Human Muscarinic Receptor Subtypes Research Articles

Identification and characterization of m1 selective muscarinic receptor antagonists1.

A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been synthesized and characterized as potential m1 selective muscarinic receptor antagonists. The affinity of these compounds for the five human muscarinic receptor subtypes (Hm1-Hm5) was determined by the displacement of [3H]-NMS binding using membranes from transfected Chinese hamster ovarian cells. One of the most potent and selective compounds of this series is an analogue of I [11, R1 = (CH2)5CH3], which has an IC50 value of 27.3 nM at the m1 receptor and possesses 100-fold (m2), 48-fold (m3), 74-fold (m4), and 19-fold (m5) selectivities at the other receptors. Thus, this analogue appears to be more selective on the basis of binding than the prototypical m1 antagonist, pirenzepine. Functional data, such as the inhibition of carbachol-stimulated phosphatidylinositol hydrolysis, on selected analogues confirmed the muscarinic antagonistic properties of this chemical series.

Stereoselective Increase in Cholinergic Transmission by R-(+)-hyoscyamine

R-(+)-hyoscyamine, the dextro enantiomer of atropine, has been shown to amplify cholinergic transmission. R-(+)-hyoscyamine, unlike S-(—)-hyoscyamine, was able to increase acetylcholine release both in vitro and in vivo at a range of concentrations (10 −14 to 10 −12 M) and doses (5 μg/kg i.p.) which were inadequate for blocking muscarinic receptors. The increase over control values in ACh release was 15.9 ± 2.1% in in vitro experiments performed in rat phrenic nerve-hemidiaphragm preparations ( n = 6), and 63.3 + 16.3% in cortical microdialysis performed in free-moving rats ( n = 5). The maximum ACh release was reached 60 min after R-(+)-hyoscyamine administration in in vivo experiments. At the same doses and concentrations, R-(+)-hyoscyamine was also able to elicit: antinociception of a cholinergic type (55.6–112.7% depending on the test used); complete prevention of scopolamine- and dicyclomine-induced amnesia; potentiation of muscular contractions electrically evoked in isolated guinea-pig ileum (16.7 ± 3.6%) and in rat phrenic nerve-hemidiaphragm (19.9 ± 3.2%) preparations. Antinociception was performed using the hot-plate and acetic acid abdominal constriction tests in mice, and the paw pressure test in rats, while prevention of induced amnesia was evaluated in mice using the passive-avoidance test. The respective affinities (p A 2) for R-(+)- and S-(—)-hyoscyamine vs M 1 (rabbit vas deferens), M 2 (rat atrium) and M 3 (rat ileum) receptor subtypes were as follows: 7.05 ± 0.05/9.33 ± 0.03 for M 1; 7.25 ± 0.04/8.95 ± 0.01 for M 2; 6.88 ± 0.05/9.04 ± 0.03 for M 3. The respective p K i values for R-(+)- and S-(—)-hyoscyamine vs the five human muscarinic receptor subtypes expressed in Chinese hamster oocytes (CHO-K 1) were as follows: 8.21 ± 0.07/9.48 ± 0.18 for m 1; 7.89 ± 0.06/9.45 ± 0.31 for m 2; 8.06 ± 0.18/9.30 ± 0.19 for m 3; 8.35 ± 0.11/9.55 ± 0.13 for m 4; 8.17 ± 0.08/9.24 ± 0.30 for m 5. © 1997 Elsevier Science Ltd. All rights reserved.

Evidence for the Presence of Regional Differences in the Subtype Specificity of Muscarinic Receptors in Rabbit Lower Urinary Tract

To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

WAL 2014 FU (talsaclidine): A preferentially neuron activating muscarinic agonist for the treatment of Alzheimer's disease

The functional selectivity of WAL 2014 FU with regard to stimulation of the neuronal muscarinic M1 receptor subtype in vitro and in vivo is shown in different receptor preparations, isolated organ models, whole animal testing, and finally humans. From receptor binding experiments in membrane preparations from rat tissues and from Chinese hamster ovary cells expressing human muscarinic receptor subtypes, it can be delineated that the ratio between M1 and M2 (hm1 and hm2) is shifted in favour of the M1 receptor affinity, when compared to several classic muscarinic agonists such as carbachol, arecoline, and oxotremorine. The intermediate GTP-shift of 7.5 for WAL 2014 FU in a M2 muscarinic receptor preparation (rat heart) indicates only partial agonistic activity at this subtype, carbachol (e.g., shows a shift of 51). Moreover, the ratio from agonist to antagonist receptor binding comparing the affinities using [3H]cis-methyldioxolane and [3H]N-methylscopolamine as radioligands, suggests only a partial agonist behaviour at M2 receptors, too. In functional assays in vitro, such as phosphoinositide breakdown measured in cerebral cortex slices from guinea pig brain, WAL 2014 FU is as effective (27.8% compared to carbachol 100%) as arecoline (27.6%), but is more effective than RS 86 (22.1%), oxotremorine (21.6%) or McN-A-343 (14.7%). WAL 2014 FU is about as effective as RS 86 and McN-A-343 in stimulating the secretion of N-acetyl-glucosaminidase from RBL cells transfected with the human m1 receptor subtype. Investigating the proton efflux from CHO cells expressing hm1, hm2, and hm3 muscarinic receptor with cytosensor technology WAL 2014 FU was clearly more effective at human m1 receptors (66.3% compared to carbachol 100%) than at hm3 receptors (18.6%) and showed no measurable effect at hm2 receptor expressing cells. The efficacy in isolated organ preparations and in whole animals has already been reported [Ensinger et al., 1993] and shows the functional selectivity of WAL 2014 FU quite impressively. The general receptor profile of WAL 2014 FU demonstrates a nearly specific interaction with muscarinic receptors, having only weak binding affinity for a1- and nicotinic receptors. Results from pharmacokinetic studies in rats and humans indicate high oral bioavailability (>95% in rats) and very low interindividual variation in plasma levels. Furthermore, good penetration of the blood brain barrier with 2.5- to 3-fold higher concentrations of WAL 2014 FU in brain than in plasma could be measured. The advantageous and stable pharmacokinetic profile of WAL 2014 FU and its functional M1 selectivity establish WAL 2014 FU as a promising candidate for drug treatment of Alzheimer's disease. In addition to its role for symptomatic treatment, WAL 2014 FU may also have a disease-modifying potential because of its stimulatory effects on APPs secretion. Drug Dev. Res. 40:144–157, 1997. © 1997 Wiley-Liss, Inc.

P-444 - Binding affinities of muscarinic antagonists for human muscarinic receptor subtypes expressed in Sf9 insect cells and muscarinic receptors in rat peripheral organs

P-444 - Binding affinities of muscarinic antagonists for human muscarinic receptor subtypes expressed in Sf9 insect cells and muscarinic receptors in rat peripheral organs

Pharmacology of muscarinic acetylcholine receptor subtypes (m1–m5): high throughput assays in mammalian cells

Based on the ability of many receptors to amplify NIH 3T3 cells, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme β-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. We used this assay to evaluate the pharmacology of agonist and antagonist ligands for five cloned human muscarinic receptor subtypes (m1–m5). When cells were transfected with subtypes that prefer the G-protein G q (m1, m3, m5) robust increases in enzyme activity were observed. The subtypes that prefer G i (m2 and m4) only induced β-galactosidase when co-expressed with a chimera between the G-proteins G q and G i (G q−i5). Overall, the rank-order of potency and intrinsic activity of most of the tested ligands were in remarkably good agreement with earlier results using cloned cell lines and isolated tissues. These data demonstrate that a high throughput colorimetric assay performed in 96-well plates can be used to evaluate subtle differences the pharmacology of ligands for cloned muscarinic receptor subtypes.

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 microM sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N-[3H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 microM. Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [3H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.

Binding of muscarinic toxins MT×1 and MT×2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M, subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3 H- N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M 1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for ml receptors: K for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and K i for MTx2 were 364 nM at m1 and 1.2μM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M 1-agonists M cN-A-343 and CPCP (4-[ N-(4-chlorophenyl)carba-moyloxy]-4-pent-2-ynyl-trimethylammonium iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors apears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.

Antimuscarinic properties of vamicamide, a novel compound for the treatment of pollakiuria

AbstractThe antimuscarinic profile of vamicamide, a novel compound for the treatment of pollakiuria, was investigated in both in vitro and in vivo preparations. Vamicamide (10 nM–10 μM) inhibited the contractile response of isolated guinea‐pig detrusor muscle to transmural electrical stimulation. In isolated guinea‐pig detrusor muscle, vamicamide also inhibited contractile response to carbachol with an IC50 value of 0.71 μM, whereas it had little or no effect on detrusor contractions induced by KCl, BaCl2, or α, β‐methylene ATP. In binding studies with dog membrane preparations, the dissociation constant (Ki) of vamicamide against tritiated N‐methylscopolamine ([3H]NMS) binding was 172 ± 8 nM for the urinary bladder, which was lower (P < 0.01) than those for the heart (322 ± 31 nM), stomach (317 ± 29 nM), and salivary gland (321 ± 32 nM). The Ki value for the cerebral cortex (95 ± 4 nM) tended to be lower than that for the urinary bladder. In binding studies with cloned human muscarinic receptor subtypes (m1–m3), the dissociation constant (Ki) of vamicamide against [3H]NMS binding was 89.0 ± 3.5 nM for the m3 receptor subtype, which was lower than those for the m1 (235 ± 5 nM, P < 0.05) and m2 (593 ± 49 nM, P < 0.01) receptor subtypes. In anesthetized dogs, vamicamide (3.2–100 μg/kg; 11–336 nmol/kg, iv) inhibited the carbachol‐induced contractile responses of the urinary bladder with an ID50 value of 16.1 μg/kg (54 nmol/kg), the stomach with a value of 31.3 μg/kg (105 nmol/kg), the descending colon with a value of 10.1 μg/kg (34 nmol/kg), and secretory response of the salivary gland to carbachol with a value of 43.3 μg/kg (146 nmol/kg); the inhibitory effects of the compound on the stomach and salivary gland were weaker (P < 0.01) than that on the urinary bladder. Furthermore, duration of the action of vamicamide was longer on the urinary bladder and descending colon than those on the stomach and salivary gland. These results suggest that vamicamide has a selective binding affinity to the muscarinic m3 receptor subtype and exhibits greater and longer inhibitory action on the urinary bladder than the other organs examined. © Wiley‐Liss, Inc.

Binding profile of the selective muscarinic receptor antagonist tripitramine

The binding selectivity of the muscarinic antagonist tripitramine has been tested on the five cloned human muscarinic receptor subtypes (Hm 1 to Hm 5) expressed in chinese hamster ovary (CHO-K1) cells. The results indicate that tripitramine binds to the muscarinic Hm 2 receptor with a K i value of 0.27 ± 0.02 nM. Tripitramine distinguishes Hm 2 vs. Hm 4 by a factor of 24 and vs. Hm 3 and Hm 5 by a factor of 142 and 125, respectively. A lower affinity ratio, about 6-fold, was found between muscarinic Hm 2 and Hm 1 receptors. A comparative study with the well-known selective muscarinic M 2 receptor antagonist methoctramine indicates that tripitramine has gained both potency and selectivity for the muscarinic Hm 2 receptor subtype.

Antagonism of the five cloned human muscarinic cholinergic receptors expressed in CHO-K1 cells by antidepressants and antihistaminics

Based on molecular cloning studies, five different muscarinic receptor subtypes exist: m1, m2, m3, m4, and m5. We determined the affinity and selectivity of binding for sixteen antidepressants, two of their metabolites, and three antihistaminics (h 1) at these subtypes. Using Chinese hamster ovary cells (CHO-K1) transfected with genes for the human muscarinic receptor subtypes, we obtained equilibrium dissociation constants ( K d s) from competitive radioligand binding studies with [ 3H]-quinuclidinyl benzilate ([ 3H]QNB) and membranal preparations of these cells. QNB was the most potent compound studied ( K d 30–80 pM). Mequitazine ( K d 6–14 nM) and amitriptyline ( K d 7–16 nM) exhibited the highest affinity among the antihistaminics and antidepressants, respectively. Among the antidepressants examined were the serotonin-selective drugs sertraline and fluoxetine, both of which displayed K d values > 1 μM. The remaining antidepressants were moderate to weak antagonists with some eliciting no radioligand competition at high concentrations. The compounds studied showed no significant selectivity among the five cloned subtypes.

Mutations of aspartate 103 in human muscarinic receptor subtype m2 expressed in cos-7 cells: Effects on receptor binding and signal transduction

Mutations of aspartate 103 in human muscarinic receptor subtype m2 expressed in cos-7 cells: Effects on receptor binding and signal transduction

A Method for Radioligand Binding Assays Using a Robotic Workstation

Radioligand binding assays provide a powerful tool for screening drug candidates at many receptors. We present a method utilizing a robotic workstation, the Biomek 1000, which automates the tedious and repetitive tasks of these assays. First, the robot handles the serial dilution of up to 8 drugs with 11 concentrations per drug. The sequential addition of diluting buffer, non-radioactively labeled ligand, radioactive ligand, and finally the tissue homogenate or membrane preparation, is fully automated. A novel rack design allows the use of tubes with a maximum capacity of 2 ml, providing a total assay volume of 1 ml. Final filtration on a Brandel cell harvester outfitted with a uniquely designed head allows for processing of 48 samples simultaneously from the rack holder. We have employed this method for the determination of equilibrium dissociation constants (Kds) for drugs at the 5 human muscarinic acetylcholine receptor subtypes expressed in cultured cells, as well as histamine H1, dopamine D2, serotonin 5HT1A, alpha 1- and alpha 2-adrenergic receptors in human brain tissue homogenates. Our results compare favorably with manual methods reported for these receptors, and exhibit a very high degree of reproducibility and throughput, with a minimum of operator input.

Comparisons between human muscarinic receptor subtypes coupled to phospholipase C and those coupled to adenylyl cyclase: Effect of receptor reserve

Comparisons between human muscarinic receptor subtypes coupled to phospholipase C and those coupled to adenylyl cyclase: Effect of receptor reserve