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 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 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.

Ligand induced changes in stability and distribution of acetylcholine receptors on surface membranes of muscle cells

Life Sciences ◽  
1979 ◽  
Vol 24 (18) ◽  
pp. 1713-1718 ◽  
Author(s):  
Joav Prives ◽  
Lynne Hoffman ◽  
Rebeca Tarrab-Hazdai ◽  
Sara Fuchs ◽  
Abraham Amsterdam
Keyword(s):  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Induced Changes

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.

scholarly journals Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

1980 ◽  
Vol 86 (2) ◽  
pp. 388-401 ◽  
Author(s):  
MW Cohen ◽  
PR Weldon
Keyword(s):  
Acetylcholine Receptors ◽  
Specific Property ◽  
Neuronal Cell ◽  
Muscle Cells ◽  
Sympathetic Ganglia ◽  
Receptor Density ◽  
Synaptic Ultrastructure ◽  
Cell Processes ◽  
Nerve Muscle ◽  
Myotomal Muscle

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.

scholarly journals Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

1984 ◽  
Vol 99 (5) ◽  
pp. 1769-1784 ◽  
Author(s):  
M J Anderson ◽  
F G Klier ◽  
K E Tanguay
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Synapse Formation ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Receptor Aggregation ◽  
Nerve Muscle ◽  
Alpha Bungarotoxin

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

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.

Effects Of Doxycycline On Mice Neuromuscular Junction, In Situ

2021 ◽  
Vol 21 ◽  
Author(s):  
Natália Tribuiani ◽  
Jocimar de Souza ◽  
Marcos Antônio de Queiroz Junior ◽  
Denicezar Angelo Baldo ◽  
Valéria de Campos Orsi ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Synaptic Cleft ◽  
Diaphragm Muscle ◽  
Na Channel ◽  
Muscle Histology ◽  
Casearia Sylvestris ◽  
Nerve Muscle ◽  
Neuromuscular Preparation

Background: The antibacterial mechanism of doxycycline is known, but on the nerve-muscle apparatus is yet unclear. Objective: To combine molecular targets of the neuromuscular machinery using the neuronal blocker effect doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations, in situ. Methods: Doxycycline was assessed at the neurotransmission; presynaptic; synaptic cleft; and postsynaptic, including the muscle fiber, using the traditional myographic technique. Preliminarily, doxycycline showed an "all or nothing" effect, being "all" obtained with 4 µM and "nothing", with 1-3 µM. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 µM doxycycline such as F55-6 (Casearia sylvestris), CaCl2 (or Ca2+), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity, the diaphragm muscle histology, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary. Results: Doxycycline does not affect cholinesterase activity nor cause damage to skeletal muscle diaphragm; acts on ryanodine receptor, sarcolemmal membrane, and on neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors. Conclusions: In conclusion, using the blocker effect we showed that doxycycline acts on multiple targets, among them, is antagonized by F55-6, a neuronal Na+-channel agonist and Ca2+, but not by neostigmine.

Regulation of Na+ pump expression by vascular smooth muscle cells

2001 ◽  
Vol 280 (4) ◽  
pp. H1869-H1874 ◽  
Author(s):  
Aslihan Aydemir-Koksoy ◽  
Julius C. Allen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Mammalian Cells ◽  
Muscle Cells ◽  
Transport Processes ◽  
Western Blots ◽  
Cytoplasmic Transport ◽  
Low K

The Na+ pump and its regulation is important for maintaining membrane potential and transmembrane Na+gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K+ (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [3H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either α1- or β1-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K+ media, the pump site upregulation was inhibited. These data suggest that the low-K+-induced upregulation of Na+ pump number can occur by translocation of preformed pumps from intracellular stores.

scholarly journals Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

2010 ◽  
Vol 299 (3) ◽  
pp. G742-G750 ◽  
Author(s):  
P. Cong ◽  
V. Pricolo ◽  
P. Biancani ◽  
J. Behar
Keyword(s):  
Small Interfering Rna ◽  
Muscle Cells ◽  
High Cholesterol ◽  
Western Blots ◽  
Human Gallbladder ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Δ Opioid Receptor ◽  
Interfering Rna ◽  
Bulk Plasma

The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a δ-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.

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