The binding of a 2-chloroethylamine derivative of oxotremorine (BM 123) to muscarinic receptors in the rat cerebral cortex

The interaction of a mustard analogue of oxotremorine, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), with muscarinic receptors in the rat cerebral cortex was investigated using 3H-ligand-binding methods. When cortical homogenates were preincubated with BM 123 (1.0 mM), washed extensively, and then assayed for the binding of the specific muscarinic antagonist, [3H](-)-N-methylscopolamine, a decrease in binding capacity was noted without an accompanying change in affinity. The rate at which BM 123 alkylated muscarinic receptors was sensitive to temperature, with little or no receptor alkylation occurring at 0 degree. Thus, it was possible to estimate the affinity of BM 123 and its transformation products for muscarinic receptors by measuring their ability to competitively inhibit 3H-ligand binding to cortical homogenates at 0 degree. When measured by competitive inhibition of [3H]oxotremorine-M and [3H](-)-N-methylscopolamine binding, the concentrations of the aziridinium ion of BM 123 required to displace 50% of specific 3H-ligand binding were 3.5 nM and 4.5 microM, respectively. In contrast, the parent 2-chloroethylamine and its alcoholic hydrolysis product were much less active. The kinetics of the alkylation of muscarinic receptors by BM 123 were consistent with a model in which the aziridinium ion rapidly forms reversible complexes with superhigh high and low affinity sites which slowly convert to covalent complexes. The rate of alkylation of the superhigh affinity site was slowest whereas the converse was true for the low affinity site. It was possible to alkylate the high and low affinity sites selectively with BM 123 by taking advantage of kinetic differences in the rates of alkylation of these two sites. Atropine, oxotremorine, and oxotremorine-M antagonized the rate of alkylation of muscarinic receptors in a manner that was consistent with competitive inhibition.     

Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.     

Inactivation of brain cortex muscarinic receptors by 4-diphenylacetoxy-1-(2-chloroethyl) piperidine mustard.

We demonstrated in this study that 4-DAMP [4-diphenylacetoxy-1-(2- chloroethyl) piperidine] mustard, which cyclizes to the aziridinium ion, behaved as a non-selective, non-competitive inhibitor of muscarinic receptors in rat brain cortex. It inactivated to the same extent the M1, M2 and M4 muscarinic receptors present in this tissue, as well as receptors accessible or not accessible to quaternary antimuscarinic drugs. Under mild incubation conditions, the muscarinic receptors in a state with super high affinity for agonists (SH receptors) were less affected by preactivated 4-DAMP mustard than the receptors in the states with lower affinity for agonists (H and L receptors).     

Tertiary 2-haloethylamine derivatives of the muscarinic agent McN-A-343, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride

4-[(2-Chloroethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (2) and 4-[(2-bromoethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (3) were synthesized. Compounds 2 and 3 cyclized at neutral pH to an aziridinium ion (4). The rate constants for the cyclization of 2 and 3 at 37 degrees C were about 0.01 and 0.4 min-1, respectively, as measured by titrimetric analysis and by 1H NMR spectroscopy. The aziridinium ion had 1/4 the potency of McN-A-343 (1) as a ganglionic muscarinic stimulant in the anesthetized, pentolinium-treated rat but showed no muscarinic effects on the isolated guinea pig ileum. It caused alkylation of muscarinic receptors in homogenates of the rat cerebral cortex. An irreversible blockade of central muscarinic receptors was also observed after intravenous administration of 3 to mice. Because of its selectivity, irreversible actions, and ability to pass into the central nervous system, 3 should become a valuable tool in studies of muscarinic receptors.     

Selective inactivation of muscarinic M2 and M3 receptors in guinea-pig ileum and atria in vitro.

1. The role of muscarinic M2 and M3 receptors in ileal smooth muscle has been evaluated by use of selective receptor alkylation. The alkylating agents, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP mustard) was studied for effects against (+)-cis-dioxolane, at muscarinic M2 and M3 receptors in guinea-pig atria or ileum, respectively. 4-DAMP mustard (10 nM, 40 min exposure) did not discriminate between these muscarinic receptors. In ileum, 4-DAMP mustard, at 100 nM, resulted in a large dextral shift (197 fold) and depression in maxima. In atria there was a smaller dextral shift (14 fold) but no depression in maxima. 2. The muscarinic antagonists, atropine (non-selective), methoctramine (M2-selective) and para-fluorohexahydro-siladiphenidol (pFHHSiD; M3 selective) were studied in protection studies against alkylation by phenoxybenzamine. Washout studies following equilibration of the tissues with atropine (30 nM), methoctramine (0.3 microM) or pFHHSiD (3 microM), showed the compounds to be reversible. No temporal changes in sensitivity to (+)-cis-dioxolane were observed. 3. Exposure, for 20 min, of atria and ileum to phenoxybenzamine (3 and 10 microM respectively) caused dextral shifts and depressions in the maxima of the concentration-response curve to (+)-cis-dioxolane. These effects were inhibited by prior equilibration with atropine (30 nM) and methoctramine (0.1 microM) in atria or atropine (30 nM) and pFHHSiD (3 microM) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist. 4. These data were consistent with muscarinic M2 receptors mediating responses in atria and M3 receptors mediating responses in ileum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkylating partial muscarinic agonists related to oxotremorine. N-[4-[(2-haloethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidones

N-[4-[(2-Chloroethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidone (3) and N-[4-[(2-bromoethyl)methylamino]-2-butynyl]-5-methyl-2- pyrrolidone (4) were synthesized. Compounds 3 and 4 cyclized in neutral aqueous solution to an aziridinium ion (4A). The rate constants for the cyclization of 3 and 4 at 37 degrees C were 0.025 and 0.89 min-1, respectively. The aziridinium ion was equipotent with carbachol as a muscarinic agonist on the isolated guinea pig ileum. It was more potent than the corresponding 2-pyrrolidone derivative (2A) in alkylating muscarinic receptors in homogenates of the rat cerebral cortex. This higher potency was due to greater receptor affinity of 4A as compared to 2A rather than to greater rate constant for alkylation of muscarinic receptors. These properties of 3 and 4 and their low toxicity should make them valuable tools for receptor inactivation studies in vivo and in vitro.     

Some differential effects of 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride on guinea-pig atria and ileum

4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (I) cyclizes at neutral pH to form an aziridinium salt. The formation and breakdown of the salt depend on the temperature (in the range 25 to 37 degrees C). In solution at 30 degrees C, peak levels, corresponding to 60-80% conversion, are reached after around 60 min and the half-life exceeds 100 min. In the presence of 0.9% NaCl conversion was reduced to 45-60%. I blocks muscarinic receptors in guinea-pig ileum and atria irreversibly and it is possible to produce dose-ratios on ileum with 10 nM I which are about 100 times those on atria. After about 30 min exposure to solutions of I (prepared 15-20 min previously so that formation of aziridinium ions is well-established) the graph of log (dose-ratio) against time is linear and similar plots were obtained with two different agonists, carbachol and ethoxyethyltrimethylammonium. With results for the ileum, extrapolation of the line suggests that it does not start from zero (dose-ratio = 1): this is because of an initial relatively rapid reversible block. This early phase is similar to that seen on ileum with 10 nM 4DAMP methobromide, which is a competitive antagonist, so is probably caused by competitive block by the aziridinium ion, which closely resembles 4DAMP metho-salts. The subsequent irreversible phase should be caused by alkylation of the receptors. I is easy to make and should be a valuable tool for the study of muscarinic receptors.     

Investigating the interaction of McN-A-343 with the M2 muscarinic receptor using its nitrogen mustard derivative

We investigated whether the aziridinium ion formed from a nitrogen mustard derivative (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; BR384) structurally related to McN-A-343 (4-(trimethyl-amino)-2-butynyl N-(3-chlorophenyl)carbamate) interacts allosterically or orthosterically with the M₂ muscarinic receptor. Chinese hamster ovary cells expressing the human M₂ muscarinic receptor were incubated with the aziridinium ion of BR384 in combination with McN-A-343 or other known orthosteric and allosteric ligands for various incubation times. After removing unreacted ligands, we measured the binding of [³H]N-methylscopolamine to residual unalkylated receptors. Affinity constants, rate constants for alkylation, and cooperativity constants were estimated for the interacting ligands using a mathematical model. Receptor alkylation by BR384 was consistent with a two-step process. After rapidly equilibrating with the receptor (step one), the aziridinium ion-receptor complex became covalently linked with a first order rate constant of about 0.95 min⁻¹ (step two). McN-A-343, acetylcholine and N-methylscopolamine competitively protected the M₂ receptor from irreversible alkylation by BR384. In contrast, the allosteric modulators, gallamine and WIN 51,708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]pyrimido[1,2-α]benzimidazole), allosterically inhibited or had no effect on, respectively, receptor alkylation by BR384. There was good agreement between affinity constants estimated from the kinetics of receptor alkylation and by displacement of [³H]N-methylscopolamine binding. Our results suggest that BR384 covalently binds to the orthosteric site of the M₂ receptor and that McN-A-343 binds reversibly at the same locus. Our method of analyzing allosteric interactions does not suffer from the limitations of more conventional approaches and can be adapted to detect allosteric interactions at receptors other than the muscarinic subtypes.     

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.     

Receptor binding profiles of some selective muscarinic antagonists

The binding of hexahydrosiladifenidol, procyclidine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) and AF-DX 116 to muscarinic receptors in the heart, ileum, urinary bladder, parotid gland and cerebral cortex from guinea pig was studied in competition experiments with (-)-[3H]QNB. The affinity of AF-DX 116 was higher in the heart than in the cortex and it was extremely low in the parotid gland. The affinities of hexahydrosiladefinidol, procyclidine and 4-DAMP were higher in the cortex and parotid gland than in the heart, bladder and ileum. Hexahydrosiladifenidol and 4-DAMP recognized two classes of muscarinic binding sites in the cortex. However, in contrast to functional data, binding results showed that 4-DAMP hexahydrosiladifenidol and procyclidine did not distinguish between the sites in the smooth muscles and those in the heart. Nevertheless, the present data support the view that the putative M2-receptors are heterogeneous, since the four drugs examined were found to distinguish between the muscarinic binding sites in the parotid gland and those in smooth muscles and heart.     

Binding properties of nine 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP) analogues to M1, M2, M3 and putative M4 muscarinic receptor subtypes.

1. We compared the binding properties of 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and nine analogues of this compound on muscarinic receptors of human neuroblastoma NB-OK1 cells (M1 subtype), rat heart (M2 subtype), rat pancreas (M3 subtype) and to the putative M4 subtype in striatum. 2. The requirements for high affinity binding were somewhat different for the four receptor subtypes. In general, the requirements of M3 receptors were more stringent than for M1, M2 or putative M4 receptors. 3. The abilities of the compounds to discriminate muscarinic receptor subtypes were not correlated with their affinities at any subtype. 4. The temperature-dependence of binding of 4-DAMP analogues to M2 receptors varied with the drug structure. In particular, the increased affinity of the alpha-methyl derivative of 4-DAMP could be ascribed to van der Waals interactions. 5. The affinities of most 4-DAMP analogues for M2 and M3 receptors were similar to their pharmacological potencies on atrial and ileum preparations, respectively. 6. At concentrations above 1 microM, all 4-DAMP analogues as well as atropine, reduced the [3H]-N-methyl scopolamine ([3H]-NMS) dissociation rate from cardiac muscarinic receptors, with no obvious structure-activity relationship.     

Characterization of muscarinic receptors mediating vasodilation in guinea-pig ileum submucosal arterioles by the use of computer-assisted videomicroscopy.

Muscarinic receptors of resistance vessels (submucosal arterioles, outside diameter 50-75 microns) from the guinea-pig small intestine were investigated in vitro using a computer-assisted videomicroscopy system (Diamtrak). The muscarinic receptor which mediates vasodilation of precontracted [U-46619 (300 nM) or (-)-noradrenaline (10 microM)] arterioles was characterized with several muscarinic agonists and subtype-selective antagonists. The following agonists all produced equivalent maximum vasodilation (given in rank order of potency): acetylcholine = arecaidine propargyl ester (APE) greater than oxotremorine = (+/-)-muscarine = (+/-)-methacholine greater than carbachol greater than 4-[[N-(4-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). 4-[[N-(3-Chlorophenyl)-carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A-343) and N-ethyl-guvacine propargyl ester (NEN-APE) produced minimal or no arteriolar vasodilation. The muscarinic antagonists pirenzepine, (+-)-5,11-dihydro-11-[[[2-[2-((dipropylamino)methyl)-1-piperidinyl] ethyl]amino]-carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one (AF-DX 384), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one (AQ-RA 741), p-fluorohexahydro-sila-difenidol (p-F-HHSiD), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and (R)- and (S)-hexahydro-difenidol [(R)-HHD, (S)-HHD] shifted the muscarine, methacholine or carbachol dose-response curve to the right in a competitive manner. Schild analysis of the data yielded pA2 values for pirenzepine (6.74/6.9), AF-DX 384 (6.72), AQ-RA 741 (6.58), p-F-HHSiD (7.53/7.57), 4-DAMP (9.06), (R)-HHD (7.88/8.32) and (S)-HHD (5.52/5.88). Thus, it can be concluded that submucosal arterioles possess only the M3 functional muscarinic receptor, the activation of which causes blood vessel dilation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.     

Muscarinic receptor subtypes in the human colon: lack of evidence for atypical subtypes

Characteristics of muscarinic receptors were investigated in circular muscle from normal human colon. In saturation studies (n=18), binding of [3H]quinuclidinyl benzylate (QNB) was of high affinity (K(d) 87.3 pM) and capacity (B(max) 362+/-27 fmol/mg protein), with no differences between ascending and sigmoid colon. Kinetic studies gave a K(d) of 55 pM. Methoctramine and darifenacin displayed biphasic binding profiles, the high affinity components being compatible with a population of approximately 80+/-5% M(2) and 13+/-2% M(3) muscarinic receptors, respectively. Pirenzepine, mamba toxin 1 and mamba toxin 3 were very weak competitors, indicating negligible expression of muscarinic M(1) and M(4) receptors. Six other subtype-preferring antagonists exhibited K(i) values typical of those reported at cloned human muscarinic M(2) receptors. In the presence of methoctramine, pre-treatment with alkylating agent 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard) inhibited [3H]quinuclidinyl benzylate binding to 26% of sites. Following alkylation of muscarinic M(3) receptors, darifenacin bound to a single low affinity site, indicating binding to muscarinic M(2) receptors.     

Evidence of paired M2 muscarinic receptors

Binding assays involving various antagonists, including N-[3H] methylscopolamine, [3H]quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat "brainstem" (medulla, pons, and colliculi). However, competition curves between N-[3H]methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of [3H]-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with [3H]quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.     

The irreversible binding of acetylcholine mustard to muscarinic receptors in intestinal smooth muscle of the guinea-pig.

1. Acetylcholine mustard (N-2-chloroethyl-N-methyl-2-acetoxyethylamine), a potent muscarinic agonist, binds virtually irreversibly to muscarinic receptors in longitudinal muscle strips from guinea-pig small intesting, as shown by the inhibition of the binding of E13-H]-propylbenzilycholine mustard ([3-H-PrBCM), an affinity label for the muscarinin receptor. 2. A value for the apparent binding affinity of acetylcholine mustard and a value for the rate constant for the receptor alkylation reaction have been deduced from the rate of onset of the inhibition of [3-H]-PrBCM binding. 3. The kinetic constants obtained may refer largely to the interaction between acetylcholine mustard and the desensitized receptor. 4. At high concentrations acetylcholine mustard practically abolishes the contractile response to carbachol. At the concentrations acetylcholine mustard appears to have multiple actions on the tissue.     

Investigating the interaction of McN-A-343 with the M1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

Background and purpose:

We investigated how McN-A-343 inhibited the alkylation of the M₁ muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically.

Experimental approach:

We incubated the M₁ muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [³H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model.

Key results:

The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [³H]N-methylscopolamine binding.

Conclusions and implications:

Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M₁ muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.     

Chimeric m2/m3 muscarinic receptors: role of carboxyl terminal receptor domains in selectivity of ligand binding and coupling to phosphoinositide hydrolysis

The cloning and expression of five mammalian muscarinic receptor genes (m1-m5) have shown that the individual receptor subtypes differ in their functional and ligand-binding properties. To study the role of the carboxyl terminal receptor domains in this pharmacological diversity, we constructed chimeric m2/m3 receptors in which a region comprising part of transmembrane domain VI, the third extracellular loop, transmembrane region VII, and the cytoplasmic tail (collectively referred to as C-terminal domains) was exchanged between the human m2 and the rat m3 receptor. The ability of the cloned receptors to mediate stimulation of phosphoinositide hydrolysis and to bind subtype-selective muscarinic ligands was studied after their transient expression in COS-7 cells. Whereas wild-type m3 strongly stimulated phosphoinositide breakdown, wild-type m2 gave only a poor response. Exchange of the C-terminal domains between m2 and m3 had no significant effect on the magnitude of these responses. In N-[3H]methylscopolamine competition binding studies, the muscarinic antagonists AF-DX 116 and methoctramine showed 11- and 23-fold higher affinities, respectively, for m2 than for m3, whereas hexahydro-silad-ifenidol (HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displayed the reverse selectivity profile, having approximately 10-fold higher affinities for m3. In comparison with wild-type m3, the mutant m3 receptor containing the C-terminal domains of m2 displayed 2.5- and 8-fold higher affinities for AF-DX 116 and methoctramine but 7- and 3-fold lower affinities for HHSiD and 4-DAMP, respectively. The mutant m2 receptor with the C-terminal domains of m3 showed 2-3-fold lower affinities for AF-DX 116 and methoctramine but 2-3-fold higher affinities for HHSiD and 4-DAMP, as compared with wild-type m2. These data suggest that the C-terminal domains of the muscarinic receptors are not involved in conferring selectivity of coupling to phosphoinositide hydrolysis but contain major structural determinants of antagonist binding selectivity.     

Kinetics of phosphoramide mustard hydrolysis in aqueous solution

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.