scholarly journals Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium

1997 ◽  
Vol 138 (1) ◽  
pp. 159-165 ◽  
Author(s):  
James P. O'Malley ◽  
Charlotte T. Moore ◽  
Miriam M. Salpeter
Keyword(s):  
Neuromuscular Junction ◽  
Half Life ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Muscle Membrane ◽  
Intracellular Camp ◽  
Signaling Systems ◽  
Camp Analogue ◽  
Degradation Rates ◽  
The Stability

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ∼1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ∼3–5 d) and a stabilized form (half-life ∼10 d), depending upon the state of innervation of the muscle. Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca2+, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

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.

scholarly journals Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction.

1986 ◽  
Vol 103 (4) ◽  
pp. 1399-1403 ◽  
Author(s):  
M M Salpeter ◽  
D L Cooper ◽  
T Levitt-Gilmour
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Degradation Rate ◽  
Postsynaptic Membrane ◽  
Nerve Crush ◽  
Adult Mice ◽  
Vertebrate Muscle ◽  
Degradation Rates ◽  
Alpha Bungarotoxin ◽  
Two Populations

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.

scholarly journals From synaptic input to muscle contraction: arm muscle cells of Octopus vulgaris show unique neuromuscular junction and excitation–contraction coupling properties

2019 ◽  
Vol 286 (1909) ◽  
pp. 20191278 ◽  
Author(s):  
Nir Nesher ◽  
Federica Maiole ◽  
Tal Shomrat ◽  
Benyamin Hochner ◽  
Letizia Zullo
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Calcium Release ◽  
Close Association ◽  
Muscle Cells ◽  
Octopus Vulgaris ◽  
Motion Generation ◽  
Cell Contraction ◽  
Receptor Complexes ◽  
Lower Amplitude

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation–contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na + and K + contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca 2+ channels. We show that cell contraction can be mediated directly by the inward Ca 2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca 2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog

1972 ◽  
Vol 181 (1065) ◽  
pp. 431-440 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Connective Tissue ◽  
Neuromuscular Junction ◽  
Muscle Fibre ◽  
Nerve Terminal ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Muscle Membrane ◽  
Muscle Fibres ◽  
Nerve Terminals

1. The acetylcholine (ACh) sensitivity of muscle fibres at the neuromuscular junction of the frog was investigated in preparations in which the nerve terminals could be clearly seen. 2. ACh released iontophoretically from a micropipette that was precisely positioned at various points along the muscle fibre in the vicinity of the synapse showed that the peak chemosensitivity (up to 1900 mV/nC) is confined to an area of postsynaptic membrane within a few micra of the nerve terminal; a tenfold decline in sensitivity was obtained when the ACh was released only 5 to 10 μm from the terminal’s edge. It is estimated that most of the response obtained when ACh is released within 40 μm from the terminal (the area covered in this study) is due to diffusion to the immediate postsynaptic area. The extrasynaptic chemosensitivity of the muscle membrane was too low to be measured with the present methods. 3. The accuracy with which micropipettes could be positioned in synaptic areas and the clarity of viewing nerve terminals were improved by bathing the tissue in collagenase, which reduced the amount of connective tissue. The distribution of chemosensitivity remained unchanged by such treatment. The ACh response was not detectably altered when nerve terminals were lifted off the muscle, exposing the subsynaptic muscle surface.

scholarly journals Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers.

1983 ◽  
Vol 97 (5) ◽  
pp. 1396-1411 ◽  
Author(s):  
M J Anderson ◽  
D M Fambrough
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Heparan Sulfate ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Muscle Cells ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Skeletal Muscle Fibers

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [35S]methionine or [35S]sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 125I-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord. During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.

scholarly journals Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

1995 ◽  
Vol 128 (4) ◽  
pp. 625-636 ◽  
Author(s):  
M Gesemann ◽  
A J Denzer ◽  
M A Ruegg
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Neuromuscular Junction ◽  
Motor Neurons ◽  
Active Site ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Effective Concentration ◽  
Achr Clustering

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH-terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.

scholarly journals Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

2011 ◽  
Vol 195 (7) ◽  
pp. 1171-1184 ◽  
Author(s):  
Nadine Schmidt ◽  
Mohammed Akaaboune ◽  
Nadesan Gajendran ◽  
Isabel Martinez-Pena y Valenzuela ◽  
Sarah Wakefield ◽  
...  
Keyword(s):  
Nervous System ◽  
Synaptic Transmission ◽  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Neuromuscular Synapse ◽  
Muscle Membrane ◽  
Erbb Signaling ◽  
Cultured Myotubes ◽  
The Stability ◽  
And Function

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.

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.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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