The liver-brain-gut neural arc maintains the Treg cell niche in the gut.

Recent clinical and experimental evidence has evoked the concept of the gut-brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1-4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver-brain-gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver-brain-gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut.

Transgenic rice seeds expressing altered peptide ligands against the M3 muscarinic acetylcholine receptor suppress experimental sialadenitis-like Sjögren's syndrome.

Objective: We previously reported that Rag1-/- mice inoculated with splenocytes from M3 muscarinic acetylcholine receptor (M3R) knockout mice immunized with an M3R peptide mixture developed sialadenitis-like Sjögren's syndrome (M3R-induced sialadenitis [MIS]). We also found that intravenous administration of altered peptide ligand (APL) of N-terminal 1 (N1), which is one of the T-cell epitopes of M3R, suppressed MIS. In this study, we aimed to evaluate the suppressive ability and its mechanisms of rice seeds expressing N1-APL7 against MIS.Methods: Rice seeds expressing N1 and N1-APL7 were orally administered to MIS mice for 2 weeks. The changes in saliva flow and sialadenitis (salivary gland inflammation) were analyzed. The M3R-specific T-cell response in the spleen and the expression of regulatory molecules in the cervical lymph nodes and mesenteric lymph nodes were also analyzed.Results: Oral administration of N1-APL7-expressing rice seeds significantly recovered reduction in saliva flow and suppressed sialadenitis when compared with treatment with nontransgenic rice seeds and N1 rice seeds. IFNγ production from M3R-reactive T cells tended to decline in the N1-APL7 rice-treated group as compared with those in the other groups. In the N1-APL7 rice-treated group, the mRNA expression levels of Foxp3 in the cervical-lymph-node CD4+ T cells were higher than those in the other groups.Conclusion: Oral administration of N1-APL7-expressing rice suppressed MIS via suppression of M3R-specific IFNγ and IL-17 production and via enhancement of regulatory molecule expression.Key messagesWe generated N1-peptide- or N1-APL7-expressing rice seeds. Oral administration of N1-APL7-expressing rice seeds significantly recovered the reduction of saliva flow and suppressed sialadenitis via the suppression of M3R specific IFNγ and IL-17 production and via enhancement of regulatory T (Treg) cells.

Differences among muscarinic agonists in M1 receptor-mediated nonselective cation channel activation and TASK1 channel inhibition in adrenal medullary cells.

Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 µM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 µM muscarine was completely suppressed by 100 µM quinine, whereas 30 µM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 µM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M1 receptor. The KD value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition.

The anergy induction of M3 muscarinic acetylcholine receptor-reactive CD4+ T cells suppresses experimental sialadenitis-like Sjögren's syndrome.

OBJECTIVE: Autoreactive CD4+ T cells are involved in the pathogenesis of Sjögren's syndrome (SS). The aim of the present study was to clarify the dominant T cell epitopes of M3 muscarinic acetylcholine receptor (M3R) and to establish a new antigen-specific therapy for SS using an experimental mouse model. METHODS: Production of cytokines from M3R-reactive CD4+ T cells, after culture with various M3R peptides, was analyzed by enzyme-linked immunosorbent assay. Adoptive cell transfer was performed using splenocytes from M3R(-/-) mice that were immunized with M3R peptides or phosphate buffered saline plus H37Ra as a control. Rag1(-/-) mice were inoculated with the splenocytes and examined for the development of sialadenitis. Altered peptide ligands (APLs) of the T cell epitopes, with substitutions in amino acid residues at T cell receptor contact sites, were synthesized, and the ability of the APLs to suppress sialadenitis was evaluated. The mechanisms underlying such effects were assessed. RESULTS: CD4+ M3R-reactive T cells produced interleukin-17 (IL-17) and interferon-γ (IFNγ) in response to the N-terminal 1 (N1) and 1st extracellular loop peptides of M3R, and Rag1(-/-) mice that received N1- and/or 1st peptide-immunized splenocytes developed sialadenitis. Among the designed APLs, N1-APL7 (N→S at amino acid 15) significantly suppressed IFNγ production in vitro, and also suppressed sialadenitis in vivo. Levels of early growth response 2 in CD4+ T cells from the cervical lymph nodes of N1-APL7-treated mice were significantly higher than those of control mice, and cell proliferation was reversed by administration of exogenous IL-2. Levels of the anergy-related molecules itchy homolog E3 ubiquitin-protein ligase, Casitas B-lineage lymphoma b, gene related to anergy in lymphocytes, and Deltex-1 were significantly higher in CD4+ T cells cultured with N1-APL7. CONCLUSION: The major T cell epitopes were from the N1 and 1st peptide regions. Moreover, N1-APL7, selected as the antagonistic APL in vitro, also suppressed sialadenitis through the induction of anergy. This is a potentially useful strategy for regulating pathogenic T cell infiltration in SS.

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion.

BACKGROUND AND PURPOSE: Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation. EXPERIMENTAL APPROACH: To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry. KEY RESULTS: Muscarinic M1, M4 and M5 receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M1, but not M3, M4 or M5, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M1 receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M1 receptor. CONCLUSIONS AND IMPLICATIONS: Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M1 receptor alone is responsible for muscarine-induced catecholamine secretion.

Mice lacking inositol 1,4,5-trisphosphate receptors exhibit dry eye.

Tear secretion is important as it supplies water to the ocular surface and keeps eyes moist. Both the parasympathetic and sympathetic pathways contribute to tear secretion. Although intracellular Ca2+ elevation in the acinar cells of lacrimal glands is a crucial event for tear secretion in both the pathways, the Ca2+ channel, which is responsible for the Ca2+ elevation in the sympathetic pathway, has not been sufficiently analyzed. In this study, we examined tear secretion in mice lacking the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3 (Itpr2-/-;Itpr3-/-double-knockout mice). We found that tear secretion in both the parasympathetic and sympathetic pathways was abolished in Itpr2-/-;Itpr3-/- mice. Intracellular Ca2+ elevation in lacrimal acinar cells after acetylcholine and epinephrine stimulation was abolished in Itpr2-/-;Itpr3-/- mice. Consequently, Itpr2-/-;Itpr3-/- mice exhibited keratoconjunctival alteration and corneal epithelial barrier disruption. Inflammatory cell infiltration into the lacrimal glands and elevation of serum autoantibodies, a representative marker for Sjögren's syndrome (SS) in humans, were also detected in older Itpr2-/-;Itpr3-/- mice. These results suggested that IP3Rs are essential for tear secretion in both parasympathetic and sympathetic pathways and that Itpr2-/-;Itpr3-/- mice could be a new dry eye mouse model with symptoms that mimic those of SS.

Distinct roles of M1 and M3 muscarinic acetylcholine receptors controlling oscillatory and non-oscillatory [Ca2+]i increase.

We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 µM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 µM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.

Measurement of postvoid residual urine in conscious mice using high-frequency transrectal ultrasound.

The aim of this study was to develop a novel technique for a minimally invasive ultrasound measurement of postvoid residual urine (PVR) in conscious mice using a miniature ultrasound probe and a transrectal approach. The PVR was determined by the ellipsoid formula in the maximum sectional image of the bladder visualized with a 20-MHz ultrasound probe (2 mm in diameter) inserted into the rectum. The accuracy, including the intra- and interobserver reproducibilities, of the ultrasonic PVR measurements (in 10 5- to 50-week-old mice) was evaluated, which revealed excellent internal consistency. In M(3) muscarinic acetylcholine receptor knockout male mice, a chronological evaluation of the PVR identified abnormal urinary retention present at infancy and exacerbated with aging, suggesting significant voiding dysfunction. Our technique for the measurement of PVR in conscious mice was accurate and useful for identifying the voiding dysfunction in mice.

Autoimmunity against M2muscarinic acetylcholine receptor induces myocarditis and leads to a dilated cardiomyopathy-like phenotype.

Patients with dilated cardiomyopathy (DCM) often have autoantibodies against cardiac antigens including the M(2) muscarinic acetylcholine receptor (M(2)R). To elucidate the role of autoimmunity against M(2)R in disease development, we induced an immune response against M(2)R by adoptive transfer into Rag2(-/-) mice of splenocytes from M(2)R(-/-) mice immunized with a recombinant M(2)R protein. T lymphocytes transiently infiltrated the heart in recipient mice followed by morphological changes in cardiomyocytes. These mice produced IgG antibodies against M(2)R, which bound to cardiomyocytes in vivo and decreased the amplitude of calcium signals in isolated rat cardiomyocytes in vitro. Recipient mice showed increased heart weights associated with increased intraventricular diameter, decreased systolic function, and increased action potential duration, which are characteristics of DCM. Our results suggest that myocarditis and DCM associated with the presence of anti-M(2)R antibodies are autoimmune diseases with a risk of progressing to the terminal stage. Our mouse model will be useful in the analysis of the molecular mechanisms of disease progression and the development of new therapies for DCM.

Contractions of the mouse prostate elicited by acetylcholine are mediated by M(3) muscarinic receptors.

Increased smooth muscle tone in the human prostate contributes to the symptoms associated with benign prostatic hyperplasia. In the mouse prostate gland, cholinergic innervation is responsible for a component of the nerve-mediated contractile response. This study investigates the muscarinic receptor subtype responsible for the cholinergic contractile response in the mouse prostate gland. To characterize the muscarinic receptor subtype, mouse prostates taken from wild-type or M(3) muscarinic receptor knockout mice were mounted in organ baths. The isometric force that tissues developed in response to electrical-field stimulation or exogenously applied cholinergic agonists in the presence or absence of a range of muscarinic receptor antagonists was evaluated. Carbachol elicited reproducible and concentration-dependent contractions of the isolated mouse prostate, which were antagonized by the presence of muscarinic receptor antagonists. Calculation of antagonist affinities (pA(2) values) indicated a rank order of antagonist potencies in the mouse prostate of: darifenacin (9.08) = atropine (9.07) = 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (9.02) > cyclohexyl-hydroxy-phenyl-(3-piperidin-1-ylpropyl)silane (7.85) > cyclohexyl-(4-fluorophenyl)-hydroxy-(3-piperidin-1-ylpropyl)silane (7.39) > himbacine (7.19) > pirenzipine (6.88) > methoctramine (6.20). Furthermore, genetic deletion of the M(3) muscarinic receptor inhibited prostatic contractions to electrical-field stimulation or exogenous administration of acetylcholine. In this study we identified that the cholinergic component of contraction in the mouse prostate is mediated by the M(3) muscarinic receptor subtype. Pharmacological antagonism of the M(3) muscarinic receptor may be a beneficial additional target for the treatment of benign prostatic hyperplasia in the human prostate gland.

Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis.

The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögren's syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)→Rag1(-/-)) mice. M3R(-/-)→Rag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)→Rag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.

Role of immune response to M3 muscarinic acethylcholine receptor in the pathogenesis of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary glands, in which CD4(+) T cells are predominant. These infiltrating T cells play a crucial role in the generation of SS. Previous studies showed that autoantibodies and auto-reactive T cells against M3 muscarinic acethylcholine receptor (M3R) were detected in patients with SS. In this study, to reveal the pathological mechanisms underlying immune response against M3R, we tried to induce SS like sialoadenitis. M3R knockout (M3R(-/-)) mice were immunized with murine M3R peptides. Their splenocytes were isolated and transferred into Rag1 knockout (Rag1(-/-)) mice. Mononuclear cells infiltration was detected in salivary glands of Rag1(-/-) mice inoculated splenocytes of M3R(-/-) mice immunized with M3R peptides. Moreover we transferred CD3(+) cells from splenocytes of M3R(-/-) mice immunized with M3R peptides into Rag1(-/-) mice. In their salivary glands, mononuclear infiltration was also detected. These findings suggest that the immune response to M3R plays a crucial role in the generation of SS like sialoadenitis.

Preferential localization of muscarinic M1 receptor on dendritic shaft and spine of cortical pyramidal cells and its anatomical evidence for volume transmission.

Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called "volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M(1), the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M(1) was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M(1) was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M(1) and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M(1) is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M(1)-mediated cortical cholinergic signaling.

Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses.

To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

M1 receptors mediate cholinergic modulation of excitability in neocortical pyramidal neurons.

ACh release into the rodent prefrontal cortex is predictive of successful performance of cue detection tasks, yet the cellular mechanisms underlying cholinergic modulation of cortical function are not fully understood. Prolonged ("tonic") muscarinic ACh receptor (mAChR) activation increases the excitability of cortical pyramidal neurons, whereas transient ("phasic") mAChR activation generates inhibitory and/or excitatory responses, depending on neuron subtype. These cholinergic effects result from activation of "M1-like" mAChRs (M1, M3, and M5 receptors), but the specific receptor subtypes involved are not known. We recorded from cortical pyramidal neurons from wild-type mice and mice lacking M1, M3, and/or M5 receptors to determine the relative contribution of M1-like mAChRs to cholinergic signaling in the mouse prefrontal cortex. Wild-type neurons in layer 5 were excited by tonic mAChR stimulation, and had biphasic inhibitory followed by excitatory, responses to phasic ACh application. Pyramidal neurons in layer 2/3 were substantially less responsive to tonic and phasic cholinergic input. Cholinergic effects were largely absent in neurons from mice lacking M1 receptors, but most were robust in neurons lacking M3, M5, or both M3 and M5 receptors. The exception was tonic cholinergic suppression of the afterhyperpolarization in layer 5 neurons, which was absent in cells lacking either M1 or M3 receptors. Finally, we confirm a role for M1 receptors in behavior by demonstrating cue detection deficits in M1-lacking mice. Together, our results demonstrate that M1 receptors facilitate cue detection behaviors and are both necessary and sufficient for most direct effects of ACh on pyramidal neuron excitability.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Comparison of muscarinic receptor selectivity of solifenacin and oxybutynin in the bladder and submandibular gland of muscarinic receptor knockout mice.

Solifenacin is a novel selective antagonist of M(3) muscarinic receptor developed for the treatment of overactive bladder. The current study was undertaken to characterize in vivo muscarinic receptor subtype selectivity of solifenacin in the bladder and submandibular gland by using muscarinic receptor subtype knockout (KO) mice. Muscarinic receptors in the bladder and submandibular gland of wild type, M(2)R KO and M(3)R KO mice under in vitro and after oral administration of solifenacin and oxybutynin were measured by radioligand binding assay using [N-methyl-(3)H]scopolamine ([(3)H]NMS). There was little difference between the bladder and submandibular gland of M(2)R KO mice in the receptor binding activities of oxybutynin and solifenacin in vitro, suggesting equal affinity for residual (predominantly M(3) subtype) muscarinic receptors in both tissues. In contrast, compared with oral oxybutynin, oral administration of solifenacin exerted a significantly greater activity to bind muscarinic receptors in the bladder of M(2)R KO mice, while exhibiting a significantly less activity to bind those in the submandibular gland. In the bladder and submandibular gland of M(3)R KO mice, the binding activity of solifenacin and oxybutynin showed no significant difference. Plasma concentrations of solifenacin and oxybutynin after oral administration differed little among wild type, M(2)R KO and M(3)R KO mice. The results indicate that oral solifenacin, unlike oral oxybutynin, may selectively bind to the muscarinic M(3) subtype in the bladder compared with such receptors in the submandibular gland in vivo. Oral solifenacin may be advantageous for the treatment of overactive bladder, in terms of high affinity for M(3) receptors in the bladder.

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

BACKGROUND AND PURPOSE: To compare loss in binding to muscarinic receptor (mAChR) subtypes with their known functions, the total density of muscarinic receptors was measured in peripheral tissues from wild type (WT) and mAChR knockout (KO) mice. EXPERIMENTAL APPROACH: Binding parameters of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were determined in 10 peripheral tissues of WT and M1-M5 receptor KO mice. Competition between [3H]NMS and darifenacin (selective M3 receptor antagonist) was also measured. KEY RESULTS: There was an extensive loss of [3H]NMS-binding sites (maximal number of binding sites, Bmax) in heart and smooth muscle from M2KO mice, compared with WT mice. Smooth muscle from M3KO mice also showed a moderate loss of Bmax. Bmax fell in pancreas and bladder of M4KO mice and in prostate in M1KO and M3KO mice. There was a large loss of Bmax in exocrine and endocrine glands of M3KO mice with a moderate decrease in M2KO mice. Darifenacin inhibited specific [3H]NMS binding in submandibular gland and bladder of WT, M2KO and M3KO mice. Ki (inhibition constant) values for darifenacin in the submandibular gland were the same in WT and M2KO mice but increased in M3KO mice. However, Ki values in bladder were decreased in M2KO mice and increased in M3KO mice. CONCLUSIONS AND IMPLICATIONS: Single mAChR KO mice exhibit a loss of mAChR in peripheral tissues that generally paralleled the reported loss of function. Quantitative analysis of data, however, also suggested that, in some instances, normal expression of a receptor subtype depended on expression of other subtypes.