Muscarinic and nicotinic receptors stimulation by vagus nerve stimulation ameliorates trastuzumab-induced cardiotoxicity via reducing programmed cell death in rats.

Despite its efficacy in human epidermal growth factor receptor 2 positive cancer treatment, trastuzumab-induced cardiotoxicity (TIC) has become a growing concern. Due to the lack of cardiomyocyte regeneration and proliferation in adult heart, cell death significantly contributes to cardiovascular diseases. Cardiac autonomic modulation by vagus nerve stimulation (VNS) has shown cardioprotective effects in several heart disease models, while the effects of VNS and its underlying mechanisms against TIC have not been found. Forty adult male Wistar rats were divided into 5 groups: (i) control without VNS (CSham) group, (ii) trastuzumab (4 mg/kg/day, i.p.) without VNS (TSham) group, (iii) trastuzumab + VNS (TVNS) group, (iv) trastuzumab + VNS + mAChR blocker (atropine; 1 mg/kg/day, ip, TVNS + Atro) group, and (v) trastuzumab + VNS + nAChR blocker (mecamylamine; 7.5 mg/kg/day, ip, TVNS + Mec) group. Our results showed that trastuzumab induced cardiac dysfunction by increasing autonomic dysfunction, mitochondrial dysfunction/dynamics imbalance, and cardiomyocyte death including apoptosis, autophagic deficiency, pyroptosis, and ferroptosis, which were notably alleviated by VNS. However, mAChR and nAChR blockers significantly inhibited the beneficial effects of VNS on cardiac autonomic dysfunction, mitochondrial dysfunction, cardiomyocyte apoptosis, pyroptosis, and ferroptosis. Only nAChR could counteract the protective effects of VNS on cardiac mitochondrial dynamics imbalance and autophagy insufficiency. Therefore, VNS prevented TIC by rebalancing autonomic activity, ameliorating mitochondrial dysfunction and cardiomyocyte death through mAChR and nAChR activation. The current study provides a novel perspective elucidating the potential treatment of VNS, thus also offering other pharmacological therapeutic promises in TIC patients.

BDNF/TrkB signalling, in cooperation with muscarinic signalling, retrogradely regulates PKA pathway to phosphorylate SNAP-25 and Synapsin-1 at the neuromuscular junction.

BACKGROUND: Protein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M1 and M2 (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1. METHODS: To explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation' changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry. RESULTS: While TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cß, it regulates PKA regulatory subunits RIα and RIIß, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M1 and M2 mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation. CONCLUSION: This complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication.

Cholinergic Mechanisms in Gastrointestinal Neoplasia.

Acetylcholine-activated receptors are divided broadly into two major structurally distinct classes: ligand-gated ion channel nicotinic and G-protein-coupled muscarinic receptors. Each class encompasses several structurally related receptor subtypes with distinct patterns of tissue expression and post-receptor signal transduction mechanisms. The activation of both nicotinic and muscarinic cholinergic receptors has been associated with the induction and progression of gastrointestinal neoplasia. Herein, after briefly reviewing the classification of acetylcholine-activated receptors and the role that nicotinic and muscarinic cholinergic signaling plays in normal digestive function, we consider the mechanics of acetylcholine synthesis and release by neuronal and non-neuronal cells in the gastrointestinal microenvironment, and current methodology and challenges in measuring serum and tissue acetylcholine levels accurately. Then, we critically evaluate the evidence that constitutive and ligand-induced activation of acetylcholine-activated receptors plays a role in promoting gastrointestinal neoplasia. We focus primarily on adenocarcinomas of the stomach, pancreas, and colon, because these cancers are particularly common worldwide and, when diagnosed at an advanced stage, are associated with very high rates of morbidity and mortality. Throughout this comprehensive review, we concentrate on identifying novel ways to leverage these observations for prognostic and therapeutic purposes.

Muscarinic Receptors in Cardioprotection and Vascular Tone Regulation.

Muscarinic acetylcholine receptors are metabotropic G-protein coupled receptors. Muscarinic receptors in the cardiovascular system play a central role in its regulation. Particularly M2 receptors slow down the heart rate by reducing the impulse conductivity through the atrioventricular node. In general, activation of muscarinic receptors has sedative effects on the cardiovascular system, including vasodilation, negative chronotropic and inotropic effects on the heart, and cardioprotective effects, including antifibrillatory effects. First, we review the signaling of individual subtypes of muscarinic receptors and their involvement in the physiology and pathology of the cardiovascular system. Then we review age and disease-related changes in signaling via muscarinic receptors in the cardiovascular system. Finally, we review molecular mechanisms involved in cardioprotection mediated by muscarinic receptors leading to negative chronotropic and inotropic and antifibrillatory effects on heart and vasodilation, like activation of acetylcholine-gated inward-rectifier K+-currents and endothelium-dependent and -independent vasodilation. We relate this knowledge with well-established cardioprotective treatments by vagal stimulation and muscarinic agonists. It is well known that estrogen exerts cardioprotective effects against atherosclerosis and ischemia-reperfusion injury. Recently, some sex hormones and neurosteroids have been shown to allosterically modulate muscarinic receptors. Thus, we outline possible treatment by steroid-based positive allosteric modulators of acetylcholine as a novel pharmacotherapeutic tactic. Keywords: Muscarinic receptors, Muscarinic agonists, Allosteric modulation, Cardiovascular system, Cardioprotection, Steroids.

Further investigations on the influence of protein phosphatases on the signaling of muscarinic receptors in the atria of mouse hearts.

The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include Gi/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct Gi/o protein-coupled activation of ACh-activated potassium current (IKACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM-10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after ß-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of ß-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Effect of spirocyclopiperazinium salt compound LXM-15 on spinal nerve injury in rats.

BACKGROUND: Currently available therapies for neuropathic pain show limited efficacy. This study aimed to investigate the anti-nociceptive effect of the spirocyclopiperazinium salt compound LXM-15 in spinal nerve ligation (SNL) rats and to explore the potential mechanisms. METHODS: Mechanical allodynia and thermal hyperalgesia tests were used to evaluate the effects of LXM-15 in SNL rats. The expression of CaMKIIα, CREB, JAK2, STAT3, c-fos and TNF-α was detected by western blotting, ELISA or qRT-PCR analysis. Receptor blocking test was performed to explore possible target. RESULTS: Administration of LXM-15 (1, 0.5, 0.25 mg/kg, i.g.) dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in rats subjected to SNL (p < 0.01, p < 0.05), and the effects were completely blocked by peripheral α7 nicotinic or M4 muscarinic receptor antagonist (p > 0.05). LXM-15 significantly decreased the overexpression of phosphorylated CaMKIIα, CREB, JAK2 and STAT3 proteins and the mRNA levels of TNF-α and c-fos (p < 0.01, p < 05). All of the effects could be blocked by α7 or M4 receptor antagonist. Furthermore, LXM-15 reduced the protein expression of TNF-α and c-fos (p < 0.01, p < 0.05). No significant acute toxicity or abnormal hepatorenal function was observed. CONCLUSIONS: This is the first study to report that LXM-15 exerts significant anti-nociceptive effect on SNL rats. This effect may occur by activating peripheral α7 nicotinic and M4 muscarinic receptors, further inhibiting the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and finally inhibiting the expression of TNF-α and c-fos. SIGNIFICANCE: Existing treatments for neuropathic pain show limited efficacy with severe adverse reactions. This paper is the first to report that LXM-15, a new spirocyclopiperazinium salt compound, exerts a significant anti-nociception in SNL rats without obvious toxicity. The underlying mechanisms include activating peripheral α7 nicotinic and M4 muscarinic receptors, then inhibiting the signalling pathways of CaMKIIα/CREB and JAK2/STAT3 and the expressions of TNF-α and c-fos. This study sheds new light on the development of novel analgesic drugs with fewer side effects.

Activation of M1 muscarinic acetylcholine receptors by proline-rich oligopeptide 7a (

Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability.(AU)

Muscarinic receptor agonist-induced ßPix binding to ß-catenin promotes colon neoplasia.

M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.

Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines.

BACKGROUND: Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. METHODS: We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) cells. RESULTS: Increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK. CONCLUSIONS: The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.

Muscarinic Acetylcholine Receptor M3 Expression and Survival in Human Colorectal Carcinoma-An Unexpected Correlation to Guide Future Treatment?

Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78; p = 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99; p = 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%, p = 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (p = 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.

Myorelaxant and antispasmodic effects of the essential oil of Artemisia campestris L., and the molecular docking of its major constituents with the muscarinic receptor and the L-type voltage-gated Ca2+channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal disorders are among the most common diseases that cause discomfort to people who are affected. In Morocco, aromatic and medicinal plants are widely used to calm these pains and eliminate their symptoms. Among these plants, Artemisia campestris L. which is used in eastern Morocco to treat digestive system problems. AIM OF THE STUDY: Our study aimed to experimentally verify the traditional use of this plant by evaluating the myorelaxant and antispasmodic effects of the essential oil of Artemisia campestris L. (EOAc). MATERIALS AND METHODS: Gas Chromatography-Mass Spectrometry analysis (GC-MS) was performed to identify the compounds present in the EOAc. Then, these molecules were subjected to the in silico study for molecular docking. The myorelaxant and antispasmodic evaluation of the EOAc were tested in vitro on an isolated rabbit and rat jejunum mounted on an organ bath. Then, an isotonic transducer connected to an amplifier recorded the graph related to intestinal contractility. RESULTS: GC-MS analysis of the essential oil of Artemisia campestris L. showed the presence of m-Cymene (17.308%), Spathulenol (16.785%), ß Pinene (15.623%), α Pinene (11.352%), α.-Campholenal (8.848%) as main constituents. The EOAc gave a dose-dependent and reversible myorelaxant effect on the spontaneous contractions of jejunum isolated from rabbits, with an IC50 equal to 72.16 ± 15.93 µg/mL. This effect did not occur through adrenergic receptors. The EOAc has an antispasmodic effect on the contractions of rat jejunal induced by a medium with low (25 mM) or high concentration (75 mM) of KCl, and carbachol 10-6 M. The obtained inhibitory effects are comparable to those of a non-competitive antagonist of cholinergic receptors. The major compounds of EOAc allowed the establishment of a relationship between these phytoconstituents and the antispasmodic effect found by the EOAc. The obtained results are also supported by a docking study. CONCLUSION: The obtained results confirm favorably the use of Artemisia campestris L. in traditional Moroccan medicine for the treatment of digestive tract illness, which gives us a new route to valorize the effects obtained by a phytomedicine specific for the digestive tract.

Treating Lower Urinary Tract Symptoms in Older Adults: Intravesical Options.

This article provides an overview of the diagnosis and the treatment of lower urinary tract symptoms in older adults complicated by the neurodegenerative changes in the micturition reflex and further confounded by age-related decline in hepatic and renal clearance raising the propensity of adverse drug reactions. The first-line drug treatment for lower urinary tract symptoms, orally administered antimuscarinics, fails to reach the equilibrium dissociation constant of muscarinic receptors even at their maximum plasma concentration and tends to evoke a half-maximal response at a muscarinic receptor occupancy of just 0.206% in the bladder with a minimal difference from exocrine glands, which raises the adverse drug reaction risk. On the contrary, intravesical antimuscarinics are instilled at concentrations 1000-fold higher than the oral maximum plasma concentration and the equilibrium dissociation constant erects a downhill concentration gradient that drives passive diffusion and achieves a mucosal concentration around ten-fold lower than the instilled concentration for a long-lasting occupation of muscarinic receptors in mucosa and sensory nerves. A high local concentration of antimuscarinics in the bladder triggers alternative mechanisms of action and is supposed to engage retrograde transport to nerve cell bodies for neuroplastic changes that underlie a long-lasting therapeutic effect, while an intrinsically lower systemic uptake of the intravesical route lowers the muscarinic receptor occupancy of exocrine glands to lower the adverse drug reaction relative to the oral route. Thus, the traditional pharmacokinetics and pharmacodynamics of oral treatment are upended by intravesical antimuscarinics to generate a dramatic improvement (~ 76%) noted in a meta-analysis of studies enrolling children with neurogenic lower urinary tract symptoms on the primary endpoint of maximum cystometric bladder capacity as well as the secondary endpoints of filling compliance and uninhibited detrusor contractions. The therapeutic success of intravesical multidose oxybutynin solution or oxybutynin entrapped in the polymer for sustained release in the pediatric population bodes well for patients with lower urinary tract symptoms at the other extreme of the age spectrum. Though generally used to predict oral drug absorption, Lipinski's rule of five can also explain the ten-fold lower systemic uptake from the bladder of positively charged trospium over oxybutynin, a tertiary amine. Chemodenervation by an intradetrusor injection of onabotulinumtoxinA is merited for patients with idiopathic overactive bladder discontinuing oral treatment because of a lack of efficacy. However, age-related peripheral neurodegeneration potentiates the adverse drug reaction risk of urinary retention that motivates the quest of liquid instillation, delivering larger fraction of onabotulinumtoxinA to the mucosa as opposed to muscle by an intradetrusor injection can also probe the neurogenic and myogenic predominance of idiopathic overactive bladder. Overall, the treatment paradigm of lower urinary tract symptoms in older adults should be tailored to individual's overall health status and the risk tolerance for adverse drug reactions.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation. Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method. Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level. Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia.

Xanomeline restores endogenous nicotinic acetylcholine receptor signaling in mouse prefrontal cortex.

Cholinergic synapses in prefrontal cortex are vital for attention, but this modulatory system undergoes substantial pre- and post-synaptic alterations during adulthood. To examine the integrated impact of these changes, we optophysiologically probe cholinergic synapses ex vivo, revealing a clear decline in neurotransmission in middle adulthood. Pharmacological dissection of synaptic components reveals a selective reduction in postsynaptic nicotinic receptor currents. Other components of cholinergic synapses appear stable, by contrast, including acetylcholine autoinhibition, metabolism, and excitation of postsynaptic muscarinic receptors. Pursuing strategies to strengthen cholinergic neurotransmission, we find that positive allosteric modulation of nicotinic receptors with NS9283 is effective in young adults but wanes with age. To boost nicotinic receptor availability, we harness the second messenger pathways of the preserved excitatory muscarinic receptors with xanomeline. This muscarinic agonist and cognitive-enhancer restores nicotinic signaling in older mice significantly, in a muscarinic- and PKC-dependent manner. The rescued nicotinic component regains youthful sensitivity to allosteric enhancement: treatment with xanomeline and NS9283 restores cholinergic synapses in older mice to the strength, speed, and receptor mechanism of young adults. Our results reveal a new and efficient strategy to rescue age-related nicotinic signaling deficits, demonstrating a novel pathway for xanomeline to restore cognitively-essential endogenous cholinergic neurotransmission.

Dopaminergic and cholinergic modulation of the amygdala is altered in female mice with oestrogen receptor ß deprivation.

The amygdala is modulated by dopaminergic and cholinergic neurotransmission, and this modulation is altered in mood disorders. Therefore, this study was designed to evaluate the presence/absence of quantitative alterations in the expression of main dopaminergic and cholinergic markers in the amygdala of mice with oestrogen receptor ß (ERß) knock-out which exhibit increased anxiety, using immunohistochemistry and quantitative methods. Such alterations could either contribute to increased anxiety or be a compensatory mechanism for reducing anxiety. The results show that among dopaminergic markers, the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine D2-like receptor (DA2) is significantly elevated in the amygdala of mice with ERß deprivation when compared to matched controls, whereas the content of dopamine D1-like receptor (DA1) is not altered by ERß knock-out. In the case of cholinergic markers, muscarinic acetylcholine type 1 receptor (AChRM1) and alpha-7 nicotinic acetylcholine receptor (AChRα7) display overexpression while the content of acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) remains unchanged. In conclusion, in the amygdala of ERß knock-out female the dopaminergic and cholinergic signalling is altered, however, to determine the exact role of ERß in the anxiety-related behaviour further studies are required.

Acetylcholine Receptors in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

Muscarinic Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs' relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.

Cold stress-induced bladder overactivity in type 2 diabetic mellitus rats is mitigated by the combination of a M3 -muscarinic antagonist and a ß3 -adrenergic agonist.

OBJECTIVES: Goto-Kakizaki (GK) rats with type 2 diabetes mellitus respond to low temperature (LT) environments with bladder overactivity, including increased voiding frequency and decreased voiding interval and micturition volume. We determined if bladder overactivity could be inhibited by treatment with the combination of a M3 -muscarinic receptor antagonist and a ß3 -adrenergic receptor agonist. METHODS: Ten-week-old female GK rats were fed a high-fat diet for 4 weeks. Cystometric investigations were conducted at room temperature (RT, 27 ± 2°C). The rats were then intraperitoneally administered the vehicle, the M3 -muscarinic receptor antagonist solifenacin, the ß3 -adrenergic agonist mirabegron, or a combination of solifenacin and mirabegron. Ten minutes after the administrations, the rats were transferred to the LT environment (4 ± 2°C), where the cystometric measurements were continued. The expressions of both M3 -muscarinic and ß3 -adrenergic receptors were investigated. RESULTS: After transfer from RT to LT, both voiding interval and bladder capacity of the vehicle-, solifenacin-, or mirabegron-treated rats were significantly decreased. However, the combination of solifenacin and mirabegron significantly mitigated the bladder overactivity. While both M3 -muscarinic and ß3 -adrenergic receptors were detected, the expression of M3 -muscarinic receptor mRNA was significantly higher than that of ß3 -adrenergic receptor mRNA. CONCLUSIONS: The cold stress-induced bladder overactivity was not improved by either the M3 -muscarinic receptor antagonist or the ß3 -adrenergic receptor agonist alone. However, the combined treatment mitigated the cold stress responses. Combined therapy with M3 -muscarinic antagonists and ß3 -adrenergic agonists could reduce side effects and improve the quality of life for diabetic patients with bladder overactivity.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aß1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aß1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aß1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.