Appearance Of Acetylcholine Receptors Research Articles

Nuclear clustering in myotubes: a proposed role in acetylcholine receptor mRNA expression

We investigated the functional relationship between nuclear topology, as expressed by degree and type of nuclear aggregation, and appearance of acetylcholine receptor (AChR) subunit mRNAs. Embryonic chick muscle cell cultures treated with the muscle activity blocking agents decamethonium (DCM), d-tubocurare (TBC), and tetrodotoxin (TTX) or co-cultured with cholinergic neurons were examined for the influence of muscle activity on nuclear aggregation and its effects on AChR α-, γ-, and δ-subunit message expression. mRNA was measured by in situ hybridization and nuclei were visualized by bis-benzimide DNA staining. DCM and TBC treatments, as well as neuronal co-culture, resulted in increased nuclear clustering within myotubes and a per nucleus upregulation in mRNA expression relative to control for each subunit. The pattern of nuclear aggregation was treatment dependent, with more and larger aggregates found when myotubes were co-cultured with neurons. Moreover, as nuclear aggregates became larger: (1) nearly all nuclei within active aggregates expressed mRNA and (2) local accumulation (mRNA per unit area) was elevated relative to single nuclei, while per nucleus mRNA production decreased. To determine whether mRNA expression was transient and did not result in steady-state upregulation of AChR receptor protein, we performed a double labeling of surface AChRs with 125 I -α-bungarotoxin ( 125 I -α-BTX) concomitant to the in situ hybridization for mRNA quantification on TTX treated muscle cells. Surface receptor expression tracked mRNA expression for all types of nuclear topology observed, indicating that message levels are in fact reliable indicators of receptor population on the plasma membrane surface in myotubes. We propose that nuclear clustering is an organelle-level, accessory mechanism whereby cells concentrate relatively large amounts of AChR mRNA/protein in specific myotube regions.

Eye-Extract Factors Promote the Expression of Acetylcholine Sensitivity in Chick Dorsal Root Ganglion Neurons

We have examined the development of acetylcholine (ACh) responses in chick sensory neurons and their regulation by growth factors. Between Embryonic Days E13 and E18 in vivo, the proportion of freshly dissociated dorsal root ganglion (DRG) neurons that were appreciably sensitive to 500 μM ACh increased from ≈40 to >90% and the size of the ACh response per unit membrane area increased nearly 10-fold. Nerve growth factor (NGF) supports the survival, growth, and differentiation of central, sympathetic, and sensory neurons. NGF also promotes ACh sensitivity in PC12 cells and nodose ganglion neurons grown in cell culture. When E13 chick DRG neurons were maintained in culture with NGF for 4-6 days, however, only ≈50% had appreciable ACh sensitivity, and large responses comparable to those observed at E18 in vivo were rare. Chick-eye extract contains a 21.5-kDa trophic factor termed growth-promoting activity (GPA) that supports survival of sympathetic and DRG neurons for 24 hr in culture and an activity of ≈50 kDa that promotes increases in choline acetyltransferase activity, ACh sensitivity, and ACh receptors (AChRs) in ciliary ganglion neurons (Nishi and Berg, 1981; Halvorsen et al. , 1991 ). In the present study, whole-eye extract supported full survival of E13 DRG neurons for up to 6 days in culture and promoted ACh sensitivity in >90% of the neurons tested. GPA-containing eye-extract fractions and NGF individually supported full DRG neuron survival for up to 6 days in culture, but neither promoted ACh sensitivity. Adding eye-extract fractions containing material of ≈50 kDa for 4-6 days to either GPA- or NGF-supplemented media led to appreciable ACh sensitivity in ≈90% of the DRG neurons tested. The levels of ACh sensitivity observed in DRG neurons maintained either in whole-eye extract or in fractions containing 50-kDa material were close to those observed at an equivalent developmental age in vivo. The results indicate that components in the 50-kDa eye-extract fraction promote the ACh sensitivity of DRG neurons in culture and suggest that similar factors may influence the appearance of AChRs on the neurons in vivo.

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in Vitro

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.

Inhibitory actions of phospholipase A2 and saponins including ginsenoside Rb1 and glycyrrhizin on the formation of nicotinic acetylcholine receptor clusters on cultured mouse myotubes

AbstractThe effects of saponin(ICN), ginsenoside Rb1(G‐Rb1; 20‐S‐protopanaxadiol aglycon), ginsenoside Rg1(G‐Rg1; 20‐S‐protopanaxatriol aglycon), and glycyrrhizin (GLR) on acetylcholine receptor (AChR) cluster formation were investigated in developing mouse myotubes co‐cultured with spinal cord explants. Peak and total intensity of relative fluorescence in fluorescein isothiocyanate α‐bungarotoxin stain were measured as markers of AChR cluster formation and AChR appearance, respectively. Saponin (ICN) (1.0 μg/mL) inhibited both the cluster formation and the appearance of AChR on the plasma membranes of the myotubes. The effect of saponin (ICN) was closely related to extracellular Ca2+ ions, because its inhibition was abolished by treatment with 2 mM EGTA. G‐Rb1 also inhibited both markers at a higher concentration (122 μM), whereas G‐Rg1 had no effect. The effect of G‐Rb1 was not dependent on extracellular Ca2+ ions. GLR (122 μm) inhibited both markers, and its effect was decreased by 2 mM EGTA. At a lower concentration of 0.3 μ/mL, saponin (ICN) showed no direct effect; it did not affect inhibition of both markers by phospholipase A2(PLA2). In contrast, at a lower concentration (36 μM) without direct effect, both G‐Rb1 and GLR abolished the inhibition induced by PLA2. The modes of inhibitory actions of saponins on PLA2/Ca2+ interaction for the formation of AChR clusters were classified into three different interactions such as saponin (ICN) with the influx of external Ca2+, G‐Rb1 with PLA2, and GLR with PLA2/Ca2+/ calmodulin interaction.

Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor.

A glycoprotein purified from chick brain, of relative molecular mass 42,000, increases the rate of appearance of acetylcholine receptors (AChRs) on the surface of chick myotubes. RNase protection assays have shown that this AChR-inducing activity (ARIA) increases the amount of mRNA encoding the alpha-subunit of the AChR, with little or no effect on the amounts of gamma- and delta-mRNAs2. Here, we report that the mRNAs encoding the alpha- and gamma-subunits of the receptor detected by in situ hybridization are concentrated around nuclei in cultured myotubes. Consistent with previous results, ARIA selectively increased the amount of alpha-subunit mRNA, but we now find that all nuclei were not activated to the same extent, with a substantial number not responding at all. Assuming that ARIA is released by motor nerve terminals, our results indicate that only a subset of muscle nuclei are capable of contributing to the accumulation of AChRs at developing neuromuscular junctions.

Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways.

In cultured chicken myotubes, calcitonin gene-related peptide (CGRP), a peptide present in spinal cord motoneurons, increased by 1.5-fold the number of surface acetylcholine receptors (AChRs) and by threefold AChR alpha-subunit mRNA level without affecting the level of muscular alpha-actin mRNA. Cholera toxin (CT), an activator of adenylate cyclase, produced a similar effect, which did not add up with that of CGRP. In contrast, tetrodotoxin, a blocker of voltage-sensitive Na+ channels, elevated the level of AChR alpha-subunit mRNA on top of the increase caused by either CGRP or CT. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, markedly decreased the cell surface and total content of [125I]alpha BGT-binding sites and reduced the rate of appearance of AChR at the surface of the myotubes without reducing the level of AChR alpha-subunit mRNA. Moreover, TPA inhibited the increase of AChR alpha-subunit mRNA caused by tetrodotoxin without affecting that produced by CGRP or CT. Under the same conditions, TPA decreased the level of muscular alpha-actin mRNA and increased that of nonmuscular beta- and gamma-actins mRNA. These data suggest that distinct second messengers are involved in the regulation of AChR biosynthesis by CGRP and muscle activity and that these two pathways may contribute to the development of different patterns of AChR gene expression in junctional and extrajunctional areas of the muscle fiber.

Muscle of adult rats and mice express the Thy-1 glycoprotein on denervation

Adult skeletal muscles when denervated undergo extensive changes in biochemical and physiological properties. Many of these changes, for example the appearance of acetylcholine receptors along the length of the fibres, are characterized by a reacquisition of features normally found in developing muscles. This generalization can now be extended to the surface glycoprotein, Thy-1, which is expressed in myoblasts and transiently in newly fused myotubes in culture 7, 16; for we have found that denervation leads to extensive production of Thy-1 in hindlimb muscles of adult rats and mice where its expression is normally very low. In addition, there is a slight transient rise in Thy-1 in muscles contralateral to the denervation.

Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent.

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.

Eclipse of Coxsackievirus Infectivity: the Restrictive Event for a Non-fusing Myogenic Cell Line

Coxsackieviruses A2, A5 and B3 did not replicate in L8CL3-U cells (a non-fusing variant of the rat L8 myogenic cell line) although these cells possessed a common receptor for coxsackieviruses A2 and A5, and a different receptor for coxsackievirus B3. The restriction in replication was identified as a block in viral eclipse, since 6 M-LiCl treatment permitted recovery of the coxsackievirus A2 inoculum from L8CL3-U cells after 2 h at 37 degrees C, and the cells could be transfected by viral RNA. Cellular fusion which was induced in L8CL3-U cultures by herpes simplex virus type 1 (HF strain) facilitated coxsackievirus A2 and A5 replication. Differentiating myogenic L8 cells acquired full susceptibility to infection concurrently with the appearance of acetylcholine receptors, the muscle-specific isoenzyme of creatine phosphokinase, prominent myotube formation and the acquired capacity of the cells to eclipse virus.

Changes in the distribution of the extrajunctional acetylcholine sensitivity along muscle fibers during development and following cordotomy in the rat

Acetylcholine sensitivity along the entire length of muscle fibers was studied during postnatal development and following transection of the spinal cord in the rat. During postnatal development, the acetylcholine sensitivity in the soleus and extensor digitorum longus muscles decreased faster at the juxtajunctional region than near the tendons. Thus, the adult pattern of low acetylcholine sensitivity at the extrajunctional membrane was achieved through the uneven change of acetylcholine sensitivity during normal development. This uneven pattern of the sensitivity was found to appear in both muscles in older rats after cordotomy, and is in striking contrast to the uniform pattern in denervated muscles. The uneven appearance of the sensitivity could not be explained by changes in input resistance or resting membrane potential. In the soleus muscle whose nerve was implanted at an ectopic site, the lowest sensitivity also appeared at the ectopic juxtajunctional region after cordotomy. These results indicate that the motor nerve exerts regionally different effects along a fiber with respect to the appearance of acetylcholine receptors.

Appearance of acetylcholine receptors in the myotome cells of the early chick embryo.

Appearance and distribution of acetylcholine receptor (AChR) in the myotome cells of the chick embryo were investigated by autoradiography using 3H-α-bungarotoxin (α-BGT). Acetylcholinesterase (AChE) histochemistry was also performed on the same sections.In the 1, 5 day-old embryo (H-H stage 11), the myotome was not formed and the specific labeling of the toxin was not found on any tissue components. In the 2 day-old embryo (stage 13), when the myotome first formed in the dorsomedial angle of the dermatome, some silver grains were demonstrated on the AChE-positive cells constituting the myotome, and thereafter, the grains on the myotome increased in number as well as in density, as development progressed. On the other hand, the specific labeling of the toxin was not observed on the neural tube, sclerotome (mesenchyme), dermatome or spinal nerves.These findings indicate that since the AChE-positive myotome cells are no longer capable of proliferation (26), AChR appears in the myotome cells shortly after their final cell division.

Isolation and characterization of human muscle cells.

We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations of muscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, alpha-actin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.

Concanavalin a inhibits fusion of myoblasts and appearance of acetylcholine receptors in muscle cultures

Concanavalin a inhibits fusion of myoblasts and appearance of acetylcholine receptors in muscle cultures

On the appearance of acetylcholine receptors in denervated rat diaphragm, and its dependence on nerve stump length

Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 μg/ml of ACh and extrajunctional binding of [ 125I]alpha-bungarotoxin ([ 125I]α-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [ 125I]α-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [ 125I]α-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [ 125I]α-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.

The appearance of acetylcholine receptors triggered by fusion of myoblasts in vitro

The appearance of acetylcholine receptors triggered by fusion of myoblasts in vitro

Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro

During differentiation of embryonic chick skeletal muscle in culture, elaboration of acetylcholine receptor (AChR) and acetylcholinesterase occurs shortly after myoblast fusion. During further development, AChR was found to decrease markedly on the myotube surface, while acetylcholinesterase continued to increase. Surface distribution of AChR, as followed by autoradiography using 125I-α-bungarotoxin, was homogeneous in newly fused myotubes. With further differentiation, clusters of AChR appeared on the surface of the myotubes, and their subsequent disappearance paralleled a decrease in overall AChR levels. Quantitative autoradiography showed a reduction of over 75% in the density of AChR on the surface of well differentiated, cross-striated myotubes. Thus the appearance of AChR on the cell surface, its condensation into clusters, and finally its depletion seem to be sequential events in the differentiation of skeletal muscle in culture in the absence of direct neuronal influence.

Appearance of acetylcholine receptors in cultured myoblasts prior to fusion.

The development of the acetylcholine receptors in chick embryo myoblasts from 11-day old embryos was studied in vitro. Using the purified alpha-bungarotoxin labeled with radioactive iodide, a high concentration of acetylcholine receptors was found in the prefusing myoblasts; most of these receptors were located in the interior of the myoblasts. However, upon the completion of myoblasts fusion, the majority of the acetylcholine receptors appeared on the external cell surface of the myotubes.

The synthesis and stability of cytoplasmic messenger RNA during myoblast differentiation in culture.

The synthesis of poly(A)-containing cytoplasmic RNA was examined in primary myoblast cultures prepared from skeletal muscle of fetal calves. After a period of cell division, these cells undergo fusion, with concomitant appearance of acetylcholine receptor and subsequent myosin synthesis. In the dividing myoblast there is a high level of messenger RNA synthesis, including a 26S RNA, the size of a putative messenger for the large subunit of myosin. In the transition period prior to fusion, there are quantitative changes in RNA synthesis. At this time, there is a pronounced production of 26S RNA, which diminishes during fusion. The possibility that 26S RNA is accumulated in the dividing myoblast was investigated by chase experiments. At fusion, there is a marked increase in the half-lives of a number of messenger RNA species, including 26 S, which increases from about 10 hr in the dividing cell to a value of more than 50 hr. The identity of the more rapidly turning over 26 S in the myoblasts, compared to that of the 26 S at fusion, was examined in terms of polysomal distribution, migration on gels, and hybridization with complementary DNA for the myosin message. The results of these analyses suggest that the 26S species are identical. Thus, it would appear that in a predetermined cell like the myoblast, the transition to the differentiated state of myotube that is synthesizing muscle specific proteins is effected by the stabilization of messenger already being actively transcribed: terminal differentiation, with respect to myosin synthesis, is preceded by the stabilization of 26S RNA.

Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle.

It is now well established that primary cultures of embryonic muscle tissue (1, 2) and muscle cell lines (2, 3) elaborate acetylcholine receptor during the process of muscle formation . However, the precise timing of the appearance of receptor with regard to the cessation of DNA synthesis, cell fusion, and the synthesis of the cytoplasmic and contractile proteins, major events that mark the course of muscle differentiation, has yet to be elucidated . Previous studies (1-3) have been hampered by the lack of rapid fusion kinetics . We have measured the appearance of acetylcholine receptor in rapidly differentiating cultures of embryonic chick skeletal muscle, under normal and fusion-arrested conditions (4-6), using iodine125labeled neurotoxins from elapid snake venoms as specific markers (8) . This method has been used to assay receptor in adult muscle (9-11), electrogenic tissue (12), and cultures of embryonic muscle (1-3) . This paper demonstrates that the elaboration of receptor in differentiating muscle, unlike other muscle proteins which have been previously assayed during differentiation (4-7), is not dependent upon the fusion process and can proceed in the absence of fiber formation .

Appearance of acetylcholine receptors during differentiation of a myogenic cell line.

Acquisition of acetylcholine receptors during differentiation of a clonal myoblast cell line was monitored with a neurotoxin isolated from venom of the Indian Cobra Naja naja. Toxin bound specifically and reversibly to acetylcholine receptors of the differentiated cells. Specificity of the binding reaction was assayed by measurement of the ability of various cholinergic agonists and antagonists to compete with neurotoxin for its binding site. The rate of toxin binding paralleled the rate of inactivation of functional acetylcholine receptors, as measured by iontophoretic application of acetylcholine. Bound toxin was released from the cells with a half-life of about 7 hr. This release was not associated with a decrease in the total number of toxin-binding sites. A slow hyperpolarizing response to acetylcholine seen in myoblasts was insensitive to toxin; the appearance of toxin-binding sites parallels the appearance of fused fibers during differentiation of the muscle cells in tissue culture.