Functional analysis of muscarinic acetylcholine receptors using knockout mice

Abstract

Because of the low selectivity of available ligands, pharmacological approaches to elucidate the functional difference among muscarinic acetylcholine receptor (mAChR) subtypes have been problematic. As an alternative approach, we have established a series of mutant mouse lines deficient in each mAChR subtype (mAChR KO mice). The systematic analyses of these mice have been useful in revealing the functional difference among mAChR subtypes. Here, we review our prior research on these mutant mice and also some notable findings reported by other research groups.

Introduction

Hammer et al. (1980) found that pirenzepine distinguishes different subclasses of mAChRs in the brain, salivary glands, heart, and smooth muscle. During 1980–1983, several laboratories reported similar subclasses using pharmacological methods. In these reports, the nomenclatures “M₁, M₂, M₃” and “m1, m2, m3” were used in inconsistent manners. During 1986–1988, molecular cloning studies identified five distinct genes, which encode putative mAChRs. The first cloning of mAChR cDNA was reported by a group of Numa (Kubo et al., 1986a). Subsequently, several groups reported cDNAs for the other subtypes (Kubo et al., 1986b, Peralta et al., 1987, Bonner et al., 1987, Bonner et al., 1988). Upon these discoveries, the proteins translated from the cloned cDNAs were designated as m1, m2, m3, m4 and m5, while pharmacologically defined subclasses were designated as M₁, M₂, M₃, M₄, and M₅. Through the subsequent pharmacological characterization of these gene products, it was recognized that M₁, M₂, M₃, M₄, and M₅ affinities are attributable reasonably to m1, m2, m3, m4, and m5, respectively. Therefore, it is now recommended to use “M₁, M₂, M₃, M₄, and M₅,” when referring to both proteins and pharmacological activities.

mAChRs are therapeutic targets in various diseases. For example, cognitive deficits in Alzheimer's disease can be alleviated by elevation of ACh levels in the brain, which can be achieved by the systemic administration of cholinesterase inhibitors. Antimuscarinic agents are used widely for the treatment of Parkinson's disease, bronchial asthma, peptic ulcer, and overactive bladder. Muscarinic agonists have therapeutic efficacy in facilitating salivation in patients suffering from dry mouth. It has recently been proposed that an unbalanced autonomic nervous system may be a major cause of the metabolic syndrome (Kreier et al., 2003). Despite this wide range of therapeutic potential, the clinical use of muscarinic and antimuscarinic drugs is often limited by undesirable side effects. In considering that most of the available agents lack high subtype selectivity, detailed information on the roles of each subtype is likely to foster the development of novel subtype-specific drugs with fewer side effects.

mAChRs belong to the seven transmembrane-spanning receptor family and are distributed widely in both central and peripheral nervous systems. Among the five subtypes, the M₁, M₃, and M₅ receptors are usually coupled to the G_q/11 protein, which activates phospholipase C, whereas the M₂ and M₄ receptors are mainly coupled to the G_i/o protein, which inhibits adenylate cyclase activity.

The distribution of mAChR subtypes in central and peripheral tissues has been investigated mainly by pharmacological studies with subtype selective agents and also by molecular, immunohistochemical and immunoprecipitational studies with measurements of the expression of mRNA levels and receptor protein (Dörje et al., 1991, Wall et al., 1991, Levey, 1993, Wei et al., 1994, Bymaster et al., 2003, Wess et al., 2003). According to previous reports with the measurements of mRNA and receptor protein in rat brain by the immunohistochemical assay (Levey et al., 1991, Wall et al., 1991, Levey, 1993, Vilaro et al., 1990, Wei et al., 1994), the M₁ receptor is abundantly expressed in all forebrain areas including the cerebral cortex, hippocampus and corpus striatum, where this subtype consists of 40∼50% of the total mAChRs. Consistent with these results, Miyakawa et al. (2001) have found a 50% decrease in the total number of [³H]QNB binding sites in the striatum of M₁ KO mice. The predominant localization of M₄ receptor in the rat striatum was verified by immunoprecipitation experiments with selective polyclonal antisera (Yasuda et al., 1993). The M₄ receptor is co-localized with distinct dopamine receptor subtypes on striatal projection neurons (Bernard et al., 1992, Ince et al., 1997). The pharmacological antagonism of muscarinic agonist-mediated inhibition of adenylate cyclase activity in the corpus striatum is consistent with an M₄ mechanism (Ehlert et al., 1989, Olianas et al., 1996). The immunoprecipitation studies also showed that the majority (84%) of mAChRs in the rat brainstem was the M₂ subtype (Levey et al., 1991). The immunoprecipitation and in situ hybridization studies have detected M₅ receptor protein/mRNA in several brain regions including midbrain regions (substantia nigra and ventral tegmental area), but the expression level is very low, representing less than 2% of the total mAChR population (Yasuda et al., 1993, Vilaro et al., 1990, Weiner et al., 1990). Thus, it is likely that M₅ receptor subtype is distributed at very low levels through the CNS.

Dörje et al. (1991) have demonstrated the presence of four (M₁–M₄) distinct receptor proteins in rabbit peripheral tissues using an immunoprecipitation assay with subtype-specific antisera, with notable differences in their tissue distribution. The M₂ receptor was the major subtype in the sympathetic ganglia, ileum, uterus and atrium. The M₃ subtype was abundant in the submaxillary gland, and the M₂ and M₄ subtypes were present in the lung. It has been shown that the M₅ receptor subtype may be expressed in blood lymphocytes (Costa et al., 1995, Tayebati et al., 1999), skin fibroblasts (Buchli et al., 1999), smooth muscles of the iris sphincter (Gil et al., 1997) and esophagus (Preiksaitis et al., 2000), and parotid gland (Bockman et al., 2001), where they are co-localized with other mAChR subtypes.

To elucidate subtype-specific functions of mAChRs, some research groups have generated mutant mice deficient in mAChRs (mAChR KO mice). In the first report on mAChR KO mice (Hamilton et al., 1997), the generation and general characterization of M₁ KO mice were described. M₁ KO mice were also generated by three other research groups of Wess (Miyakawa et al., 2001), Tonegawa (Gerber et al., 2001) and Matsui (Ohno-Shosaku et al., 2003), respectively. Wess and his colleagues reported M₂ KO mice and M₄ KO mice (Gomeza et al., 1999a, Gomeza et al., 1999b) and, subsequently, Matsui et al. also reported these mice (Matsui et al., 2002, Karasawa et al., 2003). The groups of Matsui et al. (2000) and Wess (Yamada et al., 2001b) reported M₃ KO mice independently and some of the conclusions are contradictory (see below). Wess et al. (Yamada et al., 2001a) and Yeomans et al. (Takeuchi et al., 2002) reported mutant mice for the M₅ gene. Matsui et al. also generated M₅ KO mice recently (Nakamura et al., 2004).

In addition to the single knockout mice deficient in one of the five subtypes described above, mutant mice lacking more than one subtype are useful because multiple subtypes are co-localized in various organs. To date, the following compound mutant mice have been reported: M₁/M₃ mutants (Ohno-Shosaku et al., 2003), M₁/M₅ mutants (Nakamura et al., 2004), M₂/M₃ mutants (Matsui et al., 2002), and M₂/M₄ mutants (Zhang et al., 2002, Fukudome et al., 2004).

In all of the reported mAChR KO mice, null mutations of the corresponding genes were introduced by conventional homologous recombination methods in the embryonic stem cells. Conditional targeting strategy to eliminate mAChRs in a spatiotemporal manner has not been reported.

It is well known that administration of mAChR antagonists such as scopolamine causes amnesia in animals. In Alzheimer's disease, there is a selective loss of cholinergic neurons, and systemic administration of cholinesterase inhibitors improves cognitive function possibly through the stimulation of both nicotinic and muscarinic acetylcholine receptors (Terry and Buccafusco, 2003). Therefore, it is of particular interest whether mAChR KO mice have deficits in learning and memory.

Miyakawa et al. (2001) reported that hippocampus-dependent learning was intact in M₁ KO mice using Morris water maze and contextual fear conditioning. Later, Anagnostaras et al. (2003) found that the M₁ KO mice were impaired in non-matching-to-sample working memory as well as consolidation. In this report, the authors proposed that the M₁ receptor is critical in memory processes involving an interaction between the cerebral cortex and hippocampus. In the M₂ KO mice, an impaired performance in the passive avoidance test has been reported (Tzavara et al., 2003). Recently, we have reported that endocannabinoid signaling is modulated by M₁ and M₃ receptors in the hippocampus (Ohno-Shosaku et al., 2003, Fukudome et al., 2004).

Because the deficits in learning and memory performance in these single KO mice seem rather moderate, it is of interest to investigate whether some of the compound KO mice show further deterioration. Considering that mAChRs are distributed among various areas in the brain, region-specific KO mice would be a powerful tool to elucidate the roles of each subtype in a specific brain area, such as hippocampus and cerebral cortex. The possible involvement of each mAChR subtype in Alzheimer's disease pathology has never been studied in any of the mAChR KO mice.

In the initial report of the M₃ KO mice (Matsui et al., 2000), multiple apparent abnormalities were described. These include growth retardation around weaning, impaired salivation, distended urinary bladders, and dilated pupils.

Although the growth of the M₃ KO pups is almost normal before two weeks of age, it becomes retarded significantly thereafter. Around four-five weeks of age, the average body weight of the M₃ KO pups is only about half of that of WT, and some of the M₃ KO pups become sick and die. Notably, the surviving pups catch up gradually in their growth with their WT littermates. At fifteen weeks of age, the body weight of the M₃ KO males becomes almost the same as that of the WT littermates, whereas the M₃ KO females are still smaller than the WT littermates. Another group reported similar growth pattern independently (Yamada et al., 2001b). However, the authors designated this phenotype as “leanness” rather than “growth retardation.” In their report, even males did not catch up with WT in their body weight.

Regarding the proposed mechanisms of the growth retardation (or leanness) phenotype, there are considerable differences between the two research reports. Matsui et al. proposed that the growth retardation was caused mainly by insufficient food intake resulting from reduced salivation. This interpretation was supported by three observations. First, the growth failure was not evident before weaning. Second, salivation induced by a moderate dose of pilocarpine (1 mg/kg) was nearly abolished. Third, wet paste feeding improved the growth of the pups. In contrast, Wess et al. hypothesized that the lean phenotype was most probably caused by central anorexia resulting from hypothalamic dysfunction, not by peripheral abnormalities such as salivary insufficiency. They showed that the salivary function was not impaired by strong muscarinic stimulation (pilocarpine, 15 mg/kg). In addition, they found that acute intracerebroventricular infusions of agouti-related peptide failed to stimulate food intake, which implies a disruption in the signaling cascade in central feeding centers. Thus, it remains unanswered whether the salivation in the M₃ KO mice induced by strong mAChR stimulation represents physiological salivation or not. Recently, we reported that M₃ plays a critical role in physiological salivation in mice (Nakamura et al., 2004). We conducted Ca²⁺ imaging experiments using dispersed salivary gland cells. The transient rise in intracellular Ca²⁺ elicited by carbachol with a wide concentration range was very small in the M₃ KO mice and was totally abolished in the M₁/M₃ KO mice. This confirms the primary importance of M₃ in mediating parasympathetic signals in the salivary gland cells. Furthermore, we noticed that the M₃ KO mice showed characteristic behavior during eating; they approached water nozzle very frequently when they ate dry pellet food. Because this frequent drinking behavior was not observed when they ate wet paste, it is suggested that the M₃ KO mice did not secrete enough saliva that was necessary to eat dry pellets. Thus, the lack of M₃-mediated signaling in the periphery leads to severe salivary insufficiency that causes considerable health problems.

Various additional abnormalities were reported in the M₃ KO mice. Stengel et al. (2002) found that the stomach of the M₃ KO mice was defective in smooth muscle contractility. Aihara et al. (2003) reported that secretion of gastric acid was impaired in the M₃ KO mice. Taken together, it is probable that these dysfunctions in the digestive organs make additional contributions to the malnutrition phenotype of the M₃ KO mice. Recently, impairment in insulin secretion has been shown in vitro (Zawalich et al., 2004).

The distension of the urinary bladder is a prominent phenotype in the abdomen, especially in males. In adult M₃ KO males, the mean diameter of the bladder was about twice of that of the WT males. Contractility of the detrusor muscle strip to carbachol was reduced to only 5% of the WT strip in both sexes. Although urodynamic studies are necessary to address the underlying mechanism of the urinary retention phenotype, it is possible that the mild urinary retention in females reflects lower outflow resistance.

Whereas a number of pharmacological studies have shown that the M₃ subtype is responsible for most of the cholinergic contraction of smooth muscles, there is no apparent sign of constipation or ileus in the M₃ KO mice. It was reported that the transit speed of charcoal meal was normal in the M₃ KO mice (Yamada et al., 2001b). The contractility of ileal longitudinal smooth muscles to carbachol was reduced to about 25% of that of the WT muscles (Matsui et al., 2000). This indicates that M₃ is responsible for 75% of the carbachol-induced contraction. Our subsequent study showed that the residual 25% contraction is mediated by the M₂ receptor (Matsui et al., 2002; see below). There is a modest increase in the contractile sensitivity of the ileum to PGF_2α in M₃ KO mice suggesting a compensatory mechanism that offsets the large decrease in cholinergic function (Griffin et al., 2004).

The size of the pupils is under dual regulation of the sympathetic and parasympathetic nervous systems. Sympathetic nerves innervate pupillary dilator muscle, whereas parasympathetic nerves innervate pupillary constrictor muscle. Blockade of parasympathetic signaling by muscarinic antagonist such as atropine causes dilation of pupils (mydriasis). Consistent with this well-known phenomenon, the pupil size of the M₃ KO mice was significantly greater than that of the WT mice. Some of the subtypes other than M₃ are likely to play additional roles in pupillary constriction because atropine instillation resulted in further pupillary dilation in the M₃ KO mice.

In contrast to these multiple apparent phenotypes in the M₃ KO mice, the other receptor KO mice, i.e. the M₁ KO, M₂ KO, M₄ KO, and M₅ KO mice appear healthy and are indistinguishable from their WT littermates without specific experimentations.

Previous pharmacological studies have shown that the major excitatory input to smooth muscle is cholinergic. The M₃ receptors mediate most of the contractile responses to cholinergic agents, whereas M₂ is the most abundant subtype in the smooth muscle. Previous pharmacological studies have shown that the M₂ receptor is involved in smooth muscle contraction indirectly or by a mechanism contingent upon activation of another contractile receptor, like the M₃. For example, it has been shown that stimulation of the M₂ receptor inhibits the relaxant effects of various agents that increase cAMP levels on the contraction elicited by the M₃ muscarinic receptor and the H₁ histamine receptor (Ehlert et al., 1999). It was also reported that the M₂ receptor may potentiate M₃-mediated contractions (Ehlert et al., 1999). Because the contraction mediated by the M₃ receptor is so dominant and no highly M₃-specific antagonists are available, it is very difficult to detect a direct contractile role of the M₂ receptor and dissociate this potential role of the M₂ receptor from that of the M₃. It remained unknown whether stimulation of the M₂ receptor causes a direct contraction. Therefore, the use of KO mice was expected to overcome the limitation of the pharmacological approach to reveal a possible direct contractile role of M₂ in smooth muscle organs and to substantiate its indirect role in contraction.

To answer the above questions, we generated M₂/M₃-compound KO mice and compared their phenotype with those of the M₂ KO and M₃ KO mice (Matsui et al., 2002). Because the expression of the other subtypes (M₁, M₄, M₅) is undetectable in the smooth muscle tissues, lack of both the M₂ and the M₃ receptors was expected to cause severe disorders of smooth muscle organs, such as paralytic ileus. However, the intestinal appearance of these mutant mice was normal and indistinguishable from that of the WT mice. Because the ileal longitudinal smooth muscles prepared from the M₂/M₃ KO mice showed virtually no cholinergic contraction in vitro, it is suggested that the coordination of peristaltic movements can be maintained by local mediators and by motor neurons releasing neurotransmitters other than ACh. The smooth muscles prepared from the ileum and the urinary bladder deficient in the M₃ receptor showed weak but significant cholinergic contractions, which should be mediated by M₂. This indicates that stimulation of M₂ elicits significant contractile responses without any pretreatments. M₂-mediated proportion of the cholinergic contractility was estimated to be about 25% and 5% in the ileum longitudinal muscles and the detrusor muscles, respectively. Stengel et al. (2000) also reported that smooth muscle contractility of M₂ KO mice is impaired partially. We also showed that relaxant effects of forskolin and isoproterenol were increased against muscarinic agonist-induced smooth muscle contractions in the M₂ KO mice (Matsui et al., 2003). These results show that activation of M₂ receptors opposes the relaxant effects of agents that increase cAMP on M₃ receptor-mediated contractions. Interestingly, muscarinic agonist-mediated heterologous desensitization was abolished in the M₂ KO mice as well as in the M₃ KO mice, indicating that both M₂ and M₃ are required for this phenomenon (Griffin et al., 2004). These latter two studies confirm prior studies in the guinea pig ileum using pharmacological procedures (Thomas et al., 1993, Shehnaz et al., 2001, Ehlert et al., 2001).

The above findings indicate that M₂ and M₃ cooperate to elicit contractile responses in smooth muscle organs. However, with regard to the regulation of pupil size, the roles of M₂ and M₃ appear to be rather opposite. The pupil diameter of the M₂/M₃ KO mice was always smaller than that of the M₃ KO mice. This suggests that M₂ has a possible role in the relaxation of the pupillary constrictor muscle. Whereas the precise mechanism for this “M₂-mediated mydriatic effect” is unknown, it may represent a more central mechanism regulating outflow to the ciliary nerve.

Inhibition of striatal mAChR-mediated signaling can relieve extrapyramidal symptoms caused by hypofuction of dopaminergic neurons. In addition, muscarinic antagonists are used to treat catalepsy, which is caused by antagonizing dopaminergic receptors. The site of action of muscarinic antagonists is thought to be in the striatum, where M₁ and M₄ receptors predominate. Karasawa et al. (2003) used the bar test on M₄ KO mice after haloperidol administration to evaluate the expression of catalepsy. While the M₄ KO mice developed the cataleptic response normally, systemic administration of scopolamine did not suppress the cataleptic responses. These results suggest that acute, but not chronic, blockade of M₄ receptors plays an important role in the therapeutic effect of antimuscarinic agents. It should be also noted that this analysis was possible in the DBA/2J, but not in C57BL/6J, background, probably because the C57BL/6J strain is resistant to haloperidol in causing catalepsy. We should be aware that the popular C57BL/6J strain is not always useful in evaluation of the mAChR functions.

Because the general expression level of the M₅ receptor is quite low, the physiological significance of the M₅ receptor has long been uncertain. In addition, elucidation of the role of the M₅ receptor has been hampered by the lack of selective ligands for the M₅ receptor. Therefore, the use of knockout mice is the key practical approach to identify the function of this subtype. So far, knockout studies have revealed two major roles for the M₅ receptor. One is in blood flow regulation in the brain (Yamada et al., 2001a), and the other is in the reward system (Basile et al., 2002, Fink-Jensen et al., 2003). These findings will facilitate the development of M₅-selective agents, which may help treat brain ischemia and addictive disorders.

Several groups including ours have generated mutant mice deficient in mAChRs and analyzed their phenotypes to identify the role of each mAChR subtype. It is now clear that this approach using KO mice is quite powerful in elucidating the subtype-specific functions. Possible future technical improvements will include development of double or triple KO mice and conditional KO mice. Phenotypic differences among strains might be important because cholinergic activity may vary among mouse strains. The information on the precise distribution of each mAChR subtype together with that on its physiological function should contribute to the development of selective therapeutic agents targeting specific mAChR subtypes.