Muscarinic Cholinergic Binding in Rat Brain

Abstract

Binding sites with high affinity and spec- Identification of neurotransmitter receptor sites by chemical measurements of direct binding has been reported for nicotinic cholinergic receptors of invertebrate electric organs (1), mammalian neuromuscular junction (2) and central nervous system (3), and for the glycine receptor in the mammalian central nervous system (4). Attempts to study the muscarinic cholinergic receptor biochemically have involved measuring the binding of atropine to the guinea pig intestine (5) and subcellular fractions of rat brain (6). Recently an alkylating agent derived from the muscarinic antagonist benzilylcholine has been employed in the guinea pig intestine (7) and rat brain (8). 3-Quinuclidinyl benzilate (QNB) has been reported to be a potent central muscarinic antagonist (9, 10). Moreover, in the periphery, QNB has been shown to antagonize the acetylcholine-induced contractile response of the guinea pig ileum 50% at 0.01 AoMI (11). Since QNB possesses potency, specificity, and persistence of action, it appears to be a suitable agent for receptor labeling. We now report a simple and sensitive assay for specific muscarinic cholinergic binding in the rat central nervous system using QNB, and describe kinetic properties of the binding, its regional and subcellular localization, and the relative affinity of cholinergic and noncholinergic drugs. MATERIALS AND METHODSQNB was labeled by catalytic tritium exchange at New England Nuclear Corp., Boston, Mass. Fifty milligrams of QNB dissolved in 0.3 ml of glacial acetic acid were mixed with 25 mg of platinum catalyst and 10 Ci of 3H20. After stirring 18 hr at 800, labile 3H was removed in vacuo with methanol as a solvent and, after filtration from the solvent, the product was dissolved in 10 ml of methanol. In our laboratory, the product was purified by thin-layer chromatography in Silica-Gel F-254 plates, 0.

Muscarinic Receptors in Rat Sympathetic Ganglia

“…The electrophysiological consequences of muscarinic receptor activation in central neurones show a qualitative similarity to those in ganglia (see, for example, Krnjevic & Phillis, 1963;Curtis & Ryall, 1966;Krnjevic, 1969;Krnjevic, Pumain & Renaud, 1971;Dingledine, Dodd & Kelly, 1977), but are more difficult to quantify. In consequence, central receptors have so far been characterized primarily from measurements of radiolabelled ligand-binding (Yamamura & Snyder, 1974a. Although these are difficult to correlate directly with the pharmacological response of intact central neurones, our experiments on sympathetic ganglia suggest that the ligand-binding properties of the central muscarinic receptor probably reflect their functional characteristics fairly accurately.…”

Brain ischemia decreases phosphatidylcholine-phospholipase D but not phosphatidylinositol-phospholipase C in rats.

“…5 The rank order of PLD activity in rat brain was hippocampus>brain stem>cortex>cerebellum (data not shown), and this result is compatible with the previous report. 23 Interestingly, this distribution pattern resembles that of choline acetyltransferase 24 rather than that of PLC, 16 suggesting the significance of PLD in acetylcholine synthesis in brain. The decrease in PLD activity after brain ischemia also might cause an imbalance of acetylcholine turnover.…”

<b>Solubilization of the muscarinic acetylcholine receptor by sodium cholate: Stabilization of the receptor by muscarinic li</b><b>gands </b>

“…The muscarinic receptor has been studied in detail with respect to its binding with muscarinic ligands by using [3H]-labeled ligands (3,12) and several subclasses with different affinities for agonists (2) or selective antagonists (8) have been reported. It is not known, however, whether these subclasses represent several types of receptors which differ as chemical entities, or several states of a single receptor which differ in conformation and/or in interaction with other components in the membranes.…”