High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Solubilization by various detergents, of the muscarinic cholinoreceptor and its complex with quinuclidinyl benzilate

The possibilities to solubilize the rat brain cortex muscarinic acetylcholine receptor and its complex with [3H]-L-quinuclidinyl benzilate (QNB) were studied, using 14 detergents. It was shown that the native muscarinic cholinoreceptor was solubilized in addition to digitonin, also by CHAPS, with a 6% yield. Besides, the receptor-QNB complex was solubilized with the detergents Triton X-100, -102, -114, -165 (with 30% and 50% yields) and within a narrow concentration range with sodium dodecyl sulfate (50% yield). Some detergents of the Tween series, e.g., Triton X-45 and -305, as well as sodium deoxycholate and sodium oxycholate, did not solubilize the native receptor and its complex with QNB. It was found that yield of receptor solubilization did not exceed half of the total number of the receptor sites in the membranes, despite the fact that different concentrations of detergents were applied. The solubilization yield did not increase, when different mixtures of detergents were used. It was assumed that incomplete solubilization of the receptor protein reflects its heterogeneity in the membrane structure.     

Structure-binding relationship of quinuclidinyl benzilate analogs on N4TG1 neuroblastoma muscarinic receptors

By Scatchard plot analysis of [³H]QNB (quinuclidinyl benzilate) binding, there are 2×10⁵ muscarinic sites/cell with aK _d about 10 nM in N4TG1 neuroblastoma cells. We have now examined a group of compounds structurally related to aprophen and QNB for their ability to compete with the binding of QNB to the muscarini receptor. Using this structure-inhibition relationship, the functional groups of the muscarinic ligand necessary for binding were partially characterized. It was found that the quinuclidinyl ring structure of QNB can be substituted by either alkane, H, or pyrrolidine at the N without loosing their ability to bind. The addition to the quinuclidinyl ring increases the bulk of the structure and decreases binding. Like the benzilate in QNB, a similar hydrophobic structure is apparently required for the binding.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Kinetic aspects of l-quinuclidinyl benzilate interaction with muscarinic receptor

l-Quinuclidinyl benzilate is undoubtedly the most widely used radioactive reporter ligand for studies on muscarinic receptor. In the present Commentary the kinetic aspects of the interaction of this ligand with muscarinic receptor are summarized. On the basis of these results a kinetic mechanism has been proposed involving consequential isomerization of the receptor-ligand complex and cooperative regulation of this process by the excess of the ligand. In addition, the data give evidence of at least two different types of binding sites on the receptor. Owing to the solubilization of the receptor protein there occur remarkable changes in its kinetic properties. The kinetic analysis points to inadequacy of the simple one-step equilibrium binding scheme for l-quinuclidinyl benzilate interaction with muscarinic receptor, which may explain the apparently contradictory data in the literature, such as the large scattering of the K_d values and the different regularities described in the case of the receptor-ligand complex dissociation reaction. That points to the conclusion that l-quinuclidinyl benzilate is an “inconvenient” ligand for receptor studies, which call for true equilibrium conditions of the system.     

Toxins from the venom of the green mamba Dendroaspis angusticeps that inhibit the binding of quinuclidinyl benzilate to muscarinic acetylcholine receptors

Two protein toxins that displace the muscarinic antagonist quinuclidinyl benzilate from rat cortex synaptosomal membranes have been isolated from the green mamba (Dendroaspis angusticeps) venom by gel filtration on sephadex G-50, chromatography on the ion-exchangers Bio-Rex 70 and Sulphopropyl-Sephadex C-25 and reversed-phase HPLC. Toxin 1 has 64 amino acids and four disulfides and a formula weight of 7200 and the corresponding values for toxin 2 are 63, 4 and 6840, respectively. Ultracentrifugation gave a molecular weight of 6900 for toxin 1 and 6700 for toxin 2, Quinuclidinyl benzilate that binds to all types of muscarinic cholinergic receptor was displaced to about 50% by both toxins. This partial displacement indicates that the toxins might be specific for one subtype of receptor.     

Effect of aging on the interaction of quinuclidinyl benzilate,N-methylscopolamine,pirenzepine, and gallamine with brain muscarinic receptors

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]N-methylscopolamine ([³H]NMS) as ligands in young (3months), middle-age (10months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M₁ and M₂ muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [³H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age.     

Specific binding of the muscarinic antagonist [3H]quinuclidinyl benzilate is not associated with preganglionic motor neurons in the dorsal motor nucleus of the vagus

The present study evaluates the binding of [³H]quinuclidinyl benzilate, [³H]QNB, as a measure of cholinergic muscarinic binding in six areas of the rat medulla oblongata associated with the cranial nerves. In an experimental group, the right vagus nerve was severed in the neck in order to determine whether the specific muscarinic binding sites might be located on cells that contribute efferent fibers to the vagus nerve. The level of activity of choline acetyltransferase (ChAT) also was determined in the same six areas. Additional experiments utilizing the retrograde transport of toxic ricin, a 60,000 dalton agglutinin that acts as a potent ribosomal toxin, was carried out to further evaluate localization of specific muscarinic binding in the DMN after destruction of the preganglionic efferent cells. These results support the conclusion that specific binding of the muscarinic antagonist [³H]QNB observed in the DMN of the vagus of the rat is not associated with the large cells that contribute efferent fibers into the vagus nerve. We suggest that the specific cholinergic muscarinic binding is located on interneuronal cell surfaces, on afferent terminals of local circuit neurons, or on afferent terminals of long projection axons which arise from neurons in the brainstem, hypothalamus, or forebrain.This issue is dedicated to Donald B. Tower.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Effect of lysophasphatidylcholine on sensitivity of the heart to acetylcholine and binding of quinuclidinyl benzilate to myocardial membranes

Chemical nature of the blood serum factor exerting cholinolytic effect on the animal heart, was determined. The effect is due to the serum lipid component: lysophosphatidylcholine. The latter in concentration 1-25 microM suppresses the sensitivity of the frog perfused heart ventricle to acetylcholine. Phosphatidylcholine was found to protect the heart from the lysophosphatidylcholine effect. Lysophosphatidylcholine also affected the binding of the acetylcholine high affinity antagonist [3H]-quinuclidinylbenzilate to the rabbit atria cell membranes. The mechanism of this effect is related to an increased ability of muscarinic receptors to form oligomeric complexes in presence of lysophosphatidylcholine, the latter manifesting a positive cooperativity on binding to ligand.     

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.     

Anomalous binding of [3 H] N-methyl-quinuclidinyl benzilate methyl chloride to human lymphocyte muscarinic receptors

Using the muscarinic cholinergic ligand [³ H] N-methyl quinuclidinyl benzilate methyl chloride ([³ H] NM-QNB), we demonstrated that intact, viable human lymphocytes posses specific muscarinic binding sites. Equilibrium binding studies show that muscarinic acethylcholine receptor are devided into two subtype; high affinity (Ms) and low affinity types (Mw) for the ligand.     

N-substituted derivatives of 4-piperidinyl benzilate: affinities for brain muscarinic acetylcholine receptors

N-Substituted derivatives of 4-piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4-Piperidinyl benzilate exhibited a Ki value of 2.0 nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2 nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The Ki values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p-fluorobenzyl, 3.0; phenethyl, 8.0; p-nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions (e.g., benzyl or phenethyl) as well or better than straight chain or branched alkyl substitutions (e.g., n-propyl or isopropyl).