scholarly journals Nerve-Muscle Interaction In Vitro

1973 ◽  
Vol 62 (3) ◽  
pp. 255-270 ◽  
Author(s):  
J. H. Steinbach ◽  
A.J. Harris ◽  
J. Patrick ◽  
D. Schubert ◽  
S. Heinemann
Keyword(s):  
Nerve Cell ◽  
Muscle Cell ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Acetylcholine Sensitivity ◽  
Release Of Acetylcholine ◽  
Clonal Lines ◽  
Nerve Muscle ◽  
A Site

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with α-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.

scholarly journals Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation

1988 ◽  
Vol 254 (3) ◽  
pp. C345-C364 ◽  
Author(s):  
R. J. Bloch ◽  
D. W. Pumplin
Keyword(s):  
Cell Surface ◽  
Muscle Cell ◽  
Acetylcholine Receptors ◽  
Membrane Skeleton ◽  
Postsynaptic Membrane ◽  
Molecular Assembly ◽  
Trophic Factors ◽  
Achr Cluster ◽  
Nerve Muscle ◽  
Achr Clustering

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane of newly innervated muscle fibers is one of the earliest events in the development of the vertebrate neuromuscular junction. Here, we describe two hypotheses that can account for AChR clustering in response to innervation. The "trophic factor" hypothesis proposes that the neuron releases a soluble factor that interacts with the muscle cell in a specific manner and that this interaction results in the local accumulation of AChR. The "contact and adhesion" hypothesis proposes that the binding of the nerve to the muscle cell surface is itself sufficient to induce AChR clustering, without the participation of soluble factors. We present a model for the molecular assembly of AChR clusters based on the contact and adhesion hypothesis. The model involves the sequential assembly of three distinct membrane domains. The first domain to form serves to attach microfilaments to the cytoplasmic surface of the muscle cell membrane at sites of muscle-nerve adhesion. The second domain to form is clathrin-coated membrane; it serves as a site of insertion of additional membrane elements, including AChR. Upon insertion of AChR into the cell surface, a membrane skeleton assembles by anchoring itself to the AChR. The skeleton, composed in part of actin and spectrin, binds and immobilizes significant numbers of AChR, thereby forming the third membrane domain of the AChR cluster. We make several predictions that should distinguish this model of AChR clustering from one that invokes soluble, trophic factors.

scholarly journals Acetylcholinesterase Clustering at the Neuromuscular Junction Involves Perlecan and Dystroglycan

1999 ◽  
Vol 145 (4) ◽  
pp. 911-921 ◽  
Author(s):  
H. Benjamin Peng ◽  
Hongbo Xie ◽  
Susanna G. Rossi ◽  
Richard L. Rotundo
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Cell Types ◽  
Specific Form ◽  
Lateral Migration ◽  
Extracellular Matrix Molecules ◽  
A Cell ◽  
Nerve Muscle

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve– muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake α-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve–muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve–muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through α-dystroglycan. Therefore, the dystroglycan–perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.

Glucose responsiveness and acetylcholine sensitivity of pancreatic beta-cells after vagotomy

1984 ◽  
Vol 246 (6) ◽  
pp. R985-R993 ◽  
Author(s):  
L. A. Campfield ◽  
F. J. Smith ◽  
R. E. Eskinazi
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Acetylcholine Receptors ◽  
Sham Operation ◽  
Neural Input ◽  
Acetylcholine Sensitivity ◽  
Long Evans ◽  
In Vitro Insulin Secretion

The chronic effects of removal of parasympathetic neural input to the pancreas on in vitro insulin secretion were assessed. Groups of Wistar and Long-Evans rats received total subdiaphragmatic vagotomy or sham operation. Four to ten weeks later, after the return of food intake and body weight in the vagotomized groups to values similar to the sham-operated groups, pancreatic islets were isolated and statically incubated with selected concentrations of glucose and acetylcholine. Two experimental protocols were used. In the first experiment, insulin secretion in response to basal (5 mM) glucose was 59 +/- 15 (SE) and 65 +/- 13% greater in islets from the vagotomized Wistar and Long-Evans groups, respectively, than in the corresponding sham groups. The enhancement of insulin secretion by several doses of acetylcholine observed in islets from sham-operated groups was totally absent in islets from both vagotomized strains. In the second experiment, insulin secretion was determined in response to selected glucose concentrations by using islets from Wistar rats. An upward and leftward shift of the dose-response curve was observed in the vagotomized group causing 5 mM to become a stimulatory glucose concentration and increasing the stimulatory potency of 10 mM glucose. These results suggest that interruption of vagal input to pancreatic beta-cells may induce a compensatory increase in responsiveness to glucose and a functional suppression of acetylcholine receptors. These data provide further support for the hypothesis that vagal input plays a functionally important role in the control of insulin secretion and maintenance of acetylcholine sensitivity.

scholarly journals Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis.

1995 ◽  
Vol 129 (4) ◽  
pp. 1093-1101 ◽  
Author(s):  
M W Cohen ◽  
C Jacobson ◽  
E W Godfrey ◽  
K P Campbell ◽  
S Carbonetto
Keyword(s):  
Acetylcholine Receptors ◽  
Culture Medium ◽  
Muscle Cells ◽  
Immunofluorescent Staining ◽  
Western Blots ◽  
Xenopus Embryos ◽  
Alpha Dystroglycan ◽  
Nerve Muscle ◽  
Induced Changes

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve-induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.

scholarly journals A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Binyam Mogessie ◽  
Daniel Roth ◽  
Zainab Rahil ◽  
Anne Straube
Keyword(s):  
Cell Differentiation ◽  
Cell Fusion ◽  
Muscle Cell ◽  
Cell Elongation ◽  
Myogenic Differentiation ◽  
Muscle Cells ◽  
Microtubule Array ◽  
Muscle Cell Differentiation ◽  
Microtubule Transport

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes.

scholarly journals The Rate of Cellular Energy Production of Muscle Cells Is Attenuated By Carnitine Intracellular Deficiency Caused By Imatinib Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3609-3609
Author(s):  
Pavel Burda ◽  
Alzbeta Hlavackova ◽  
Vaclava Polivkova ◽  
Nikola Curik ◽  
Hana Klamová ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Muscle Cell ◽  
Energy Production ◽  
Conflicts Of Interest ◽  
Muscle Cells ◽  
Imatinib Treatment ◽  
Atp Levels ◽  
Intracellular Concentrations

Abstract Introduction: Previous works identified that imatinib intake through the carnitine-specific OCTN2 (SLC22A5) transporter resulted in a significant decrease of carnitine intracellular concentrations in chronic myeloid leukemia (CML) and muscle cell lines. On contrary, even high doses of carnitine in preincubation did not influence imatinib cell intake capacity. Specifically performed inhibition of OCTN2 activity by vinorelbine resulted in block of carnitine cell intake, while imatinib intake was only slightly reduced (13-30%). This observation is in line with the knowledge that imatinib is transported also through other known SLC transporters. OCTN2 transporter is the major transporter for carnitine, an essential compound in cell energy metabolism. Presented work follows a hypothesis that non-equal competition between imatinib and carnitine intake through OCTN2 can lead to the carnitine intracellular deficiency, which can be in CML patients manifested by a disruption of skeletal muscle mitochondrial density and cause side effects like fatigue, muscle pain and cramps reported up to 80% of patients treated with imatinib (Kekale et al., 2015). Methods: Muscle cell HTB-153 (human rhabdomyosarcoma, ATCC HS 729), CML cell line KCL-22 (DSMZ ACC 519) were used for in vitro experiments. Intracellular concentration of imatinib, carnitine and metabolites were measured by chromatographic separation using XBridge Amide column (50x2.1mm, 3.5µm; Waters, Milford (MA), USA) and ZIC-pHILIC column (50x2.1mm, 5 µm; Merck, Darmstadt, Germany) coupled to tandem mass spectrometer (QTRAP 4000; Sciex, USA). Results: Carnitine, resp. L-carnitine transports long-chain fatty acids to mitochondria and its high rate is required especially in energetically demanding tissues such as skeletal and cardiac muscles. The concentrations of citric acid cycle (CAC) metabolites (citrate, malate, alpha-ketoglutarate, succinate, fumarate, 2-hydroxyglutarate, cis-aconitate), glycolysis (phosphoenolpyruvate, 3- phosphoglycerate, lactate), production of ATP, ADP and AMP were measured in HTB-153 cells 3 and 24 hours after imatinib treatment in vitro. The significant decrease of malate (CAC), lactate (glycolysis) and ATP levels were found at both time points after imatinib treatment compared to baseline. The same observations were found in KCL-22, which was used for comparison as BCR-ABL1 positive cell line. Additionally, significant decrease of succinate and 2-hydroxyglutarate (CAC) was detected in KCL-22 after imatinib treatment. Next, HTB-153 was incubated with imatinib (1-8 µM) for 24 hours and carnitine (8 µM) was supplied for last 3 hours of incubation, i.e., after 21 hours of imatinib treatment start. No significant changes were found in any metabolites of CAC and glycolysis. Production of ATP, ADP and AMP was not changed as well. Conclusions: Imatinib treatment of muscle (rhabdomyosarcoma) and CML cell lines caused a significant decrease of intracellular concentrations of carnitine. Significant decrease of ATP levels and of certain metabolites of CAC and glycolysis outlined that cells struggle from attenuated mitochondria energy production after imatinib treatment. This has not happened, if carnitine was supplied to the culture for final 3 hours of 24 hours incubation with imatinib. Observed data strongly support the hypothesis that decreased carnitine intake to the muscle cells due to competition with imatinib transport through OCTN2 attenuated mitochondria energy production. Interestingly, the clinical trial NCT03426722 (Chae H et al. 2019) showed that L-carnitine could effectively relieve imatinib-related muscle cramps and significantly increase QoL in patients with advanced gastrointestinal stromal tumor. Supported by GACR18-18407S, MZCR00023736 Disclosures No relevant conflicts of interest to declare.

Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells

2003 ◽  
Vol 285 (6) ◽  
pp. L1296-L1304 ◽  
Author(s):  
Deborah A. Sawatzky ◽  
Paul J. Kingham ◽  
Niamh Durcan ◽  
W. Graham McLean ◽  
Richard W. Costello
Keyword(s):  
Acetylcholine Receptors ◽  
In Vitro Model ◽  
Acetylcholine Release ◽  
Prolonged Exposure ◽  
Receptor Expression ◽  
Initial Increase ◽  
Cholinergic Nerve ◽  
Release Of Acetylcholine ◽  
Vitro Model

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 ± 2% of control at 10-7 M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 ± 23% of control at 10-5 M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 ± 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 ± 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.

scholarly journals A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction

2020 ◽  
Vol 9 (10) ◽  
pp. 3238
Author(s):  
Jasdeep Saini ◽  
Alessandro Faroni ◽  
Adam J. Reid ◽  
Kamel Mamchaoui ◽  
Vincent Mouly ◽  
...  
Keyword(s):  
Motor Unit ◽  
Motor Neurons ◽  
Acetylcholine Receptors ◽  
In Vivo Studies ◽  
Cholinergic Antagonists ◽  
Myoblast Cell ◽  
Nerve Muscle ◽  
And Function

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. Results: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.

Induction of a specialized muscle basal lamina at chimaeric synapses in culture

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 51-61 ◽  
Author(s):  
L.E. Swenarchuk ◽  
S. Champaneria ◽  
M.J. Anderson
Keyword(s):  
Monoclonal Antibodies ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Synaptic Cleft ◽  
Sulfate Proteoglycan ◽  
Cellular Origin ◽  
Initial Deposition ◽  
Species Specific ◽  
Nerve Muscle

To identify mechanisms that regulate the formation of the neuromuscular junction, we examined the cellular origin of a heparan sulfate proteoglycan (HSPG) that becomes highly concentrated within the synaptic cleft during the initial deposition of the junctional basal lamina. Using cultured nerve and muscle cells from anuran and urodele embryos, we prepared species-chimaeric synapses that displayed spontaneous cholinergic potentials, and eventually developed organized accumulations of acetylcholine receptors and HSPG at the sites of nerve-muscle contact. To determine the cellular origin of synaptic HSPG molecules, these chimaeric junctions were stained with both species-specific and cross-reactive monoclonal antibodies, labeled with contrasting fluorochromes. Our results demonstrate that synaptic HSPG is derived almost exclusively from muscle. Since it has already been shown that muscle cells can assemble virtually all of the known constituents of the junctional basal lamina into organized surface accumulations, without any input from nerve cells, we consider the possibility that the specialized synaptic basal lamina may be generated primarily by the myofibre, in response to another ‘inductive’ positional signal at the site of nerve-muscle contact.

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