation of 5HT2 receptor binding sites. We found that this agonist up-regulation was homologous and required new RNA and protein synthesis. The up-regulation was independent of the production of inositol trisphosphate and diacylglycerol or the activation of PKC. However, the up-regulation required the influx of calcium and the activity of calmodulin-activated enzymes which probably involved calmodulin kinase. We also found that the structural integrity of microtubules is necessary for this agonist-induced up-regulation. Possible mechanisms underlying this up-regulation are discussed.
