| The muscarinic acetylcholine receptor is the major protein that mediates cholinergic transmission in central and parasympathetic nervous tissues. This thesis has examined the biochemical characteristics and molecular adaptability of muscarinic acetylcholine receptors.;Sustained cholinergic stimulation specifically modulated features of agonist-receptor interaction. After stimulation, the receptor system had reduced affinity for agonists and a reversed response to guanine nucleotides. Differences between control and stimulated receptor were eliminated by solubilization. The molecular basis for agonist-specific modulation may be a biochemical modification of receptor protein, since the chronically stimulated receptors had a lower isoelectric point. The data suggest that molecular adaptability of the muscarinic receptor system in response to sustained receptor activity may include alterations in the coupling mode of receptor to adenylate cyclase. A sequential two-step model is proposed to account for agonist-receptor interactions both with and without chronic cholinergic stimulation.;Cellular mechanisms underlying the long-term adaptive response of the muscarinic receptor system in cultured NG108-15 cells were investigated. Filipin completely blocked the carbachol-induced decrease in receptor number. Furthermore, filipin increased 3H-N-methylscoploamine binding by 100% in control and by 400% in carbachol-treated cells. Muscarinic receptor regulation was shown to be independent of membrane potential and the ionic composition of extracellular medium. Treatment of the cells with tunicamycin, an inhibitor of glycosylation, caused decreases in receptor number for both control and carbachol-treated cultures. In the continued presence of tunicamycin the receptor level reached a new equilibrium by 24 hours, suggesting that the turnover rate of the receptor was altered by tunicamycin treatment.;Biochemical characteristics of muscarinic receptors from brain tissue were examined. The receptors from calf and mouse brain examined by SDS-PAGE were shown to have molecular weights of 77,000 and 83,000 daltons, respectively. Calf brain muscarinic receptors had a sedimentation coefficient of 10.3 S and isoelectric point of 4.3. The receptor protein covalently labeled with 3H-Propylbenzyl choline mustard (3H-PrBCM) was purified 25-fold by gel filtration on a HPLC (High Performance Liquid Chromatography) column. |
