Acetylcholine Secretion Research Articles

The Contribution of L-Type Calcium Channels to Acetylcholine Secretion in Frog and Mouse Neuromuscular Junctions with Active and Inactivated Voltage-Gated Potassium Channels

We report here experiments addressing the contribution of L-type Ca2+ channels to evoked acetylcholine secretion from frog and mouse motor nerve endings with active and inactivated voltage-gated K+ channels. These studies evaluated the effects of the specific L-type Ca2+ channel blocker nitrendipine on the quantum composition of endplate currents and the time course of the secretion of acetylcholine quanta in intact preparations and after preliminary blockade of voltage-gated K+ channels with 4-aminopyridine (4-AP) in medium with depressed and physiological Ca2+ levels. A fluorescence method was used to measure calcium transients reflecting the integral influx of Ca2+ into nerve endings; computer modeling was applied to the processes underlying exocytosis in the presence of the two types of Ca2+ channel (N and L) and with different durations of nerve ending action potentials. In frog synapses, L-type Ca2+ channels were found to contribute to evoked acetylcholine secretion in the presence of active K+ channels, but only in the presence of a depressed Ca2+ level in the medium; on inactivation of voltage-gated K+ channels, the contribution of L-type channels to the secretory process became less significant. At a physiological Ca2+ level, the involvement of L-type channels in evoked acetylcholine secretion was apparent, as in mouse synapses, only in conditions of blockade of voltage-gated K+ channels.

Calcium Transients and Transmitter Secretion in Different Parts of Frog Nerve Endings in Different Conditions of Calcium Ion Influx

Experiments on frog neuromuscular preparations were performed to study the characteristics of the calcium response and the quantum secretion of acetylcholine in different pats of extended nerve terminals in different conditions of calcium influx. A calcium-sensitive fluorescent dye was used to analyze Ca2+ influx (Ca2+ transients) into the proximal and distal parts of nerve endings in conditions of increased K+ ion content, in response to blockers of N- and L-type calcium channels, and on blockade of calcium-activated potassium channels. These studies showed that at a uniform distribution density of voltage-gated calcium channels along nerve endings, the proximal-to-distal decrement in calcium transients and quantum secretion intensity persisted in conditions of additional opening of voltage-gated calcium channels by potassium depolarization, on “thinning” of these channels using specific blockers, but changed on blockade of calcium-activated potassium channels.

Quaternary-Ammonium-Modulated Surface-Enhanced Raman Spectroscopy Effect: Discovery, Mechanism, and Application for Highly Sensitive In Vitro Sensing of Acetylcholine.

Quaternary ammonium (QA) plays multiple roles in biological functions, whose dysregulation may result in multiple diseases. However, how to efficiently detect QA-based materials such as acetylcholine (ACh) still remains a great challenge, especially in complex biological environments. Here, a new effect [called quaternary-ammonium-modulated surface-enhanced Raman spectroscopy (QAM-SERS) effect] is discovered, showing that the existence of QA will modulate the intensity of SERS signals in a concentration-dependent manner. When the QAM-SERS effect is used, a new method is easily developed for in vitro detection of ACh with an extremely high sensitivity and an ultrawide dynamic range. Particularly, the linear dynamic range can be freely tuned to adapt for various physiological samples. As a proof-of-concept experiment, the time-dependent secretion of ACh from PC12 cells was successfully monitored using the QAM-SERS method, which were under either the stimulation of potassium ions or the incubation of drugs. The discovery of the QAM-SERS effect provides an easy and universal strategy for detecting ACh as well as other QA-contained molecules, which can also inspire new insights into the roles that QA could play in biology and chemistry.

Adrenaline Facilitates Synaptic Transmission by Synchronizing Release of Acetylcholine Quanta from Motor Nerve Endings.

The long history of studies on the effect of catecholamines on synaptic transmission does not answer the main question about the mechanism of their action on quantal release in the neuromuscular junction. Currently, interest in catecholamines has increased not only because of their widespread use in the clinic for the treatment of cardiovascular and pulmonary diseases but also because of recent data on their possible use for the treatment of certain neurodegenerative diseases, muscle weakness and amyotrophic sclerosis. Nevertheless, the effects and mechanisms of catecholamines on acetylcholine release remain unclear. We investigated the action of noradrenaline and adrenaline on the spontaneous and evoked quantal secretion of acetylcholine in the neuromuscular junction of the rat soleus muscle. Noradrenaline (10μM) did not change the spontaneous acetylcholine quantal release, the number of released quanta after nerve stimulation, or the timing of the quantal secretion. However, adrenaline at the same concentration increased spontaneous secretion by 40%, increased evoked acetylcholine quantal release by 62%, and synchronized secretion. These effects differ from those previously described by us in the synapses of the frog cutaneous pectoris muscle and mouse diaphragm. This indicates specificity in catecholamine action that depends on the functional type of muscle and the need to take the targeted type of muscle into account in clinical practice.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses.

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.

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.

Mechanism of gamete shedding in starfish: Involvement of acetylcholine in extracellular Ca2+-dependent contraction of gonadal walls

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.

Striatal Acetylcholine Helps to Preserve Functional Outcomes in a Mouse Model of Stroke

Acetylcholine (ACh) has been suggested to facilitate plasticity and improve functional recovery after different types of brain lesions. Interestingly, numerous studies have shown that striatal cholinergic interneurons are relatively resistant to acute ischemic insults, but whether ACh released by these neurons enhances functional recovery after stroke is unknown. We investigated the role of endogenous striatal ACh in stroke lesion volume and functional outcomes following middle cerebral artery occlusion to induce focal ischemia in striatum-selective vesicular acetylcholine transporter-deficient mice (stVAChT-KO). As transporter expression is almost completely eliminated in the striatum of stVAChT-KO mice, ACh release is nearly abolished in this area. Conversely, in other brain areas, VAChT expression and ACh release are preserved. Our results demonstrate a larger infarct size after ischemic insult in stVAChT-KO mice, with more pronounced functional impairments and increased mortality than in littermate controls. These changes are associated with increased activation of GSK-3, decreased levels of β-catenin, and a higher permeability of the blood–brain barrier in mice with loss of VAChT in striatum neurons. These results support a framework in which endogenous ACh secretion originating from cholinergic interneurons in the striatum helps to protect brain tissue against ischemia-induced damage and facilitates brain recovery by supporting blood–brain barrier function.

The Role of BDNF and proBDNF in the Regulation of Parameters of Spontaneous Acetylcholine Secretion in Mature and Newly-Formed Mouse Motor Synapses

The Role of BDNF and proBDNF in the Regulation of Parameters of Spontaneous Acetylcholine Secretion in Mature and Newly-Formed Mouse Motor Synapses

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling.

For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter "taste" substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca2+]i in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca2+]i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.

Diverse Effects of Noradrenaline and Adrenaline on the Quantal Secretion of Acetylcholine at the Mouse Neuromuscular Junction

Despite the long history of investigations of adrenergic compounds and their biological effects, specific mechanisms of their action in distinct compartments of the motor unit remain obscure. Recent results have suggested that not only skeletal muscles but also the neuromuscular junctions represent important targets for the action of catecholamines. In this paper, we describe the effects of adrenaline and noradrenaline on the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal release in the motor nerve endings of the mouse diaphragm. Noradrenaline and adrenaline decreased the frequency of the spontaneous release of acetylcholine quanta. The effect of noradrenaline was prevented by the β adrenoreceptor blocker propranolol, whereas the action of adrenaline was abolished by the α adrenoreceptor antagonist phentolamine.Noradrenaline did not alter the quantal content of endplate potentials, while adrenaline suppressed the evoked release of acetylcholine. Blocking the α adrenoreceptors prevented the decrease in quantal secretion caused by adrenaline. Quantal release became more asynchronous under noradrenaline, as evidenced by a greater dispersion of real synaptic delays; in contrast, adrenaline synchronized the release process.Our data suggest an involvement of α and β adrenoreceptors in the diverse modulation of the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal secretion in the mouse neuromuscular junction. Moreover, the adrenoblockers affected both the evoked and spontaneous quantal release of acetylcholine, suggesting the presence of endogenous catecholamines in the vicinity of cholinergic synapses.

Dendrimer grafted albumin nanoparticles for the treatment of post cerebral stroke damages: A proof of concept study

Stroke is the second largest disease of mortality. The biggest hurdle in designing effective brain drug delivery systems is offered by the blood-brain barrier (BBB), which is highly impermeable to many drugs. Albumin nanoparticles (NP) have gained attention due to their multiple ligand binding sites and long circulatory half-life. Citicoline (CIT) is reported to enhance the acetylcholine secretion in the brain and also helps in membrane repair and regeneration. However, the poor BBB permeation of CIT results in lower levels of CIT in the brain. This demands the development of a suitable delivery platform to completely realize the therapeutic benefit of CIT in stroke therapy. This investigation reports the synthesis and characterization of second generation (2.0 G) dendrimer Amplified Albumin (dAA) biopolymer by FTIR, MALDI-TOF, and surface charge (mV). Further, the synthesized biopolymer has been utilized to develop a CIT nanoformulation using a commercially translatable one-pot process. Release of CIT from biopolymer was performed within an acetate buffer at pH 5 and Phosphate buffer at pH 7.4. Further, we investigated the ability of biopolymer to permeate BBB by in vitro permeability assay in bEnd.3 cells. MTT assay of CIT-dAA-NP, CIT-ANP, and 2.0 G PAMAM dendrimers was performed in bEnd.3 cells. Therapeutic efficacy of the synthesized biopolymer was determined by VEGF gene expression within an in vitro hypoxia model in PC12 cells. Thus, this investigation resulted in biopolymers that can be used to deliver any therapeutic agent by altering the permeability of the BBB. Also, cationization by dendrimer grafting is one such strategy that may be used to cationize any other negatively charged polymer, such as albumin. The synthesized biopolymer is not limited to deliver molecules to the brain, but can also be used to increase the loading of negatively-charged drug molecules, siRNA, or any other oligonucleotide.

Abstract 2064: CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel

Abstract Background: Chemotherapy-resistant prostate cancer (PCa) remains a leading cause of cancer-related deaths in advanced PCa patients. The current study investigates the role of acetylcholine (ACh) signaling in the development and maintenance of chemotherapy resistance in PCa. Methods: Human PCa cell cultures were used to harvest total RNA and protein for qPCR and Western blot analyses, respectively. The chemotherapy drug docetaxel’s (DTX) ability to induce apoptosis was measured with crystal violet survival assays and Western blot analysis of pro-apoptotic proteins. Results: Treatment of the androgen-independent human PCa cell lines 22Rv1 and DU145 with a low concentration of DTX at 1 nM increases the expression of the key ACh production and secretion enzymes choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and the high-affinity choline transporter 1 (CHT1), which is accompanied by a 4-fold increase in ACh secretion. These genes also show increased expression in DTX-resistant 22Rv1 cells. The muscarinic receptor system further shows alterations in gene expression with low-concentration DTX treatment and acquired DTX resistance. Specifically, across all 5 muscarinic cholinergic receptors (CHRM1-5), the expression of CHRM1 is increased in both circumstances. Treatment of both DTX-sensitive and -resistant cell lines with a combination of DTX and the CHRM1 antagonist dicyclomine decreases cell survival, lowers DTX IC50 values (DU145: 62%; 22Rv1: 48%; DTX-resistant 22Rv1: 23%), and enhances the expression of the pro-apoptotic proteins cleaved caspase-3 and cleaved PARP1 compared to treatment with DTX alone. This is paralleled by the observations that treatment with the ACh mimetic carbachol (CCh) protects cells from DTX treatment. Mechanistically, ACh signaling protects PCa cells from DTX through activation of the extracellular signal-regulated kinase / cAMP response element binding protein 1 (ERK/CREB) pathway. Treatment of 22Rv1 cells with CCh increases levels of the active phosphorylated forms of both ERK1/2 and CREB within 30 min. This increase is inhibited by the pan muscarinic receptor antagonist atropine and is not affected by the pan nicotinic receptor antagonist tubocurarine. Treatment of 22Rv1 cells with a combination of DTX and either the ERK inhibitor UO-126 or the CREB inhibitor 666-15 decreases cell survival in a synergistic manner. Conclusion: DTX induces an enhancement of ACh signaling in PCa cells, which may contribute to the development and maintenance of DTX resistance through CHRM1 and the downstream ERK/CREB pathway. Antagonism of CHRM1 and its downstream effectors may be useful in preventing and reverting chemotherapy resistance in PCa. Citation Format: Tyler Bland, Karen Vo, Baron Bechtold, Boyang Wu. CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2064.

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells.

Background and ObjectivesMesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation.Methods and ResultsWe cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride.ConclusionsOur findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Endocrine regulation of the thymic function in European sea bass: Interactions between environmental factors and the reproductive system development

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Establishment of efficient differentiation conditions from tonsil-derived mesenchymal stem cells to motor neuron cells

Background & Aim Tonsil-derived MSCs (T-MSCs) present typical features of MSCs, including the ability to differentiate into tissues of the three primary germ layers, and have excellent proliferation capacity and ease of source supply are useful for stem cell therapy in various disease states. Previously, we reported that T-MSCs can be differentiated into skeletal myocytes and Schwann-like cell, which are therefore a promising candidate in cell therapies for neuromuscular disease. Motor neurons are differentiated cells that control voluntary actions and are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs). MNDs may present with a range of symptoms deriving from muscular weakness/atrophy and leading to death, but no effective treatment exists for these illnesses. Therefore, we investigated the potential of motor neuron-like cells (MNCs) differentiated from T-MSCs for overcome of MNDs. Methods, Results & Conclusion After the MN differentiating in our own methods, we confirmed that the expression of MN-related markers, including ISL1, HB9, ChAT in T-MSC-MNCs has increased compared to T-MSCs. Also, to test their functional efficiency, the change of acetylcholine secretion in the culture medium under the differentiation into T-MSC-MNCs was measured. Furthermore, by confirming the acetylcholine receptor clusters when co-culturing T-MSC-MNCs and human skeletal muscle cells, which was demonstrated the T-MSC-MNCs could form neuromuscular junctions. The functional improvements afforded by these T-MSC-MNCs are potentially useful in the treatment of human MNDs caused by genetic, viral, and environmental problems.

Differentiation of human glioblastoma U87 cells into cholinergic neuron

To facilitate research methodologies for investigating the role of cholinergic nerves in many diseases, establishing an in vitro cholinergic neuron model is necessary. In this study, we investigated whether human glioblastoma U87 cells could be differentiated into cholinergic neurons in vitro. Sodium butyrate was used as the differentiation agent. The differentiated cells established by inducing U87 cells with sodium butyrate were named D-U87 cells. Immunofluorescence was used to label the neuronal markers MAP2, NF-M, and ChAT and the glial marker GFAP in D-U87 cells. Flow cytometry was used to measure cell cycle distribution in D-U87 cells. PCR, protein chip, and western blot assays were used to measure the expression levels of muscarinic cholinergic receptor 1 (M1), M4, ChAT, SYP and Akt. ELISA was used to measure neurotransmitter levels. As a result, we found that sodium butyrate induced U87 cell differentiation into cells with neuronal characteristics and increased not only the expression levels of the cholinergic neuron-related proteins M1, M4, ChAT and SYP in D-U87 cells but also the acetylcholine neurotransmitters in D-U87 cells. Moreover, the Akt protein expression in D-U87 cells was increased compared with that in U87 cells. Finally, we found that M1, M4, ChAT and SYP protein expression and acetylcholine secretion levels were significantly decreased in D-U87 cells after treatment with the Akt inhibitor MK-2206. These results demonstrate that D-U87 cells exhibit cholinergic neuron characteristics and that sodium butyrate induced U87 cell differentiation into cholinergic neuron partially through Akt signaling.

Вклад кальциевых каналов L-типа в секрецию ацетилхолина в нервно-мышечных соединениях лягушки и мыши при активных и инактивированных потенциал-зависимых калиевых каналах

In this work, the contribution of L-type Ca2+ channels to the evoked secretion of acetylcholine from the frog and mouse motor nerve endings with active and inactivated potential-dependent K+ channels was studied. The effects of the specific Ca2+ blocker of the L-type channels nitrendipine on the quantal content of endplate currents and the timing of acetylcholine quanta secretion in intact preparations and after the preliminary blockade of potential-activated K+ channels with 4-aminopyridine (4-AP) were evaluated under conditions of a decreased and physiological level of ambient Ca2+. Fluorescent measurement of calcium transient reflecting the integral input of Ca2+ into the nerve ending was performed as well as the computer simulation of the processes underlying exocytosis assuming the presence of two types of Ca2+ channels (N- and L-types) and varying duration of the nerve action potential. It was shown that in the frog synapses, L-type calcium channels contribute to the evoked secretion of acetylcholine at active K+ channels only under conditions of decreased Ca2+ level; after inactivation of potential-dependent K+ channels, the contribution of L-type channels to the secretory process becomes less significant. At the physiological level of ambient Ca2+, the involvement of L-type channels in the evoked acetylcholine secretion, as in mice synapses, is manifested only under conditions of inactivated potential-dependent K+ channels.