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Muscarinic acetylcholine receptor M5 is involved in spermatogenesis through the modification of cell-cell junctions.

ABSTRACT: Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell-cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell-cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell-cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

SUBMITTER: Han X

PROVIDER: S-EPMC8183636 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cholinergic responses of ophthalmic arteries in M3 and M5 muscarinic acetylcholine receptor knockout mice.

Project description:PURPOSE:To determine the functional role of M(3) and M(5) muscarinic acetylcholine receptor subtypes in ophthalmic arteries using gene-targeted mice. METHODS:Muscarinic receptor gene expression was quantified in murine ophthalmic arteries using real-time PCR. To test the functional relevance of M(3) and M(5) receptors, ophthalmic arteries from mice deficient in either subtype (M3R(-/-), M5R(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal vessel diameter in response to muscarinic and nonmuscarinic receptor agonists were measured by video microscopy. RESULTS:With the use of real-time PCR, all five muscarinic receptor subtypes were detected in ophthalmic arteries. However, mRNA levels of M(1), M(3), and M(5) receptors were higher than those of M(2) and M(4) receptors. In functional studies, after preconstriction with phenylephrine, acetylcholine and carbachol produced concentration-dependent dilations of ophthalmic arteries that were similar in M5R(-/-) and wild-type mice. Strikingly, cholinergic dilation of ophthalmic arteries was almost completely abolished in M3R(-/-) mice. Deletion of either M(3) or M(5) receptor did not affect responses to nonmuscarinic vasodilators such as bradykinin or nitroprusside. CONCLUSIONS:These findings provide the first evidence that M(3) receptors are critically involved in cholinergic regulation of diameter in murine ophthalmic arteries.

| S-EPMC4111104 | biostudies-literature

Expression of muscarinic acetylcholine receptors M3 and M5 in osteoporosis.

Project description:BACKGROUND: Cholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model. MATERIAL AND METHODS: Osteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR. RESULTS: Relative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups. CONCLUSIONS: M3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis.

| S-EPMC4049973 | biostudies-literature

Extrasynaptic acetylcholine signaling through a muscarinic receptor regulates cell migration.

Project description:Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73 This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.

| S-EPMC7817160 | biostudies-literature

Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia.

Project description:Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5-/- mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5-/- mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5-/- mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.

| S-EPMC123277 | biostudies-literature

Microscopic binding of M5 muscarinic acetylcholine receptor with antagonists by homology modeling, molecular docking, and molecular dynamics simulation.

Project description:By performing homology modeling, molecular docking, and molecular dynamics (MD) simulations, we have developed three-dimensional (3D) structural models of the M5 muscarinic acetylcholine receptor (mAChR) and two complexes for M5 mAChR binding with antagonists SVT-40776 and solifenacin in the environment of lipid bilayer and solvent water. According to the simulated results, each of the antagonists is oriented horizontally in the binding pocket formed by transmembrane helices 2, 3, and 5-7. The cationic headgroup of each of the antagonists interacts with a negatively charged residue, Asp110, through electrostatic and hydrogen-bonding interactions. The simulated results also reveal some significant difference between the binding modes of SVT-40776 and solifenacin. In particular, SVT-40776 is persistently hydrogen bonded with the side chain of residue Tyr458, whereas solifenacin cannot form a similar hydrogen bond with residues around its carbonyl group. Such significant difference in the binding structures is consistent with the fact that SVT-40776 has a much higher binding affinity (K(d) = 0.4 nM) to M5 mAChR than that of solifenacin (K(d) = 31 nM) with the same reeptor. The calculated binding free energy change (-2.3 ± 0.3 kcal/mol) from solifenacin to SVT-40776 is in good agreement with the experimentally derived binding free energy change (-2.58 kcal/mol), suggesting that our modeled M5 mAChR structure and its complexes with the antagonists are reliable. The new structural insights obtained from this computational study are expected to stimulate further biochemical and pharmacological studies on the detailed structures of M5 and other subtypes of mAChRs.

| S-EPMC3257414 | biostudies-literature

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium.

Project description:Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

| S-EPMC5506046 | biostudies-literature

The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation

Project description:The M3 muscarinic acetylcholine receptor (CHRM3) is predominantly expressed in the basal epidermal layer where it mediates the effects of the auto/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to CHRM3 and show alterations in keratinocyte adhesion, proliferation and differentiation, suggesting that CHRM3 controls these cellular functions. Chrm3 mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. Here, we characterized the cellular and molecular mechanisms whereby CHRM3 controls epidermal structure and function. We used single cell (sc)RNA-seq to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chrm3 KO neonatal mice.

2022-05-06 | GSE201885 | GEO

Discovery of the first highly M5-preferring muscarinic acetylcholine receptor ligand, an M5 positive allosteric modulator derived from a series of 5-trifluoromethoxy N-benzyl isatins.

Project description:This report describes the discovery and initial characterization of the first positive allosteric modulator of muscarinic acetylcholine receptor subtype 5 (mAChR5 or M5). Functional HTS, identified VU0119498, which displayed micromolar potencies for potentiation of acetylcholine at M1, M3, and M5 receptors in cell-based Ca(2+) mobilization assays. Subsequent optimization led to the discovery of VU0238429, which possessed an EC(50) of approximately 1.16 microM at M5 with >30-fold selectivity versus M1 and M3, with no M2 or M4 potentiator activity.

| S-EPMC3875304 | biostudies-literature

Fine Tuning Muscarinic Acetylcholine Receptor Signaling Through Allostery and Bias.

Project description:The M1 and M4 muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, in particular for Alzheimer's disease and schizophrenia. Due to high sequence homology, selective targeting of any of the M1-M5 mAChRs through the endogenous ligand binding site has been notoriously difficult to achieve. With the discovery of highly subtype selective mAChR positive allosteric modulators in the new millennium, selectivity through targeting an allosteric binding site has opened new avenues for drug discovery programs. However, some hurdles remain to be overcome for these promising new drug candidates to progress into the clinic. One challenge is the potential for on-target side effects, such as for the M1 mAChR where over-activation of the receptor by orthosteric or allosteric ligands can be detrimental. Therefore, in addition to receptor subtype selectivity, a drug candidate may need to exhibit a biased signaling profile to avoid such on-target adverse effects. Indeed, recent studies in mice suggest that allosteric modulators for the M1 mAChR that bias signaling toward specific pathways may be therapeutically important. This review brings together details on the signaling pathways activated by the M1 and M4 mAChRs, evidence of biased agonism at these receptors, and highlights pathways that may be important for developing new subtype selective allosteric ligands to achieve therapeutic benefit.

| S-EPMC7878563 | biostudies-literature

Novel allosteric effects of amiodarone at the muscarinic M5 receptor.

Project description:Allosteric sites on muscarinic receptors may present superior therapeutic targets for several central nervous system disorders, due to the potential of allosteric ligands to provide more selective modulation and to preserve the spatiotemporal patterning that is characteristic of synaptic transmission. We have found that the antiarrhythmic drug amiodarone interacts allosterically with M(1) and M(5) muscarinic receptors. At both M(1) and M(5), amiodarone was only able to partially inhibit the binding of the orthosteric antagonist [(3)H]N-methylscopolamine (NMS). In addition, amiodarone was able to alter the rate of dissociation of [(3)H]NMS from M(1) and M(5) receptors. These findings suggest that NMS and amiodarone are able to bind to the receptor simultaneously. The pharmacology of the effect on NMS dissociation demonstrated that amiodarone was not interacting at the "common" site at which gallamine, obidoxime, and many other muscarinic allosteric ligands are known to bind. In functional studies, amiodarone enhanced the ability of acetylcholine (at EC(20)) to activate the M(5) receptor; however, under the same conditions, amiodarone did not enhance M(1) activation. More detailed studies at M(5) found that the effect of amiodarone was to enhance the efficacy of acetylcholine, without increasing its potency. This report describes the first demonstration of allosteric enhancement of efficacy at the M(5) receptor, and the first demonstration of enhancement of efficacy but not potency at any muscarinic receptor. In summary, amiodarone has been shown to be a novel positive allosteric modulator of muscarinic receptors that is selective for the M(5) subtype, relative to M(1).

| S-EPMC2912050 | biostudies-literature

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