Antagonist 4-DAMP Research Articles

Ethanolic Extracts of Cupressaceae Species Conifers Provide Rapid Protection against Barium Chloride-Induced Cardiac Arrhythmia.

Sudden cardiac death (SCD) is responsible for a high percentage of cardiovascular fatalities, with ventricular arrhythmias being the most common cause. Despite numerous clinically available antiarrhythmic drugs (AADs), AADs retain some undesirable arrhythmic effects, and their inappropriate use can lead to severe adverse reactions. The exploration of new therapeutic options against arrhythmias with fewer unreceptive effects is of utmost importance. The ethanolic extracts of seven Cupressaceae species, namely, Chamaecyparis obtusa, Juniperus chinensis (L.) Ant., Sabina chinensis (L.) Ant. cv. Kaizuca, Platycladus orientalis (L.) Franco, Juniperus sabina L., Fokienia hodginsii, and Juniperus chinensis 'Pyramidalis' were investigated for their pharmacological effects on barium chloride (BaCl2)-induced arrhythmia using normal II lead electrocardiogram (ECG) measurements in a mouse model. According to the ECG profiles, pretreatment with C. obtusa, P. orientalis, and J. sabina extracts provoked dose-dependent protection against BaCl2-induced arrhythmia, while pretreatment with the other four species and amiodarone did not exert cardioprotective effects. The treatment effects were confirmed using a rat model. The therapeutic effects of C. obtusa, P. orientalis, and J. sabina extracts on the M2 and M3 receptors but not the M1 receptor were mediated by the inhibition of the M2 receptor blocker (methoctramine tetrahydrochloride), M3 antagonist (4-DAMP), or M1 receptor blocker (pirenzepine dihydrochloride). This first-line evidence illustrates that certain Cupressaceae species possess active antiarrhythmic components. The first line of key findings revealed that active components of certain Cupressaceae species have cardioprotective effects, suggesting that these innovative phytochemicals have promising potential for preventing the occurrence of cardiac arrhythmia and reducing sudden cardiac death.

Characterizing Muscarinic Receptor Subtype Roles in Inspiratory Burst Modulation of Hypoglossal Motoneurons in Mice

Loss of tongue muscle (innervated by hypoglossal (XII) motoneurons) tone is responsible for airway obstruction during sleep. Contributing factors include loss of noradrenergic drive and activation of muscarinic acetylcholine receptors (mAChRs). While noradrenergic effects are well studied, less is known about muscarinic effects. In mice, XII motoneurons express four mAChRs; M1, M3, and M5 are excitatory while M2 is inhibitory. We hypothesize that M2 is upregulated with postnatal maturation whereas M1, M3, and M5 are downregulated. We therefore expect that activation of M1, M3, and M5 receptors will potentiate inspiratory bursting early in postnatal development whereas activation of M2 receptors will inhibit inspiratory bursting later in postnatal development. To test this, we utilized rhythmic medullary slice preparations from mice at postnatal days 0-5 (P0-5) and P9-14, which contained preBötzinger Complex (site of inspiratory rhythm generation) and XII motoneurons. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarinic potentiation of inspiratory bursting in P0-5 mice at 100 μM (168±23% at 10 μM and 129±16% at 100 μM in muscarine [p<0.05] vs 183±34% muscarine (100 μM) control response, n=9). Activating M1 receptors locally with cevimeline (1 mM) for 30s or 60s did not mediate potentiation of inspiratory bursting as seen with muscarine (116±7% at 30s and 117±7% at 60s vs 181±40% control muscarine response [n=6, p<0.05]). Bath application of M2 antagonist methoctramine (MTH) in P0-5 mice at 1 μM had no effect on muscarine-potentiation of inspiratory bursting (207±109% at 1 μM MTH in muscarine vs 212±84% muscarine control response, n=9). Preliminary analysis of the P9-14 age group using 1 μM MTH suggests there may be an increase in muscarine-mediated potentiation of inspiratory bursting with M2 blockade (170±56% at 1 μM MTH in muscarine vs 152±56% muscarine control response, n=6, p>0.5). Bath application of M3 receptor antagonist 4-DAMP in P0-5 mice at 10 nM had no effect on muscarine-potentiated inspiratory bursting (172±12% vs 178±30% muscarine control), whereas application at 100 nM decreased muscarine-mediated inspiratory burst potentiation compared to control (133±12%, n=4, p<0.05). However, bath application with another M3 receptor antagonist, J Fumarate 104129, had no significant effect on muscarinic modulation of inspiratory bursting in P0-5 mice (188±46% at 0.6 nM [n=6], 185±32% at 6 nM [n=6], and 144±24% at 60 nM [n=4] vs 176±34% muscarine control [n=6]). Overall, our data partially supports a role of M1 receptors contributing to muscarinic potentiation of inspiratory bursting in XII motoneurons, with the contributions of M2 and M3 appearing insignificant early in postnatal life. Preliminary analysis suggests that M2 has a larger contribution later in maturation. Future research will evaluate the contribution of muscarinic receptor subtypes to inspiratory burst modulation later in postnatal life. NIH/NHLBI Funding: R15HL148870. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

The local GLP-1 system in the olfactory bulb is required for odor-evoked cephalic phase of insulin release in mice

ObjectiveThe olfactory bulb (OB) codes for sensory information and contributes to the control of energy metabolism by regulating foraging and cephalic phase responses. Mitral cells are the main output neurons of the OB. The glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) system in the OB (GLP-1OB) has been shown to be a major regulator of mitral cell activity but its function in vivo is unclear. Therefore, we investigated the role of GLP-1OB in foraging behavior and odor-evoked Cephalic Phase Insulin Release (CPIR). Methods and resultsBy fluorescent labeling, we confirmed the presence of GLP-1 producing neurons and the expression of GLP-1R in the mouse OB. In response to food odor presentation, we collected blood, quantified plasma insulin by ELISA and showed the existence of an odor-evoked CPIR in lean mice but its absence in obese animals. Expression of shRNA against preproglucagon mRNA in the OB resulted in blunted CPIR in lean mice. Injecting Exendin (9-39), a GLP-1R antagonist, into the OB of lean mice also resulted in decreased CPIR. Since parasympathetic cholinergic input to the pancreas is known to be partly responsible for CPIR, we systemically administered the muscarinic M3 receptor antagonist 4-DAMP which resulted in a reduced odor-evoked CPIR. Finally, local injection of Exendin (9-39) in the OB extinguished olfactory foraging in lean mice whereas the injection of the GLP-1R agonist Exendin-4 rescued the loss of foraging behavior in obese mice. ConclusionsOur results demonstrate that GLP-1OB controls olfactory foraging and is required for odor-evoked CPIR. We describe a new crucial brain function for GLP-1 and GLP-1R expressed within the brain.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of β-Adrenoceptors.

Beta-adrenoceptor (β-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting β-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of β-ARs. M2R-dependent contractions were apparent at low frequency (2Hz)electric field stimulation (EFS) and short (10s) stimulus intervals.The β1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 sand was more effective at inhibiting contractions evoked by EFS at 2than 20 Hz.Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the β3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of β1-ARs and that genetic ablation of M2Rs decreased the efficacy of β-AR agonists on cholinergic contractions.

Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction.PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the β2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect.In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

Activation of M3-AChR and IP3/Ca2+/PKC signaling pathways by pilocarpine increases glycine-induced currents in ventral horn neurons of the spinal cord

Our study aimed to determine the effects of pilocarpine and the mechanisms involving muscarinic acetylcholine receptors (mAChRs) on glycine receptors (GlyRs) in neurons of the spinal cord ventral horn. An enzymatic digestion combined with acute mechanical separation was applied to isolate neurons from the spinal cord ventral horn. Patch-clamp recording was then used to investigate the outcomes of pilocarpine. Our results indicate that pilocarpine increased the glycine currents in a concentration-dependent manner, which was blocked by the M3-AChR selective antagonists 4-DAMP and J104129. Pilocarpine also enhanced the glycine currents in nominally Ca2+-free extracellular solution. Conversely, the enhancement of glycine currents by pilocarpine disappeared when intracellular Ca2+ was chelated by BAPTA. Heparin and Xe-C, which are IP3 receptor antagonists, also totally abolished the pilocarpine effect. Furthermore, Bis-IV, a PKC inhibitor, eliminated the pilocarpine effect. Additionally, PMA, a PKC activator, mimicked the pilocarpine effect. These results indicate that pilocarpine may increase the glycine currents by activating the M3-AChRs and IP3/Ca2+/PKC pathways.

Contribution of Postjunctional M2 Muscarinic Receptors to Cholinergic Nerve-Mediated Contractions of Murine Airway Smooth Muscle.

Postjunctional M2Rs on airway smooth muscle (ASM) outnumber M3Rs by a ratio of 4:1 in most species, however, it is the M3Rs that are thought to mediate the bronchoconstrictor effects of acetylcholine. In this study, we describe a novel and profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM at low stimulus frequencies.. Contractions induced by 2Hz EFS were augmented by > 2.5-fold when the stimulus interval was reduced from 100 to 10 s. This effect was reversed by the M2R antagonists, methoctramine, and AFDX116, and was absent in M2R null mice. The M3R antagonist 4-DAMP abolished the entire response in both WT and M2R KO mice. The M2R-mediated potentiation of EFS-induced contractions was not observed when the stimulus frequency was increased to 20 Hz. A subthreshold concentration of carbachol enhanced the amplitude of EFS-evoked contractions in WT, but not M2R null mice. These data highlight a significant M2R-mediated potentiation of M3R-dependent contractions of ASM at low frequency stimulation that could be relevant in diseases such as asthma and COPD.

Cholinergic Modulation by Activation of Muscarinic Acetylcholine Receptor Subtypes Modulates Inspiratory Bursting in Hypoglossal Motoneurons in Mice in Vitro

During wakefulness, tonic and respiratory input provides drive to upper airway dilatory muscles, and in particular genioglossus, the main tongue protruder, which stiffens the airway and keeps airway resistance low. These respiratory muscles experience decrements in activity during rapid eye movement sleep. Disruption in motor output and insufficient muscle tone of the tongue due to inhibition, or loss of excitation, of hypoglossal motoneurons (which innervate tongue muscles) contributes to sleep-disordered breathing. Recent data from adult rats in vivo suggest that muscarinic inhibition of hypoglossal motoneurons may be a major mechanism leading to loss of tongue muscle tone during sleep. However, available data indicate that there are contrasting effects of muscarinic modulation: in neonatal rhythmic slice preparations, muscarine applied within the hypoglossal nucleus has an excitatory effect on inspiratory burst amplitude. Muscarinic receptor subtypes can be broadly divided into those that are coupled to excitatory G-protein coupled receptors (M1, M3, M5) and those that are coupled to inhibitory G-protein coupled receptors (M2, M4). Hypoglossal motoneurons express M1, M2, M5, and possibly M3 receptor subtypes. Many modulatory systems undergo postnatal maturation, which we hypothesize underlies the switch in muscarinic modulation from net excitation to net inhibition of inspiratory bursting behavior at hypoglossal motoneurons. As an initial test of our hypothesis, our goal was to determine the contribution of excitatory and inhibitory muscarine receptor subtypes to the muscarinic modulation of inspiratory burst output from hypoglossal motoneurons in neonatal mice. Using the in vitro rhythmic slice preparation, we first investigated inspiratory behavior by observing the net effects of muscarine locally applied into the hypoglossal nucleus (100 mM, 30 s) on inspiratory burst amplitude in neonatal CD-1 mice (postnatal day, P0-P5). Our data confirm that in mice ages P0-5, local application of muscarine increased inspiratory burst amplitude (range: 53-180% potentiation, n = 9) compared to control inspiratory burst activity. Preliminary results suggest that the local application of M2 antagonist methoctramine in addition to muscarine (range: 20%-135% potentiation, n=4) had little effect on inspiratory behavior compared to baseline effects of muscarine (range: 4%-143% potentiation). Next, we tested the contribution of M3 receptors to muscarinic modulation on inspiratory burst amplitude. At baseline, locally applied muscarine increased inspiratory burst amplitude (range: 20%-63% potentiation); the muscarinic potentiation was attenuated during bath application of the M3 receptor-preferring antagonist 4-DAMP (range: 17%-25% potentiation) and recovered upon washout (range: 13%-117% potentiation, n=3). These preliminary results indicate muscarinic excitation is mediated in part by the M3-receptor subtype. Future experiments will determine how the M2 and M3 receptor contributions change with postnatal maturation.

Huperzine A lowers intraocular pressure via the M3 mAChR and provides retinal neuroprotection via the M1 mAChR: a promising agent for the treatment of glaucoma.

BackgroundGlaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer’s disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma.MethodsThe ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD).ResultsHupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA.ConclusionsHupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Intestinal Electrical Stimulation Enhances Release of Postprandial Incretin Hormones Via Cholinergic Mechanisms.

Intestinal electrical stimulation (IES) has been reported to reduce body weight and improve glucose tolerance in obese and diabetic rats. Our study aimed to investigate possible IES mechanisms involving incretin hormones using intraduodenal glucose infusion in rats. We hypothesized that the enhanced release of postprandial glucagon-like peptide-1 (GLP-1) at early phase by IES was mediated through neuro/paracrine mechanisms involving the vagal nerve and glucose-dependent insulinotropic peptide (GIP). Fifteen normal male Sprague-Dawley rats chronically implanted with duodenal electrodes for IES, and an intra-duodenum catheter for the infusion of glucose were studied in a series of sessions with IES of different parameters with and without atropine and M3 receptor antagonist. Blood samples were collected via the tail vein for the measurement of blood glucose, and plasma GLP-1, and GIP. (1) Compared to sham-IES, IES of 0.3ms reduced blood glucose by 16.5-28.4% between 30 and 120min (all time points p < 0.05), and IES of 3-ms reduced blood glucose at 60 (12.6%) and 90min (11.8%). IES of 0.3ms showed a greater hypoglycemic effect than 3ms (p = 0.024) at 30min. (2) IES elevated plasma GLP-1 with 0.3ms (p = 0.001) and with 3ms p = 0.03). (3) IES substantially elevated plasma GIP with 0.3ms (p = 0.002) and with 3ms (p < 0.001). (4) Pretreatment of atropine and the M3 receptor antagonist 4-DAMP blocked the effects of IES on GLP-1, GIP, and blood glucose. IES reduces postprandial blood glucose by enhancing the release of GLP-1 and GIP mediated via the cholinergic mechanism.

Breakdown of phospholipids and the elevated nitric oxide are involved in M3 muscarinic regulation of acetylcholine secretion in the frog motor synapse

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.

Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway.

Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+thyroid cancer cells through the activation of the CD133-Akt pathway.

Participation of acetylcholine and its receptors in the contractility of inflamed porcine uterus

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10−5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10−5 M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10−5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10−5 M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10−7, 10−6 M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10−7 M) and ENDO/MYO (ACh: 10−5 M) than in the SAL group. MR2 or MR3 antagonists and ACh (10−5 M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10−6, 10−5 M) than in other groups. Both antagonists and ACh (10−5 M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.

Acetylcholine Release Inhibits Distinct Excitatory Inputs Onto Hippocampal CA1 Pyramidal Neurons via Different Cellular and Network Mechanisms.

In hippocampal CA1, muscarinic acetylcholine (ACh) receptor (mAChR) activation via exogenous application of cholinergic agonists has been shown to presynaptically inhibit Schaffer collateral (SC) glutamatergic inputs in stratum radiatum (SR), and temporoammonic (TA) and thalamic nucleus reuniens (RE) glutamatergic inputs in stratum lacunosum-moleculare (SLM). However, steady-state uniform mAChR activation may not mimic the effect of ACh release in an intact hippocampal network. To more accurately examine the effect of ACh release on glutamatergic synaptic efficacy, we measured electrically evoked synaptic responses in CA1 pyramidal cells (PCs) following the optogenetic release of ACh in genetically modified mouse brain slices. The ratio of synaptic amplitudes in response to paired-pulse SR stimulation (stimulus 2/stimulus 1) was significantly reduced by the optogenetic release of ACh, consistent with a postsynaptic decrease in synaptic efficacy. The effect of ACh release was blocked by the M3 receptor antagonist 4-DAMP, the GABAB receptor antagonist CGP 52432, inclusion of GDP-β-S, cesium, QX314 in the intracellular patch clamp solution, or extracellular barium. These observations suggest that ACh release decreased SC synaptic transmission through an M3 muscarinic receptor-mediated increase in inhibitory interneuron excitability, which activate GABAB receptors and inwardly rectifying potassium channels on CA1 pyramidal cells. In contrast, the ratio of synaptic amplitudes in response to paired-pulse stimulation in the SLM was increased by ACh release, consistent with presynaptic inhibition. ACh-mediated effects in SLM were blocked by the M2 receptor antagonist AF-DX 116, presumably located on presynaptic terminals. Therefore, our data indicate that ACh release differentially modulates excitatory inputs in SR and SLM of CA1 through different cellular and network mechanisms.

Activation of M3 Muscarinic Acetylcholine Receptors Delayed Cardiac Aging by Inhibiting the Caspase-1/IL-1β Signaling Pathway

Background/Aims: Because the prevalence of age-related cardiac impairment increases as the human lifespan increases, it is important to combat the effects of aging. Recently, the cardiac M3 muscarinic acetylcholine receptor (M3-mAChR) has been demonstrated to play important roles in cardiac development and in the pathogenesis of cardiac diseases. However, the role of M3-mAChR in aging remains largely unknown. Therefore, the aim of this study was to investigate the involvement of M3-mAChR in the progression of cardiac aging. Methods: We established a cardiac aging model in mice through subcutaneous injection with D-galactose at a dose of 100 mg/kg/day for 6 weeks. D-galactose was also used to induce aging in primary cultured neonatal mouse cardiomyocytes. The myocardium from mice was stained with hematoxylin and eosin for histological analysis. The protein expression levels of p53 and p21 were determined using western blotting. The mRNA and protein expression levels of M3-mAChR, caspase-1, and interleukin (IL)-1β were determined using real-time PCR, immunohistochemical staining, and western blotting. Results: The expression of M3-mAChR was down-regulated in the myocardium from aged mice and D-galactose-treated mice, while the expression levels of caspase-1 and its downstream molecule IL-1β were significantly increased. The M3-mAChR agonist choline reduced the increase in caspase-1 in cardiomyocytes induced by D-galactose, which was reversed by the M3-mAChR antagonist 4-DAMP. Moreover, 4-DAMP promoted D-galactose-induced cardiomyocyte aging, which was attenuated by a caspase-1 inhibitor. Conclusion: Activation of M3-mAChR delayed cardiac aging by inhibiting the caspase-1/IL-1β signaling pathway.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10−6-10−3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10−6–10−3 M) and acetylcholine (10−9-10−4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10−5 M), selective M1 mAChR antagonist pirenzepine (5 × 10−5 M) and selective M3 mAChR antagonist 4-DAMP (10−7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10−6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10−2 M) and cystathionine-β-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10−3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.

Muscarinic cholinergic receptor antagonists in the VTA and RMTg have opposite effects on morphine-induced locomotion in mice

The ventral tegmental area (VTA) and the rostromedial tegmental nucleus (RMTg) each contribute to opiate reward and each receive inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic cell groups. We compared the contributions of VTA or RMTg muscarinic cholinergic receptors to locomotion induced by morphine infusions into the same sites. VTA co-infusion of atropine completely blocked VTA morphine-induced locomotion providing additional support for the important role of VTA muscarinic cholinergic receptors in the stimulant effects of opiates. By contrast, RMTg co-infusion of atropine increased RMTg morphine-induced locomotion. Furthermore, RMTg co-infusion of the M3-selective antagonist 4-DAMP, but not the M4-selective antagonist Tropicamide, strongly increased RMTg morphine-induced locomotion. RMTg infusions of 4-DAMP, but not of Tropicamide, by themselves strongly increased drug-free locomotion. Muscarinic cholinergic receptors in the RMTg thus also contribute to the stimulant effects of morphine, but in a way opposite to those in VTA. We suggest that the net effect of endogenous cholinergic input to the RMTg on drug-free and on RMTg morphine-induced locomotion is inhibitory.

Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation

Acetylcholine (ACh), known as a neurotransmitter, regulates the functions of numerous fundamental central and peripheral nervous system. Recently, emerging evidences indicate that ACh also plays an important role in tumorigenesis. However, little is known about the role of ACh in gastric cancer. Here, we reported that ACh could be auto-synthesized and released from MKN45 and BGC823 gastric cancer cells. Exogenous ACh promoted cell proliferation in a does-dependent manner. The M3R antagonist 4-DAMP, but not M1R antagonist trihexyphenidyl and M2/4 R antagonist AFDX-116, could reverse the ACh-induced cell proliferation. Moreover, ACh, via M3R, activated the EGFR signaling to induce the phosphorylation of ERK1/2 and AKT, and blocking EGFR pathway by specific inhibitor AG1478 suppressed the ACh induced cell proliferation. Furthermore, the M3R antagonist 4-DAMP and darifenacin could markedly inhibit gastric tumor formation in vivo. 4-DAMP could also significantly enhance the cytotoxic activity of 5-Fu against the MKN45 and BGC823 cells, and induce the expression of apoptosis-related proteins such as Bax and Caspase-3. Together, these findings indicated that the autocrine ACh could act through M3R and the EGFR signaling to promote gastric cancer cells proliferation, targeting M3R or EGFR may provide us a potential therapeutic strategy for gastric cancer treatment.

Cholinergic modulation of the parafacial respiratory group.

This study investigates the effects of cholinergic transmission on the expiratory oscillator, the parafacial respiratory group (pFRG) in urethane anaesthetized adult rats. Local inhibition of the acetyl cholinesterase enzyme induced activation of expiratory abdominal muscles and active expiration. Local application of the cholinomimetic carbachol elicited recruitment of late expiratory neurons, expiratory abdominal muscle activity and active expiration. This effect was antagonized by local application of the muscarinic antagonists scopolamine, J104129 and 4DAMP. We observed distinct physiological responses between the more medial chemosensitive region of the retrotrapezoid nucleus and the more lateral region of pFRG. These results support the hypothesis that pFRG is under cholinergic neuromodulation and the region surrounding the facial nucleus contains a group of neurons with distinct physiological roles. Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem: inspiration driven by a neuronal network located in the preBötzinger complex (preBötC) and expiration driven by a neuronal network located in the parafacial respiratory group (pFRG). While continuous activity of the preBötC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in the presence of increased respiratory drive (e.g. hypoxia, hypercapnia, exercise and through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state-dependent fashion, frequently occurring during periods of REM sleep. We hypothesized that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre-application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow.