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Cholinergic Stimulation of Salivary Secretion Studied with M₁ and M₃ Muscarinic Receptor Single- and Double-Knockout Mice

Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland (D.G., T.H., Y.C., L.B., J.W.) and Veterinary Resources Program, National Center for Research Resources, National Institutes of Health, Bethesda, Maryland (G.M.)

Received January 15, 2004; accepted May 20, 2004

	Abstract

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Identification of the specific muscarinic acetylcholine receptor (mAChR) subtypes mediating stimulation of salivary secretion is of considerable clinical interest. Recent pharmacological and molecular genetic studies have yielded somewhat confusing and partially contradictory results regarding the involvement of individual mAChRs in this activity. In the present study, we re-examined the roles of M₁ and M₃ mAChRs in muscarinic agonist-mediated stimulation of salivary secretion by using M₁ and M₃ receptor single-knockout (KO) mice and newly generated M₁/M₃ receptor double-KO mice. When applied at a low dose (1 mg/kg, s.c.), the muscarinic agonist pilocarpine showed significantly reduced secretory activity in both M₁ and M₃ receptor single-KO mice. However, when applied at higher doses, pilocarpine induced only modestly reduced (5 mg/kg, s.c.) or unchanged (15 mg/kg, s.c.) salivation responses, respectively, in M₁ and M₃ receptor single-KO mice, indicating that the presence of either M₁ or M₃ receptors is sufficient to mediate robust salivary output. Quantitative reverse transcriptase-polymerase chain reaction studies with salivary gland tissue showed that the inactivation of the M₁ or M₃ mAChR genes did not lead to significantly altered mRNA levels of the remaining mAChR subtypes. Strikingly, the sialagogue activity of pilocarpine was abolished in M₁/M₃ receptor double-KO mice. However, salivary glands from M₁/M₃ receptor double-KO mice remained responsive to stimulation by the -adrenergic receptor agonist, (S)-isoproterenol. Taken together these studies support the concept that a mixture of M₁ and M₃ receptors mediates cholinergic stimulation of salivary flow.

The major salivary glands, the submandibular, sublingual, and parotid glands, are innervated by both parasympathetic and sympathetic nerves (Baum, 1993). Activation of the parasympathetic fibers innervating the salivary glands leads to a copious flow of saliva, mediated by acetylcholine-induced activation of acinar cell muscarinic acetylcholine receptors (mAChRs) (Baum, 1993). In fact, mAChR-mediated stimulation of saliva output is considered the major mechanism by which salivary fluid secretion is regulated physiologically (Baum, 1993; Turner and Sugiya, 2002; Scully, 2003).

Stimulation of mAChRs located on salivary acinar cells is known to lead to the activation of G proteins of the G_q family (Baum, 1993; Luo et al., 2001) which mediate an increase in intracellular calcium levels and the opening of calcium-activated Cl^– and K⁺ channels (Baum, 1993; Turner and Sugiya, 2002). Recent studies with gene knockout (KO) mice demonstrated that muscarinic agonist-induced salivary secretion is strongly reduced in mice lacking the aquaporin-5 water channel (Ma et al., 1999) or the Na⁺/K⁺/2Cl^– cotransporter (Evans et al., 2000), indicating that these two proteins represent key components of the signaling pathway stimulating salivary flow.

Identification of the specific mAChR subtype(s) mediating stimulation of salivary secretion is of considerable interest for the development of more selective muscarinic sialagogues or of drugs with reduced side effects. The mAChR family consists of five molecularly distinct members (M₁-M₅) that can be subdivided, based on their G protein-coupling profiles, into two major functional subclasses (Caulfield, 1993; Wess, 1996; Caulfield and Birdsall, 1998). Whereas the odd-numbered receptors (M₁, M₃, and M₅) are selectively coupled to G proteins of the G_q family, the even-numbered mAChR subtypes (M₂ and M₄) are preferentially linked to G proteins of the G_i class (Caulfield, 1993; Wess, 1996; Caulfield and Birdsall, 1998).

Pharmacological, biochemical, and molecular genetic studies indicate that the M₃ receptor subtype plays a key role in mediating increased salivary flow (Laniyonu et al., 1990; Dai et al., 1991; Caulfield, 1993; Watson et al., 1996; Moriya et al., 1999; Matsui et al., 2000; Bockman et al., 2001; Bymaster et al., 2003). However, pharmacological and biochemical studies suggest that M₁ (Watson and Culp, 1994; Culp et al., 1996; Luo et al., 2001; Tobin et al., 2002) and M₅ mAChRs (Flynn et al., 1997; Meloy et al., 2001; Tobin et al., 2002) may also play a role in muscarinic agonist-mediated salivary secretion. Moreover, two recent studies reported that muscarinic agonist-induced stimulation of salivary output was impaired not only in M₃ receptor KO mice but also in M₁ and M₄ receptor KO mice (Bymaster et al., 2003) and M₅ receptor KO mice (Takeuchi et al., 2002). Taken together, these studies suggest that multiple mAChRs (M₁, M₃, M₄, and M₅) may be involved in cholinergic stimulation of salivary flow.

To learn more about the functional roles of the M₁ and M₃ receptor subtypes as well as of non-M₁/M₃ receptors in cholinergic stimulation of salivary secretion in vivo, we used M₁ and M₃ receptor single-KO mice and newly generated M₁/M₃ receptor double-KO mice as novel experimental tools. All mutant mice and the corresponding WT controls received three different doses (1, 5, and 15 mg/kg, s.c.) of the nonselective muscarinic agonist pilocarpine, and the magnitudes of the resulting salivation responses were recorded. These studies were complemented by radioligand binding and quantitative reverse transcriptase (RT)-PCR studies (TaqMan), primarily to exclude the possibility that inactivation of one specific mAChR gene causes altered expression levels of the remaining mAChR subtypes.

Whereas high doses of pilocarpine led to robust salivary flow in M₁ and M₃ receptor single-KO mice, pilocarpine-induced salivary secretion was abolished in M₁/M₃ receptor double-KO mice. This finding is consistent with the concept that mAChR-mediated stimulation of salivary secretion is mediated by a mixture of M₁ and M₃ receptors and that other glandular mAChRs are unlikely to contribute to this activity to a significant extent. These observations should be of considerable relevance for the design of more selective sialagogues or of muscarinic and nonmuscarinic drugs with reduced side effects on salivary flow.

	Materials and Methods

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Animals. The generation of homozygous M₁ and M₃ receptor KO mice (129SvEv [50%] x CF1 [50%]) has been described previously (Yamada et al., 2001; Miyakawa et al., 2001; Fisahn et al., 2002). To generate mice deficient in both M₁ and M₃ mAChRs, homozygous M₁ receptor KO mice were mated with homozygous M₃ receptor KO mice. The resulting F1 compound heterozygotes were then inter-crossed to generate F2 offspring. According to Mendelian inheritance, 1/16 of the F2 pups would be expected to be homozygous for both the M₁ and the M₃ receptor gene disruptions (M₁/M₃ receptor double-KO mice). M₁/M₃ receptor double-KO mice were obtained at a frequency of about 4.7%, which is slightly below the predicted Mendelian frequency of 6.25%. The M₁/M₃ receptor double-KO mice were interbred to generate mice used for the experiments described in this study. In parallel, the wild-type (WT) F2 mice were interbred to obtain WT control mice. Thus, both M₁/M₃ receptor double-KO mice and the corresponding WT control mice had a genetic background that was identical to that of the M₁ and M₃ single-KO mice (129SvEv [50%] x CF1 [50%]). The identity of the various mutant mouse strains was verified by Southern blotting and PCR analysis of mouse tail DNA as described previously (Yamada et al., 2001; Fisahn et al., 2002).

The generation of M₂/M₃ (Struckmann et al., 2003) and M₂/M₄ (Duttaroy et al., 2002) receptor double-KO mice has been described previously. These mutant mice had the following mixed genetic background: 129J1 [50%] x CF1 [50%]. M₁/M₄ receptor double-KO mice were generated in a fashion identical to that described above for the M₁/M₃ receptor double-KO mice (J. Wess, unpublished results). The M₁/M₄ receptor double-KO mice had the same mixed genetic background as the M₁/M₃ receptor double-KO mice (129SvEv [50%] x CF1 [50%]).

All experiments were performed during the light cycle using adult male mice that were 10 to 18 weeks old. In all experiments, KO mice and age-matched WT mice of the same genetic background were run in parallel. All animal studies were conducted according to the National Institute of Diabetes and Digestive and Kidney Diseases guidelines for standard animal care and usage.

Salivation Studies. For salivation studies, WT and mAChR mutant mice were anesthetized with pentobarbital (50 mg/kg, i.p.). Salivation responses to pilocarpine hydrochloride (1, 5, and 15 mg/kg, s.c.) or (S)-isoproterenol bitartrate (1 mg/kg, s.c.) (both compounds were purchased from Sigma) were quantitated in 5-min intervals over a 35-min observation period by collecting saliva on filter paper using a method similar to that described by Parkes and Parks (1972). In brief, mice were placed face down on a slightly inclined surface (about 5° to the horizontal), with the heads facing downwards to allow free flow of saliva. After the injection of secretagogues, a preweighed piece of circular Whatman filter paper (diameter, 125 mm) was placed immediately below the mouth area of each mouse. Fresh pieces of preweighed filter paper were added in 5-min intervals (last change of filter paper was 30 min after injection of the secretagogue). Filter papers were weighed for the quantitation of saliva production.

Radioligand Binding Studies. Submandibular salivary glands were removed from individual mice and frozen immediately in liquid nitrogen. Tissue samples were homogenized for 30 s in 50 volumes of binding buffer (50 mM sodium phosphate buffer, pH 7.4, containing 2 mM MgCl₂, and 100 mM NaCl) by using a Brinkmann homogenizer. The homogenates were then filtered through four layers of cheesecloth and centrifuged once at 50,000 x g. The membrane pellets were resuspended in binding buffer at 60 mg/ml (tissue wet weight) and binding assays were carried out in triplicate according to a published method (Bymaster et al., 2003). In brief, membrane homogenates (0.4 mg of protein/tube) were incubated in a 0.2-ml total volume of binding buffer for 2 h at room temperature (22°C). The non–subtype-selective muscarinic antagonist [³H]N-methylscopolamine ([³H]NMS; specific activity, 83.5 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA), served as a radioligand (final concentration in most assays was 3 nM). In saturation binding assays using submandibular gland membranes prepared from WT mice, six different concentrations of [³H]NMS (0.02 to 3 nM) were tested. Binding data from saturation bindings studies were analyzed using the nonlinear curve-fitting program Prism 3.0 (GraphPad Software, San Diego, CA).

Nonspecific binding was determined in the presence of 10 µM atropine. Bound and free ligand were separated by vacuum filtration over GF/B filters (Whatman, Clifton, NJ), pretreated with 0.3% polyethylenimine for 1 h. The filters were washed three times with 5 ml of ice-cold phosphate buffer, dried, and placed in vials with 7 ml of Bio-Safe II scintillation cocktail (Research Products International, Mt. Prospect, IL). Radioactivity bound to the filters was determined after 18 h of extraction.

Quantitative Real-Time PCR (TaqMan) Analysis of Salivary Gland cDNA from WT and mAChR Mutant Mice. Three age-matched WT, M₁ receptor KO, and M₃ receptor KO mice were sacrificed, and the sublingual and submandibular glands were excised and immediately snap frozen in liquid nitrogen. Total RNA was obtained from the individual gland tissues by homogenization in TRIzol (Invitrogen) according the manufacturer's instructions. The RNA preparations were further processed using the RNA cleanup protocol of the RNeasy Mini Kit (QIAGEN, Valencia, CA), including on-column DNase treatment using the RNase-free DNase set (QIAGEN), to obtain total RNA free of contaminating genomic DNA. RNA quantity and quality were assessed by measuring the absorbance at 260 and 280 nm using an Eppendorf Biophotometer (Eppendorf–5 Prime, Inc., Boulder, CO). Salivary gland RNA (1 µg) was reverse-transcribed in a final volume of 100 µl by using the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

Real-time PCR (TaqMan) was carried out on a 7900 HT sequence detection system (Applied Biosystems) using a 96-well format (final volume of reaction mixtures, 50 µl). 18S rRNA (TaqMan RNA control reagents, VIC Probe; Applied Biosystems) was used as an internal standard for normalization of mAChR mRNAs in each reaction. Specific mouse M₁–M₅ receptor primers and TaqMan probes (Table 1) were designed from GenBank sequences using Primer Express 1.0 software (Applied Biosystems) and were synthesized by Applied Biosystems. The subtype specificity of the primers/probes used was verified in control reactions using plasmid DNA containing the coding sequences of the mouse M₁–M₅ mAChRs (data not shown). The amplification efficiencies of the individual primer-probe pairs were determined by serial dilution of cDNA and plotting of threshold cycle (C_t) values versus log input amount of cDNA. The amplification efficiencies of all primer-probe pairs were found to be between 1.9 and 2.0 (data not shown).

TaqMan reaction mixtures contained 5 µl from the cDNA reaction (corresponding to 50 ng of total RNA), 1x TaqMan universal master mix (Applied Biosystems), and all primers and probes at a final concentration of 200 nM. The following cycling conditions were used: 10 min at 95°C for activation of AmpliTaq Gold DNA polymerase, followed by 40 cycles of 15 s at 95°C for denaturation and 1 min at 60°C for annealing and amplification. Control reactions using RNA samples that had not been treated with RT were included as negative controls, confirming that the RNA samples were free of contaminating genomic DNA (data not shown). All RT reactions and TaqMan assays were carried out in duplicate. Relative mAChR expression levels were compared between samples from WT versus M₁ or M₃ receptor KO mice by comparing C_t values (C_t[mAChR] – C_t[control=18S rRNA]) (Livak and Schmittgen, 2001).

Morphological Studies. Salivary glands were harvested from WT and M₁/M₃ receptor double-KO mice. Tissues were fixed in 10% neutral buffered formalin, processed into paraffin blocks, sectioned at 6 µm, and stained with hematoxylin and eosin. Stained sections were examined by light microscopy (BX41; Olympus, Tokyo, Japan). Photographs were taken with an Olympus DP12 digital camera, using the 20x oil immersion lens.

Statistics. Statistical significance between two or more groups was determined by Student's t tests or one-way analysis of variance using post hoc t tests (Bonferroni's method).

	Results

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Dose-Dependence of the Sialagogue Activity of Pilocarpine in M₁ and M₃ Receptor Single-KO Mice. At first, we examined the ability of three different doses of pilocarpine (1, 5, and 15 mg/kg, s.c.) to stimulate salivary flow in M₁ and M₃ receptor single-KO mice (Fig. 1). Administration of the lowest dose of pilocarpine (1 mg/kg) resulted in a significant reduction in saliva output in both M₁ and M₃ receptor single-KO mice, compared with the corresponding WT control mice. The reduction in the magnitude of salivary secretion was clearly more pronounced in the M₃ receptor KO than in the M₁ receptor KO mice (reduction in saliva output by 90 and 50%, respectively, measured over the entire 35 min observation period; Fig. 1A).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Pilocarpine-induced salivation in WT and M1 and M3 receptor single-KO mice. Pilocarpine (A, 1 mg/kg; B, 5 mg/kg; C, 15 mg/kg) was administered s.c. to WT and M1 and M3 receptor single-KO mice. Cumulative salivary secretion for the entire observation period was measured for up to 35 min after pilocarpine administration, as described under Materials and Methods. Data are given as means ± S.E.M. (five or six mice per group). *, P < 0.05; **, P < 0.001, compared with the corresponding WT value.

In contrast, administration of the intermediate pilocarpine dose (5 mg/kg) produced only a rather modest reduction in salivary flow in the M₁ and M₃ receptor single-KO mice (reduction in saliva output by 20% measured over the entire 35-min observation period; Fig. 1B). This decrease proved to be statistically significant only in the case of the M₃ receptor KO mice (Fig. 1B). Finally, the highest pilocarpine dose used (15 mg/kg) induced robust salivation responses in both M₁ and M₃ receptor single-KO mice that did not differ significantly from the corresponding WT responses (Fig. 1C).

Analysis of mAChR Gene Expression in Mouse Salivary Glands by Quantitative Real-Time PCR (TaqMan). As described in the previous paragraph, pilocarpine (5 and 15 mg/kg) retained the ability to mediate robust salivation responses in M₁ and M₃ receptor single-KO mice (Fig. 1). To exclude the possibility that the robustness of these responses was caused by altered mAChR gene expression patterns (e.g., overexpression of M₁ receptors in M₃ receptor KO mice or, vice versa, overexpression of M₃ receptors in M₁ receptor KO mice), we used quantitative real-time PCR (TaqMan) to determine the relative expression levels of the M₁-M₅ receptor transcripts in salivary glands from WT and M₁ and M₃ receptor single-KO mice (for experimental details, see Materials and Methods). Relative mAChR expression levels were compared between WT samples and samples from M₁ or M₃ receptor single-KO mice by comparing C_t values (C_t[mAChR] – C_t[internal control=18S rRNA]) (Livak and Schmittgen, 2001).

Quantitative real-time PCR analysis of cDNA prepared from mouse submandibular glands indicated that M₁ receptor transcripts were present at similar levels in WT and M₃ receptor KO mice (Fig. 2). Likewise, M₃ receptor transcripts were expressed at similar levels in WT and M₁ receptor KO mice (Fig. 2). In addition, TaqMan analysis showed that submandibular gland tissue from WT mice also contained low levels of M₂, M₄, and M₅ receptor mRNA (note the significant increase in C_t values compared with the corresponding M₁ and M₃ receptor values). The relative mRNA expression levels of the M₂, M₄, and M₅ receptors remained unaffected by the lack of M₁ or M₃ receptors (Fig. 2). Very similar results were obtained using cDNA prepared from sublingual gland tissue from WT and M₁ and M₃ receptor KO mice (data not shown). Taken together, these data indicate that inactivation of the M₁ or M₃ mAChR genes does not result in significantly altered mRNA levels of the remaining mAChRs expressed in salivary gland tissue.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Quantitative RT-PCR analysis (TaqMan) of M1-M5 mAChR expression in submandibular gland tissue from WT and mAChR mutant mice. Quantitative RT-PCR analysis (TaqMan) was used to determine the relative levels of M1-M5 mAChR transcripts in submandibular gland tissue from WT and M1 and M3 receptor single-KO mice. The sequences of the receptor subtype-selective TaqMan primers and probes used are given in Table 1. Relative mAChR expression levels were expressed as Ct values (Ct[mAChR]-Ct[internal control=18S rRNA]) (Livak and Schmittgen, 2001). Comparison of Ct values indicates that mAChR expression levels were not significantly affected by the lack of M1 or M3 receptors. Very similar results were obtained with cDNA prepared from sublingual gland tissue from WT and M1 and M3 receptor KO mice (data not shown). Data are presented as means ± S.D. from three different experiments (sets of mice), each carried out in duplicate.

Generation of M₁/M₃ mAChR Double-KO Mice. To test the hypothesis that pilocarpine-induced salivary secretion is mediated by a mixture of M₁ and M₃ mAChRs, we generated M₁/M₃ receptor double-KO mice as novel experimental tools (for experimental details, see Materials and Methods). The M₁/M₃ receptor double-KO mice were fertile, seemed healthy, and showed no obvious behavioral abnormalities. However, like the M₃ receptor single-KO mice (Yamada et al., 2001), adult M₁/M₃ receptor double-KO mice weighed significantly less (30–40%) than the corresponding WT control mice. We also noted that intermating of male and female M₁/M₃ receptor double-KO mice resulted in somewhat reduced litter sizes (average number of pups per litter: WT, 8.4 ± 0.9; M₁/M₃ double KO, 5.4 ± 0.7; n = 14). The reason underlying this phenomenon remains unclear at present.

Pilocarpine-Induced Salivary Secretion Is Abolished in M₁/M₃ Receptor Double-KO Mice. We next tested the ability of pilocarpine (1, 5, and 15 mg/kg, s.c.) to stimulate salivary outflow in M₁/M₃ receptor double KO and their corresponding WT control mice. These studies showed that pilocarpine-mediated salivary secretion was essentially abolished in M₁/M₃ receptor double-KO mice (Fig. 3), independent of the pilocarpine dose used. However, (S)-isoproterenol (1 mg/kg, s.c.), an agent that stimulates salivary output via activation of -adrenergic receptors, retained its stimulatory effect on saliva production in M₁/M₃ receptor double-KO mice (Fig. 4), indicating that the absence of both M₁ and M₃ mAChRs did not interfere with the integrity of downstream signal transduction cascades triggering salivary secretion. In fact, (S)-isoproterenol-mediated saliva outflow was even slighter higher in M₁/M₃ receptor double-KO mice than in WT mice at several time points (p < 0.05; Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Pilocarpine-induced salivation in WT and M1/M3 receptor double-KO mice. Pilocarpine (A, 1 mg/kg; B, 5 mg/kg; C, 15 mg/kg) was administered s.c. to WT and M1/M3 receptor double-KO mice. Cumulative salivary secretion for the entire observation period was measured for up to 35 min after pilocarpine administration, as described under Materials and Methods. Note that pilocarpine failed to stimulate salivary secretion in M1/M3 receptor double-KO mice. Data are given as means ± S.E.M. (five to eight mice per group).

View larger version (16K): [in this window] [in a new window]   Fig. 4. (S)-Isoproterenol-induced salivation in WT and M1/M3 receptor double-KO mice. (S)-Isoproterenol (1 mg/kg, s.c.) was administered to WT and M1/M3 receptor double-KO mice. Cumulative salivary secretion for the entire observation period was measured for up to 35 min after drug administration, as described under Materials and Methods. Data are given as means ± S.E.M. (five or six mice per group; *, P < 0.05, compared with the corresponding WT value).

In addition, light microscopic studies showed that the morphology of submandibular, sublingual, and parotid salivary glands from M₁/M₃ receptor double-KO mice did not differ from that of the corresponding WT control mice (Fig. 5).

View larger version (88K): [in this window] [in a new window]   Fig. 5. Histological profile of salivary gland tissues from WT and M1/M3 receptor double-KO mice. Sections of submandibular, sublingual, and parotid glands from WT (A–C) and M1/M3 double-KO mice (D–F) were stained by hematoxylin/eosin and observed by light microscopy (20x).

[³H]NMS Binding Studies with Salivary Gland Tissue. We next performed [³H]NMS binding studies to estimate the densities of M₁ and M₃ receptors expressed by mouse salivary gland tissue. In particular, we labeled membranes prepared from submandibular glands from M₁ and M₃ receptor single KO and M₁/M₃ receptor double-KO mice and their corresponding WT strains with a high concentration (3 nM) of the non–subtype-selective muscarinic antagonist [³H]NMS. In gland tissue from WT mice, [³H]NMS labeled 351 ± 51 fmol of receptors/mg of membrane protein (the WT receptor density was set equal to 100% in each individual experiment; Fig. 6). The number of detectable [³H]NMS binding sites was reduced by 25% and 65% in M₁ and M₃ receptor single-KO mice, respectively (Fig. 6), suggesting that 25% of all mAChRs present in the mouse submandibular gland represent M₁ and 65% represent M₃ receptors. Only residual [³H]NMS binding activity (8% of WT binding) remained in gland tissue from M₁/M₃ receptor double-KO mice (Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Number of [3H]NMS binding sites in submandibular gland tissue from WT and mAChR KO mice. Membranes were prepared from submandibular glands of M1 receptor KO, M3 receptor KO, and M1/M3 receptor double-KO mice and the corresponding WT control mice. Membranes were then incubated with 3 nM of the non–subtype-selective muscarinic antagonist [3H]NMS, as described under Materials and Methods. In each individual experiment, the number of binding sites observed with the WT mice was set equal to 100% (absolute number of binding sites, 351 ± 51 fmol/mg; n = 6). Data are presented as means ± S.E.M. (three to five mice per KO group). *, P < 0.05; **, P < 0.01 compared with WT.

Saturation binding studies with membranes prepared from submandibular glands from WT mice showed that [³H]NMS bound to submandibular gland mAChRs with a K_D of 0.357 ± 0.007 nM (n = 3). The [³H]NMS concentration of 3 nM used for the studies described above therefore labels about 90% of the total mAChR population.

Pilocarpine-Induced Salivation in M₁/M₄, M₂/M₃, and M₂/M₄ Receptor Double-KO Mice. Besides the M₁/M₃ receptor double-KO mice described in detail above, we recently also generated M₂/M₃ (Struckmann et al., 2003), M₂/M₄ (Duttaroy et al., 2002), and M₁/M₄ receptor double-KO mice (J. Wess, unpublished results). As shown in Fig. 7, pilocarpine (15 mg/kg, s.c.) induced the flow of similar amounts of saliva in M₁/M₄, M₂/M₃, and M₂/M₄ receptor double-KO mice, compared with their corresponding WT control mice (saliva output was measured over the entire 35-min observation period).

View larger version (29K): [in this window] [in a new window]   Fig. 7. Pilocarpine-induced salivation in WT and M1/M4, M2/M3, and M2/M4 receptor double-KO mice. Pilocarpine (15 mg/kg) was administered s.c. to M1/M4 receptor double-KO mice and the corresponding WT control mice (A) and to M2/M3 and M2/M4 receptor double-KO mice and the corresponding WT control mice (B). The amount of saliva secreted over the entire 35-min observation period was measured as described under Materials and Methods. Data are given as means ± S.E.M. (four mice per group).

	Discussion

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Muscarinic antagonists are used clinically in many pathophysiological conditions including, for example, the treatment of Parkinson's disease and the reduction of increased bronchial, gastrointestinal, and bladder smooth muscle tone (Eglen et al., 1999; Wess, 2001). A major side effect of these drugs is impaired salivary flow (dry mouth), predicted to be caused by blockade of glandular mAChRs. Impaired salivary flow is also a frequent side effect of many other classes of drugs including the tricyclic antidepressants, antipsychotics, -adrenergic receptor blockers, and antihistaminergic drugs (Scully, 2003). Muscarinic agonists such as pilocarpine are successfully used in the treatment of xerostomia (Wiseman and Faulds, 1995; Fox et al., 2001). Thus, determination of the specific mAChR subtypes involved in mediating enhanced salivary flow is of considerable therapeutic interest.

To learn more about the functional roles of the M₁ and M₃ mAChR subtypes in muscarinic agonist-induced salivary secretion in vivo, we used M₁ and M₃ receptor single-KO mice and newly generated M₁/M₃ receptor double-KO mice as novel experimental tools. All mutant mice and the corresponding WT controls received three different doses (1, 5, and 15 mg/kg, s.c.) of the nonselective muscarinic agonist pilocarpine.

Studies with M₁ and M₃ receptor single-KO mice showed that the magnitudes of the observed salivation responses were critically dependent on the administered pilocarpine dose. The salivation response to the lowest dose of pilocarpine (1 mg/kg) was drastically reduced (by 90%), but not abolished, in M₃ receptor single-KO mice (Fig. 1A). A similar effect was observed when M₃ receptor single-KO mice were treated with another muscarinic agonist, oxotremorine (0.3 mg/kg, s.c.) (Bymaster et al., 2003). In contrast, Matsui et al. (2000) reported that pilocarpine (1 mg/kg, s.c.)-mediated salivary secretion was completely abolished in M₃ receptor single-KO mice. A possible reason for the discrepancy between this and our study may be that Matsui et al. (2000) used a different method to measure salivary flow and employed mice of a somewhat different genetic background. It should also be noted that we found previously that 1 mg/kg (s.c.) of pilocarpine had little sialagogue activity (<2 mg saliva/g measured over a 30-min observation period) in either WT or M₃ receptor KO mice (Yamada et al., 2001). Possible explanations for the relatively low pilocarpine sensitivity of the mice used in this previous study may include differences in mouse age (present study, 10 weeks; previous study, 24 weeks) or mouse genetic background (present study, 129SvEv x CF1; previous study, 129SvEV x C57BL/6J). In any case, our present results are consistent with the concept that the M₃ receptor plays a key role in mediating cholinergic stimulation of salivary secretion (Laniyonu et al., 1990; Dai et al., 1991; Caulfield, 1993; Watson et al., 1996; Moriya et al., 1999; Matsui et al., 2000; Bockman et al., 2001; Bymaster et al., 2003).

It is interesting that administration of the lowest dose of pilocarpine (1 mg/kg) also resulted in a significant reduction (by 50%) in salivary output in M₁ receptor single-KO mice (Fig. 1A), indicating that M₁ receptor activation also contributes to cholinergic stimulation of salivary flow. Likewise, Bymaster et al. (2003) recently reported that oxotremorine (0.3 mg/kg, s.c.) also showed significantly reduced sialagogue activity in M₁ receptor KO mice (reduction in saliva output by 35% measured over a 15-min observation period). Pharmacological and biochemical techniques have also shown that M₁ receptors (as well as M₃ receptors) mediate stimulation of mucous acinar exocrine secretion in rat sublingual glands (Culp et al., 1996; Luo et al., 2001).

In contrast with the lowest pilocarpine dose used (1 mg/kg, s.c.), the intermediate pilocarpine dose employed (5 mg/kg, s.c.) produced only a modest reduction (by 20%) in salivary flow in the M₁ and M₃ receptor KO mice, which proved to be statistically significant only in the case of the M₃ receptor KO mice (Fig. 1B). Finally, the highest pilocarpine dose used (15 mg/kg, s.c.) induced WT-like salivation responses in both M₁ and M₃ receptor single-KO mice (Fig. 1C), indicating that the presence of either M₁ or M₃ receptors is sufficient to mediate robust salivary output. Consistent with this concept, pilocarpine (15 mg/kg, s.c.) induced the flow of similar amounts of saliva in M₁/M₄, M₂/M₃, and M₂/M₄ receptor double-KO mice, compared with their corresponding WT control mice (Fig. 7). The observation that a high dose of a muscarinic agonist can induce a maximum secretion response in both the M₁ and M₃ receptor KO mice can be explained best by assuming that M₁ and M₃ receptors are coexpressed by the saliva-producing acinar cells. If M₁ and M₃ receptors were expressed by different subpopulations of acinar cells, one would expect to observe reduced salivation responses in both the M₁ and M₃ receptor single-KO mice, even at maximum agonist doses.

Quantitative real-time RT-PCR experiments (TaqMan) showed that genetic inactivation of the M₁ or M₃ mAChR genes had no significant effect on the mRNA levels of the remaining mAChR subtypes in salivary gland tissues (Fig. 2). It is therefore unlikely that the robust salivation responses observed in the M₁ and M₃ receptor single-KO mice after the administration of high doses of pilocarpine are caused by the compensatory overexpression of other mAChR subtypes. However, we cannot completely rule out minor changes in receptor protein expression levels or changes in receptor coupling properties that may occur despite unchanged receptor expression levels.

As discussed above, the pattern of salivation responses obtained with the lowest pilocarpine dose used (1 mg/kg) indicates that the M₃ receptor plays a key role in mediating stimulation of salivary flow in vivo, consistent with previous studies (Laniyonu et al., 1990; Dai et al., 1991; Caulfield, 1993; Watson et al., 1996; Moriya et al., 1999; Matsui et al., 2000; Bockman et al., 2001; Bymaster et al., 2003). The predominant functional role of M₃ receptors in stimulating salivary secretion (at least at low agonist concentrations) is most probably a consequence of the preferential expression of M₃ receptors in salivary gland tissue. In the present study, [³H]NMS binding studies indicated that 65% of the mAChRs present in submandibular gland tissue are M₃ receptors, 25% are M₁ receptors, and <10% represent other mAChR subtypes (Fig. 6). Quantitative RT-PCR studies indicated that the small population of non-M₁/M₃ mAChRs probably represents a mixture of M₂, M₄, and M₅ receptors (Fig. 2). It should also be noted in this context that immunoprecipitation studies have shown that rat sublingual gland tissue expresses approximately similar numbers of M₁ and M₃ receptors (Watson and Culp, 1994). Moreover, radioligand binding studies suggest that human salivary gland tissues also express a mixture of M₁ and M₃ mAChRs (Giraldo et al., 1988; Mei et al., 1990).

On the other hand, radioligand binding (Hammer et al., 1980; Dai et al., 1991; Bockman et al., 2001), functional (Dai et al., 1991; Bockman et al., 2001), and immunoprecipitation studies (Dai et al., 1991) indicate that the rat parotid gland almost exclusively expresses M₃ receptors. The contribution of M₁ receptors to muscarinic agonist (acetylcholine)-mediated stimulation of salivary secretion (see below) may therefore be mediated predominantly by M₁ receptors present in the submandibular and sublingual glands.

Several recent studies suggest that non-M₁/M₃ mAChRs may also play a role in cholinergic stimulation of glandular secretion. For example, it has been proposed based on studies using subtype-preferring muscarinic antagonists that M₅ receptors may be present/functional in salivary gland tissue (Flynn et al., 1997; Meloy et al., 2001; Tobin et al., 2002). Moreover, Takeuchi et al. (2002) reported that salivary secretion induced by pilocarpine (1 mg/kg, s.c.) was slightly reduced (by 10–20%) in M₅ receptor KO mice. Finally, Bymaster et al. (2003) recently found that administration of a single dose of oxotremorine (0.3 mg/kg, s.c.) resulted in a reduced salivation response in M₄ receptor single-KO mice (reduction in saliva output by 35% measured over a 15-min observation period).

To test the hypothesis that non-M₁/M₃ mAChRs play a significant role in cholinergic stimulation of salivary secretion in vivo, we generated and analyzed mice in which both the M₁ and M₃ receptor genes had been inactivated (M₁/M₃ receptor double-KO mice). It was remarkable that pilocarpine, at any of the three doses tested, was no longer able to stimulate salivary secretion in M₁/M₃ receptor double-KO mice (Fig. 3). However, the -adrenergic receptor agonist (S)-isoproterenol (1 mg/kg, s.c.) retained the ability to stimulate saliva production in M₁/M₃ receptor double-KO mice with high efficacy (Fig. 4), indicating that the functional deficits displayed by the M₁/M₃ receptor double-KO mice were receptor-specific and not caused by changes in the expression levels of downstream signaling components. Our observation that pilocarpine-mediated stimulation of salivary flow in vivo is abolished in the M₁/M₃ receptor double-KO mice indicates that non-M₁/M₃ receptors, such as M₄ and M₅ receptors, are unlikely to make a significant contribution to muscarinic agonist-induced saliva production. The functional deficits displayed by the M₄ (Bymaster et al., 2003) and M₅ receptor single-KO mice (Takeuchi et al., 2002) are therefore most probably caused by other factors, such as mouse genetic background or altered expression levels of other signaling proteins.

Takakura et al. (2003) recently showed that pilocarpine, which readily enters the brain, can stimulate salivary secretion by activating central mAChRs, probably by increasing parasympathetic outflow. Because the M₄ and M₅ mAChRs are preferentially expressed in the CNS (Wess, 1996; Caulfield and Birdsall, 1998), the reduction in pilocarpine- or oxotremorine-induced stimulation of salivary function observed in M₄ (Bymaster et al., 2003) and M₅ (Takeuchi et al., 2002) mAChR KO mice could therefore also be caused by the lack of neuronal rather than glandular M₄ or M₅ receptors.

Although our data suggest that drugs devoid of activity at M₁ and M₃ receptors should lack adverse effects on salivation, clinical studies indicate that relative potency at M₃ mAChRs may not be a reliable predictor of xerostomia. For example, administration of the muscarinic antagonist solifenacin, which is endowed with a moderate degree of M₃ receptor selectivity, seems to cause less pronounced drymouth symptoms than tolterodine (nonselective) or darifenacin, another M₃ receptor-preferring antagonist (Ikeda et al., 2002; Chapple et al., 2004a,b; Kobayashi et al., 2004). These differences in drug effects on salivary gland function observed in clinical studies may be caused by differences in pharmacokinetics, drug stability, or other as-yet-unknown parameters that affect drug activity at salivary glands in vivo.

In conclusion, our results indicate that muscarinic agonists trigger salivary secretion in vivo by activating a mixture of glandular M₁ and M₃ mAChRs. Other glandular mAChR subtypes are unlikely to be involved in mediating this activity, at least not in the mouse. These findings should be highly relevant for the development of novel, clinically useful muscarinic drugs.

	Acknowledgements

 
We thank A. Duttaroy for advice and helpful discussions.

	Footnotes

 
ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; KO, knockout; WT, wild type; RT, reverse transcriptase; PCR, polymerase chain reaction; C_t, threshold cycle; NMS, N-methyl scopolamine.

Address correspondence to: Dr. Jürgen Wess, Molecular Signaling Section, Lab. of Bioorganic Chemistry, NIH-NIDDK, Bldg. 8A, Room B1A-05, 8 Center Drive MSC 0810, Bethesda, MD 20892-0810. E-mail: jwess{at}helix.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 
Baum BJ (1993) Principles of saliva secretion. Ann NY Acad Sci 694: 17–23.[Medline]

Bockman CS, Bradley ME, Dang HK, Zeng W, Scofield MA, and Dowd FJ (2001) Molecular and pharmacological characterization of muscarinic receptor subtypes in a rat parotid gland cell line: comparison with native parotid gland. J Pharmacol Exp Ther 297: 718–726.[Abstract/Free Full Text]

Bymaster FP, Carter PA, Yamada M, Gomeza J, Wess J, Hamilton SE, Nathanson NM, McKinzie DL and Felder CC (2003) Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis and pilocarpine-induced seizure activity. Eur J Neurosci 17: 1403–1410.[CrossRef][Medline]

Caulfield MP (1993) Muscarinic receptors— characterization, coupling and function. Pharmacol Ther 58: 319–379.[CrossRef][Medline]

Caulfield MP and Birdsall NJ (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50: 279–290.[Abstract/Free Full Text]

Chapple CR, Arano P, Bosch JL, De Ridder D, Kramer AE, and Ridder AM (2004a) Solifenacin appears effective and well tolerated in patients with symptomatic idiopathic detrusor overactivity in a placebo- and tolterodine-controlled phase 2 dose-finding study. BJU Int 93: 71–77.[CrossRef][Medline]

Chapple CR, Rechberger T, Al-Shukri S, Meffan P, Everaert K, Huang M, and Ridder A (2004b) Randomized, double-blind placebo- and tolterodine-controlled trial of the once-daily antimuscarinic agent solifenacin in patients with symptomatic overactive bladder. BJU Int 93: 303–310.[CrossRef][Medline]

Culp DJ, Luo W, Richardson LA, Watson GE, and Latchney LR (1996) Both M₁ and M₃ receptors regulate exocrine secretion by mucous acini. Am J Physiol 271: C1963–C1972.

Dai YS, Ambudkar IS, Horn VJ, Yeh CK, Kousvelari EE, Wall SJ, Li M, Yasuda RP, Wolfe BB, and Baum BJ (1991) Evidence that M₃ muscarinic receptors in rat parotid gland couple to two second messenger systems. Am J Physiol 261: C1063–C1073.

Duttaroy A, Gomeza J, Gan JW, Siddiqui N, Basile AS, Harman WD, Smith PL, Felder CC, Levey AI, and Wess J (2002) Evaluation of muscarinic agonist-induced analgesia in muscarinic acetylcholine receptor knockout mice. Mol Pharmacol 62: 1084–1093.[Abstract/Free Full Text]

Eglen RM, Choppin A, Dillon MP, and Hegde S (1999) Muscarinic receptor ligands and their therapeutic potential. Curr Opin Chem Biol 3: 426–432.[CrossRef][Medline]

Evans RL, Park K, Turner RJ, Watson GE, Nguyen HV, Dennett MR, Hand AR, Flagella M, Shull GE, and Melvin JE (2000) Severe impairment of salivation in Na⁺/K⁺/2Cl^– cotransporter (NKCC1)-deficient mice. J Biol Chem 275: 26720–26726.[Abstract/Free Full Text]

Fisahn A, Yamada M, Duttaroy A, Gan JW, Deng CX, McBain CJ, and Wess J (2002) Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents. Neuron 33: 615–624.[CrossRef][Medline]

Flynn DD, Reever CM, and Ferrari-DiLeo G (1997) Pharmacological strategies to selectively label and localize muscarinic receptor subtypes. Drug Dev Res 40: 104–116.

Fox RI, Konttinen Y, and Fisher A (2001) Use of muscarinic agonists in the treatment of Sjogren's syndrome. Clin Immunol 101: 249–263.[CrossRef][Medline]

Giraldo E, Martos F, Gomez A, Garcia A, Vigano MA, Ladinsky H, and Sanchez de La Cuesta F (1988) Characterization of muscarinic receptor subtypes in human tissues. Life Sci 43: 1507–1515.[CrossRef][Medline]

Hammer R, Berrie CP, Birdsall NJ, Burgen AS, and Hulme EC (1980) Pirenzepine distinguishes between different subclasses of muscarinic receptors. Nature (Lond) 283: 90–92.[CrossRef][Medline]

Ikeda K, Kobayashi S, Suzuki M, Miyata K, Takeuchi M, Yamada T, and Honda K (2002) M₃ receptor antagonism by the novel antimuscarinic agent solifenacin in the urinary bladder and salivary gland. Naunyn Schmiedeberg's Arch Pharmacol 366: 97–103.[CrossRef][Medline]

Kobayashi S, Ikeda K, and Miyata K (2004) Comparison of in vitro selectivity profiles of solifenacin succinate (YM905) and current antimuscarinic drugs in bladder and salivary glands: a Ca²⁺ mobilization study in monkey cells. Life Sci 74: 843–853.[CrossRef][Medline]

Laniyonu A, Sliwinski-Lis E, and Fleming N (1990) Muscarinic M₃ receptors are coupled to two signal transduction pathways in rat submandibular cells. Eur J Pharmacol 188: 171–174.[CrossRef][Medline]

Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2^-CT. Methods 25: 402–408.[CrossRef][Medline]

Luo W, Latchney LR, and Culp DJ (2001) G protein coupling to M₁ and M₃ muscarinic receptors in sublingual glands. Am J Physiol 280: C884–C896.

Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS (1999) Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem 274: 20071–20074.[Abstract/Free Full Text]

Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, Takahashi S, and 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]

Mei L, Roeske WR, Izutsu KT, and Yamamura HI (1990) Characterization of muscarinic acetylcholine receptors in human labial salivary glands. Eur J Pharmacol 176: 367–370.[CrossRef][Medline]

Meloy TD, Daniels DV, Hegde SS, Eglen RM, and Ford AP (2001) Functional characterization of rat submaxillary gland muscarinic receptors using microphysiometry. Br J Pharmacol 132: 1606–1614.[CrossRef][Medline]

Miyakawa T, Yamada M, Duttaroy A, and Wess J (2001) Hyperactivity and intact hippocampus-dependent learning in mice lacking the M₁ muscarinic acetylcholine receptor. J Neurosci 21: 5239–5250.[Abstract/Free Full Text]

Moriya H, Takagi Y, Nakanishi T, Hayashi M, Tani T, and Hirotsu I (1999) Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland. Life Sci 64: 2351–2358.[CrossRef][Medline]

Parkes MW and Parks JC (1972) Supersensitivity of salivation in response to pilocarpine after withdrawal of chronically administered hyoscine in the mouse. Br J Pharmacol 46: 315–323.[Medline]

Scully C (2003) Drug effects on salivary glands: dry mouth. Oral Dis 9: 165–176.[CrossRef][Medline]

Struckmann N, Schwering S, Wiegand S, Gschnell A, Yamada M, Kummer W, Wess J, and Haberberger RV (2003) Role of muscarinic receptor subtypes in the constriction of peripheral airways: studies on receptor-deficient mice. Mol Pharmacol 64: 1444–1451.[Abstract/Free Full Text]

Takakura AC, Moreira TS, Laitano SC, De Luca LA Jr, Renzi A, and Menani JV (2003) Central muscarinic receptors signal pilocarpine-induced salivation. J Dent Res 82: 993–997.[Abstract/Free Full Text]

Takeuchi J, Fulton J, Jia ZP, Abramov-Newerly W, Jamot L, Sud M, Coward D, Ralph M, Roder J, and Yeomans J (2002) Increased drinking in mutant mice with truncated M5 muscarinic receptor genes. Pharmacol Biochem Behav 72: 117–123.[CrossRef][Medline]

Tobin G, Giglio D, and Gotrick B (2002) Studies of muscarinic receptor subtypes in salivary gland function in anaesthetized rats. Auton Neurosci 100: 1–9.[CrossRef][Medline]

Turner RJ and Sugiya H (2002) Understanding salivary fluid and protein secretion. Oral Dis 8: 3–11.[CrossRef][Medline]

Watson GE and Culp DJ (1994) Muscarinic cholinergic receptor subtypes in rat sublingual glands. Am J Physiol 266: C335–C342.

Watson EL, Abel PW, DiJulio D, Zeng W, Makoid M, Jacobson KL, Potter LT, and Dowd FJ (1996) Identification of muscarinic receptor subtypes in mouse parotid gland. Am J Physiol 271: C905–C913.

Wess J (1996) Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 10: 69–99.[Medline]

Wess J (2001) Muscarinic acetylcholine receptors, in Encyclopedia of Molecular Medicine (Creighton TE ed) pp 2150–2154, Wiley, New York.

Wiseman LR and Faulds D (1995) Oral pilocarpine: a review of its pharmacological properties and clinical potential in xerostomia. Drugs 49: 143–155.[Medline]

Yamada M, Miyakawa T, Duttaroy A, Yamanaka A, Moriguchi T, Makita R, Ogawa M, Chou CJ, Xia B, Crawley JN, et al. (2001) Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature (Lond) 410: 207–212.[CrossRef][Medline]

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