Benzilylcholine Mustard Research Articles

Homologues of benzilylcholine mustard.

Abstract Seven homologues of the alkylating muscarinic antagonist benzilylcholine mustard (BCM) have been prepared. The rate of reaction of the aziridinium ion derivatives with water is largely independent of the structure, whereas the rate of reaction with the muscarinic receptor drops markedly when the N-alkyl group is larger than n-propyl. PrBCM is practically equiactive with BCM. Recovery from block caused by PrBCM was first-order with a half-time of 32 h at 37°.

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.

The action of acetylcholine and other drugs on the efflux of potassium and rubidium from smooth muscle of the guinea-pig intestine. 1968.

. A method is described for measuring continuously the efflux of potassium or rubidium from smooth muscle of the guinea‐pig. . Muscarinic drugs cause at maximum a 100‐fold increase in the efflux rate, due to a direct increase in permeability and only to a minor extent secondary to depolarization. With acetylcholine the dose response curve for producing efflux is displaced to 1,000 times higher concentrations than that for contraction. . The shift varies with different agonists. The efflux and contractile responses to agonists are antagonized to an equivalent extent by atropine and several other reversible antagonists but benzhexol has a relatively greater effect on efflux. An estimate of spare receptors was obtained with benzilylcholine mustard and was similar for both responses. Dibenamine and local anaesthetics led to a parallel shift of the contraction dose response curve but a depression without shift in the efflux response. . The most satisfactory explanation of these results is that there are two types of the muscarinic receptor in the smooth muscle of the guinea‐pig intestine.

Two populations of muscarinic binding sites in the chick heart distinguished by affinities for ligands and selective inactivation

1. By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtype-specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DX116 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2. Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX116 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3. It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DX116 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population.4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.

Protection by Alcuronium of Muscarinic Receptors Against Chemical Inactivation and Location of the Allosteric Binding Site for Alcuronium

We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3H]methyl-N-scopolamine ([3H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p-nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, and N-ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [3H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [3H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.

Studies of muscarinic receptor reserve linked to phosphoinositide hydrolysis in parotid gland and cerebral cortex.

Hydrolysis of inositol phospholipids caused by muscarinic agonists was studied in the guinea-pig parotid gland (PG) and cerebral cortex (CX). The present study describes the effect of two muscarinic agonists, carbachol and oxotremorine, on stimulation of phosphoinositide hydrolysis and on binding of [3H]NMS in the presence of the irreversible muscarinic antagonist benzilyl choline mustard (BCM). Carbachol and oxotremorine stimulated the formation of inositol phosphates in PG, pD2(Carb) = 5.3 +/- 0.1, pD2(Oxo) = 5.9 +/- 0.1 and in CX, pD2(Carb) = 4.3 +/- 0.2, pD2(Oxo) = 5.8 +/- 0.2. In the present study slices from both tissues have been exposed to 0.1 microM BCM for 2, 5 and 10 min. Treatment for 10 min caused a 75-85% reduction in specific [3H]N-methyl scopolamine ([3H]NMS) binding sites in both PG and CX. Following 2 min BCM treatment of PG a marked decrease in pD2 value of the carbachol-stimulated inositol phosphate formation was found. This effect was not found in CX. The results showed that a 30-40% reduction in binding sites shifted the carbachol concentration response curve to the right by one order of magnitude and reduced the oxotremorine-induced release of inositol phosphates by approximately 20%. In PG, the BCM-induced reduction of the carbachol-stimulated inositol phosphate formation was paralleled by the reduction in receptor binding sites. Similar treatment, but in CX, showed a lower reduction of the carbachol-stimulated inositol phosphate formation as compared to the reduction in receptor-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle.

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.

The mechanism of hypothermia-induced supersensitivity of guinea pig left atria to carbachol

Hypothermia has previously been demonstrated to induce supersensitivity (defined as a decrease in the ED 50) of guinea pig left atria to the negative inotropic effect of carbachol. In the present investigation, the dissociation constant (pK A or −log K A) for carbachol, determined using benzilylcholine mustard, was found to be significantly increased at 25°C compared to 37°C. However, the increase in pD 2 (−log ED 50) for carbachol at 25°C was much less than would be predicted from the increase in pK A. Increasing the extracellullar Ca 2+ concentration or the frequency of stimulation, both of which, like hypothermia, are believed to increase Ca 2+ influx into cardiac cells, resulted in a decrease in sensitivity to carbachol. Carbachol had no significant effect on cAMP or cGMP levels at either 37°C or at 25°C. These results suggest that the hypothermia-induced increase in sensitivity of left atria to carbachol can be explained by an increase in the affinity of the muscarinic receptor for this agonist. However, the expression of this increased affinity appears to be limited. This may be due to a concurrent decrease in the efficacy of the carbachol-muscarinic receptor complex.

Aging and cholinergic responses in bovine trachealis muscle.

1. The relative potencies of muscarinic agonists on bovine tracheal smooth muscle were unchanged as a consequence of aging and were carbachol greater than oxotremorine greater than muscarine greater than pilocarpine greater than McNeil A-343. 2. During aging, the potencies of carbachol, oxotremorine, McNeil A-343 and pilocarpine, but not muscarine, were reduced. 3. Maximal induced tensions to all the agents studied were reduced as a consequence of age. 4. Irreversible antagonism with benzilylcholine mustard showed that agonist efficacy was significantly reduced during aging. 5. Estimated receptor occupancy at the EC50 was significantly greater in tracheal tissues from the mature versus immature cows for every agonist studied. 6. The dissociation constants for full agonists (carbachol, oxotremorine and methacholine) were decreased with maturation while the converse was observed with partial agonists (McNeil A-343, pilocarpine). 7. We conclude that there are significant changes in the properties and coupling of muscarinic receptors during aging. These changes may contribute to the reduced airway reactivity seen in vivo.

1,4-Dithiothreitol-induced changes in histamine H1-agonist efficacy and affinity in the longitudinal smooth muscle of guinea-pig ileum.

The effect of 1,4-dithiothreitol (DTT) on histamine H1-receptor agonist affinity and efficacy has been investigated in longitudinal muscle strips of guinea-pig ileum. Exposure of ileal smooth muscle to DTT significantly increased the maximal responses to the partial agonists SKF71473 and DE-2PEA, indicative of an increase in agonist efficacy. This effect was paralleled by a small decrease in EC50 values. In contrast, DTT produced a parallel displacement of the concentration-response curve to the full agonist histamine in the same muscle strips. Studies in which phenoxybenzamine and benzilylcholine mustard were used to reduce the maximum response to histamine suggested that DTT altered both agonist affinity and efficacy. The affinity constant for histamine, calculated by the method of Furchgott & Bursztyn (1967), increased by 2.7 fold in the presence of DTT. Furthermore, agonist efficacy also appeared to increase in the presence of DTT since the maximum response to histamine following phenoxybenzamine treatment increased on application of DTT. [3H]-mepyramine binding studies confirmed that DTT increased agonist affinity. DTT produced a significant parallel shift to the left of the displacement curves for histamine, 2-methylhistamine, 2-pyridylethylamine and 2-thiazolylethylamine. The results of this study therefore suggest that DTT potentiates H1-receptor-mediated contractile activity in guinea-pig ileum by increasing both agonist efficacy and affinity.

The muscle chemoreceptors of the ascidian halocynthia aurantium

The body wall muscle of the ascidian, Halocynthia aurantium, is sensitive to both nicotinomimetics and muscarinomimetics. Atropine (4 X 10(-11 M) and benzilylcholine mustard (1 X 10(-5) M for 15 min) selectively block responses to muscarinomimetics. d-Tubocurarine (2 X 10(-6) M) and hexadecamethylene-bis-trimethylammonium (2 X 10(-7) M) prevent contractions induced by both nicotinomimetics and muscarinomimetics. In the cold season of the year the sensitivity of the muscle to muscarinomimetics disappears while responses to nicotinomimetics do not change throughout the year. The cooling of the water in the experimental bath results in an increase in the muscle contractions caused by muscarinomimetics while the responses to nicotinomimetic propionylcholine do not change. Catecholamines induce a decrease of the ascidian muscle responses to acetylcholine, cGMP and papaverine produce a similar effect.

Evidence against the presence of spare muscarinic receptors in the rabbit urinary bladder

AbstractIn a recent study by Anderson and Marks [1982], indirect evidence was presented for the existence of “150‐fold muscarinic receptor excess” in the rabbit urinary bladder. This conclusion was based on the quantitative comparison of the ability of carbamylcholine to both directly contract bladder strips and to inhibit (quinuclidinyl benzilate) binding. In order to investigate the presence of “spare receptors” in the bladder directly, we have determined the effect of benzilylcholine mustard (a noncompetitive cholinergic inhibitor) on both bethanechol stimulation of muscle‐strip contraction and on [3H]QNB binding (muscarinic receptor density). The results of these studies indicate that there is no significant “muscarinic receptor excess” in the rabbit urinary bladder.

Benzilycholine mustard and spare receptors in guinea pig ileum

A comparison was made of muscarinic receptor occupancy by the irreversible antagonist benzilylcholine mustard (BCM) as determined from shifts in the dose-response curve to a muscurinic agonist and from 3H-QNB binding to homogenates of BCM-treated tissue. Major discrepancies were found. A low concentration of BCM (3×10 −8M/15 min.) produced a parallel dose-response curve shift corresponding to 98–99% receptor occupancy by BCN, whereas 3H-QNB binding revealed only 48% receptor occupancy. Possible origins of this discrepancy are discussed. High concentrations of BCM (5×10 −5M, min.) fail to completely alkylate all 3H-QNB binding sites even though response is completely lost. Although significant (64%) recovery of response occurs after prolonged tissue washing (240 min.) this is not accompanied by an increase in 3H-QNB binding. The small fraction (∼5%) of sites inaccessible to BCM and with reduced affinity for 3H-QNB may represent a subpopulation of muscarinic receptors.

Pharmacological study of two kinds of cholinoreceptors on the membrane of identified completely isolated neurones of Planorbarius corneus

Completely isolated identified neurones (LPed-2 and LPed-3) of the left pedal ganglion of Planorbarius corneus were shown to have two kinds of cholinoreceptors (ChR) on their membrane. One kind of ChR is a classical nicotinic receptor which is sensitive to nicotinomimetics and can be blocked by tubocurarine; the depolarization caused by activation of this ChR type is chloride-dependent. The other kind of ChR, which mediates a potassium-dependent hyperpolarization, has some common features with muscarinic (M) ChR of vertebrates, being sensitive to the muscarinomimetics, dioxolane F-2268, methylfurmethide, mecholyl, and arecoline, although insensitive to oxotremorine. The sensitivity of this receptor to muscarine, itself, was not tested. Like the muscarinic ChR of vertebrates, it can be blocked by benzilylcholine mustard, but, in contrast to vertebrate muscarinic receptors, it cannot be blocked by atropine and cannot distinguish between the optical isomers of F-2268. TEA, mytolon, and cooling prevent the hyperpolarization caused by ACh and muscarinomimetics. The two kinds of ChR in P. corneus neurones seem to be similar to those found by Kehoe 24–26 on the medial cells of Aplysia pleural ganglia, both in their pharmacological characteristics and in the ionic permeability changes which they control.

The number of muscarinic receptors in chick amnion muscle.

1. [(3)H]-Propyl benzilylcholine mustard ([(3)H]-PrBCM) an irreversible specific antagonist of muscarinic acetylcholine receptors, has been used to label the muscarinic receptors in the aneuronal muscle of isolated 11 day amniotic membranes of chick embryos.2. This muscle was found to have about 9 fmol/mg dry weight of receptor material. This is only 1-2% of the amount found in intestinal muscle.3. Pharmacological studies with isolated amniotic membranes using PrBCM show that labelling is to functional receptors and that the receptor reserve is small.4. The significance of the difference in receptor reserve in innervated and aneuronal plain muscle is discussed.

Interaction of benzilylcholine mustard, benzilylcholine and lachesine with the histamine receptor in the longitudinal muscle of guinea-pig ileum.

1. The histamine receptors of longitudinal muscle strips from guinea-pig ileum were inactivated on extended exposure to concentrations of benzilylcholine mustard (BCM) above 10(-6)M. These concentrations are 3 orders of magnitude greater than those required for inactivation of the muscarinic receptors. Mepyramine (3 x 10(-8)M) afforded complete protection for the histamine receptor against the effects of BCM.2. Recovery from block at 30 degrees C was very slow with a first-order rate constant of approximately 10(-7) s(-1).3. The rate constant, k(2), for the alkylation reaction in which the reversible BCM-receptor complex is converted into an irreversible complex is much greater than the rate constant, k(-1), for dissociation of the reversible complex. The value of the rate constant, k(1), for formation of the reversible complex was 1.1 +/- 0.2 x 10(3)M(-1) s(-1).4. These observations suggested that k(1) was probably not much greater than for BCM acting on the muscarinic receptor, even though the value of k(1) was reduced by more than 2 orders of magnitude. This effect has been established for benzilylcholine and lachesine, reversible analogues of BCM. For both drugs k(1) for the histamine receptor was approximately 3 orders of magnitude less than for the muscarinic receptor, whereas k(1) was greater by a factor of only 6.

The influence of calcium on the activity of full and partial muscarinic agonists

The effect of gradually decreasing the Ca 2+ concentration in a Mg 2+-free Krebs solution on the dose-effect curves of acetylcholine, hexyl-trimethyl-ammonium and pilocarpine was qualitatively and quantitatively studied on the isolated longitudinal muscle of the guinea-pig ileum. Changes in the dose-effect curves were analogous to those observed after irreversible blockade of the receptors. However they were not additive with the changes induced by dibenamine and were reversed by restoring the “normal” Ca 2+ concentration. The observed changes agree with the hypothesis that the receptor mobilizes calcium and renders it accessible, in an ionic form, to the cell contractile mechanism and that the amount of Ca 2+ mobilized by each receptor activation is constant for each drug at each calcium level and is the limiting factor of the induced effect. These changes also permit calculation of agonist equilibrium constants. The values obtained do not depend on the calcium concentration and do not differ statistically from those determined by the use of dibenamine or benzilylcholine mustard.

The action of acetylcholine and other drugs on the efflux of potassium and rubidium from smooth muscle of the guinea‐pig intestine

1. A method is described for measuring continuously the efflux of potassium or rubidium from smooth muscle of the guinea-pig.2. Muscarinic drugs cause at maximum a 100-fold increase in the efflux rate, due to a direct increase in permeability and only to a minor extent secondary to depolarization. With acetylcholine the dose response curve for producing efflux is displaced to 1,000 times higher concentrations than that for contraction.3. The shift varies with different agonists. The efflux and contractile responses to agonists are antagonized to an equivalent extent by atropine and several other reversible antagonists but benzhexol has a relatively greater effect on efflux. An estimate of spare receptors was obtained with benzilylcholine mustard and was similar for both responses. Dibenamine and local anaesthetics led to a parallel shift of the contraction dose response curve but a depression without shift in the efflux response.4. The most satisfactory explanation of these results is that there are two types of the muscarinic receptor in the smooth muscle of the guinea-pig intestine.