Activation Of M1 Muscarinic Receptors Research Articles

M1 muscarinic receptor activation reverses age-related memory updating impairment in mice

Previously consolidated memories can become temporarily labile upon reactivation. Reactivation-based memory updating is chiefly studied in young subjects, so we aimed to assess this process across the lifespan. To do this, we developed a behavioural paradigm wherein a reactivated object memory is updated with contextual information; 3-month-old and 6-month-old male C57BL/6 mice displayed object memory updating, but 12-month-old mice did not. We found that M1 muscarinic acetylcholine receptor signaling during reactivation was necessary for object memory updating in the young mice. Next, we targeted this mechanism in an attempt to facilitate object memory updating in aging mice. Remarkably, systemic pharmacological M1 receptor activation reversed the age-related deficit. Quantification of cholinergic system markers within perirhinal cortex revealed subtle cellular changes that may contribute to differential performance across age groups. These findings suggest that natural cholinergic change across the lifespan contributes to inflexible memory in the aging brain.

Voltage dependence of M2 muscarinic receptor antagonists and allosteric modulators

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs’ voltage dependence and may have pharmacological implications.

Acetylcholine Engages Distinct Amygdala Microcircuits to Gate Internal Theta Rhythm.

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOM→CCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOM→CCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

Zuojin Pill enhances gastrointestinal motility by modulating the pacemaker potentials in interstitial cells of Cajal through multiple signaling pathways.

Zuojin Pill (ZJP) is a traditional herbal preparation used to treat various gastrointestinal (GI) disorders. The purpose of this study was to elucidate the underlying cellular and molecular mechanisms by evaluating the effects of ZJP on the pacemaker activity of isolated interstitial cells of Cajal (ICCs) and on in vivo GI motility in mice. We isolated ICCs from mouse small intestine and measured pacemaker potentials by whole-cell patch clamping as well as intracellular calcium signaling by microfluorometry. Intestinal transit rate (ITR) was measured by Evans Blue migration. Administration of ZJP depolarized ICCs and reduced the amplitude and frequency of pacemaker potentials. Pretreatment with the 5-HT4 receptor antagonist RS39604, administration of the muscarinic M3 receptor antagonist 4-DAMP, or intracellular perfusion of the G-protein inhibitor GDP-β-S blocked ZJP-induced ICCs depolarization. In addition, external calcium-free medium and administration of the Ca2+-ATPase inhibitor thapsigargin, which depletes intracellular calcium stores, also blocked ZJP-induced ICCs depolarization. Moreover, ZJP-induced ICCs depolarization was inhibited in the presence of the phospholipase C (PLC) inhibitor U-73122, IP3-dependent Ca2+ release inhibitor xestospongin C, or various mitogen-activated protein kinase (MAPK) inhibitors. Alternatively, ZJP-induced ICCs depolarization in the presence of protein kinase C (PKC) inhibitors. Furthermore, ZJP reversed the reduction of ITR caused by loperamide (Imodium) and normalized the ITR abnormality of two etiologically distinct GI motility disorder (GMD) mouse models. Finally, ZJP increased serum concentrations of the pro-peristalsis factors motilin and substance P. Our results suggest that ZJP depolarizes ICCs via 5-HT4 or muscarinic M3 receptor activation and G-protein dependent calcium-, PLC-, inositol triphosphate-, and MAPK signaling pathways (but not PKC-dependent pathways), leading to enhanced GI motility.

Selective Activation of M1 Muscarinic Receptors Attenuates Human Colon Cancer Cell Proliferation.

M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.

The Partial M1 Muscarinic Cholinergic Receptor Agonist, CDD-0102A, Differentially Modulates Glutamate Efflux in Striatal Subregions during Stereotyped Motor Behavior in the BTBR Mouse Model of Autism.

The BTBR T+ Itpr3tf/J (BTBR) mouse displays elevated repetitive motor behaviors. Treatment with the partial M1 muscarinic receptor agonist, CDD-0102A, attenuates stereotyped motor behaviors in BTBR mice. The present experiment investigated whether CDD-0102A modifies changes in striatal glutamate concentrations during stereotyped motor behavior in BTBR and B6 mice. Using glutamate biosensors, change in striatal glutamate efflux was measured during bouts of digging and grooming behavior with a 1 s time resolution. Mice displayed both decreases and increases in glutamate efflux during such behaviors. Magnitude of changes in glutamate efflux (decreases and increases) from dorsomedial and dorsolateral striatum were significantly greater in BTBR mice compared to those of B6 mice. In BTBR mice, CDD-0102A (1.2 mg/kg) administered 30 min prior to testing significantly reduced the magnitude change in glutamate decreases and increases from the dorsolateral striatum and decreased grooming behavior. Conversely, CDD-0102A treatment in B6 mice potentiated glutamate decreases and increases in the dorsolateral striatum and elevated grooming behavior. The findings suggest that activation of M1 muscarinic receptors modifies glutamate transmission in the dorsolateral striatum and self-grooming behavior.

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled Gq protein cycle

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation–deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gβγ subunits. We also find that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled G protein cycle.

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the reversible activation-deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a new Förster resonance energy transfer (FRET) tool for human M3 muscarinic acetylcholine receptor, we found that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results revealed that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gβγ subunits. We also found that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovered the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human epithelial ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer-related deaths in females. Many ovarian tumor cell lines express muscarinic receptors (mAChRs), and their expression is correlated with reduced survival of patients. We have characterized the expression of mAChRs in two human ovarian carcinoma cell lines (SKOV-3, TOV-21G) and two immortalized ovarian surface epithelium cell lines (iOSE-120, iOSE-398). Among the five subtypes of mAChRs (M1-M5 receptors), we focused our attention on the M2 receptor, which is involved in the inhibition of tumor cell proliferation. Western blot analysis and real-time PCR analyses indicated that the levels of M2 are statistically downregulated in cancer cells. Therefore, we investigated the effect of arecaidine propargyl ester hydrobromide (APE), a preferential M2 agonist, on cell growth and survival. APE treatment decreased cell number in a dose and time-dependent manner by decreasing cell proliferation and increasing cell death. FACS and immunocytochemistry analysis have also demonstrated the ability of APE to accumulate the cells in G2/M phase of the cell cycle and to increase the percentage of abnormal mitosis. The higher level of M2 receptors in the iOSE cells rendered these cells more sensitive to APE treatment than cancer cells. The data here reported suggest that M2 has a negative role in cell growth/survival of ovarian cell lines, and its downregulation may favor tumor progression.

Colocalization of M1 muscarinic cholinergic receptors and NMDA receptors in dendrites and spines of pyramidal neurons of the mouse basolateral amygdala: An ultrastructural analysis

The cholinergic innervation of the neocortex, hippocampus, and basolateral amygdala is critical for higher cognitive functions, including attention and memory. One action of ACh in the hippocampus is the potentiation of NMDA receptor (NMDAR) currents in pyramidal neurons (PNs) by M1 muscarinic receptors (M1Rs). The increase in these currents enhances long-term potentiation (LTP), an important mechanism for memory formation. Ultrastructural observations in the hippocampus revealed that M1Rs and NMDARs were colocalized in hippocampal PN dendrites, consistent with electrophysiological studies demonstrating M1R-NMDAR interactions. Similar to the hippocampus, activation of M1Rs have been shown to be critical for mnemonic functions in the anterior basolateral nucleus of the amygdala (BLa). In the present study dual-labeling immunoelectron microscopy was used to determine if there was colocalization of M1Rs and NMDARs in neurons of the mouse BLa. We found extensive colocalization of these receptors in dendrites and spines of BLa PNs, and most of the M1Rs were membrane-associated where they could be activated by released acetylcholine. These results suggest that M1Rs in BLa PNs could be targeted by M1R positive allosteric modulators (PAMs), resulting in amelioration of memory impairments in neuropsychiatric disorders, such as Alzheimer’s disease, by potentiating NMDAR currents in the amygdala.

Target-specific control of olfactory bulb periglomerular cells by GABAergic and cholinergic basal forebrain inputs.

The olfactory bulb (OB), the first relay for odor processing in the brain, receives dense GABAergic and cholinergic long-range projections from basal forebrain (BF) nuclei that provide information about the internal state and behavioral context of the animal. However, the targets, impact, and dynamic of these afferents are still unclear. How BF synaptic inputs modulate activity in diverse subtypes of periglomerular (PG) interneurons using optogenetic stimulation and loose cell-attached or whole-cell patch-clamp recording in OB slices from adult mice were studied in this article. GABAergic BF inputs potently blocked PG cells firing except in a minority of calretinin-expressing cells in which GABA release elicited spiking. Parallel cholinergic projections excited a previously overlooked PG cell subtype via synaptic activation of M1 muscarinic receptors. Low-frequency stimulation of the cholinergic axons drove persistent firing in these PG cells, thereby increasing tonic inhibition in principal neurons. Taken together, these findings suggest that modality-specific BF inputs can orchestrate synaptic inhibition in OB glomeruli using multiple, potentially independent, inhibitory or excitatory target-specific pathways.

Reduced expression of the psychiatric risk gene DLG2 (PSD93) impairs hippocampal synaptic integration and plasticity

Copy number variants indicating loss of function in the DLG2 gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability. DLG2 encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear. Here, hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygous Dlg2+/− rat model using ex vivo patch-clamp electrophysiology, pharmacology, and computational modelling. Dlg2+/− rats had reduced supra-linear dendritic integration of synaptic inputs resulting in impaired associative long-term potentiation. This impairment was not caused by a change in synaptic input since NMDA receptor-mediated synaptic currents were, conversely, increased and AMPA receptor-mediated currents were unaffected. Instead, the impairment in associative long-term potentiation resulted from an increase in potassium channel function leading to a decrease in input resistance, which reduced supra-linear dendritic integration. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in the Dlg2+/− rats. These findings demonstrate a biological phenotype that can be reversed by compound classes used clinically, such as muscarinic M1 receptor agonists, and is therefore a potential target for therapeutic intervention.

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction

Heterozygous missense variants in KCNQ2, which encodes the potassium channel subunit Kv7.2, are among the most common genetic causes of severe neonatal-onset epileptic encephalopathy. Because about 20% of known severe Kv7.2 missense changes lie within the intracellular C-terminal region, improving understanding of the underlying pathogenic mechanisms is important. We analyzed the basis for the severe phenotypes of Kv7.2 A337T and A337G, variants in the C-terminal’s calmodulin (CaM)-binding Helix A. When expressed heterologously in mammalian cells, alone or in combination with wild type Kv7.2 or with wild type Kv7.2 and Kv7.3, both variants strongly suppressed channel currents. A337T channels expressed alone exhibited significantly reduced protein half-life and surface trafficking and co-immunoprecipitated less CaM. For both variants, increasing cellular phosphatidylinositol 4,5-bisphosphate (PIP2) by overexpression of PI(4)P5-kinase restored current densities. For both variants, the fraction of current suppressed by activation of M1 muscarinic receptors with 10 μM oxotremorine methiodide, which depletes PIP2, was less than for controls. During voltage-sensitive phosphatase-induced transient PIP2 depletion and resynthesize, potassium current inhibition and recovery kinetics were both markedly slowed. These results suggest that these variants may reduce currents by a mechanism not previously described: slowing of PIP2 migration between the bulk membrane and binding sites mediating channel electromechanical coupling. A novel Kv7.2/3-selective opener, SF0034, rescued current amplitudes. Our findings show that these two Helix A variants suppress channel current density strongly, consistent with their severe heterozygous phenotypes, implicate impairment of CaM and PIP2 regulation in KCNQ2 encephalopathy pathogenesis, and highlight the potential usefulness of selective Kv7 openers for this distinctive pathogenic mechanism and patient subgroup.

Diacylglycerol kinases regulate TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is activated by heat and by capsaicin, the pungent compound in chili peppers. Calcium influx through TRPV1 has been shown to activate a calcium-sensitive phospholipase C (PLC) enzyme and to lead to a robust decrease in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels, which is a major contributor to channel desensitization. Diacylglycerol (DAG), the product of the PLC-catalyzed PI(4,5)P2 hydrolysis, activates protein kinase C (PKC). PKC is known to potentiate TRPV1 activity during activation of G protein-coupled receptors, but it is not known whether DAG modulates TRPV1 during desensitization. We found here that inhibition of diacylglycerol kinase (DAGK) enzymes reduces desensitization of native TRPV1 in dorsal root ganglion neurons as well as of recombinant TRPV1 expressed in HEK293 cells. The effect of DAGK inhibition was eliminated by mutating two PKC-targeted phosphorylation sites, Ser-502 and Ser-800, indicating involvement of PKC. TRPV1 activation induced only a small and transient increase in DAG levels, unlike the robust and more sustained increase induced by muscarinic receptor activation. DAGK inhibition substantially increased the DAG signal evoked by TRPV1 activation but not that evoked by M1 muscarinic receptor activation. Our results show that Ca2+ influx through TRPV1 activates PLC and DAGK enzymes and that the latter limits formation of DAG and negatively regulates TRPV1 channel activity. Our findings uncover a role of DAGK in ion channel regulation.

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres by microglia in vitro. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain. No effect of CNO on cytokine expression was observed in DREADD-negative mice. Interestingly, CNO activation of M3Dq in microglia was able to attenuate LPS-mediated decrease in social interactions. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, can trigger an inflammatory-like response that preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases.

SAT-306 Muscarinic and Adrenergic Receptor Cooperativity in a Human Adrenocortical Carcinoma Cell Line

The role of autonomic receptors in the regulation of the adrenal cortex is poorly understood. We recently showed that activation of M3 muscarinic receptors stimulates intracellular calcium oscillations, aldosterone production, and expression of CYP11B2 (1). The present study explores the relationship between muscarinic and adrenergic receptors in corticosteroid production. Using live-cell fluorescence imaging of HAC15 adrenocortical cells with the calcium-sensitive probe Fluo-4, we have shown that stimulation of adrenergic receptors with the endogenous, non-selective adrenergic agonist norepinephrine (10μM) enhances intracellular Ca2+ oscillations caused by the cholinergic agonist carbachol (1μM). However, Ca2+ is not affected by norepinephrine alone. Adrenergic enhancement of carbachol-induced Ca2+ oscillations is blocked by the ⍺ adrenergic receptor antagonist phentolamine, but not by the β adrenergic receptor antagonist propanolol. Specifically, ⍺2 and β2 antagonists (such as yohimbine and butoxamine, respectively) significantly suppressed the norepinephrine effect, but ⍺1 and β1 antagonists (such as tamsulosin and metoprolol, respectively) had no effect. RT qPCR identified ⍺2A receptors as the most abundant adrenergic receptor in HAC15 cells. Saturation experiments using 3H-NMS and 3H-Rauwolscine confirmed the presence of muscarinic M2 and M3 receptors as well as ⍺2A receptors. Using competition radiolabeled binding assays we explored the cooperation between M2/M3 and ⍺2A adrenergic receptors. Our results suggest that autonomic regulation of intracellular Ca2+ depends on an interplay of M3 and ⍺2A receptors. Additional experiments will use ELISA methods to determine the functional impact of autonomic receptor cooperativity on steroid synthesis and secretion.

M2 Receptor Activation Counteracts the Glioblastoma Cancer Stem Cell Response to Hypoxia Condition.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

Design and synthesis of two new steroid derivatives with biological activity on heart failure via the M2-muscarinic receptor activation

Several drugs have been prepared to treat of heart failure using some protocols which require dangerous reagents and specific conditions. The aim of this study was to synthesize a series of steroid derivatives (compounds 2 to 18) using some chemical strategies. The biological activity of steroid derivatives against heart failure was evaluated using an ischemia/reperfusion model. In addition, the effect exerted by compounds 4 or 5 on left ventricular pressure was evaluated in the absence or presence of yohimbine, butaxamine and methoctramine. The results showed that 1) both compounds 4 or 5 significantly decrease the heart failure (translated as infarct area) compared with the compounds 2, 3 and 6–18. In addition, the compound 4 and 5 decreased the left ventricular pressure in a dose-dependent manner and this effect was significantly inhibited in the presence of methoctramine (p = 005). In conclusion, the compounds 4 or 5 decrease both the infarct area and left ventricular pressure via M2-muscarinic receptor activation.

Impairment of agonist-induced M2 muscarinic receptor activation by autoantibodies from chagasic patients with cardiovascular dysautonomia

Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.