M3R Activation Research Articles

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

Potential Role for Combined Subtype-Selective Targeting of M1 and M3 Muscarinic Receptors in Gastrointestinal and Liver Diseases.

Despite structural similarity, the five subtypes comprising the cholinergic muscarinic family of G protein-coupled receptors regulate remarkably diverse biological functions. This mini review focuses on the closely related and commonly co-expressed M1R and M3R muscarinic acetylcholine receptor subtypes encoded respectively by CHRM1 and CHRM3. Activated M1R and M3R signal via Gq and downstream initiate phospholipid turnover, changes in cell calcium levels, and activation of protein kinases that alter gene transcription and ultimately cell function. The unexpectedly divergent effects of M1R and M3R activation, despite similar receptor structure, distribution, and signaling, are puzzling. To explore this conundrum, we focus on the gastrointestinal (GI) tract and liver because abundant data identify opposing effects of M1R and M3R activation on the progression of gastric, pancreatic, and colon cancer, and liver injury and fibrosis. Whereas M3R activation promotes GI neoplasia, M1R activation appears protective. In contrast, in murine liver injury models, M3R activation promotes and M1R activation mitigates liver fibrosis. We analyze these findings critically, consider their therapeutic implications, and review the pharmacology and availability for research and therapeutics of M1R and M3R-selective agonists and antagonists. We conclude by considering gaps in knowledge and other factors that hinder the application of these drugs and the development of new agents to treat GI and liver diseases.

Pancreas-specific CHRM3 activation causes pancreatitis in mice.

Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein–coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.

Allosteric modulation of β-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic β cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured β cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by β-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in β cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Targeting M3 Muscarinic Receptors for Colon Cancer Therapy.

Expression and activation of subtype-3 muscarinic receptors (M3R) plays an important role in the progression of colorectal neoplasia. Herein, we describe the role of muscarinic receptors in colon cancer, focusing specifically on M3R, illustrate how M3R over-expression and activation of post-receptor signaling pathways potentiates tumor progression, and explore the efficacy and safety of a variety of therapeutic approaches that can target the molecules involved. Colon cancers overexpress M3R mRNA (CHRM3) and protein, and post-M3R signaling stimulates cell proliferation. Post-M3R signal transduction is complex, involving interplay between epidermal growth factor receptors (EGFR)/ERK and protein kinase C (PKC)/p38 mitogen-activated protein (MAP) kinase signaling pathways. In particular, the development of an invasive and metastatic phenotype requires that these signaling interactions augment cellular release of a key collagenase, matrix metalloproteinase-1 (MMP1). Blocking either M3R activation or post-M3R signaling attenuates MMP1 release and colon cancer invasiveness. Parsing the complexities of these signaling interactions is important, not only to understand these mechanisms of cancer initiation and progression, but also to develop novel treatment modalities. Since the vast majority of persons with colon cancer die from disseminated disease, preventing or reversing metastatic spread of cancer cells by targeting M3R, post-M3R signaling, or MMP1 has therapeutic potential.

Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility.

The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.

Cortical nNOS/NK1 Receptor Neurons are Regulated by Cholinergic Projections From the Basal Forebrain.

Cholinergic (ACh) basal forebrain (BF) neurons are active during wakefulness and rapid eye movement (REM) sleep and are involved in sleep homeostasis. We have previously shown in adult animals that cortical neurons that express neuronal nitric oxide synthase (nNOS) and the receptor for Substance P (NK1R) are activated during non-REM (NREM) sleep in proportion to homeostatic sleep drive. Here, we show that BF neurons modulate cortical nNOS/NK1R cells. In vitro optogenetic stimulation of BF terminals both activated and inhibited nNOS/NK1R neurons. Pharmacological studies revealed cholinergic responses mediated by postsynaptic activation of muscarinic receptors (mAChRs; M3R > M2/4R > M1R) and that presynaptic M3R and M2R activation reduced glutamatergic input onto nNOS/NK1R neurons whereas nicotinic receptor (nAChR)-mediated responses of nNOS/NK1R neurons were mixed. Cholinergic responses of nNOS/NK1R neurons were largely unaffected by prolonged wakefulness. ACh release, including from BF cells, appears to largely excite cortical nNOS/NK1R cells while reducing glutamatergic inputs onto these neurons. We propose that cholinergic signaling onto cortical nNOS/NK1R neurons may contribute to the regulation of cortical activity across arousal states, but that this response is likely independent of the role of these neurons in sleep homeostasis.

A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren’s syndrome

Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren’s syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction.

Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium.

The role of muscarinic receptors in mucosal homeostasis, response to enteric pathogens, and modulation of immune cell function is undefined. The contribution of type 3 muscarinic receptors (M3R) to mucosal homeostasis within the colon and host defense against Citrobacter rodentium was determined in uninfected and C. rodentium-infected WT and M3R-deficient (Chrm3) mice. In addition, WT and Chrm3 bone marrow-derived macrophages were studied to determine the ability of M3R to modulate macrophage phenotype and function. In Chrm3 mice, clearance of C. rodentium was delayed despite an amplified TH1/TH17 response. Delayed clearance of C. rodentium from Chrm3 mice was associated with prolonged adherence of bacteria to colonic mucosa, decreased goblet cell number, and decreased mucin 2 gene expression. Treatment of bone marrow-derived macrophages with bethanechol, a muscarinic-selective agonist, induced a classically activated macrophage phenotype, which was dependent on M3R expression. Chrm3 bone marrow-derived macrophages retained their ability to attain a classically activated macrophage phenotype when treated with the TH1 cytokine IFN-γ. In Chrm3 mice, mucin production is attenuated and is associated with prolonged adherence of C. rodentium to colonic mucosa. The immune response, as characterized by production of TH1/TH17 cytokines, in C. rodentium-infected Chrm3 mice is intact. In addition, M3R activity promotes the development of classically activated macrophages. Our data establish a role for M3R in host defense against C. rodentium through effects on goblet cell mucus production and in the modulation of macrophage phenotype and function.

Alterations in adenylyl cyclase sensitivity to hormones in the brain, myocardium, and testes of rats immunized with BSA-conjugated peptide 269–280 of type 3 melanocortin receptor

Binding of the type 3 melanocortin receptors (M3R) functionally coupled to Gs proteins with α-melanocyte-stimulating hormone (α-MSH) or M3R agonist γ-MSH results in the activation of adenylyl cyclase (AC) in the brain, which is responsible for the M3R-mediated regulation of physiological and biochemical processes in the central and peripheral nervous systems. A decrease in the M3R activity leads to energy metabolism disorders, metabolic syndrome, and functional disturbances of the nervous and cardiovascular systems. However, molecular mechanisms of these alterations and the role of the adenylyl cyclase signaling system (ACSS) are yet poorly understood. The aim of this work was to study the effects of a long-term (13 months, 8 injections) immunization of male rats with BSA-conjugated peptide A-[PTNPYCICTTAH269–280]-A (A-[269–280]-A), corresponding to the third extracellular loop of rat M3R, on the functional activity of ACSS and its regulation by neurotransmitters and hormones in rat brain, testes, and myocardium. At the end of the experiment, the body weight of animals decreased, the specific mass of adipose tissue increased, dyslipidemia was observed, and the sensitivity to insulin was reduced in spite of the increase in its secretion in response to a glycemic load. In the brain of immunized rats, the AC-stimulating effects of γ-MSH, noradrenaline, serotonin and PACAP-38 decreased, the stimulating effect of dopamine and AC-inhibiting effects of serotonin and 5-nonyloxytryptamine (an agonist of 5-hydroxytryptamine receptor (5-HTR) of the 1B/1D subtype) increased, while the effects of the M3R/M4R agonist α-MSH and the M4R agonist THIQ remained unchanged. In myocardial membranes, stimulation of AC by the agonists of β3-adrenergic receptors (β3-AR) was enhanced, while the AC-stimulating effects of β1- and β2-agonists were attenuated and the AC effect of relaxin weakened. In testicular membranes, the AC-stimulating effects of chorionic gonadotropin and PACAP-38 decreased, as well as the effect of GppNHp (non-hydrolysable GTP analog). These data indicate that the attenuation of the M3R signaling leads to considerable alterations in the ACSS sensitivity to the hormones playing a key role in the functioning of the nervous, cardiovascular and reproductive systems. The alterations in the ACSS sensitivity observed in brain, myocardium and testes are characterized by hormonal and receptor specificity. Identification of ACSS disorders is one of the approaches to studying etiology and pathogenesis of diseases associated with inhibition of M3R-melanocortin signaling system of the brain.

Monitoring the Dynamic Change of Inositol Lipid Pools upon EGFR and M3R Activation in Live Cells

Phosphoinositides (PI) in the plasma membrane (PM) can dynamically change upon hormonal stimulation which can influence several cellular processes thus measuring the level of PIs can help us to better understand their distinct functions. We developed a highly sensitive method which enables us to follow the dynamic change of these lipids in living cells. We performed BRET measurements between various luciferase‐labeled PI‐binding domains and a PM‐targeted Venus in HEK 293T cells. To monitor the inositol lipid pools the followings were used: the 2xP4M domain of SidM for PI(4)P, the PH domain of PLCδ1 for PI(4,5)P2, the PH domain of BTK for PI(3,4,5)P3 and an intramolecular IP3 BRET sensor based on the ligand binding domain of the type‐1 IP3 receptor. Stimulation of cells with 100 ng/ml EGF resulted in a sustained increase of PI(3,4,5)P3, a slowly developing IP3 signal a transiently decreased PI(4,5)P2 level and an unexpected increase of PI(4)P. We also tested the lipid changes in case of M3R activation and we found that 100 µM carbachol causes more robust increase in the IP3, and decrease in the PI(4,5)P2 and PI4P levels however decreasing the carbachol concentration to 100 nM resulted in a very similar lipid changes as were seen in case of the EGF stimulation. Using different PI4K inhibitors we found that the increase in the PI(4)P level was caused by the activation of PI4KIIIα, and could be prevented by PKC inhibitors. With the help of our highly sensitive approach we were able to follow the dynamic inositol lipid changes upon stimulation of the cells with various compounds, and we could identify a possible signaling route how the cells can resynthesize PIs.

Divergent effects of muscarinic receptor subtype gene ablation on murine colon tumorigenesis reveals association of M3R and zinc finger protein 277 expression in colon neoplasia

BackgroundM3 and M1 subtype muscarinic receptors are co-expressed in normal and neoplastic intestinal epithelial cells. In mice, ablating Chrm3, the gene encoding M3R, robustly attenuates intestinal tumor formation. Here we investigated the effects of Chrm1 gene ablation, alone and in combination with Chrm3 ablation.MethodsWe used wild-type, Chrm1-/-, Chrm3-/- and combined Chrm1-/-/Chrm3-/- knockout (dual knockout) mice. Animals were treated with azoxymethane, an intestine-selective carcinogen. After 20 weeks, colon tumors were counted and analyzed histologically and by immunohistochemical staining. Tumor gene expression was analyzed using microarray and results validated by RT-PCR. Key findings were extended by analyzing gene and protein expression in human colon cancers and adjacent normal colon tissue.ResultsAzoxymethane-treated Chrm3-/- mice had fewer and smaller colon tumors than wild-type mice. Reductions in colon tumor number and size were not observed in Chrm1-/- or dual knockout mice. To gain genetic insight into these divergent phenotypes we used an unbiased microarray approach to compare gene expression in tumors from Chrm3-/- to those in wild-type mice. We detected altered expression of 430 genes, validated by quantitative RT-PCR for the top 14 up- and 14 down-regulated genes. Comparing expression of this 28-gene subset in tumors from wild-type, Chrm3-/-, Chrm1-/- and dual knockout mice revealed significantly reduced expression of Zfp277, encoding zinc finger protein 277, in tissue from M3R-deficient and dual knockout mice, and parallel changes in Zfp277 protein expression. Notably, mRNA and protein for ZNF277, the human analogue of Zfp277, were increased in human colon cancer compared to adjacent normal colon, along with parallel changes in expression of M3R.ConclusionsOur results identify a novel candidate mouse gene, Zfp277, whose expression pattern is compatible with a role in mediating divergent effects of Chrm3 and Chrm1 gene ablation on murine intestinal neoplasia. The biological importance of this observation is strengthened by finding increased expression of ZNF277 in human colon cancer with a parallel increase in M3R expression. The role of zinc finger protein 277 in colon cancer and its relationship to M3R expression and activation are worthy of further investigation.

Type 3 Muscarinic Receptors Modulate Macrophage Phenotype in the Presence of Th2 Cytokines

Purpose: Macrophages (Mφ) play a critical role in IBD. Resident Mφs within the intestinal lamina propria are exposed to many antigens, but are tolerant to stimulation and prevent inflammation (Heinsbroek & Gordon, 2009). We showed previously that stimulation of type 3 (M3R) muscarinic receptors (MR) encoded by Chrm3 promoted the development of pro-inflammatory classically activated Mφs (CAM) (McLean, et al, 2012). Whether M3R plays a role in the development of alternatively activated Mφs (AAM), which promote wound healing and tissue repair, is unclear. Our aim was to test the hypothesis that M3R plays a role in AAM development. Methods: Bone marrow cells were isolated from WT and Chrm3-/- mice and differentiated in the presence of monocyte colony stimulating factor (M-CSF) for 7 days. Flow cytometry was used to analyze cell populations. After 24 hour treatment with a muscarinic receptor-selective agonist, bethanechol (BTH), +/- the Th2 cytokine IL-4, cells were harvested and mRNA expression of the AAM marker Arg-1 assess by real-time PCR. Results: We confirmed that ˜95% of our cell population was positive for both CD11b and F4/80, indicating that these cells are Mφs. These Mφs express mRNA for M1R, M2R, M3R and M4R. In WT Mφs, BTH treatment significantly attenuated Arg-1 expression, indicating a constitutive bias against AAM development. Treatment of naïve WT Mφs with IL-4 significantly up-regulated Arg-1 expression; addition of BTH to IL-4 did not enhance this effect. In contrast, in Chrm3-/- Mφs up-regulation of Arg-1 in response to IL-4 was significantly greater than that observed in WT Mφs. Moreover, addition of BTH to IL-4 potentiated this up-regulation of Arg-1 in Chrm3-/- Mφs. Conclusion: Treatment with BTH alone attenuated the development of AAMs from naïve WT Mφs. As expected, treatment of WT Mφs with IL-4 induced the development of AAMs. In WT Mφs, addition of BTH to IL-4 did not modulate this effect. In M3R-deficient Mφs, IL-4 further polarized Mφs to the AAM phenotype. Notably, exposing M3R-deficient Mφs to both IL-4 & BTH further enhances AAM polarization. We previously reported that M3R expression exerts pro-inflammatory effects on Mφs. Our findings suggest that in the presence of IL-4, M3R activation attenuates the development of AAMs and, in the presence of IL-4, further polarizes M3R-deficient Mφs to an AAM phenotype, possibly by acting at M1R, M2R, and/or M4R. This effect is not observed in WT Mφs, likely due to M3R activity. These data further support a pro-inflammatory role of M3R. Pharmacologic attempts to manipulate Mφ phenotypes should focus on blocking M3R expression or activation to promote AAMs, a strategy that may reduce disease activity or maintain remission in IBD patients.

Immunohistochemical and functional studies for M3 muscarinic receptors and cyclo‐oxygenase‐2 expressed in the mouse atrium

In mouse atrium, M₂ and M₃ muscarinic receptors (M₂R and M₃R) are involved in biphasic (negative and positive) inotropic actions of muscarinic agonists, and the positive inotropic action is reduced by indomethacin. The aim of our study was to determine the localization of M₂R, M₃R and cyclo-oxygenase (COX) in mouse atrium and to characterize muscarinic receptor-mediated positive inotropy. M₂R immunoreactivity was found only on atrial myocardium, but M₃R immunoreactivity was localized on both the myocardium and endocardial endothelium. COX-1 and COX-2 immunoreactivities were identified in both myocardial and endocardial endothelium. In electrically stimulated left atria, carbachol caused M₂R-mediated negative inotropy followed by M₃R-mediated positive inotropy. Removal of atrial endothelium reduced the positive inotropy without affecting the negative inotropy, suggesting that stimulation of endothelial M₃R mediates the positive inotropy. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398, COX-2 inhibitor) decreased the carbachol-induced positive inotropy; however, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560, COX-1 inhibitor), 1-[[4,5-bis(4-methoxyphenyl)-2-thiazolyl]carbonyl]-4-methylpiperazine (FR122047, COX-1 inhibitor) and L-nitroarginine methylester did not affect the inotropic response. M₃R activation caused positive chronotropy in spontaneously beating right atria when M₂R-mediated negative chronotropy was suppressed and rate of contraction was low, <350 beats min⁻¹. Our results indicate that although M₃Rs are located on both myocardial cells and endocardial endothelial cells, only endothelial M₃Rs mediate positive inotropy in response to muscarinic agonists via activation of COX-2 in the mouse atrium. M₃R-mediated positive chronotropy counteracting M₂R-mediated negative chronotropy was also demonstrated.

Spinophilin as a novel regulator of M 3 muscarinic receptor‐mediated insulin release in vitro and in vivo

Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic β cells. In this study, we examined the ability of SPL to modulate the activity of β-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving β-cell function for the treatment of type 2 diabetes.

Autoantibody activation of beta-adrenergic and muscarinic receptors contributes to an “autoimmune” orthostatic hypotension

Background Orthostatic hypotension (OH) is characterized by an abnormal autonomic response to upright posture. Activating autoantibodies to β1/2-adrenergic (AAβ1/2AR) and M2/3 muscarinic receptors (AAM2/3R) produce vasodilative changes in the vasculature that may contribute to OH. Methods Immunoglobulin (Ig)G from 6 patients with idiopathic OH harboring autoantibodies and from 10 healthy control subjects were examined for: 1) β1AR and M2R activity with a perfused Purkinje fiber assay and PKA assay in H9c2 cells and 2) vasodilator β2AR and M3R activity using a pressurized cremaster resistance arteriole assay. Changes in IgG activity with and without propranolol, atropine, and L-NAME were used to estimate AAβAR, AAM2R, and AAM3R activation of their respective functions. Results All six patients had elevated enzyme-linked immunosorbent assay titers to at least one of the receptors compared with controls. βAR-mediated contractility activity and M2R activity were increased in five of the six patients. IgG from all six patients produced a direct vasodilator effect on cremaster arterioles. βAR and nitric oxide synthase blockade led to near normalization of IgG-induced vasodilation. Conclusion AAβ1/2AR and AAM2/3R are present in some patients with idiopathic OH compatible with an in vivo effect. These autoantibodies and their cardiovascular effects provide new mechanistic insights into the pathophysiology of OH.

Regulation of M3 Muscarinic Receptor Expression and Function by Transmembrane Protein 147

The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.

Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.

Brain-gut-liver-immune axis during non-inflammatory stress in rat

Patients with systemic sclerosis (SSc) have impairments in gastrointestinal smooth muscle function. The disorder has been associated with circulating antibodies to cholinergic muscarinic the type-3 receptor (M3-R). We investigated whether it is possible to neutralize these antibodies with pooled human IgGs (pooledhIgG).We studied the effects of IgGs purified from patients with SSc (SScIgGs) on cholinergic nerve stimulation in rat colon tissues. We also examined the effects of SScIgGs on M3-R activation by bethanechol (BeCh), M3-R occupancy, and receptor binding using immunofluorescence, immunoblot, and enzyme-linked immunosorbent analyses of human internal anal sphincter (IAS) smooth muscle cells, before and after administration of pooledhIgG. Functional displacement of M3-R occupancy by the SScIgGs was compared with that of other IgGs during the sustained phase of BeCh-induced contraction of intact smooth muscles from rats.SScIgG significantly attenuated neurally mediated contraction and acetylcholine release in rat colon as well as BeCh-induced sustained contraction of the IAS smooth muscle. In immunofluorescence analysis, SScIgG co-localized with M3-R. In immunoblot and enzyme-linked immunosorbent analyses, M3-R loop-2 peptide and human IAS SMC membrane lysates bound significant amounts of SScIgG, compared with IgGs from healthy individuals and pooledhIgG. Binding was attenuated significantly by application of pooledhIgG, which by itself had no significant effect. Incubation of samples with pooledhIgG, or mixing pooledhIgG with SScIgG before administration to tissues, significantly reduced binding of SScIgG, indicating that pooledhIgG prevents SScIgG blockade of M3-R.In studies of rat and human tissues, pooled human IgG prevent and reverses the cholinergic dysfunction associated with the progressive gastrointestinal manifestations of SSc by neutralizing functional M3-R antibodies present in the circulation of patients with SSc.