The endogenous opioid pentapeptides [Met5]enkephalin (H-TyrGly-Gly-Phe-Met-OH) and [Leu5]enkephalin (H-Tyr-Gly-GlyPhe-Leu-OH) have been shown to interact with several classes of opioid receptors (1-3) that may mediate different physiological responses. Elucidation of the roles of the individual receptor classes has been hampered by the general lack of enkephalin analogs with a high degree of selectivity for a single receptor type. The vast majority of analogs crossreact extensively with the different receptors, making it difficult to define receptor roles. This situation has been in part ameliorated by recent reports of an enkephalin analog highly selective for the ,At opioid receptor (4-6) and a nonpeptide opiate with high K receptor selectivity (7). However, analogs with corresponding selectivity for the 8 opioid receptor have not been demonstrated.One approach for the design of more selective analogs involves the incorporation of conformational restrictions. The native enkephalins, like most small, linear peptides, possess considerable conformational flexibility and by virtue of this flexibility can attain the presumably different conformational features required for interaction with different classes of opioid receptors. In principle, appropriate restriction of this flexibility can lead to analogs able to assume the conformation required to interact favorably with only one class of receptor. One method for effecting conformational restrictions is via cyclization of the peptide that constrains the resulting analog to assume a compact topography. Several active, cyclic enkephalin analogs have been reported, all of which are cyclized by either side chain to carboxyl terminus (8,9) It has previously been shown that, in aqueous solution, the tocin ring portion of Pen-containing oxytocin analogs is conformationally restricted, whereas the tocin ring of oxytocin itself is quite flexible (13-16). This difference arises from the rigidifying effect of gem-dialkyl substituents in medium-sized rings and suggests that the 8 (Fig. 1) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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.

The accumulation of [3H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [3H]choline concentrations varying from 0.5 to 100 p~. The accumulation of [3H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by doublereciprocal plots and by least squares iterative fitting of a substratevelocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K, values of 1.4 VM (corpus striaturn), and 3.1 ,UM (cerebral cortex) and a low affinity uptake system with respective K,,, values of 93 and 33 p~ for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [3H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [3H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [3H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affiity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.