Distribution Of Muscarinic Receptors Research Articles

Assessing the effect of breed, age, and sex on muscarinic receptor distribution in atria and ventricles of turkeys

Atrial and ventricular myocardium from young (6-wk-old), young adult (3-6-mo-old), and aged (14-15-mo-old) meat-type (B.U.T. Big 6) and wild-type (Cröllwitzer) turkeys were used to study the influence of age and sex on cholinergic muscarinic receptors using [3H]-N-methyl-scopolamine (3H-NMS) binding studies. In both breeds, saturation experiments indicated the presence of regional-, sex-, and age-related differences in the density of cholinergic muscarinic receptors (Bmax), that is, a decrease or increase. Except for right atria, Bmax was decreased in both male and female B.U.T. Big 6 hearts with increasing age. Similarly, a negative correlation between Bmax and age could be seen in female and male atria of Cröllwitzer turkeys, while positive correlation could be seen in right and left ventricles of male, and only right ventricles of female Cröllwitzer turkeys. The affinity of the receptor (KD) was not affected by age, sex and breed. In all cardiac chamber tissues, the M2-subtype was shown to be predominant followed by the M3-subtype and to a lesser extent the M1-subtype. Aspects of this age-dependent remodeling of the heart differ between sexes, resulting in maladaptive changes in older turkeys with a high degree of frailty. These observations may help explain why males and females are susceptible to different cardiovascular diseases as they age and why frail older adults are most often affected by these diseases.

P90 | Differential breed‐, age‐ and sex‐regulated muscarinic receptor distribution in atria and ventricles of turkeys

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 128-129 POSTER PRESENTATIONS P90 | Differential breed-, age- and sex-regulated muscarinic receptor distribution in atria and ventricles of turkeys First published: 11 June 2023 https://doi.org/10.1111/jvp.13369Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 128-129 RelatedInformation

Expression and Potential Clinical Significance of Urothelial Cytodifferentiation Markers in the Exstrophic Bladder

We characterize the urothelium from patients with classic bladder exstrophy-epispadias complex for the expression of proteins associated with urothelial differentiation, and discuss a potential impact of urothelial phenotype on the structural and functional properties of the bladder template following bladder closure. From 2005 to 2010 bladder biopsies from 32 infants with bladder exstrophy-epispadias complex obtained at primary bladder closure were collected. After histological assessment immunochemistry was used to investigate the expression of uroplakin IIIa, cytokeratin differentiation restricted antigens CK13 and CK20, and tight junction protein claudin 4. Overall tissue morphology showed gross alterations with inflammatory, proliferative and metaplastic changes in most specimens. Sections of intact epithelium were present in 78% of biopsies. With respect to urothelial phenotype, CK13 was expressed in all specimens, whereas UPIIIa and CK20 were absent in 76% of the tissues examined. Of the biopsies 52% revealed an irregular expression pattern of tight junction protein Cl-4. This is the first study to our knowledge to characterize the urothelium from infants with bladder exstrophy-epispadias complex for the expression of urothelial differentiation associated antigens. Our findings suggest urothelial differentiation changes in a majority of exstrophic bladders, at least at primary bladder closure. Although the underlying etiology remains to be established, abnormal urothelial differentiation may result in a dysfunctional urothelial barrier with implications for the structural and functional properties of the bladder template. Despite the study limitations, our preliminary findings provide a platform for further investigation of the significance of the urothelium for the exstrophic bladder.

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.

Muscarinic receptors on rat ileal villus and crypt cells.

Ileal villus and crypt cells exhibit morphological and biochemical differences which may be responsible for functional differences in relation to ion transport. Cholinergic agonists act directly on epithelial cell muscarinic receptors, but it is not known if these receptors exist on both villus and crypt cells. Using the potent muscarinic antagonist [3H](-)-quinuclidinyl benzilate (QNB) we have determined the distribution of muscarinic receptors in rat ileal villus and crypt cells. Plasma membrane preparations from ileal villus and crypt cells possessed a specific, saturable, and high affinity QNB binding site with apparent dissociation constants of 0.23 +/- 0.05 and 0.21 +/- 0.04 nM (mean +/- s.e., n = 6) and densities of 92.2 +/- 2.8 and 90.1 +/- 16.2 fmol (mg protein)-1, respectively. Both types of cells showed similar potencies for agonist and antagonist competition of QNB binding. The muscarinic receptors in membrane fractions from villus cells were found primarily on the basolateral membrane rather than on the brush border membrane. Secretion induced by cholinergic stimulation of the small intestine might, therefore, be due to an effect on both villus and crypt cells as both types contain muscarinic receptors. Furthermore, such stimulation also may result in mucin secretion, as goblet cells were present in the preparation we studied, and receptors on these cells may have contributed to the amount of binding of [3H]QNB.

Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

The ideal antiserum for immunohistochemical (IHC) applications contains monospecific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are "affinity" purified, but it is unknown if they meet "hard" specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. "Certified" antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers.

Antimuscarinics for the treatment of overactive bladder: A review of central nervous system effects

Antimuscarinic drugs commonly used to treat overactive bladder are often associated with central nervous system (CNS) side effects including cognitive dysfunction, memory impairment, dizziness, fatigue, and headache. New agents show reduced CNS penetrance and better selectivity for the M3 muscarinic receptor. However, changes associated with aging may lead to alterations in blood-brain barrier permeability. Therefore, use of antimuscarinics in the elderly or in patients with Alzheimer’s disease presents a significant challenge. This review highlights muscarinic receptor distribution and function in the CNS, provides a description and incidence of CNS side effects with therapy, offers information specific to currently available agents, and describes the use of antimuscarinics in special populations including children, the elderly, and patients with Alzheimer’s disease.

Antimuscarinics for the treatment of overactive bladder: A review of central nervous system effects

Antimuscarinic drugs commonly used to treat overactive bladder are often associated with central nervous system (CNS) side effects including cognitive dysfunction, memory impairment, dizziness, fatigue, and headache. New agents show reduced CNS penetrance and better selectivity for the M3 muscarinic receptor. However, changes associated with aging may lead to alterations in blood-brain barrier permeability. Therefore, use of antimuscarinics in the elderly or in patients with Alzheimer's disease presents a significant challenge. This review highlights muscarinic receptor distribution and function in the CNS, provides a description and incidence of CNS side effects with therapy, offers information specific to currently available agents, and describes the use of antimuscarinics in special populations including children, the elderly, and patients with Alzheimer's disease.

M1 Receptors Play a Central Role in Modulating AD-like Pathology in Transgenic Mice

We investigated the therapeutic efficacy of the selective M1 muscarinic agonist AF267B in the 3xTg-AD model of Alzheimer disease. AF267B administration rescued the cognitive deficits in a spatial task but not contextual fear conditioning. The effect of AF267B on cognition predicted the neuropathological outcome, as both the Abeta and tau pathologies were reduced in the hippocampus and cortex, but not in the amygdala. The mechanism underlying the effect on the Abeta pathology was caused by the selective activation of ADAM17, thereby shifting APP processing toward the nonamyloidogenic pathway, whereas the reduction in tau pathology is mediated by decreased GSK3beta activity. We further demonstrate that administration of dicyclomine, an M1 antagonist, exacerbates the Abeta and tau pathologies. In conclusion, AF267B represents a peripherally administered low molecular weight compound to attenuate the major hallmarks of AD and to reverse deficits in cognition. Therefore, selective M1 agonists may be efficacious for the treatment of AD.

Muscarinic receptors in schizophrenia.

An increasing body of evidence suggests that the muscarinic receptors may present a potential therapeutic target for the treatment of schizophrenia. This argument is supported by studies using postmortem CNS tissue and a neuroimaging study that have shown there are regionally specific decreases in selective muscarinic receptors in the CNS of subjects with schizophrenia. This raises the possibility that drugs specific to individual muscarinic receptors could have beneficial effects on the symptoms of schizophrenia, a posit supported by studies in receptor knockout/knockdown mice where it has been shown that specific behaviours affected by schizophrenia are also abnormal in mice lacking a single muscarinic receptor. Moreover, drugs have been synthesised that are partial agonists at muscarinic receptors and these drugs have been shown to improve the behavioural deficits in humans which are modulated by the muscarinic receptor family. The widespread distribution of muscarinic receptors in the human CNS and the receptor specific changes identified in postmortem CNS from subjects with schizophrenia would suggest that drugs targeting specific muscarinic receptors would also need to partition into selected CNS regions to achieve optimal responses. Some existing compounds show regional selectivity for the same muscarinic receptor in different CNS regions, suggesting that this characteristic could be engineered into muscarinic receptor targeting drugs. This review presents data from diverse areas of research to argue that it is now imperative that the therapeutic potential of manipulating the activity of muscarinic receptors for the treatment of schizophrenia is fully explored.

In vivo muscarinic 2 receptor imaging in cognitively normal young and older volunteers

The precise effects of normal aging on the cholinergic system are unknown, as both in vitro and PET studies have shown conflicting results. In vivo determination of muscarinic receptor distribution and density has been hampered by both poor subtype selectivity and/or blood-brain barrier permeability of known ligands. Previous in vitro and in vivo work with the F-18 labeled muscarinic agonist, 3-(3- (3-[(18)F]Flouropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine ((18)FP-TZTP) suggested the use of (18)FP-TZTP to selectively quantify M2 receptors in humans. In this study, we used (18)FP-TZTP to infer M2 receptor avidity in the brains of 15 healthy younger subjects (mean age = 28.3 +/- 5.5 years) and 20 healthy older subjects (mean age = 62.1 +/- 7.7 years). Corrections for subject motion during the 120-min acquisition and partial voluming (PVC) were performed. A one-tissue compartment model was used to estimate the volumes of distribution (V(T)) of (18)FP-TZTP. Within both groups of subjects, volumes of distribution (K(1)/k(2)) in cortical, subcortical, and cerebellar areas were consistent with M2 receptor topography. Compared to younger subjects older subjects had significantly higher means and standard deviations for the volumes of distribution of (18)FP-TZTP throughout much of the cerebellum, cortex, and subcortex (Global Gray V(T) = 742 +/- 163 in older subjects and 645 +/- 74 in younger subjects, P < 0.03). Across all subjects (18)FP-TZTP, regional, and Global Gray distribution volumes were significantly correlated to age (Global Gray V(T,) r = 0.41, P < 0.01). A lower concentration of acetylcholine in the synapse of some older subjects is one possible explanation for the data.

Muscarinic receptors in equine airways.

The distribution of muscarinic receptors in equine airways was investigated using autoradiography. Frozen sections of tissue from six different levels in the bronchial tree, from the trachea to the distal bronchioles, were incubated in vitro with 1.5 nmol/L of the muscarinic receptor antagonist 1-[N-methyl-3H]scopolamine methyl chloride (3H-NMS). In addition, the subtype pattern of muscarinic receptors was investigated in equine tracheal smooth muscle using radioligand binding with methoctramine, tripinamidc, 4-DAMP-methiodide and pirenzipine as competitors against the binding of 1.3 nmol/L 3H-NMS. The autoradiograms showed specific labelling indicating a high density of muscarinic receptors in smooth-muscle tissue in all levels of the airway tree investigated. Besides muscle tissue, subepithelial glands were the only structures specifically labelled. The dominating subtypes in tracheal smooth muscle investigated with radioligand binding studies were found to be M2 and M4, as both methoctramine (pKd = 8.5) and tripinamide (pKd = 8.6 and 6.7 for two different sites) showed high affinity. The density of the M3-muscarinic receptor subtype was low, but this subtype could be detected with statistical significance when methoctramine was used as the competitor against 3H-NMS binding.

Differential distribution of ionic channels and muscarinic receptors at the cat tracheal neuromuscular junction

The ionic and pharmacological properties of atropine-sensitive excitatory junction potentials (EJPs) were investigated by the double sucrose gap and microelectrode membrane potential recording methods, and compared with those of inward currents evoked by carbachol ( I CCh), in cat tracheal smooth muscle. A single and repetitive field stimulation (10–30 V, 50 μs, 20 Hz) evoked atropine-inhibitable EJPs and associated twitch-like contractions. Reduction in external Na + concentration strongly, but in the external Cl − concentration, decreased the EJP amplitude after 5 min superfusion, although prolonged exposure to low Cl − solutions attenuated the EJPs modestly. Chloride channel blockers such as 9-AC and niflumic acid (each 100 μ M), at concentrations high enough to inhibit I CCh almost completely, failed to abolish the EJPs. Pirenzepine, AF-DX116 and 4-DAMP all effectively inhibited the EJPs at their concentrations to block respective muscarinic receptor subtypes relatively specifically, while depletion of internal stores by 10 mM caffeine and/or 3 μM ryanodine caused only a partial decrease in the EJP amplitude. These properties are considerably different from those of I CCh which is activated exclusively through activation of the M 3 receptor/IP 3-mediated Ca 2+ release pathway and reflects mostly a Ca 2+-dependent Cl − current, and suggests the differential distribution of muscarinic receptors and ionic channels inside and outside of the cholinergic neuromuscular junction of this muscle.

Acetylcholine synthesis and neuron differentiation.

Development of the nervous system is dependent on the co-operation between cell determination events and the action of epigenetic factors; in addition to well known factors, e.g. growth factors, neurotransmitters have been assigned a role as "morphogens" and modulators of neuronal differentiation in an early developmental phase. The possible role of acetylcholine as a modulator of neuronal differentiation has been considered in two experimental systems. A neuroblastoma cell line, which does not synthesise any neurotransmitter, has been transfected with a choline acetyltransferase construct; activation of acetylcholine synthesis, thus achieved, is followed by a higher expression of neuronal specific traits. The presence in these cells of muscarinic receptors is consistent with the existence of an autocrine loop, which may be responsible for the more advanced differentiation state observed in the transfected cells. Expression of cholinergic markers appears as a common feature of DRG sensory neurons, independently of the neurotransmitter used. Choline acetyltransferase can be detected in DRG at early developmental stages. The distribution of muscarinic receptors in DRG has suggested that activation of acetylcholine synthesis may be related in an early developmental phase to the interaction between neurons and nonneuronal cells and to modulation of cell differentiation. Both systems suggest that acetylcholine may have a role as a modulator of neuronal differentiation.

Neuronal NOS-cGMP-dependent ACh-induced relaxation in pial arterioles of endothelial NOS knockout mice.

We evaluated the effects of superfusing 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), eNOS null (B) an inhibitor of soluble guanylyl cyclase, and 7-nitroindazole sodium (7-NI), a selective neuronal nitric oxide synthase (nNOS) inhibitor, on the acetylcholine (ACh) response in endothelial NOS (eNOS) null mice. Pial arteriolar diameter was measured by intravital microscopy through a closed cranial window under alpha-chloralose anesthesia. NOS activity was measured by [3H]arginine-to-[3H]citrulline conversion in subjacent cortex in vitro. The density and distribution of muscarinic receptors in the brain were determined by quantitative [3H]quinuclidinyl benzilate autoradiography and did not differ between the eNOS mutants and wild-type mice. ACh superfusion (1 and 10 microM) dose dependently dilated pial arterioles in eNOS null and wild-type mice. ODQ (10 microM) attenuated ACh-induced dilation in both eNOS mutants (41% decrease at 10 microM ACh, P < 0.01, n = 6) and wild-type strains (n = 5 per group). By contrast, topical superfusion of 7-NI (100 microM) attenuated the ACh response in eNOS mutants only (66%, P < 0.05, and 25% decrease, P < 0.05, at 1 and 10 microM ACh, respectively). Our findings suggest that nNOS-guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathways dilate pial arterioles by compensatory mechanisms after eNOS gene disruption.

Distribution of muscarinic receptors on the endothelium of cortical vessels in the rat brain

Functional and pharmacological studies have suggested that there are muscarinic receptors (mAChRs) on the endothelial cells of major cerebral arteries, while recent immunological studies indicate that there are no mAChRs on the endothelium of brain capillaries. This difference may be because the distribution of mAChR on the endothelium varies with the type of vessel. This paper examines the distribution of mAChR on the vascular endothelium along intraparenchymal blood vessels in the rat brain by immunolabelling and laser confocal microscopy. Sections were immunostained by combinations of an anti-mAChR antibody (M35) with antibodies to endothelial (anti-GLUT1), or to smooth muscle markers (anti-actin). Antibody labellings were detected with fluorescent second antibodies. Most of the penetrating vessels bore mAChR immunolabelling which coincided over almost all the vessel surface with endothelial labelling. The mAChR immunolabelling was less widespread over the endothelium on the medium sized vessels (diameter<50 μm) and only 50% of these vessels had mAChR staining on the endothelium. There was no mAChR immunostaining on the endothelium of the capillaries. In contrast with the basilar artery, there was no mAChR immunolabelling on the smooth muscle layer of the intracortical vessels. These data indicate that the intensity of mAChR immunolabelling decreases along the vascular tree from large conducting vessels to capillaries.

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion.

To clarify whether muscarinic acetylcholine receptor (mAChR) binding can be a viable muscarinic neuronal marker which provides therapeutic information different from perfusional information in global brain, we evaluated the discrepancy between the distribution of cerebral blood flow (CBF), mAChR and its five subtypes of messenger ribonucleic acid (mRNA) in the acute (n=9) and chronic (n=8) phases of a middle cerebral artery (MCA) occlusion model and in sham-operated controls (n=6). In the acute phase, regional CBF was markedly reduced in the MCA territory, whereas mAChR was not reduced and the mRNA was reduced only slightly. In the chronic phase, mAChR was reduced markedly in the infarcted lesion and the mRNA was also reduced. The mAChR was slightly reduced in the ipsilateral substantia nigra and pontine nucleus because of remote effects; however, regional CBF in the substantia nigra was slightly increased and did not change in the pontine nucleus. The discrepancy between CBF and mAChR was clarified, and the tendency toward a reduction in mRNA in the acute ischaemic region without a reduction in mAChR suggested the presence of cholinergic neurons which were viable but hypometabolic. It is concluded that mAChR imaging may be of value for the assessment of the viable cholinergic neuron density in vivo.

Pharmacological characterization and distribution of muscarinic receptors in human placental syncytiotrophoblast brush-border and basal plasma membranes

Based on the existence of choline acetyltransferase and acetylcholine in human placenta, we have investigated the presence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta. Radioligand binding assay, using [ 3H] N-methyl-scopolamine as tracer, showed the existence of acetylcholine muscarinic receptors in brush-border ( K d 0.28±0.04 nM; B max 9.4±1.6 fmol/mg protein) and basal plasma membranes ( K d 0.24±0.05 nM; B max 34.3±6.3 fmol/mg protein). In order to perform a pharmacological characterization of these receptors, competition binding experiments were carried out using the muscarinic receptor antagonists pirenzepine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6 H-pyrido(14)benzodiazepine (AF-DX 116), himbacine, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP), dicyclomine and hexahydro-sila-difenidol (HHSD). The results obtained showed that the muscarinic receptors in brush-border and basal plasma membranes belong to different subtypes. In brush-border membranes, the receptor found match in terms of affinity for the antagonists with the muscarinic M 1 receptor subtype ( K i pirenzepine, 13.6±8.2 nM; K i AF-DX 116, 1680±271 nM; K i himbacine, 212±6.5 nM; K i 4-DAMP, 1.5±0.4 nM; K i dicyclomine, 5.1±0.8 nM; K i HHSD, 34.3±7.3 nM), whereas the receptor in basal plasma membrane seems to be of the muscarinic M 2 receptor subtype ( K i pirenzepine, 202±48 nM; K i AF-DX 116, 124±60 nM; K i himbacine, 20.6±4.8 nM; K i 4-DAMP, 4.5±1.2 nM; K i dicyclomine, 54.6±22 nM; K i HHSD, 89.2±15.8 nM). The results obtained show the existence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta with a different distribution pattern in terms of number of receptors and distribution of different subtypes. The functional significance of these findings is as yet unknown, but these receptors probably mediate different functions as they belong to different subtypes and are coupled to different second messengers.

糖尿病ラット膀胱におけるムスカリンレセプターの分布に関する研究

We evaluated the alterations of density, localization and subtype specificity of muscarinic receptors in experimentally-induced diabetic rat bladder. Five groups of rats were maintained for sixteen weeks 1) diabetic, 2) diabetic insulin-treated (insulin started 8 weeks after the onset of diabetes), 3) sucrose-fed diuretic, 4) sucrose-removed and 5) age matched controls. We used radioligand binding technique and light microscopic autoradiography to define the density and distribution of muscarinic receptors in the rat urinary bladder. Saturation experiments showed an increase in the density of muscarinic receptors in the bladders of diabetic and diuretic rats compared with age matched controls. Insulin treatment partially reversed the up-regulatin of muscarinic receptors in rat urinary bladder. Autoradiographic studies also indicated that muscarinic receptors were located in all layers of the bladder muscularis. The muscularis of the bladder dome contained higher densities of muscarinic receptors than that of the bladder base. Lack of [3H]QNB binding to transitional epithelium, lamina propria, and tunica adventitia suggests an absence of muscarinic receptors in these regions. Inhibition of [3H]QNB binding to the bladder sections by selective muscarinic antagonists indicated existence of the M2 and M3 receptor subtypes in the muscularis of the rat bladder.