Amount Of Acetylcholine Receptor Research Articles

Photobiomodulation Triple Treatment in Peripheral Nerve Injury: Nerve and Muscle Response.

Muscle preservation or decrease in muscle degeneration and progressive atrophy are major challenges in patients with severe peripheral nerve injury (PNI). Considerable interest exists in the potential therapeutic value of laser phototherapy (photobiomodulation) for restoring denervated muscle atrophy and for enhancing regeneration of severely injured peripheral nerves. As previously published, the laser phototherapy has a protective and immediate effect in PNI. Laser phototherapy in the early stages of muscle atrophy may preserve the denervated muscle by maintaining creatinine kinase (CK) activity and the amount of acetylcholine receptor (AChR). In the present study, the effectiveness of triple treatment laser phototherapy, namely, applied simultaneously at three areas: injured area of the peripheral nerve, corresponding segments of the spinal cord, and corresponding denervated muscle (triple treatment), was evaluated for the treatment of incomplete PNI in rats with the ultimate goal of achieving improved limb function. Forty-five days after the sciatic nerve insult, all rats regained normal walking (functional sciatic index values returned to baseline); however, the long laser irradiation (7 min) group presented the fastest recovery as opposed to short laser irradiation (3 min). A histological evaluation of the nerves revealed that long laser irradiation led to a higher amount of neuronal fibers that were larger than 4 μm (543 ± 76.8, p < 0.01) than short irradiation (283 ± 35.36). A histological evaluation of muscular atrophy showed that long laser irradiation evolved with significantly less muscle atrophy (8.06% ± 1.23%, p < 0.05) than short irradiation (24.44% ± 7.26%). The present study and our previous investigations showed that the laser phototherapy increases biochemical activity and improves morphological recovery in muscle and, thus, could have direct therapeutic applications on muscle, especially during progressive atrophy resulting from PNI.

Protective Effect of Laser Phototherapy on Acetylcholine Receptors and Creatine Kinase Activity in Denervated Muscle

This study was designed to assess the status of skeletal muscles after laser treatment during long-term denervation processes, by investigating changes in the level of acetylcholine receptors (AChR) and creatine kinase (CK) activity in the denervated gastrocnemius muscle of the rat. Progressive muscle atrophy is common in patients with severe peripheral nerve injury. Denervated muscles can account for significant differences in the extent of AChR and CK activity during the denervation period. The study was conducted on 96 rats: 48 that received laser treatment and 48 untreated controls. The gastrocnemius muscle was denervated by removing a 10 mm segment of the sciatic nerve. Low power laser irradiation was delivered transcutaneously to the right gastrocnemius muscle (HeNe continuous wave [CW] laser, 632.8 nm, 35 mW, 30 min) for 14 consecutive days. Under general anesthesia, the rats were euthanized at seven time points: day 7 (n=10), day 14 (n=10), day 21 (n=10), day 30 (n=5), day 60 (n=4), day 120 (n=5), and day 210 (n=4), with and without laser treatment, respectively. AChR was quantified by the (125)I-α-bungarotoxin. CK activity was measured by a specific spectrophotometric method. Laser treatment had a significant therapeutic effect on the denervated muscle during the first 21 days for AChR and the first 30 days for CK activity. In the early stages of muscle atrophy, laser phototherapy may preserve the denervated muscle by maintaining CK activity and the amount of AChR.

Decrease of LFA-1 Is Associated with Upregulation of TGF-β in CD4+T Cell Clones Derived from Rats Nasally Tolerized against Experimental Autoimmune Myasthenia Gravis

Tolerance to experimental autoimmune myasthenia gravis by nasal administration of microgram amounts of acetylcholine receptor (AChR) has been reported. To elucidate the mechanisms behind tolerance induction via the respiratory tract and the involvement of CD4+T cells, we established AChR-specific CD4+CD8−T cell clones from nasally tolerized rats. Nasal tolerance decreased leukocyte function-associated antigen-1 (LFA-1) expression in CD4+T cells from tolerized rats. There was no difference between nasally tolerized and control rats in expression of intercellular adhesion molecule-1. The levels of transforming growth factor-β (TGF-β) mRNA-expressing cells were upregulated in CD4+T cell clones after tolerance induction. These findings suggest that decreased LFA-1 expression in CD4+T cells contributes to reduction of the infiltration of inflammatory CD4+T cells, while upregulated TGF-β may inhibit lymphocyte functions.

Clathrin‐coated membrane: A distinct membrane domain in acetylcholine receptor clusters of rat myotubes

We have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR-rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR-rich domains are independently stabilized.

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.

Muscle acetylcholine receptor biosynthesis. Regulation by transcript availability.

The expression of the muscle nicotinic acetylcholine receptor (AChR) on the cell surface entails a complex biosynthetic pathway, involving the expression and assembly of four subunits. The amount of AChR on the cell surface changes throughout muscle development and upon muscle denervation. We have examined the regulatory role of transcript levels on surface AChR expression by RNA blot analysis. During myogenesis of the fusing mouse muscle cell line C2, which expresses an embryonic type of receptor, changes in the rate of appearance of cell surface AChR have been assayed by 125I-alpha-bungarotoxin binding. The maximal increase in the rate of appearance of cell surface AChR occurs upon cell fusion, closely following the maximal increase in transcript levels for the alpha-, beta-, gamma-, and delta-AChR subunits. AChR alpha-, beta-, gamma-, and delta-subunit gene transcript levels have also been examined in innervated and denervated rat and mouse muscle. Muscle denervation results in an increase of transcripts for all four subunits. However, the amount of beta-subunit transcript in innervated rat skeletal muscle is high relative to the other subunit transcripts, and increases less than the other subunit transcripts upon denervation. Our results indicate that, during myogenesis and upon denervation, the availability of AChR subunit transcripts for translation plays a regulatory role in surface AChR appearance.

Actin at receptor-rich domains of isolated acetylcholine receptor clusters.

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR-rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin-like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.

Inhibition of myogenesis by trifluoperazine and compound [formula omitted

When trifluoperazine (TFP), a calmodulin antagonist, was given to chick or rat myoblasts in cultures, formation of multinucleated myotubes was inhibited. The inhibition of cell fusion by TFP in rat cultures prevents the normal increase in the amount of acetyl-choline receptors (AChR) and creatine kinase (CK), while the levels of these proteins in chick muscle cultures are hardly affected. Another calmodulin antagonist, compound 48 80 , inhibits fusion at doses that correspond closely to its antagonistic effects on calmodulin. Thus, our results suggest a possible role for calmodulin in the regulation of myoblast fusion, but not on the appearance of muscle proteins.

Inhibition of acetylcholine receptor synthesis by thyroid hormones.

Studies were made on the effect of thyroid hormones on the level of acetylcholine receptors (AChR) in cultured rat skeletal muscle. Treatment of differentiated myotubes in vitro with thyroxine (T4; 2 X 10(-7) mol/l) for 2-3 days caused a marked decrease in the amount of AChR (P less than 0.05) and an increase in activity of Na+-K+-ATPase (P less than 0.05). There was no significant effect of hormone treatment on other muscle proteins, such as creatine kinase and acetylcholinesterase. Measurements of the turnover rate of AChR in T4-treated myotubes showed only a very slight effect of T4 on the rate of AChR degradation. To study the mechanism by which the hormone exerts its effect, muscle cells were labelled with radioactive amino acid and the rate of its incorporation into AChR protein was measured. The AChR was then isolated using anti-AChR antibodies. The specific activity of labelled AChR was lower in hormone-treated cells. These experiments suggest that the decreased level of AChR in response to thyroid hormone treatment is due to a partial suppression of receptor synthesis.

Role of calcium in the regulation of acetylcholine receptor synthese in cultured muscle cells*.

Embroyonic muscles differentiated in vitro were used to study the effects of intracellular Ca2+ ([Ca2+1]i) variations on the amount of acetylcholine receptors ([AChR]) in the cell membrane. 2. Increased Ca2+ concentration in the growth medium ([Ca2+]o) caused a marked elevation of AChR levels, apparently through de novo synthesis. 3. Agents known to increase [Ca2+]i and its accumulation in the sarcoplasmic reticulum (SR), such as ionophore A23187, sodium dantrolene (DaNa), or high [Mg2+]o all enhanced alpha-bungarotoxin (alpha-BGT) binding after 48 h of treatment. 4. Electrical stimulation or caffeine, both affectors of SR calcium release, brought about a decrease in [AChR] probably by suppressing its synthesis. 5. The effects of simultaneous treatment with two AChR-inducing agents, namely, high [Ca2+]o in the presence of tetrodotoxin (TTX) or high [Mg2+]o were not additive, thus suggesting action via a common saturable mediator. 6. Intermediate AChR levels obtained following simultaneous treatments with opposing effects, e.g., electrical stimulation in the presence of high [Ca2+]o or DaNa, suggest contradictory actions on a common mediator. 7. All these observations indicate a strong correlation between SR calcium levels and [AChR] on myotubes; while calcium accumulation in the Sr was followed by increased AChR synthesis, calcium release was accompanied by suppression of receptor synthesis.

Autoimmune Response to Acetylcholine Receptors in Myasthenia Gravis and Its Animal Model

This chapter discusses the process of neurotransmission as well as acetylcholine receptors (AChR) structure and function to understand the pathological mechanisms involved in myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG). The studies of the autoimmune response to AChR in MG and its animal model are presented. MG is an autoimmune disease in which transmission is impaired by an autoimmune response to acetylcholine receptors in the postsynaptic membrane of the muscle. Biochemical study of pathological mechanisms in MG is dependent on development of methods for quantitating and purifying AChRs. The pathological mechanisms impairing neuromuscular transmission in MG and EAMG have turned out to be quite similar. In both cases, the primary effect of the immune response is not to produce antibodies to AChR that block AChR activity but rather to produce antibodies to AChR that cause a decrease in the amount of AChR. Patients with myasthenia gravis have similar antibodies that can be quantitated by radioimmune assays using labeled receptors obtained from human muscle.