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. 2009 Feb 17;156(7):1147–1153. doi: 10.1111/j.1476-5381.2009.00113.x

Quantitative analysis of the loss of muscarinic receptors in various peripheral tissues in M1–M5 receptor single knockout mice

Yoshihiko Ito 1, Luvsandorj Oyunzul 1, Masanao Seki 1, Tomomi Fujino (Oki) 1, Minoru Matsui 2, Shizuo Yamada 1
PMCID: PMC2697689  PMID: 19378377

Abstract

Background and purpose:

To compare loss in binding to muscarinic receptor (mAChR) subtypes with their known functions, the total density of muscarinic receptors was measured in peripheral tissues from wild type (WT) and mAChR knockout (KO) mice.

Experimental approach:

Binding parameters of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were determined in 10 peripheral tissues of WT and M1–M5 receptor KO mice. Competition between [3H]NMS and darifenacin (selective M3 receptor antagonist) was also measured

Key results:

There was an extensive loss of [3H]NMS-binding sites (maximal number of binding sites, Bmax) in heart and smooth muscle from M2KO mice, compared with WT mice. Smooth muscle from M3KO mice also showed a moderate loss of Bmax. Bmax fell in pancreas and bladder of M4KO mice and in prostate in M1KO and M3KO mice. There was a large loss of Bmax in exocrine and endocrine glands of M3KO mice with a moderate decrease in M2KO mice. Darifenacin inhibited specific [3H]NMS binding in submandibular gland and bladder of WT, M2KO and M3KO mice. Ki (inhibition constant) values for darifenacin in the submandibular gland were the same in WT and M2KO mice but increased in M3KO mice. However, Ki values in bladder were decreased in M2KO mice and increased in M3KO mice.

Conclusions and implications:

Single mAChR KO mice exhibit a loss of mAChR in peripheral tissues that generally paralleled the reported loss of function. Quantitative analysis of data, however, also suggested that, in some instances, normal expression of a receptor subtype depended on expression of other subtypes.

Keywords: peripheral muscarinic receptor subtype, distribution, knockout mice

Introduction

The family of muscarinic acetylcholine receptors (mAChRs) consists of five molecularly distinct subtypes (M1–M5). Based on their G protein-coupling properties, the five receptors can be subdivided into major functional classes. M1, M3 and M5 receptors are usually coupled to the Gq/11 protein that activates phospholipase C, whereas the M2 and M4 subtypes are mainly coupled to the Gi/o protein that inhibits adenylate cyclase activity (Caulfield, 1993).

Muscarinic acetylcholine receptors are distributed throughout peripheral tissues. The actions mediated by peripheral mAChRs are closely related to parasympathetic functions including reductions in heart rate and the stimulation of glandular secretion and smooth muscle contraction (Bymaster et al., 2003). The distribution of mAChR subtypes in peripheral tissues has been investigated mainly by pharmacological studies with subtype-selective agents and also by molecular and immuno-precipitation studies with measurements of levels of mRNA and protein (Doods et al., 1987; Peralta et al., 1987; Maeda et al., 1988; Dörje et al., 1991; Levey, 1993; Shida et al., 1993; Boschero et al., 1995; Krejčí and Tuček, 2002; Bymaster et al., 2003). However, identification of the regional distribution of pharmacologically relevant mAChR subtypes has proven a difficult task, primarily due to the lack of highly selective ligands and to the fact that most tissues or organs express multiple mAChRs. One promising approach is to utilize mice deficient in specific mAChR receptor genes (M1–M5) generated by gene-targeting technology (Wess et al., 2003; Matsui et al., 2004).

In the present study, we have measured specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in 10 peripheral tissues of wild type (WT) and mAChR subtype (M1–M5) knockout (KO) mice. The advantage of our technique is that it allows the direct estimation of physiologically or pharmacologically relevant receptors and can be adapted to investigate the interaction of acetylcholine (ACh) and selective agonists and antagonists with mAChR subtypes, as revealed by our current quantitation of central nervous system mAChR (Oki et al., 2005). The decreases in the density of [3H]NMS-binding sites in tissues of single mAChR KO mice would represent the net effect of a loss of the receptor together with any negative or positive compensatory changes. Our data have confirmed that there may be a significant tissue-based difference in the distribution of mAChR subtypes in the parasympathetic nervous system.

Methods

Animals

This study was conducted in accordance with the guide for care and use of laboratory animals as adopted by the US National Institutes of Health. Mice were housed with a 12 h light–dark cycle and fed laboratory food and water ad libitum. The generation and characterization of WT, M1KO, M2KO, M3KO, M4KO and M5KO mice was described previously (Matsui et al., 2004). The genetic background of the mice used in this study was a mixture of the 129/SvJ and C57BL/6J strains. Most of the animals used were at least N7th generation C57BL/6JJcl mice (CLEA, Japan). Accordingly, the WT mice used here are reasonably similar in genetic background to all five of the mutant lines.

Tissue preparation

Male (3–8 months of age) WT, M1KO, M2KO, M3KO, M4KO and M5KO mice were exsanguinated by taking the blood from the descending aorta after an intraperitoneal administration of pentobarbitone (161 µmol·kg−1), and the tissues were perfused with cold saline from the aorta. Then the submandibular gland, sublingual gland, lung, heart, stomach, pancreas, ileum, colon, bladder and prostate were excised, and fat and blood vessels were removed by trimming. The entire tissue was used for the measurement of mAChR subtypes. The tissues from three mice were pooled for a single determination, because of very low tissue weights. The tissues were minced with scissors and homogenized by a Kinematica Polytron homogenizer (Model K with a PTA10TS shaft) at 10 000 rpm in 19 volumes of ice-cold 30 mmol·L−1 Na+/HEPES buffer (pH 7.5) for 1 min, and the homogenates were then centrifuged at 40 000×g for 20 min. The resulting pellet was suspended in buffer for the binding assay. Protein concentrations were measured by the method of Lowry et al. (1951).

Muscarinic receptor-binding assay

The binding assay for mAChR was performed by using [3H]NMS as previously described (Ehlert and Tran, 1990; Oki et al., 2005). The tissue weight (3–10 mg per assay) and protein concentration (34–1250 µg protein per assay) used for the assay differed among tissues, taking account of the amount of specific [3H]NMS binding and on overall tissue weight. The ratios of protein to tissue weight in WT tissues were 0.06 (submandibular gland), 0.06 (sublingual gland), 0.04 (lung), 0.11 (heart), 0.07 (stomach), 0.13 (pancreas), 0.05 (ileum), 0.03 (colon), 0.06 (bladder) and prostate (0.04). The ratio in each tissue did not differ significantly between WT mice and each of the KO mice.

The crude membrane fractions of mouse tissues were incubated with different concentrations (0.06–1.0 nmol·L−1) of [3H]NMS in 30 mmol·L−1 Na+/HEPES buffer (pH 7.5). Incubation was carried out for 60 min at 25°C. The reaction was terminated by rapid filtration (Cell Harvester, Brandel Co., Gaithersburg, MD, USA) through Whatman GF/B glass fibre filters, and the filters were then rinsed two times with 3 mL of ice-cold buffer. Tissue-bound radioactivity was extracted from the filters overnight by immersion in scintillation fluid (2 L toluene, 1 L Triton X-100, 15 g 2,5-diphenyloxazole and 0.3 g 1,4-bis[2-(5-phenyloxazolyl)]benzene), and the radioactivity was measured with a liquid scintillation counter. Specific [3H]NMS binding was determined experimentally from the difference between counts in the absence and presence of 1 µmol·L−1 atropine. All assays were conducted in duplicate.

In the competitive inhibition experiments with darifenacin, the inhibitory effects of five or six different concentrations (0.3 nmol·L−1–3 µmol·L−1) of this agent on the specific binding of [3H]NMS (submandibular gland: 0.1 nmol·L−1, bladder: 0.2 nmol·L−1) in the submandibular gland and bladder of WT, M2KO and M3KO mice were examined.

Data analysis

[N-methyl-3H]scopolamine methyl chloride binding data was subjected to a non-linear regression analysis by using Graph Pad PRISM (version 4, Graph Pad Software, San Diego, CA, USA). The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]NMS were estimated. The ability of darifenacin to inhibit the specific binding of [3H]NMS was estimated from the IC50, which is the molar concentration of darifenacin necessary to displace 50% of the specific binding of [3H]NMS (determined by Graph Pad PRISM). The inhibition constant, Ki, was calculated from the equation, Ki= IC50/(1 + L/Kd), where L equals the concentration of [3H]NMS.

Statistical analysis of the data was performed by a one-way analysis of variance, followed by Dunnett's test for multiple comparison. Statistical significance was accepted at P < 0.05.

Materials

[N-methyl-3H]scopolamine methyl chloride (3.03 TBq·mmol−1) was purchased from PerkinElmer Life Sciences, Inc. (Boston, MA, USA). All other chemicals were purchased from commercial sources.

Results

[3H]NMS-binding characteristics in mAChR KO mice

The specific binding of [3H]NMS (0.06–1.0 nmol·L−1) was saturable in crude membrane fractions of 10 peripheral tissues (submandibular gland, sublingual gland, lung, heart, stomach, pancreas, ileum, colon, bladder and prostate) of WT and each (M1–M5) mAChR KO mice. Table 1 shows pKd and Bmax values for specific [3H]NMS binding in these tissues. The binding parameters in each tissue of M1–M5 subtype KO mice were compared with those of WT mice.

Table 1.

Kd and Bmax for specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in the peripheral tissues of wild type (WT) and muscarinic acetylcholine receptor (mAChR) subtype (M1–M5) knockout (KO) mice

Tissues KO type pKd Bmax (fmol·mg·protein−1)
Submandibular gland WT 9.88 ± 0.02 174 ± 9 (1.0)
M1KO 9.86 ± 0.04 169 ± 20
M2KO 9.93 ± 0.02 112 ± 8 (0.6)**
M3KO 9.96 ± 0.05 37 ± 3 (0.2)**
M4KO 9.90 ± 0.03 160 ± 12
M5KO 9.88 ± 0.03 147 ± 11
Sublingual gland WT 9.85 ± 0.05 183 ± 11 (1.0)
M1KO 9.84 ± 0.04 170 ± 23
M2KO 9.87 ± 0.04 130 ± 4
M3KO 9.94 ± 0.06 51 ± 6 (0.3)**
M4KO 9.83 ± 0.07 172 ± 15
M5KO 9.82 ± 0.04 151 ± 16
Lung WT 9.67 ± 0.04 72 ± 5 (1.0)
M1KO 9.68 ± 0.03 80 ± 7
M2KO 10.03 ± 0.02** 6.9 ± 1.2 (0.1)**
M3KO 9.62 ± 0.03 49 ± 4 (0.7)*
M4KO 9.68 ± 0.04 55 ± 6
M5KO 9.75 ± 0.03 50 ± 3 (0.7)*
Heart WT 9.44 ± 0.04 29 ± 1 (1.0)
M1KO 9.44 ± 0.07 32 ± 3
M2KO 9.29 ± 0.15 2 ± 1 (0.1)**
M3KO 9.45 ± 0.06 25 ± 3
M4KO 9.46 ± 0.03 28 ± 4
M5KO 9.43 ± 0.04 28 ± 1
Stomach WT 9.54 ± 0.05 192 ± 25 (1.0)
M1KO 9.52 ± 0.04 194 ± 11
M2KO 9.80 ± 0.12 12 ± 2 (0.1)**
M3KO 9.50 ± 0.11 144 ± 34
M4KO 9.46 ± 0.05 199 ± 11
M5KO 9.43 ± 0.08 164 ± 18
Pancreas WT 9.61 ± 0.08 19 ± 1 (1.0)
M1KO 9.76 ± 0.03 16 ± 1
M2KO 9.76 ± 0.07 12 ± 2 (0.6)**
M3KO 9.75 ± 0.08 5 ± 1 (0.3)**
M4KO 9.75 ± 0.10 15 ± 1 (0.8)*
M5KO 9.67 ± 0.05 17 ± 1
Ileum WT 9.38 ± 0.06 366 ± 43(1.0)
M1KO 9.23 ± 0.06 506 ± 59
M2KO 10.11 ± 0.07** 39 ± 8 (0.1)**
M3KO 9.37 ± 0.13 232 ± 27 (0.6)*
M4KO 9.36 ± 0.07 278 ± 40
M5KO 9.28 ± 0.03 327 ± 26
Colon WT 9.57 ± 0.08 234 ± 25 (1.0)
M1KO 9.59 ± 0.06 273 ± 29
M2KO 9.92 ± 0.17* 65 ± 9 (0.3)**
M3KO 9.62 ± 0.10 190 ± 23
M4KO 9.48 ± 0.04 233 ± 19
M5KO 9.49 ± 0.06 222 ± 40
Bladder WT 9.58 ± 0.03 128 ± 6 (1.0)
M1KO 9.49 ± 0.04 136 ± 15
M2KO 10.07 ± 0.05** 23 ± 5 (0.2)**
M3KO 9.49 ± 0.11 77 ± 10 (0.6)*
M4KO 9.40 ± 0.09 83 ± 7 (0.7)*
M5KO 9.55 ± 0.11 84 ± 15 (0.7)*
Prostate WT 9.91 ± 0.03 158 ± 7 (1.0)
M1KO 9.74 ± 0.08 60 ± 10 (0.4)**
M2KO 9.82 ± 0.04 130 ± 29
M3KO 9.91 ± 0.11 95 ± 21 (0.6)*
M4KO 9.77 ± 0.02 153 ± 10
M5KO 9.88 ± 0.02 177 ± 28
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The specific binding of [3H]NMS (0.06–1.0 nmol·L−1) was measured in peripheral tissues taken from WT and mAChR KO mice. The ages (in weeks) of these mice were 13–39 (WT), 14–46 (M1KO), 13–31 (M2KO), 12–51 (M3KO), 11–26 (M4KO) and 11–40 (M5KO). Each tissue was derived from the same animals. In addition, there was little difference in the age of each mouse used in determining [3H]NMS-binding parameters (Kd, Bmax). Values in parentheses represent the Bmax relative to WT (controls). Values represent the mean ± SEM of three to eight determinations.

Asterisks show a significant difference from the values in WT mice

*

P < 0.05

**

P < 0.01.

In the submandibular gland, sublingual gland and pancreas, the Bmax values for [3H]NMS binding were markedly (70–80%) lower in M3KO mice than in WT mice. Similarly, in M2KO mice, there were significant (40%) decreases in Bmax values in the submandibular gland and pancreas and a tendency for decrease (29%) in the sublingual gland. In the latter case, it is considered that there may be changes, but the low number of experiments did not allow us to detect the difference. In other words, the statistical power of the data was insufficient to detect a 29% difference. The M4KO mice showed a slight (20%) loss of [3H]NMS-binding sites only in the pancreas.

There was a significant (90%) decrease in Bmax values for [3H]NMS binding in the heart of M2KO mice compared with WT mice. The Bmax value for cardiac [3H]NMS binding was not significantly altered in the other mice relative to WT mice.

In the lung and ileum, there were significant (30–90%) decreases in Bmax values for [3H]NMS binding in M2KO and M3KO mice compared with WT mice. The decrease was greatest (90%) in the lung and ileum of M2KO mice and moderate (30% and 40% respectively) in M3KO mice. The Bmax values for [3H]NMS binding in the stomach and colon were markedly (90% and 70% respectively) lower in M2KO mice than WT mice. Also, there was a significant (30%) decrease in the lungs of M5KO mice but a non-significant decrease in the stomach and colon of M3KO mice.

In the bladder, the greatest (80%) decrease in Bmax values for [3H]NMS binding was in M2KO mice, with moderate (40%, 30% and 30% respectively) decreases in M3KO, M4KO and M5KO mice. These decreases were statistically significant. The binding in the bladder of M1KO mice was similar to that in WT mice.

There were significant (60% and 40% respectively) decreases in Bmax values for [3H]NMS binding in the prostate of M1KO and M3KO mice with a tendency for lower (18%) Bmax values in M2KO mice, compared with WT mice.

There was little change in the pKd values for [3H]NMS binding in peripheral tissues of any of the KO mice compared with WT mice, except in the lung, ileum, colon and bladder that showed significant (2.3-, 5.4-, 2.4- and 3.1-fold respectively) increases in M2KO mice.

Competitive inhibition by darifenacin of specific [3H]NMS binding in the submandibular gland and bladder of WT, M2KO and M3KO mice

We examined the competitive inhibition of specific [3H]NMS binding by darifenacin, a highly selective antagonist of the M3 receptor subtype, in the submandibular gland and bladder from WT, M2KO and M3KO mice. Darifenacin (0.3 nmol·L−1–3 µmol·L−1) inhibited the binding of [3H]NMS in these tissues in a concentration-dependent manner. As shown in Table 2, the Ki value of darifenacin for the competitive inhibition of specific [3H]NMS binding in the submandibular gland was not changed in the M2KO mice (4.3 nmol·L−1) compared with WT mice (3.0 nmol·L−1) and was significantly (6.3-fold) increased in M3KO mice (19 nmol·L−1). On the other hand, the Ki value of darifenacin in the bladder was markedly decreased in the M2KO mice (2.5 nmol·L−1) compared with WT mice (24 nmol·L−1) and was significantly increased in the M3KO mice. Thus, the ratios of Ki (submandibular gland)/Ki (bladder) were 0.1 (WT), 1.7 (M2KO) and 0.4 (M3KO). Further, the Hill coefficients for the competition by this agent in both tissues were significantly increased, being close to unity in M2KO and M3KO mice compared with WT mice. These results are consistent with the idea that the M3 receptor is predominant in the submandibular gland while the M2 receptor is dominant in the bladder.

Table 2.

Competitive inhibition by darifenacin of specific [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding in the submandibular gland and bladder of wild type (WT), M2 and M3 knockout (KO) mice

Submandibular gland
 Bladder
 Ki (submandibular gland)/Ki (bladder)
Ki (nmol·L−1) nH Ki (nmol·L−1) nH
WT 3.0 ± 0.6* 0.8 ± 0.1 24 ± 7 0.6 ± 0.1 0.1
M2KO 4.3 ± 0.9## 1.0 ± 0.1† 2.5 ± 0.7†,# 1.0 ± 0.2† 1.7
M3KO 19 ± 2*,†† 1.1 ± 0.1† 43 ± 10† 0.9 ± 0.1† 0.4
Open in a new tab

The submandibular gland and bladder were taken from WT and muscarinic acetylcholine receptor (M2, M3) KO mice. Specific [3H]NMS binding in the submandibular gland (0.1 nmol·L−1) and bladder (0.2 nmol·L−1) was measured in the absence and presence of five to six different concentrations (0.3 nmol·L−1–3 µmol·L−1) of darifenacin, and the values of Ki and Hill coefficients (nH) were estimated. Values represent the mean ± SEM of three to six determinations.

Symbols show a significant difference from the values for bladder in the same group of mice

*

(P < 0.05), a significant difference from the values for the same tissue in WT mice

†

(P < 0.05,

††

P < 0.001) and a significant difference from the values for the same tissue in M3KO mice

#

(P < 0.05,

##

P < 0.001).

Discussion

The binding parameters of the five mAChRs were compared in peripheral tissues of WT and mAChR subtype (M1–M5) KO mice. Table 3 summarizes the relative loss of mAChR-binding sites in tissues of mAChR KO mice based on the data in Table 1, with other information such as mRNA expression and antibody binding of mAChR subtypes from previously published work. Functional and immunological studies have consistently shown that the M3 receptor subtype plays a key role in the parasympathetic control of salivation (Caulfield, 1993; Levey, 1993; Moriya et al., 1999). In fact, severe hyposalivation that led to growth failure due to eating difficulties was previously demonstrated in M3KO mice (Matsui et al., 2000; 2004). The M3 receptor may account for at least 70% of all mAChRs present in the submandibular and sublingual glands, as estimated by the loss of exocrine [3H]NMS-binding sites in M3KO mice compared with WT mice. Also, the mRNA for the M3 receptor subtype has been found in rat exocrine glands (Maeda et al., 1988). In M2KO mice, there was moderate loss of [3H]NMS-binding sites in exocrine glands. Although there is little published to explain the physiological significance of exocrine M2 receptors, in situ hybridization by using oligonucleotide probes has shown that the mRNA for the M2 receptor subtype is expressed in the rat salivary gland (Shida et al., 1993). Takeuchi et al. (2002) found, using M5KO mice, that this subtype may be involved in the slow secretory process of pilocarpine-induced salivation, and our data in Table 1 disclose a slight loss of [3H]NMS-binding sites in both submandibular and sublingual glands of the M5KO mice.

Table 3.

Semiquantitative localization of muscarinic acetylcholine receptor (mAChR) subtypes (M1–M5) in various tissues of mice

Tissues Loss of mAChR sites in muscarinic knockout mice mRNA expression Antibody binding
Submandibular gland M3 > M2 M3 (Shida et al., 1993) M3 (42%), M1 (36%), M2 (12%), M2 (7%) (Dörje et al. 1991)
M1, M3 (Gautam et al., 2004)
Sublingual gland M3
Lung M2 > M3, M5 M1, M2, M3 (Struckmann et al., 2003) M4 (41%), M2 (40%) (Dörje et al., 1991)
Heart M2 M2 (Maeda et al., 1988) M2 (88%) (Krejčí and Tuček, 2002)
M2 (Peralta et al., 1987)
M2 (Levey, 1993)
M2 (>90%) (Krejčí and Tuček, 2002)
Stomach M2 M1–M5 (Aihara et al., 2005)
Pancreas M3 > M2, M4 M4 (Peralta et al., 1987)
M1, M3 (Boschero et al., 1995)
Ileum M2 > M3 M2 (69%), M4 (12%), M3 (4%), M1 (3%) (Dörje et al., 1991)
M2, M4 (Takeuchi et al., 2002)
Colon M2
Bladder M2 > M3, M4, M5 M2, M3 (Maeda et al., 1988) M1–M5 (Giglio et al., 2005)
Prostate M1 > M3 M3 (Kim et al., 2005) M2 (Ruggieri et al., 1995)
M2, M3 (Pontari et al., 1998)
M1 (>70%) (Ruggieri et al., 1995)
Open in a new tab

The rank order is based on the loss of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS)-binding sites measured in each single muscarinic receptor knockout mice (M1–M5), in comparison with wild type, as shown in Table 1. The distribution of mRNA and antibody binding was taken from the references shown.

A previous functional study on our mAChR KO mice had shown that the acinar cells of the submandibular glands expressed lower levels of M1 receptors than M3 receptors (Gautam et al., 2004; Nakamura et al., 2004). In the current study, however, the submandibular glands of M1KO mice showed no significant reduction of [3H]NMS binding compared with those of WT mice (Table 1). This may indicate that the numbers of M1 receptors in the submandibular glands of WT mice were so small that the difference between the genotypes was below the level detectable by our receptor-binding assay. Alternatively, the expression of residual subtypes might be up-regulated to compensate for the loss of M1 receptors in the M1KO mice.

The pancreas showed a significant (40% and 70% respectively) loss of mAChR-binding sites in M2KO and M3KO mice, compared with WT mice. The predominance of M3 receptors in the pancreas has provided convincing evidence for a functional role of this subtype in cholinergic secretory responses of insulin in pancreatic islets (Boschero et al., 1995; Zawalich Zawalich et al., 2004) and of amylase in acinar cells (Gautam et al., 2005). Further, the mRNA for the M3 receptor subtype was detected in rat pancreatic islet cells (Boschero et al., 1995). Consistent with previous observations (Dörje et al., 1991; Levey, 1993), the density of mAChR M2 receptors in cardiac and smooth muscular tissues was confirmed to be high, by using M1–M5 receptor KO mice. It is known that cardiac responses to cholinergic agonists are exclusively mediated through stimulation of the M2 receptor subtype (Caulfield, 1993; Stengel et al., 2000). The present data obtained with KO mice have shown M2 receptors to predominate in the mouse heart. This observation is fully consistent with the quantitative data for M2 receptor mRNA representing more than 90% of all mAChR mRNA in rat atria and ventricles (Krejčí and Tuček, 2002). The predominance of M2 receptor mRNA in the heart was shown also by others (Peralta et al., 1987; Maeda et al., 1988; Levey, 1993).

The mRNA for the M2 receptor subtype was expressed in smooth muscular tissues (Maeda et al., 1988; Levey, 1993). In the lung, stomach, ileum, colon and bladder, the M2 receptor predominated. Also, there were moderate amounts of the M3 receptor in the lung, ileum and bladder. These subtypes are involved in mechanical responses of smooth muscles to cholinergic agonists (Matsui et al., 2000; 2004; Struckmann et al., 2003; Wess et al., 2003). The lung mAChR is involved in bronchial parasympathetic control. The measurement of lung mAChR revealed the M2 receptor subtype to be predominant with moderate levels of the M3 and M5 receptors. The coexistence of M2 and M3 receptors in the lung has been suggested previously (Esqueda et al., 1996; Myers and Undem, 1996; Struckmann et al., 2003). Notably, the videomicroscopy and digital imaging of lung slices has shown that muscarine-mediated bronchoconstriction was diminished partly in M3KO mouse lung and completely abolished in M2/M3 double KO mouse lung, suggesting a concerted action of both subtypes in the cholinergic constriction of murine airways (Struckmann et al., 2003).

Interestingly, in the bladder, there might be significant loss of mAChR-binding sites in M4KO and M5KO mice in addition to M2KO and M3KO mice. A recent immunohistochemical study by Giglio et al. (2005) showed all subtypes of mAChR to be present in rat urinary bladder with a prominent increase in the expression of the M5 receptor subtype in both smooth muscle and urothelium from cyclophosphamide-treated rats. Hence, the M5 receptor may be significantly involved in the pathogenesis of urinary bladder disorders such as interstitial cystitis. Also, it should be noted that the negative feedback mechanism inhibiting the release of ACh in the urinary bladder may be mediated by prejunctional M4 receptors (D'Agostino et al., 1997; Zhou et al., 2002).

The Ki values (nmol·L−1) for the competitive inhibition by darifenacin of specific [3H]NMS binding in CHO-K1 cell lines expressing human mAChR subtypes were 31 (M1), 57 (M2), 2.5 (M3), 16 (M4) and 9.6 (M5) (Maruyama et al., 2006). In this case, the Ki value was significantly smaller for the M3 receptors than the M1 and M2 receptor subtypes. The Ki values in the submandibular gland of WT mice (3.0 nmol·L−1) and M2KO mice (4.3 nmol·L−1) and in the bladder of M2KO mice (2.5 nmol·L−1) (Table 2) were close to the Ki value for the human M3 receptor. The Ki values in the bladder of M2KO (2.5 nmol·L−1) and M3KO (43 nmol·L−1) mice seem to reflect the affinity of darifenacin mainly for the M3 and M2 receptor subtypes respectively. These results are consistent with the major subtype of mAChR being M3 in the submandibular gland and M2 in the bladder.

Ruggieri et al. (1995) showed the localization of the M2 receptor in rat prostate using an antibody, whereas receptor binding and pharmacological studies with selective antagonists suggested the presence of functional M3 receptors (Latifpour et al., 1991; Yazawa and Honda, 1993; Lau and Pennefather, 1998). In agreement with pharmacological observations, there was significant loss of mAChR-binding sites in the prostate of M3KO mice compared with WT mice. The greater decrease of mAChR density was also observed in this tissue in M1KO mice. To our knowledge, the existence of the M1 receptor subtype in the murine prostate is little reported, but, interestingly, an antibody study in human prostate revealed a major (>70%) presence of M1 receptors in the glandular epithelial cells (Ruggieri et al., 1995).

The M5 receptor was shown to be expressed ubiquitously throughout the brain and in non-neuronal tissues such as skin fibroblasts and keratinocytes, endothelial cells and smooth muscle of the neurovasculature and lymphocytes (Ndoye et al., 1998; Buchli et al., 1999). The M5 receptor has been the hardest mAChR subtype to study for at least two reasons: no selective ligands for the M5 receptor have been found, and no tissues have been found where M5 receptors are in higher concentrations than all other mAChRs. In M5KO mice, compared with WT mice, there were moderate decreases in Bmax values for [3H]NMS binding in the lung and bladder (Table 1).

There were significant increases in pKd values for [3H]NMS binding in the lung, ileum, colon and bladder of M2KO mice. It is reported that [3H]NMS exhibits higher affinity for M1, M3 and M4 receptors than M2 receptors as shown in Kd values of 120 pmol·L−1 (M1, NB-OK1 cells), 500 pmol·L−1 (M2, rat heart), 120 pmol·L−1 (M3, rat pancreas) and 50 pmol·L−1 (M4, rat striatum) (Waelbroeck et al., 1990). Therefore, the significant increases in pKd values in these tissues in M2KO mice may be due to the enhanced affinity of [3H]NMS for the residual receptors, following the marked loss of the M2 receptors.

The sum of the loss in mAChR-binding sites in the single KO mice greatly exceeds the binding sites in WT, indicating that in some instances, the expression of a given receptor subtype depends on the expression of the other subtypes. The sum of the reduction in all the individual Bmax values in the lung and bladder of each of the KO mice relative to WT mice was 1.5- and 1.8-fold greater, respectively, than Bmax values of WT mice (Table 1). This suggests that the loss of some mAChR subtypes causes a decrease, and not a compensatory increase, in the residual mAChR-binding sites. Although the precise mechanism of this unexpected phenomenon is unclear at present, it should be noted that exposure to chronically increased release of ACh may cause a down-regulation of mAChR expression (Yamada et al., 1983a,b). A similar consideration was noted in the corpus striatum and pons-medulla oblongata of mAChR subtype KO mice relative to WT mice (Oki et al., 2005). M2 and M4 receptors are located at the nerve terminals as autoreceptors to inhibit ACh release, and these prejunctional autoreceptors may function in the lung and bladder (Kilbinger et al., 1995; Myers and Undem, 1996; D'Agostino et al., 1997; Zhou et al., 2002; Bymaster et al., 2003). Hence, in M2KO and M4KO mice, ACh levels in the cholinergic synapses of lung and bladder are presumably elevated. Taken together, it is conceivable that the decreases in the [3H]NMS-binding sites in these tissues of M2KO and M4KO mice reflect not only loss of these subtypes but also desensitization of the other receptor subtypes caused by a hyper-cholinergic status. Alternatively, ‘cross-talk’ might occur among mAChR subtypes, resulting in compensatory changes, as has been assumed for β3 adrenoceptor KO mice (Susulic et al., 1995).

In conclusion, the comparative measurement of mAChRs in KO mouse tissues has confirmed the pharmacologically relevant M1–M5 subtypes are widely distributed throughout peripheral tissues but with significant variation. The quantitative distribution of mAChR subtypes may contribute not only to further understanding of cholinergic function but to the development of novel muscarinic therapeutic drugs, such as selective antagonists of the M4 and M5 receptors.

Acknowledgments

This work was supported in part by Grant-in-Aid 11672271 for Scientific Research in 2003 and 2004 from the Ministry of Education, Science and Culture of Japan (SY). This study was funded by Pharmacia, Detrol LA Research Grant Program from Pfizer, The Industrial Technology Research Grant Program 02A09001a from The New Energy and Industrial Technology Development Organization (NEDO) of Japan, and Grant-in-Aid for Scientific Research on Priority Areas 16067101 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT).

Glossary

Abbreviations

[3H]NMS

[N-methyl-3H]scopolamine methyl chloride

Bmax

maximal number of binding sites

Kd

apparent dissociation constant

Ki

inhibition constant

KO

knockout

mAChR

muscarinic acetylcholine receptor

WT

wild type

Conflict of interest

The authors state no conflict of interest.

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