QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (62) Citing Articles via Google Scholar Google Scholar Articles by Matsui, M. Articles by Taketo, M. M. Search for Related Content PubMed PubMed Citation Articles by Matsui, M. Articles by Taketo, M. M.

 Previous Article  |  Next Article 

The Journal of Neuroscience, December 15, 2002, 22(24):10627-10632

Mice Lacking M₂ and M₃ Muscarinic Acetylcholine Receptors Are Devoid of Cholinergic Smooth Muscle Contractions But Still Viable

Minoru Matsui^{1, 2}, Daisuke Motomura², Toru Fujikawa³, Jian Jiang³, Shin-ichi Takahashi², Toshiya Manabe¹, and Makoto M. Taketo^{2, 4}

¹ Division of Neuronal Network, Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan, ² Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan, and ³ Banyu Tsukuba Research Institute (Merck), Ibaraki 300-2611, Japan, and ⁴ Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cholinergic agents elicit prominent smooth muscle contractions via stimulation of muscarinic receptors that comprise five distinct subtypes (M₁-M₅). Although such contractions are important for autonomic organs, the role of each subtype has not been characterized precisely because of the poor selectivity of the currently available muscarinic ligands. Here, we generated a mutant mouse line (M₂/M₃/ mice) lacking M₂ and M₃ receptors that are implicated in such cholinergic contractions. The relative contributions of M₂ and M₃ receptors in vitro was ~5 and 95% for the detrusor muscle contraction and ~25 and 75% for the ileal longitudinal muscle contraction, respectively. Thus, M₁, M₄, or M₅ receptors do not seem to play a role in such contractions. Despite the complete lack of cholinergic contractions in vitro, M₂/M₃/ mice were viable, fertile, and free of apparent intestinal complications. The urinary bladder was distended only in males, which excludes a major contribution by cholinergic mechanisms to the urination in females. Thus, cholinergic mechanisms are dispensable in gastrointestinal motility and female urination. After 10 Hz electrical field stimulation, noncholinergic inputs were found to be increased in the ileum of M₂/M₃/ females, which may account for the lack of apparent functional deficits. Interestingly, the M₂/M₃/ mice had smaller ocular pupils than M₃-deficient mice. The results suggest a novel role of M₂ in the pupillary dilation, contrary to the well known cholinergic constriction. These results collectively suggest that an additional mechanism operates in the control of pupillary constriction-dilatation.

Key words: acetylcholine; muscarinic receptors; smooth muscle contraction; gene targeting; intestinal motility; ocular pupils

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Acetylcholine (ACh) is the most common neurotransmitter at the parasympathetic nerve ending to induce smooth muscle contractions. In the gastrointestinal tract, ACh is released from the primary excitatory motor neurons and mediates an immediate smooth muscle contraction (Goyal and Hirano, 1996; Furness, 2000). Activities of the motor neurons in the gut wall are coordinated by the enteric nervous system to generate functional movement, such as peristalsis. Although the excitatory enteric neurons corelease other transmitters, such as tachykinins (Holzer and Holzer-Petsche, 1997), ACh is believed to be functionally predominant in inducing contractions (Goyal and Hirano, 1996; Furness, 2000).

The cholinergic signaling is mediated by the muscarinic ACh receptor expressed on the surface of the smooth muscle cells. To date, five subtypes (M₁-M₅) of muscarinic receptors have been identified, which are variable in both the tissue distribution and signal transduction mechanisms (Caulfield and Birdsall, 1998). Delineating the role of these receptors has been a matter of considerable interest, because they are promising therapeutic targets for various diseases (Eglen et al., 2001). However, their pharmacological characterization remains inconclusive because of the poor subtype selectivity of available ligands. In the intestine, for example, M₃ is considered to be predominant in eliciting cholinergic contraction, whereas the role of the more abundant M₂ remains unclear (Ehlert et al., 1999; Eglen, 2001).

To overcome such technical problems, mutant mice deficient in each individual subtype have been constructed (Hamilton et al., 1997; Gomeza et al., 1999a,b; Matsui et al., 2000; Yamada et al., 2001a,b). Notably, all five mutant mouse lines are viable, which suggests a functional redundancy among the subtypes. The mice deficient in either M₂ or M₃ receptors show decreased responses to cholinergic stimuli (Matsui et al., 2000; Stengel et al., 2000), but precise mechanisms of the residual contractions remain to be elucidated.

Here, we generated mutant mice lacking both M₂ and M₃ receptors and studied the role of these subtypes in various smooth muscle organs. In addition, we used electrical field stimulation (EFS) and evaluated the in vivo significance of cholinergic smooth muscle contraction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Construction of a targeting vector for disruption of M₂ receptor gene. A targeting vector, pChrm2-N1 (Fig. 1A), was constructed using the mouse M₂ genomic fragments (Matsui et al., 1999). The BamHI-EcoRI (1.0 kb) and SmaI-BamHI (8.5 kb) fragments were placed upstream and downstream of the PGK-neo-bpA cassette (Soriano et al., 1991), respectively. The PGK-DTA cassette (Yagi et al., 1990) was inserted at the upstream end in reverse orientation.

View larger version (42K): [in this window] [in a new window]   Figure 1.   Generation of M2/ mice. A, Targeting strategy. Arrows MF13 and PR1 indicate the PCR primers used for homologous recombinant screening, and arrows MF26, MR22, and PR1 indicate PCR primers used for genotyping. BamHI (B), HindIII (H), EcoRI (R), and SmaI (S) sites relevant to the identification of homologous recombinant clones are shown together with the expected sizes hybridizable to the M2 and the neo probes. B, Hybridization with the M2 probe showing a 5.5 kb band specific to the targeted allele (KO) and a 10.6 kb band derived from the wild-type allele (WT). C, Hybridization with the neo probe showing a 5.5 kb band specific to the targeted allele. D, Northern analysis showing mRNA levels of M2 and M4 in the brains of wild-type, M2+/, and M2/ mice. Note that the M2 mRNA in the M2+/ brain decreased to approximately half of the wild-type brain and was absent in the M2/ brain. The mRNA levels of M4 are not different among the three genotypes. Bottom, Signals hybridized with a Gapd probe used as an internal control.

Gene targeting in embryonic stem cells. The gene targeting in embryonic stem cells (RW4; Genome Systems, St. Louis, MO) was performed as described previously (Matsui et al., 2000). G418-resistant cells were screened by PCR using primers MF13 (5'-CCC TGC TTC AAA TAC TTG TCC-3') and PR1 (5'-CAG ACT GCC TTG GGA AAA GC-3') to amplify a 1.2 kb fragment. Two independent clones (2N-16 and 2N-26) were verified for the homologous recombination (Fig. 1B,C). The 2N-26 clone contributed to chimeric males that transmitted the targeted allele to the germ line. A mutant mouse line was established in a mixed background between 129/SvJ and C57BL/6.

Genotyping. The genotyping protocol for the M₃ allele was described previously (Matsui et al., 2000). The procedure for the M₂ allele was similar, except that the primers used were MF26 (5'-GGT TGG GTG CAT TGG TTA GT-3'), PR1 (5'-CAG ACT GCC TTG GGA AAA GC-3'), and MR22 (5'-GTG TTC AGT AGT CAA GTG GC-3').

Northern analysis. An M₂ probe was prepared from a 510 bp genomic DNA fragment (SmaI-SmaI) corresponding to the mouse M₂-encoding region (129/SvJ origin). For M₄ and the glyceraldehyde-3-phosphate dehydrogenase gene (Gapd) probes, DNA fragments were amplified by PCR from cloned genomic DNA fragments of a 129/SvJ mouse and from a genomic DNA of a C57BL/6 mouse, respectively. The primers used were as follows: for M₄, M4F (5'-AGC CGC AGC CGT GTT CAC AA-3') and M4R (5'-TGG GTT GAG GGT TCG TGG CT-3'); and for Gapd, GapdF (5'-GCG TCC TGC ACC AAC TG-3') and GapdR (5'-ATG GTC CTT TAC TCG AAG TG-3').

Generation of M₂/M₃/ mice. The mouse colony was maintained in a specific pathogen-free area, and the lights in the animal room were turned on between 7:00 A.M. and 7:00 P.M. The M₂/ mice (N3 generation) and M₃/ mice (N2 generation) were crossed to obtain the M₂+/M₃+/ mice. Intercross between these mice yielded pups of various genotypes for M₂ and M₃ alleles, including M₂/M₃/ mutants. The descendants of these mutant mice were used for phenotypic analysis in this study. To improve the growth of the pups lacking M₃, hydrated paste food was fed as described previously (Matsui et al., 2000).

Blood chemistry. Blood samples were taken intracardially from anesthetized animals and analyzed with a Fuji Dri-Chem system (model 5500) at Fujimoto Biomedical Laboratories (Osaka, Japan).

Histological analyses. All organs were fixed in 10% Formalin, embedded in paraffin, and sectioned at 4 µm. Sections were stained with hematoxylin and eosin and examined under a light microscope.

In vitro responsiveness of ileal and urinary bladder smooth muscles. The urinary bladder was prepared free of serosal connective tissue and cut into four longitudinal unfolded sections. The ileum was removed, and longitudinal muscles were isolated from the segments of the ileum (5 mm long). The preparations were placed in 5 ml organ baths containing modified Krebs-Henseleit solution maintained at 32°C, aerated continuously with 95% O₂ and 5% CO₂, and connected to isometric transducers with sutures.

Mechanical responses were recorded isometrically by a multichannel polygraph. The tissue was equilibrated for at least 60 min with initial tension of 0.5 gm, and then contractions were induced by adding 50 mM KCl twice. The second contractile response to KCl was taken as a reference. The concentration-contraction curves for carbachol were obtained by cumulative addition of carbachol to the organ bath.

Trains of EFS (pulses at 1 or 10 Hz) were given for 10 sec at 5 min intervals. Each pulse was 45 V, 0.5 msec (bladder) or 40 V, 1 msec (ileum). After the muscle strips were contracted by 50 mM KCl for a reference, EFS was applied to cause basal contraction. To obtain noncholinergic contraction of wild-type muscle, atropine (10 µM) was applied subsequently.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Generation of M₂/M₃/ mice

To establish a mouse line lacking both M₂ and M₃ receptors, we first constructed a mutant mouse line deficient in M₂ (M₂/ mice) (Fig. 1). The M₂/ mice seemed healthy, consistent with another report on a similar mutant line lacking M₂ (Gomeza et al., 1999a). We crossed our M₂/ mice with the M₃/ mice (Matsui et al., 2000) to produce M₂+/M₃+/ mutants and further intercrossed these M₂+/M₃+/ mice to obtain M₂/M₃/ mutants. Among the pups, the M₂/M₃/ mutants were found at the Mendelian ratio (data not shown), excluding lethality in utero.

Although cholinergic signals are involved in the control of vital organs, such as gastrointestinal (Goyal and Hirano, 1996; Furness, 2000) and urinary (Andersson, 1998) tracts, the M₂/M₃/ pups survived to adulthood after a transient postweaning growth retardation (data not shown). The growth of M₂/M₃/ pups was improved by feeding paste food, reflecting their impaired salivation as a result of the loss of M₃-mediated signals (Matsui et al., 2000). The M₃/ mice of either sex were fertile (Matsui et al., 2000), despite the proposed roles of the muscarinic receptor in genital organs (Lepor and Kuhar, 1984; Eglen et al., 1989). Because M₂ and M₃ receptors are coexpressed in the uterus (Choppin et al., 1999) and prostate (Pontari et al., 1998), it was conceivable that M₂ receptors compensated for the lost function of M₃. However, the M₂/M₃/ mice of either sex were still fertile. Together, the M₂/M₃/ mice were unexpectedly healthy, despite the proposed importance of these receptors in the functions of various autonomic organs.

Sex difference in urinary retention phenotype

In the urinary bladder, muscarinic contraction of the detrusor muscle is considered to be the main source of voiding power (Andersson, 1998), and muscarinic antagonists have been applied for the treatment of bladder hyperactivity in humans (de Groat and Yoshimura, 2001). In our previous report (Matsui et al., 2000), the cholinergic contractility of the M₃/ detrusor muscle corresponded to only 5% of that of the wild-type muscle. Although there was no sex difference in the degree of such in vitro deficits, urinary disturbance was severe in males but only mild in females, suggesting that the contribution of M₂ is significantly larger in females. However, the urinary bladder of the M₂/ mice seemed normal in both sexes. Furthermore, the bladder diameter of the female M₂/M₃/ mice (5.9 ± 0.7 mm; n = 4) was not significantly different (p = 0.81) from that of the female M₃/ mice (5.7 ± 0.5 mm; n = 7) reported by Matsui et al. (2000). Consistent with a minor urinary retention phenotype, the kidney histology was normal (data not shown). In males, the bladder distension in the M₂/M₃/ mice (14.5 ± 1.4 mm; n = 4) was not significantly different (p = 0.29) from that in the M₃/ mice (12.3 ± 1.3 mm; n = 6) reported by Matsui et al. (2000). Urinalysis and blood chemistry data for renal functions were normal in both sexes (data not shown). Thus, M₃, but not M₂, is essential for voiding in males, but the contribution by M₂ or M₃ in female urination is rather small. Although the reason for the sex difference is unclear, it is conceivable that the roles of the central muscarinic receptors in urination (Ishiura et al., 2001) are different between the sexes. An alternative and more plausible explanation is that there is an additional, nonmuscarinic postjunctional mechanism in females that mediates urination in the M₂/M₃/ mice (see Discussion).

Normal appearance of digestive tracts

In the digestive tract, both M₂ and M₃ mediate the intestinal smooth muscle contraction (Ehlert et al., 1999; Eglen, 2001), which is considered the basis of functional gut movements, including peristalsis (Goyal and Hirano, 1996; Furness, 2000). Loss of peristalsis should be fatal, as exemplified by a clinical condition called acute colonic pseudo-obstruction, in which cholinergic dysfunction is implicated (Ponec et al., 1999). Therefore, we anticipated severe malfunctions in the gut of the M₂/M₃/ mice. Unexpectedly, they showed no signs of abnormal abdominal distension or constipation. At necropsy, the whole digestive tract of the M₂/M₃/ mice seemed normal (data not shown). Histological examinations revealed no abnormalities in the gastric, jejunal, or ileal sections (Fig. 2). Other visceral organs, such as the heart, lung, and spleen, appeared normal at gross and histological examinations (data not shown). Muscarinic receptors are implicated in gall bladder contraction (Parkman et al., 1999a) and in pancreatic endocrine (Boschero et al., 1995) and exocrine (Schmid et al., 1998) secretions. However, blood biochemistry data were normal, including those representing the hepatic and pancreatic functions (data not shown).

View larger version (143K): [in this window] [in a new window]   Figure 2.   Normal histology of the gastrointestinal tract of M2/M3/ mice. Sections of the stomach (A, D), jejunum (B, E), and ileum (C, F), stained with hematoxylin and eosin. Note that the diameter of the lumen and morphology of the muscular or mucosal layers are indistinguishable between the wild-type (A-C) and the M2/M3/ (D-F) mice. Scale bars, 0.5 mm.

In vitro contractility of smooth muscles to carbachol

Although M₂ and M₃ receptors have been implicated in cholinergic contraction (Sawyer and Ehlert, 1998; Matsui et al., 2000; Stengel et al., 2000; Giglio et al., 2001), the mice lacking both receptors showed no apparent abnormalities in the autonomic organs, except for the urinary retention in males. Because pharmacological analyses in these reports were inconclusive, it was conceivable that cholinergic signaling remained intact through other muscarinic receptor subtypes. To address this issue, we examined in vitro contractility of the detrusor muscle (Fig. 3A,B) and of the ileal longitudinal smooth muscle (Fig. 3C,D). Their contractility in response to 50 mM KCl was comparable among wild-type, M₂/, M₃/, and M₂/M₃/ tissues. Strikingly, however, the M₂/M₃/ muscles did not respond to carbachol, even at concentrations (e.g., 10⁵ M) that evoked maximal contractions in the wild-type muscles. Although application of higher dosages induced small contractions, these responses were blocked by hexamethonium, which indicated a slight contribution by nicotinic receptors (data not shown). Regarding the role of each subtype in mediating contractions, the proportions reduced by the M₃ loss (95% in the bladder, 72-77% in the ileum) reported by Matsui et al. (2000) were essentially the same as those that remained in the M₂/ tissues (Fig. 3) (Stengel et al., 2000). Thus, we conclude that M₂ and M₃ receptors are entirely responsible for the cholinergic contraction of smooth muscles in an additive manner.

View larger version (45K): [in this window] [in a new window]   Figure 3.   Abolished bladder (A, B) and ileal (C, D) smooth muscle contractions in response to carbachol in the M2/M3/ mice. Responses to carbachol are shown (mean ± SEM) as percentages of those to 50 mM KCl in the male (A, C) wild-type, M2/, M3/, and M2/M3/ mice and the female (B, D) wild-type, M2/, M3/, and M2/M3/ mice. Each symbol represents the data of four experiments. The data of the M3/ mice have been published previously (Matsui et al., 2000).

In vitro contractility of smooth muscles to EFS

When stimulated in an electrical field, intramural neurons of the smooth muscle tissue release multiple endogenous neurotransmitters that elicit muscle contractions. To estimate the possible difference in the noncholinergic component of the EFS-induced contraction, we compared the responses between the mutant tissues with those of the wild type treated with atropine.

When the male bladder strips were stimulated in an electrical field at 10 Hz frequency, the noncholinergic (i.e., atropine-resistant) contraction was significantly weaker in the mutants than in the wild type (40.3 vs 102.3%; p < 0.05) (Fig. 4B). A similar tendency was detected in the stimulation at 1 Hz frequency (8.9 vs 18.2%; p = 0.065) (Fig. 4A). This reduction may reflect a functional damage to the intramural noncholinergic neurons, which may be caused by the severe extension of the bladder wall. In the female mutant tissue, in contrast, noncholinergic components were substantially larger (24.3 vs 12.9% at 1 Hz and 115.6 vs 84.3% at 10 Hz; statistically not significant) (Fig. 4A,B). This may reflect a weak compensatory upregulation of the noncholinergic contraction mechanism.

View larger version (32K): [in this window] [in a new window]   Figure 4.   Noncholinergic contractile responses to EFS of the bladder (A, B) and ileal (C, D) smooth muscles. Responses to EFS (1 Hz, A, C; 10 Hz, B, D) are shown (mean with SEM; each bar represents the data from four mice) as percentages of those to 50 mM KCl.

In the ileum of females, the noncholinergic response elicited by EFS at 10 Hz frequency was stronger in the mutants than in the wild type (65.6 vs 25.2%; p < 0.01) (Fig. 4D). A similar tendency was detected in males as well when stimulated at 1 Hz (16.7 vs 7.3%; p = 0.23) (Fig. 4C). These findings may suggest that the noncholinergic excitatory stimulations were upregulated in a compensatory manner.

Finally, we analyzed the sex difference in the proportion of cholinergic contraction to the total contraction in the wild-type tissues. Interestingly, cholinergic components in the urinary bladder contraction were larger in males than in females. In males, the contributions of cholinergic contraction were 28% (1 Hz stimulation) to 45% (10 Hz stimulation), whereas they were only 18% (1 Hz stimulation) to 27% (10 Hz stimulation) in females. This is consistent with our interpretation of the sex difference in the urinary retention phenotype that the cholinergic mechanism in vivo is more important in males than in females.

Pupillary constriction by M₂ loss

Muscarinic stimulation is known to cause strong constriction of the pupil (Gil et al., 2001). As reported previously (Matsui et al., 2000), the M₃/ mice showed partially dilated pupils (Fig. 5C). In contrast, the pupil size of the M₂/ mice looked normal (Fig. 5B), suggesting that M₂ is dispensable for normal pupillary function. Strikingly, however, the M₂/M₃/ mice had smaller pupils (Fig. 5D) than the M₃/ mice (Fig. 5C). These results suggest a role of M₂ in dilation, rather than constriction, of pupils. An instillation of 1% atropine solution resulted in additional mydriasis (Fig. 5E), suggesting that M₁, M₄, and/or M₅ receptors might play some additional roles in constricting the pupils in the M₂/M₃/ mouse (see Discussion).

View larger version (39K): [in this window] [in a new window]   Figure 5.   Appearance of pupils of the wild-type (A), M2/ (B), M3/ (C), and M2/M3/ (D) mice. The pupils of the M2/M3/ mice (D) are smaller than those of the M3/ mice (C). Full mydriasis was achieved in the M2/M3/ mouse after instillation of atropine solution (E). The pupils of the M2/ mice (B) are indistinguishable from those of the wild-type mice (A). Scale bars, 1 mm. F, G, Pupil diameter (mean ± SEM; n = 4-30) of the mutant mice, measured in a bright room (~1000 lux). The M2/M3/ mice have smaller pupils than the M3/ mice in both sexes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Role of muscarinic receptor subtypes in cholinergic contractions

Whereas M₃ is known to play a dominant role in eliciting smooth muscle contractions, the significance of other colocalizing subtypes has been understood poorly. Thus far, the following indirect roles of M₂ have been suggested. (1) Relaxation of smooth muscles induced by isoproterenol could be reversed by the stimulation of M₂ through the inhibition of adenylate cyclase (Ehlert et al., 1999). (2) Stimulation of M₂ may have a modulatory role on the intracellular signaling of M₃ (Ehlert et al., 1999). Here, we demonstrated that M₂ has considerable potency to contract the muscle "directly," i.e., independent of the coexisting M₃ receptors or accompanying stimuli to increase cAMP levels. From the response curves shown in Figure 3, we estimated the proportion of contribution by each subtype. Contributions by M₁, M₂, M₃, M₄, and M₅ receptors were ~0, 5, 95, 0, and 0%, respectively, in detrusor muscle contraction and ~0, 25, 75, 0, and 0%, respectively, in ileal longitudinal muscle contraction. The mechanism of the direct contraction through M₂ remains unclear, but it may involve the opening of nonselective cation channels (Kotlikoff et al., 1999).

In vivo significance of cholinergic contractions in visceral organs

In humans, systemic administration of atropine delays gastric emptying (Parkman et al., 1999b) and reduces colonic motility (O'Brien et al., 1997). Therefore, muscarinic antagonists, such as atropine, are used widely as a premedication to reduce gastrointestinal movements. However, an elaborate study (Waxman et al., 1991) showed the clinical efficacy of such anticholinergic pretreatment in colonoscopy to be limited, raising a question about the role of cholinergic signaling in smooth muscle contraction in vivo.

Loss of key molecules in gastrointestinal motility can lead to the apparent bowel obstruction in the relevant mutant mice, as exemplified by enlarged stomachs of the mice deficient in neuronal nitric oxide synthase (Huang et al., 1993). However, we found no such obstruction phenotype in the gastrointestinal or female urinary tracts, despite the complete loss of cholinergic contractions in these tissues. Thus, cholinergic contractions cannot be regarded as a prerequisite for the basal function of these organs. It is worth noting that the phenotypes are essentially identical between our gene-targeted mice and the in vivo effects caused by an acute pharmacological block of the muscarinic receptors (Hardman et al., 2001). Low doses of atropine cause dryness of the mouth, and moderate doses result in mydriasis. We reported a similar phenotype in the M₃/ mice (Matsui et al., 2000). The urinary disturbance and inhibition of peristalsis become evident at relatively high dosages. Consistent with the intact gut appearance of M₂/M₃/ mutant mice, it was reported that acute administration of a high dose of atropine did not block gut motility completely (Ruwart et al., 1979; Calignano et al., 1992).

In the intestines and female bladder, neurotransmitters other than ACh should contribute significantly to the smooth muscle contractions in vivo and compensate for the loss of cholinergic signaling sufficiently. In fact, neurotransmission in the enteric nervous system is characterized by functional redundancy through multiple parallel pathways (Goyal et al., 1996; Furness, 2000). In our EFS experiment, a substantial increase in noncholinergic (i.e., atropine-resistant) contraction was detected in the female M₂/M₃/ tissues. These results suggest that excitatory inputs other than the cholinergic stimuli were upregulated in the mutant tissue and contributed to the functional compensation in vivo under certain conditions. In the intestines, such inputs may be mediated by tachykinins (Holzer and Holzer-Petsche, 1997) or novel neurotransmitters yet to be identified. In female urination, other parasympathetic neurotransmitters, such as purine (Burnstock et al., 1972), should be of primary importance, because neurotransmission blockade at the parasympathetic ganglion results in severe urinary retention (Xu et al., 1999a,b).

M₂ as a pupil dilator

We found that the pupils of the M₂/M₃/ mice are unexpectedly smaller than those of the M₃/ mice. It is worth noting that muscarinic autoreceptors inhibiting ACh release were found in the parasympathetic nerve terminals in the iris, and the subtype was presumed to be M₂ (Bognar et al., 1990). Lack of such autoreceptors would result in ACh over-release. Because topical applications of atropine caused full mydriasis in the M₃/ (Matsui et al., 2000) and M₂/M₃/ (Fig. 5E) pupils, it is conceivable that the over-released ACh stimulated the residual subtypes (M₁, M₄, and/or M₅) and caused the relative mydriasis. However, precise mechanisms of the phenomenon remain inconclusive, because atropine interferes with other receptors, such as histamine receptor H₁ at high concentrations in the range of 1-10 µM (Arunlakshana and Schild, 1959). Because the actual concentration at the smooth muscle of the iris cannot be determined, it is uncertain whether the effects of atropine are selective only to the muscarinic receptors (1% solution is ~0.03 M).

It should be noted that small amounts of M₂ are expressed in other tissues of the iris, such as the pupillary sphincter muscle (Ishizaka et al., 1998) and the sympathetic nerve terminals at the dilator muscle (Jumblatt and Hackmiller, 1994). However, our results are unlikely to be caused by the effects in such tissues. It is also possible that loss of M₂ altered cholinergic neurotransmission in the CNS and influenced the reflex control of the pupil. In fact, a tonic muscarinic inhibitory input to the Edinger-Westphal nucleus was found in the dog (Sharpe and Pickworth, 1981).

Finally, it may be speculated that other subtypes, particularly M₁ and M₅, were upregulated as a result of M₂ loss and contributed to the relative miotic phenotype of the M₂/M₃/ animals. However, this seems rather unlikely, because no compensatory changes in other subtypes have been found in any previous reports on muscarinic receptor-deficient mice (Hamilton et al., 1997; Gomeza et al., 1999a,b; Matsui et al., 2000; Yamada et al., 2001a,b).

Conclusion

We discovered that cholinergic contractions are not necessarily required for gastrointestinal or female urinary functions. These pieces of information should facilitate additional studies on other mediators and help establish a valid rationale for developing better drugs for diseases with autonomic dysregulation. Such diseases include urinary incontinence, irritable bowel syndrome, and posttraumatic stress disorder. Thus far, M₂ receptors are considered to play only minor roles in smooth muscles, despite its abundant expression. Our gene-targeted mice should be useful to further unravel the functions of this subtype. In fact, we demonstrated an unexpected role of M₂ in pupillary dilation that may help develop a novel class of muscarinic drugs for ocular diseases.

	FOOTNOTES

Received June 7, 2002; revised Sept. 17, 2002; accepted Sept. 24, 2002.

This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture (M.M., M.M.T.), by Industrial Technology Research Grant Programs in 2000 and 2002 from the New Energy and Industrial Technology Development Organization of Japan (M.M.), by a grant from the Organization for Pharmaceutical Safety and Research, Japan (M.M.T.), and by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (T.M.). We thank T. Tamai, T. Ishikawa, and K. Takaku for technical advice; A. M. Watabe and H. Morikawa for critical reading of this manuscript; I. Ishii, A. Matsunaga, and A. Yokoi for blastocyst injections; Y. Araki, S. Kobayashi, N. Matsubara, and H. Karasawa for technical assistance; and S. Ishikawa and his staff for animal care.

Correspondence should be addressed to Dr. Minoru Matsui, Division of Neuronal Network, Department of Basic Medical Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: mmatsui{at}dd.iij4u.or.jp.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Andersson KE (1998) The importance of the cholinergic system in neurourology. Eur Urol 34:6-9[Medline].
• Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol 14:48-58[Medline].
• Bognar IT, Wesner MT, Fuder H (1990) Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris. Naunyn Schmiedebergs Arch Pharmacol 341:22-29[Medline].
• Boschero AC, Szpak-Glasman M, Carneiro EM, Bordin S, Paul I, Rojas E, Atwater I (1995) Oxotremorine-m potentiation of glucose-induced insulin release from rat islets involves M3 muscarinic receptors. Am J Physiol 268:E336-E342[Medline].
• Burnstock G, Dumsday B, Smythe A (1972) Atropine-resistant excitation of the urinary bladder: possibility of transmission via nerves releasing a purine nucleotide. Br J Pharmacol 44:451-461[ISI][Medline].
• Calignano A, Capasso A, Persico P, Mancuso F, Sorrentino L (1992) Dexamethasone modifies morphine-, atropine-, verapamil-induced constipation in mice. Gen Pharmacol 23:753-756[Medline].
• Caulfield MP, Birdsall NJM (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279-290[Abstract/Free Full Text].
• Choppin A, Stepan GJ, Loury DN, Watson N, Eglen RM (1999) Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats. Br J Pharmacol 127:1551-1558[Medline].
• de Groat WC, Yoshimura N (2001) Pharmacology of the lower urinary tract. Annu Rev Pharmacol Toxicol 41:691-721[ISI][Medline].
• Eglen RM (2001) Muscarinic receptors and gastrointestinal tract smooth muscle function. Life Sci 68:2573-2578[ISI][Medline].
• Eglen RM, Michel AD, Whiting RL (1989) Characterization of the muscarinic receptor subtype mediating contractions of the guinea-pig uterus. Br J Pharmacol 96:497-499[Medline].
• Eglen RM, Choppin A, Watson N (2001) Therapeutic opportunities from muscarinic receptor research. Trends Pharmacol Sci 22:409-414[Medline].
• Ehlert FJ, Sawyer GW, Esqueda EE (1999) Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle. Life Sci 64:387-394[ISI][Medline].
• Furness JB (2000) Types of neurons in the enteric nervous system. J Auton Nerv Syst 81:87-96[ISI][Medline].
• Giglio D, Delbro DS, Tobin G (2001) On the functional role of muscarinic M(2) receptors in cholinergic and purinergic responses in the rat urinary bladder. Eur J Pharmacol 428:357-364[Medline].
• Gil D, Spalding T, Kharlamb A, Skjaerbaek N, Uldam A, Trotter C, Li D, WoldeMussie E, Wheeler L, Brann M (2001) Exploring the potential for subtype-selective muscarinic agonists in glaucoma. Life Sci 68:2601-2604[Medline].
• Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng H, Wess J (1999a) Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96:1692-1697[Abstract/Free Full Text].
• Gomeza J, Zhang L, Kostenis E, Felder C, Bymaster F, Brodkin J, Shannon H, Xia B, Deng C, Wess J (1999b) Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M4 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96:10483-10488[Abstract/Free Full Text].
• Goyal RK, Hirano I (1996) The enteric nervous system. N Engl J Med 334:1106-1115[Free Full Text].
• Hamilton SE, Loose MD, Qi M, Levey AI, Hille B, McKnight GS, Idzerda RL, Nathanson NM (1997) Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice. Proc Natl Acad Sci USA 94:13311-13316[Abstract/Free Full Text].
• Hardman JG, Limbird LE, Gilman AG (2001) In: Goodman and Gilman's the pharmacological basis of therapeutics, Ed 10. New York: McGraw-Hill.
• Holzer P, Holzer-Petsche U (1997) Tachykinins in the gut. I. Expression, release and motor function. Pharmacol Ther 73:173-217[ISI][Medline].
• Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC (1993) Targeted disruption of the neuronal nitric oxide synthase gene. Cell 75:1273-1286[ISI][Medline].
• Ishiura Y, Yoshiyama M, Yokoyama O, Namiki M, de Groat WC (2001) Central muscarinic mechanisms regulating voiding in rats. J Pharmacol Exp Ther 297:933-939[Abstract/Free Full Text].
• Ishizaka N, Noda M, Yokoyama S, Kawasaki K, Yamamoto M, Higashida H (1998) Muscarinic acetylcholine receptor subtypes in the human iris. Brain Res 787:344-347[ISI][Medline].
• Jumblatt JE, Hackmiller RC (1994) M2-type muscarinic receptors mediate prejunctional inhibition of norepinephrine release in the human iris-ciliary body. Exp Eye Res 58:175-180[Medline].
• Kotlikoff MI, Dhulipala P, Wang YX (1999) M2 signaling in smooth muscle cells. Life Sci 64:437-442[ISI][Medline].
• Lepor H, Kuhar MJ (1984) Characterization of muscarinic cholinergic receptor binding in the vas deferens, bladder, prostate and penis of the rabbit. J Urol 132:392-396[Medline].
• Matsui M, Araki Y, Karasawa H, Matsubara N, Taketo MM, Seldin MF (1999) Mapping of five subtype genes for muscarinic acetylcholine receptor to mouse chromosomes. Genes Genet Syst 74:15-21[Medline].
• Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S, Taketo MM (2000) Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci USA 97:9579-9584[Abstract/Free Full Text].
• O'Brien MD, Camilleri M, Thomforde GM, Wiste JA, Hanson RB, Zinsmeister AR (1997) Effect of cholecystokinin octapeptide and atropine on human colonic motility, tone, and transit. Dig Dis Sci 42:26-33[Medline].
• Parkman HP, Pagano AP, Ryan JP (1999a) Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions. Am J Physiol 276:G1243-G1250[Medline].
• Parkman HP, Trate DM, Knight LC, Brown KL, Maurer AH, Fisher RS (1999b) Cholinergic effects on human gastric motility. Gut 45:346-354[Abstract/Free Full Text].
• Ponec RJ, Saunders MD, Kimmey MB (1999) Neostigmine for the treatment of acute colonic pseudo-obstruction. N Engl J Med 341:137-141[Abstract/Free Full Text].
• Pontari MA, Luthin GR, Braverman AS, Ruggieri MR (1998) Characterization of muscarinic cholinergic receptor subtypes in rat prostate. J Recept Signal Transduct Res 18:151-166[ISI][Medline].
• Ruwart MJ, Klepper MS, Rush BD (1979) Evidence for noncholinergic mediation of small intestinal transit in the rat. J Pharmacol Exp Ther 209:462-465[Free Full Text].
• Sawyer GW, Ehlert FJ (1998) Contractile roles of the M2 and M3 muscarinic receptors in the guinea pig colon. J Pharmacol Exp Ther 284:269-277[Abstract/Free Full Text].
• Schmid SW, Modlin IM, Tang LH, Stoch A, Rhee S, Nathanson MH, Scheele GA, Gorelick FS (1998) Telenzepine-sensitive muscarinic receptors on rat pancreatic acinar cells. Am J Physiol 274:G734-G741[Medline].
• Sharpe LG, Pickworth WB (1981) Pharmacologic evidence for a tonic muscarinic inhibitory input to the Edinger-Westphal nucleus in the dog. Exp Neurol 71:176-190[Medline].
• Soriano P, Montgomery C, Geske R, Bradley A (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693-702[ISI][Medline].
• Stengel PW, Gomeza J, Wess J, Cohen ML (2000) M2 and M4 receptor knockout mice: muscarinic receptor function in cardiac and smooth muscle in vitro. J Pharmacol Exp Ther 292:877-885[Abstract/Free Full Text].
• Waxman I, Mathews J, Gallagher J, Kidwell J, Collen MJ, Lewis JH, Cattau Jr EL, al-Kawas FH, Fleischer DE, Benjamin SB (1991) Limited benefit of atropine as premedication for colonoscopy. Gastrointest Endosc 37:329-331[Medline].
• Xu W, Gelber S, Orr-Urtreger A, Armstrong D, Lewis RA, Ou CN, Patrick J, Role L, De Biasi M, Beaudet AL (1999a) Megacystis, mydriasis, and ion channel defect in mice lacking the 3 neuronal nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 96:5746-5751[Abstract/Free Full Text].
• Xu W, Orr-Urtreger A, Nigro F, Gelber S, Sutcliffe CB, Armstrong D, Patrick JW, Role LW, Beaudet AL, De Biasi M (1999b) Multiorgan autonomic dysfunction in mice lacking the 2 and the 4 subunits of neuronal nicotinic acetylcholine receptors. J Neurosci 19:9298-9305[Abstract/Free Full Text].
• Yagi T, Ikawa Y, Yoshida K, Shigetani Y, Takeda N, Mabuchi I, Yamamoto T, Aizawa S (1990) Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection. Proc Natl Acad Sci USA 87:9918-9922[Abstract/Free Full Text].
• Yamada M, Lamping KG, Duttaroy A, Zhang W, Cui Y, Bymaster FP, McKinzie DL, Felder CC, Deng CX, Faraci FM, Wess J (2001a) Cholinergic dilation of cerebral blood vessels is abolished in M5 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 98:14096-14101[Abstract/Free Full Text].
• Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, Felder CC, Deng CX, Wess J (2001b) Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature 410:207-212[Medline].

---

Copyright © 2002 Society for Neuroscience  0270-6474/02/222410627-06$05.00/0

This article has been cited by other articles:

				A. Shekhar, W. Z. Potter, J. Lightfoot, J. Lienemann, S. Dube, C. Mallinckrodt, F. P. Bymaster, D. L. McKinzie, and C. C. Felder Selective Muscarinic Receptor Agonist Xanomeline as a Novel Treatment Approach for Schizophrenia Am J Psychiatry, August 1, 2008; 165(8): 1033 - 1039. [Abstract] [Full Text] [PDF]

				F. J. Ehlert, S. Ahn, K. J. Pak, G. J. Park, M. S. Sangnil, J. A. Tran, and M. Matsui Neuronally Released Acetylcholine Acts on the M2 Muscarinic Receptor to Oppose the Relaxant Effect of Isoproterenol on Cholinergic Contractions in Mouse Urinary Bladder J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 631 - 637. [Abstract] [Full Text] [PDF]

				T. Takeuchi, K. Tanaka, H. Nakajima, M. Matsui, and Y.-T. Azuma M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: findings obtained with muscarinic-receptor knockout mouse Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G154 - G164. [Abstract] [Full Text] [PDF]

				Y.-K. Ng, W. C. de Groat, and H.-Y. Wu Muscarinic regulation of neonatal rat bladder spontaneous contractions Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2006; 291(4): R1049 - R1059. [Abstract] [Full Text] [PDF]

				J. A. Tran, M. Matsui, and F. J. Ehlert Differential Coupling of Muscarinic M1, M2, and M3 Receptors to Phosphoinositide Hydrolysis in Urinary Bladder and Longitudinal Muscle of the Ileum of the Mouse J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 649 - 656. [Abstract] [Full Text] [PDF]

				J. W. Wegener, V. Schulla, A. Koller, N. Klugbauer, R. Feil, and F. Hofmann Control of intestinal motility by the Cav1.2 L-type calcium channel in mice FASEB J, June 1, 2006; 20(8): 1260 - 1262. [Abstract] [Full Text] [PDF]

				H.-M. Zhang, S.-R. Chen, M. Matsui, D. Gautam, J. Wess, and H.-L. Pan Opposing Functions of Spinal M2, M3, and M4 Receptor Subtypes in Regulation of GABAergic Inputs to Dorsal Horn Neurons Revealed by Muscarinic Receptor Knockout Mice Mol. Pharmacol., March 1, 2006; 69(3): 1048 - 1055. [Abstract] [Full Text] [PDF]

				K. E. Belmonte Cholinergic Pathways in the Lungs and Anticholinergic Therapy for Chronic Obstructive Pulmonary Disease Proceedings of the ATS, November 1, 2005; 2(4): 297 - 304. [Abstract] [Full Text] [PDF]