Population Of Muscarinic Receptors Research Articles

Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus

Ca(2+) sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca(2+) sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/W(v) mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse 'volume' neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.

Characterization of Muscarinic Receptors in the Human Bladder Mucosa: Direct Quantification of Subtypes Using 4-DAMP Mustard

To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard. Muscarinic receptors in homogenates of bladder mucosa, detrusor muscle, and parotid gland were measured using the radioligand [3H]NMS. 4-DAMP mustard was used to inactivate M3 receptors irreversibly. Specific [3H]NMS binding in the homogenates of the mucosa and detrusor muscle was saturable and of high affinity as shown by dissociation constants (Kd) of 260 ±82 and 237 ±49 pM, respectively. Antimuscarinic agents (oxybutynin, propiverine, tolterodine, and darifenacin) and their active metabolites competed with [3H]NMS for the binding sites in the human mucosa in a concentration-dependent manner. These agents exhibited similar affinity in the detrusor muscle. The Bmax. values of [3H]NMS in the detrusor, bladder mucosa, and parotid gland were significantly decreased by pretreatment with 4-DAMP mustard (36%, 41% and 63%, respectively). The density and binding affinity profile of the muscarinic receptor population in the human bladder mucosa was shown to be similar to that of the detrusor muscle. The density of the M3 subtype in the mucosa was similar to that in the detrusor muscle but lower than that in the parotid gland.

The muscarinic M 4 receptor is the functionally predominant subtype in rat and mouse striatum as demonstrated using [ 35S] GTPγS binding

We have used selective muscarinic receptor antagonists and M 2 and M 4 receptor knockout (KO) mouse tissue to define the functional muscarinic acetylcholine receptor populations in rodent striatum. [ 3H] NMS binding studies in rat and mouse striatum demonstrated that approximately 30% of muscarinic acetylcholine receptors expressed are M 1 receptors. Radioligand binding studies suggest that the remaining muscarinic acetylcholine receptor population is largely M 4 with small levels of M 2. In agreement, carbachol-induced GTPγS binding studies in M 2 and M 4 receptor KO mouse striatum implicated the M 4 receptor as the predominant functional receptor subtype. Based on these data we have developed a novel, native tissue M 4 receptor [ 35S] GTPγS binding assay. Pharmacological assessment of M 4 receptor agonist and positive 3modulators revealed clear differences in the potencies observed in a human recombinant CHO-M 4 receptor [ 35S] GTPγS binding assay as compared to the native tissue [ 35S] GTPγS binding assay. These differences are believed to reflect differences in receptor reserve between the assay systems as well as differences in compound pharmacology (relative contribution of compound affinity and efficacy to observed potency). These studies have demonstrated the importance of understanding the pharmacology of test compounds in a native environment when predicting in vivo response.

Comparison of the kinetics and extent of muscarinic M 1–M 5 receptor internalization, recycling and downregulation in Chinese hamster ovary cells

We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M 1–M 5) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [ 3H]QNB and [ 3H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M 2 > M 4 = M 5 > M 3 = M 1, respectively. Unlike the M 2 receptor, M 1, M 3, M 4 and M 5 receptors recycled back to the plasma membrane after 1 h carbachol treatment. The receptor downregulation elicited to 24 h carbachol treatment was similar for M 2, M 3, M 4 and M 5 receptors, whereas that for the M 1 receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells.

Muscarinic receptor antagonism at the spinal cord level causes inhibitory effects on male rat sexual behavior

The role of cholinergic neurotransmission in male rat sexual behavior at the brain level has been studied by several researchers. However, little is known about its role at the spinal cord level. In this study, the effects of the intrathecal (IT) administration of the muscarinic receptor antagonist subtypes (MRAs) methoctramine (Meth), tropicamide (Trop) and 4-DAMP on male rat sexual behavior were evaluated during three ejaculatory series. Meth and Trop are preferring antagonists for the M2/M4 receptor subtypes, and 4-DAMP is a preferring antagonist for the M3 receptor subtype. All the MRAs tested noticeably inhibited male rat copulatory behavior, reflected by a reduction in the number of animals engaging in sexual behavior and a gradual decrease in the number of animals able to ejaculate. Significant increases in intromission latency (IL), ejaculation latency (EL) and post-ejaculatory interval (PEI) were observed. The ranking of inhibitory potency in all recorded parameters was Meth ≥ 4-DAMP > Trop. In theory, the effects of Meth and Trop could be a result of interaction with M2/M4 receptors. However, given that the M2 receptor constitutes the greatest population of muscarinic receptors at all spinal cord sites and given the high affinity for Meth on M2 receptors, the high potency in the inhibitory effects of Meth is indicative of the special role of M2 spinal receptors in the implementation of this behavior. The weaker effects of Trop could be linked to the smaller population of M4 receptors in the spinal cord, but some interaction with M2 receptors is probable. Since some differences in the pattern of inhibitory response between Meth and 4-DAMP were observed in this and a previous study, a possible role for M3 receptors must be considered. The data obtained in this study confirm the facilitating effect of acetylcholine (ACh) at the spinal cord level on male rat sexual behavior through muscarinic mechanisms, with an important influence on ejaculatory processes. These data support the hypothesis of the modulating role of ACh on male rat sexual behavior at the spinal cord level.

Altered M1 Muscarinic Acetylcholine Receptor (CHRM1)-Gαq/11 Coupling in a Schizophrenia Endophenotype

Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M(1) subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Galpha(q/11)-[35S]-GTPgammaS binding was significantly decreased in the MRDS group (pEC(50) (M)=5.69+/-0.16) compared with the control group (6.17+/-0.10) and the non-MRDS group (6.05+/-0.07). The levels of Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPgammaS binding to Galpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with schizophrenia displaying markedly decreased PZP binding (MRDS) we have shown that although agonist potency may decrease, the efficacy of CHRM1-Galpha(q/11) coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia.

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

Mammal heart tissue has long been assumed to be the exclusive domain of the M(2) subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M(2) subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M(1) muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M(1)-M(4) receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M(2) subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M(2), with the majority of these belonging to the M(1) subtype. We were also able to detect a marginal fraction (6 +/- 2%) of receptors that, based on other findings, belong mainly to the M(5) muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M(1)-M(4) receptors and can not therefore be used as a method for M(5) muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M(1) muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M(5) muscarinic receptors but not to that of the M(3) receptors.

Pharmacological characterization of muscarinic receptors in dog isolated ciliary and urinary bladder smooth muscle.

1. The pharmacological characteristics of muscarinic receptors mediating contraction of dog isolated ciliary muscle were determined and compared to those mediating contraction of dog urinary bladder smooth muscle. 2. (+)-Cis-dioxolane induced concentration-dependent contractions of ciliary muscle (pEC50=7.18+/-0.07, Emax=453+/-64 mg, n=19) and urinary bladder isolated smooth muscle (pEC50=6.55+/-0.07, Emax=11+/-1 g, n=19). These responses were antagonized by several muscarinic receptor antagonists (pKb values for the ciliary muscle and the bladder smooth muscle, respectively): atropine (8.25+/-0.14 and 9.21+/-0.09), pirenzepine (6.31+/-0.13 and 6.70+/-0.25), tolterodine (7.97+/-0.14 and 8.68+/-0.12), oxybutynin (7.40+/-0.08 and 7.88+/-0.12), zamifenacin (6.46+/-0.19 and 7.69+/-0.11), S-secoverine (6.66+/-0.14 and 8.13+/-0.07), AQ-RA 741 (6.16+/-0.15 and 7.08+/-0.23), p-F-HHSiD (7.10+/-0.27 and 7.35+/-0.07) and responses were not antagonized by PD 102807 (up to 100 nM). 3. In urinary bladder smooth muscle, the profile of antagonist pKB values correlated significantly with pK(i) values at human recombinant m3 muscarinic receptors, suggesting that M3 muscarinic receptors mediated the response. In the ciliary muscle, a significant (P<0.01) correlation was obtained with human recombinant m3 and m5 receptors. 4. Darifenacin displayed insurmountable antagonism at receptors in the bladder. At receptors in the ciliary muscle, it exhibited two phases of antagonism, comprising an initial low affinity (pKB<6) component and a high affinity phase (pKB>8). 5. The role of pigmentation in the atypical behaviour of darifenacin was examined. In blue coloured eyes, darifenacin produced apparent surmountable, competitive antagonism of the responses to (+)-cis-dioxolane (pKB=8.76+/-0.07). The antagonist profile obtained in this tissue suggested the involvement of a site which has the pharmacological attributes of the M5 receptor. 6. We suggest that the dog urinary bladder contracts in response to M3 muscarinic receptor activation. Contraction of the brown-eyed dog ciliary muscle is more complex and may include involvement of at least two receptors, possibly the M5 and M3 receptor, whereas blue-eyed dog ciliary muscle may involve a single population of M5 muscarinic receptors.

The role of M(2)-muscarinic receptors in mediating contraction of the pig urinary bladder in vitro.

1. In urinary bladder, M(2)-muscarinic receptors predominate, but it is the smaller population of M(3)-receptors which mediate detrusor contraction. This study examines the M(2) : M(3) ratio and the role of M(2)-receptors in contraction of pig urinary bladder. 2. Competition experiments with [(3)H]-QNB determined the ratio of M(2) : M(3). In functional studies, affinity values (pK(B)) for 4-DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M(3)-inactivation (incubation with 40 nM 4-DAMP mustard in the presence of 1 microM methoctramine to protect M(2)-receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 microM) or forskolin (1 microM). 3. In competition binding, displacement of [(3)H]-QNB by 4-DAMP, darifenacin and methoctramine best fitted a two-site model suggesting a predominant (70 - 80%) population of M(2)-receptors. 4. On normal detrusor in vitro, 4-DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pK(B) values of 9.37+/-0.07 and 6.05+/-0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pK(B) value being 8.61+/-0.10. 5. In tissues where the M(3)-receptors had been inactivated and cyclic AMP levels elevated, 4-DAMP and darifenacin were less potent, with apparent pK(B) values of 8.72+/-0.08 and 6.74+/-0.07. In contrast, methoctramine was more potent, the apparent pK(B) value increasing significantly to 6.86+/-0.06. 6. se data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M(3)-receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M(2)-receptors in contraction can be observed following pharmacological manipulation of the receptor population.

Efficacy of muscarinic stimulation and mode of excitation-contraction coupling in bovine trachealis muscle

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M 3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K + channel blocker, nifedipine almost completely inhibited contractions induced by the M 3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M 3 receptor is coupled to a cellular pathway linked with the activation of K + channels that exerts a potent functional antagonism against activation of voltage-dependent Ca 2+ entry.

Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats.

1. The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. 2. Competition radioligand binding studies, using uterine membranes and [3H]-NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one-site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33+/-8 and 38+/-2%) and low (67+/-8 and 62+/-2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions. 3. Carbachol induced concentration-dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, sh.; ov.): zamifenacin (9.19; 9.18), p-F-HHSiD (8. 50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6. 26; 6.61), MTx 3 (<7.00; <7.00) and PD 102807 (<7.00; <7.00). 4. The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions. 5. Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

Effects of AF64A on the mRNA levels of muscarinic receptor subtypes in the rat iris sphincter.

The reduction of parasympathetic nerve activity by the treatment with ethylcholine mustard aziridinium ion (AF64A) in vivo induced both specific and non-specific supersensitivities in the rat iris sphincter (Tanaka et al., 1999). Changes in the expression of muscarinic receptor subtypes, which could be a cause of specific supersensitivity induced by the treatment with AF64A, were examined using competitive PCR techniques. Muscarinic receptor population is composed of m2, m3, and m4 subtypes in the rat iris (Furuta et al., 1998). Interestingly, m4 mRNA was much more abundantly expressed than m2 and m3 in the rat iris sphincter. The treatment with AF64A significantly increased the mRNA levels of m2 and m3 subtypes to 370 and 330% of the control but not that of m4 (approximately 90% of the control). In addition, the total protein contents were increased to approximately 125% of the control. The up-regulation of the mRNA levels of m2 and m3 subtypes by the treatment with AF64A was significant when they were compensated for the increase in total protein contents. The down regulation of m4 mRNA expression was not significant even after being corrected for the protein content. These results suggest that the up-regulation of the mRNA levels of m2 and m3 subtypes may be, at least in part, responsible for the supersensitivity to muscarinic agonists after the treatment with AF64A in vivo.

Subtypes of the Muscarinic Receptor in Smooth Muscle

Muscarinic receptors are expressed in smooth muscle throughout the body. In most instances, the muscarinic receptor population in smooth muscle is composed of mainly the M2 and M3 subtypes in an 80% to 20% mixture. The M3 subtype mediates phosphoinositide hydrolysis and calcium mobilization, whereas the M2 subtype mediates an inhibition of cAMP accumulation. In addition, a variety of ionic conductances are elicited by muscarinic receptors. Muscarinic agonists stimulate a nonselective cation conductance that is pertussis toxin-sensitive and dependent on calcium. The pertussis toxin-sensitivity of this response suggests that it is mediated by M2 receptors. Following agonist induced depolarization of smooth muscle, voltage dependent calcium channels are activated to enable an influx of calcium. In some instances, muscarinic agonists enhance this conductance through a mechanism involving protein kinase C, whereas in other instances, muscarinic agonists suppress this calcium conductance. Smooth muscle often contains calcium activated potassium channels that tend to repolarize the membrane following calcium influx. Activation of muscarinic receptors suppresses this potassium conductance in some smooth muscles. Under standard conditions, muscarinic agonists elicit pertussis toxin-insensitive contractions through activation of the M3 receptor. When most of the M3 receptors are inactivated, it is possible to measure a pertussis toxin-sensitive contractile response to muscarinic agonists that is most likely mediated through M2 receptors. M2 receptors also cause an indirect contraction by inhibiting the relaxant effects of agents that increase cAMP (e.g., forskolin and isoproterenol).

Effect of S-adenosyl methionine on muscarinic receptors in young rats.

In the present study we have investigated the effect of a chronic administration of S-Adenosyl Methionine (SAM) on muscarinic receptor subtypes in young rat forebrain, cerebellum, heart and lacrimal gland. Saturation binding experiments were performed using 3H-N-methylscopolamine (3H-NMS) to label the total population of muscarinic receptors in plasma membranes from forebrain, cerebellum, heart and lacrimal gland. 3H-Pirenzepine (3H-Pz) was used to label the M1 subtype in plasma membranes from forebrain. The results obtained in cerebellum, heart and lacrimal gland show no changes in the affinity (Kd) nor in the number of receptors (Bmax) of the treated versus control groups. Saturation experiments in forebrain show an increase in the number of receptors of the treated versus control groups when using 3H-NMS (Bmax 2117 +/- 63 versus 1643 +/- 104 fmol/mg protein) without changes in the affinity. Saturation experiments with 3H-Pz, show an increase in the number of M1 receptors in the treated group with no changes in the affinity (Bmax 421 +/- 16 versus 225 +/- 19 fmol/mg protein). From our results, we conclude that SAM increase the number of receptors in forebrain and this increase is mainly due to changes in the number of M1 receptor subtypes.

Functional interactions between muscarinic M2 receptors and 5-hydroxytryptamine (5-HT)4 receptors and beta 3-adrenoceptors in isolated oesophageal muscularis mucosae of the rat.

1. Relaxations of isolated oesophageal muscularis mucosae of rat are mediated by 5-hydroxytryptamine (5-HT), acting at 5-HT4 receptors, and isoprenaline, principally acting via beta 3-adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M2 receptors, also present in this tissue, functionally oppose 5-HT and beta-adrenoceptor-relaxant effects in this preparation. 2. Contractions of rat oesophageal muscularis mucosae were induced, in a concentration-dependent manner, by the muscarinic receptor agonist, oxotremorine M (pEC50 = 6.7 +/- 0.1). The contractile responses to oxotremorine M were surmountably antagonized by the following compounds, (pKB values in parentheses): atropine (9.1 +/- 0.2), 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide, 8.7 +/- 0.1), p-F-HHSiD (para-fluoro-hexa-hydro-siladifenidol, 7.5 +/- 0.1), zamifenacin (8.6 +/- 0.3), himbacine (7.2 +/- 0.2), pirenzepine (6.8 +/- 0.3) and methoctramine (6.2 +/- 0.2). These data are consistent with a role for muscarinic M3 receptors mediating contractions to oxotremorine M. The contractile response was associated with a low receptor reserve, since the responses were shifted to the right and virtually abolished by the alkylating agent, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl) piperidine, 40 nM; 60 min equilibration). 3. In tissues precontracted with U46619 (0.7 microM; approx. EC90), isoprenaline (pEC50 = 8.0 +/- 0.1) and 5-HT (pEC50 = 7.5 +/- 0.2) induced concentration-dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine M (isoprenaline, pEC50 = 7.4 +/- 0.2). In contrast, the potency of 5-HT (pEC50 = 7.5 +/- 0.2), in tissues that were precontracted with 1 microM (EC90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M2 receptor antagonist, methoctramine (1 microM), there was no effect on the relaxant potencies to either 5-HT or isoprenaline. Collectively, these data suggest that muscarinic M2 receptors do not, under these conditions, modulate relaxant potencies to either 5-HT or isoprenaline. 4. In a second protocol, tissues were pre-contracted with U46619 (0.7 microM) and relaxed with either 5-HT (0.1 microM) or isoprenaline (0.1 microM). In these tissues (in which the muscarinic M3 receptor population was extensively depleted by alkylation), oxotremorine M caused concentration-dependent re-contractions (i.e. reversal of relaxations). In tissues relaxed with 5-HT, the potency of oxtremorine M was 5.9 +/- 0.2, while in tissues relaxed with isoprenaline, the potency (pEC50) = 5.6 +/- 0.3. These re-contractions were antagonized, in a surmountable fashion, by methoctramine (1 microM; pKB = 7.6 +/- 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 microM; pKB = 7.5 +/- 0.2). Under these conditions, therefore, the pKB values are consistent with activation of muscarinic M2 receptors, and inconsistent with activation of M3 receptors. 5. It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M3 receptors mediate direct contractions and are associated with a low receptor reserve. When this population is depleted, and the tissues relaxed via activation of receptors that augment adenylyl cyclase activity, a functional role for muscarinic M2 receptors is revealed.

Muscarinic receptor heterogeneity in neonatal rat ventricular myocytes in culture.

Carbachol increased ventricular automaticity in a concentration-dependent fashion from a control rate of 72 +/- 5 (mean +/- SEM) to 86 +/- 4 beats per minute at 10(-4) M carbachol. Pirenzepine, an M1-selective antagonist, and AFDX 116, an M2-selective antagonist, both at 10(-7) M, did not block the carbachol-induced positive chronotropic response. In contrast, 10(-7) M HHSiD, an M3-selective antagonist, completely blocked the positive chronotropic effect of carbachol. Carbachol stimulated the accumulation of IP1 in a concentration-dependent manner at concentrations > or = 3 x 10(-6) M. AFDX 116 had no effect on carbachol-induced IP1 accumulation. HHSiD significantly inhibited IP1 accumulation at concentrations > or = 3 x 10(-8) M, while pirenzepine inhibited IP1 accumulation only at concentrations > or = 10(-5) M. McN A343 and methacholine, two muscarinic receptor agonists with minimal M2 activities, and carbachol did not alter basal cAMP concentration, but all three agonists significantly attenuated the increase in cAMP accumulation in response to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations > or = 10(-7) M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrations, the inhibitory effects of HHSiD and AFDX-116 were similar, while that of pirenzepine was much less. Pretreatment with pertussis toxin for 24 h did not prevent the carbachol-mediated positive chronotropic response or accumulation of IP1 but completely abolished the inhibition of isoproterenol-stimulated cAMP accumulation. These results indicate that (a) neonatal ventricular myocytes in culture have a heterogeneous population of muscarinic (M2 and M3) receptors, (b) the M3 receptor is coupled to pertussis toxin-sensitive and pertussis toxin-insensitive G proteins, (c) M3 receptor stimulation activates phosphoinositide hydrolysis and increases automaticity via a pertussis toxin-insensitive G protein-dependent pathway, and (d) both M2 and M3 receptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproterenol stimulation.

Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08. 5. The pharmacological profile of antagonist affinities at the muscarinic receptor population responding to muscarinic agonist-evoked contraction is similar to that widely accepted as characterizing the activation of an M3 muscarinic receptor subtype, although pA2 values of some antagonists are lower than that seen in other investigations.

Characterization of muscarinic receptors in the rat nucleus tractus solitarius

Crude membrane preparations from the dorsomedial medulla and whole heart of adult rats were used to characterize muscarinic receptors (mChRs) in the nucleus tractus solitarius (NTS). Isotherm binding assays with [ 3H]quinuclidinyl benzilate ([ 3H]QNB) indicated a uniform population of muscarinic cholinergic receptors in both tissues with a K d of 60.3 pM and a B max of 401 fmol/mg protein for NTS membranes while heart membranes had a K d of 56.6 pM and a B max of 193 fmol/mg protein. Competitive binding assays with selective muscarinic antagonists on NTS membranes using 80 pM [ 3H]QNB showed the following relative K i values consistent with M2 mChRs: atropine, 1.9 nM; methoctramine, 7.7 nM; 4-diphenylacetoxy- N-methylpiperidine methobromide, 9.4 nM; AF-DX 116, 87.6 nM; and pirenzepine, 657 nM. Similarly, two agonists, carbachol and oxotremorine, showed K i values of 1.82 μM and 0.379 μM, respectively. All of the competitive binding assays exhibited Hill coefficients close to 1.0 and InPlot analysis showed a single binding site for each agonist and antagonist examined.

Role of muscarinic M 2 and M 3 receptors in guinea-pig trachea: effects of receptor alkylation

Muscarinic M 2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M 3 receptor population that mediates contraction of many of these tissues. The role of the majority of M 2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M 2 receptors has been demonstrated in tissues subjected to M 3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M 2 and M 3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M 2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M 2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M 3 receptor alkylation. Our results suggest (i) that there is no evidence of M 2 receptor-mediated re-contraction of tracheal smooth muscle after M 3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M 2 receptors, after M 3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M 2 receptors in guinea-pig trachea remains to be determined.

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.