An assay for antibodies to human acetylcholine receptor in serum from patients with myasthenia gravis.
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Acetylcholine↗
Biomedical subjects
J Lindstrom
Publications and source records attributed to J Lindstrom.
Modulation of acetylcholine receptor by antibody against the receptor.
Antibody against acetylcholine receptor induces an increase in the rate of degradation of acetylcholine receptors on a mouse cell line (BC(3)H-1) and cultured rat skeletal muscle. The increased rate of degradation results in a lowered density of acetylcholine receptors on muscle membrane and a lowered sensitivity to iontophoretically applied acetylcholine. The modulation of acetylcholine receptor is energy, temperature, and time dependent and may be related to antigenic modulation found in other systems. Acetylcholine noise analysis demonstrates that antibody against acetylcholine receptor reduces the channel mean conductance and mean open time slightly. It is concluded that antibody binds to the acetylcholine receptor, impairs its function, and induces receptor degradation. This results in a lowered density of acetylcholine receptor and a lowered sensitivity to acetylcholine. Patients with myasthenia gravis have antibodies to their acetylcholine receptor in their serum. Antigenic modulation of receptor in the muscle of patients with myasthenia gravis could contribute to the observed decrease in amplitudes of miniature endplate potentials and in muscle acetylcholine sensitivity, and the symptoms of muscular weakness.
Acetylcholine↗
Acetylcholine receptors and myasthenia gravis: the effect of antibodies to eel acetylcholine receptors on eel electric organ cells.
Antisera to acetylcholine receptors purified from Electrophorus electricus were tested for their ability to bind to receptors on electric organ cells, block the depolarizing response of the cells to carbamylcholine, and inhibit binding of 125-I-alpha-bungarotoxin to the cells. It was found that although antibodies could bind to most of the receptors, resulting in substantial inhibition of the depolarizing response, binding of 125-I-alpha-bungarotoxin to the cells was only slightly inhibited. This was consistent with the observation that these antibodies did not compete for the toxin binding site on detergent-solubilized receptor. These results suggest that the antibodies inhibited receptor activity primarily by interfering with the ionophore of the receptor or its regulation by the acetylcholine binding site. However, the possibilities could not be completely eliminated that blockage of a small fraction of the binding sites caused a large inhibition of the depolarizing response or that bound antibody allosterically reduced binding affinity for carbamylcholine without completely inhibiting toxin binding.
Animals↗
Reduced muscle acetylcholine sensitivity in rats immunised with acetylcholine receptor.
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Acetylcholine↗
Immunological studies of acetylcholine receptors.
Immunochemical techniques for the study of acetylcholine receptors are described. Immunization of rabbits, rats, guinea pigs, and goats with acetylcholine receptor protein purified from Electrophorus electric organ tissue results in muscular weakness and death due to impaired neuromuscular transmission. Serum from immunized animals contains high concentrations of antibodies directed at receptors from the electric organ and low concentrations of antibodies directed at receptors from skeletal muscle. The detailed similarities between the disease of receptor-immunized animals "experimental autoimmune myasthenia gravis" (EAMG), and myasthenia gravis are compared. Reactions of antisera from animals with EAMG with receptor from Electrophorus and Torpedo are studied. Antireceptor antibodies in these antisera are directed predominantly and determinants other than the acetylcholine-binding site.
Acetylcholine↗
Effect of fibroblast growth factor on the division and fusion of bovine myoblasts.
The effect of fibroblast growth factor (FGF) on the rate of proliferation and fusion of bovine myoblast has been examined. Addition to the cultures of 0.1 mug-1 mug/ml of FGF stimulates the rate of proliferation and delays the fusion of primary cultures of bovine myoblasts cultured in 10% serum. Final cell densities reached in the presence of 0.1 mug/ml of FGF were fivefold higher than in controls; with 1 mug/ml, they were 10-fold higher. Increases in cell density were paralleled by increases in acetylcholine receptor sites as measured by the binding of 125I-alpha-bungarotoxin. Both fusion and the appearance of acetylcholine receptor sites were delayed in the presence of FGF. Growth hormone, insulin and testosterone, which have been reported to be mitogenic for rat and chick embryo myoblasts, did not have significant effects on DNA synthesis in bovine myoblasts when compared to the FGF. Conversely, FGF did not stimulate the proliferation of chick embryo myoblasts, indicating that it is not active in all vertebrate species.
Acetylcholine↗
Fibroblast growth factor: its localization, purification, mode of action, and physiological significance.
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Adrenal Glands↗
Effects of swimming on dystrophic Syrian hamster heart.
Morphological and histopathological heart changes were determined for sixteen dystrophic Syrian hamsters (B10 14.6 strain) and sixteen normal hamsters. Eight animals were randomly assigned to each of the following groups: dystrophic swim (DYS-SWM), dystrophic sedentary (DSY-SED), normal swim (NOR-SWM), and normal sedentary (NOR-SED). The daily swimming program consisted of an initial 30-minute swim which was gradually extended to 60 minutes by the end of eight weeks. Weights up to 3% body weight were attached during swimming to increase the work load. Sedentary animals received no experimental treatment. Four animals in each group were sacrificed at 4 and 8 weeks after the initiation of treatments. In comparison with the two groups of sedentary animals, the NOR-SWM group had a greater heart weight/body weight ratio at both 4 and 8 weeks (P less than .05), while the DYS-SWM group had an increased ratio only at 8 weeks (P less than .05). Subjective histopathological evaluation of heart lesions showed that the DYS-SED group had many large areas of inflammatory reaction with infrequent diffuse areas of calcification. In contrast, the DYS-SWM group had fewer and smaller areas of inflammatory reaction with moderate amounts of calcification.
Animals↗
Autoimmune response to acetylcholine receptor.
Injection of rabbits with acetylcholine receptor highly purified from the electric organ of Electrophorus electricus emulsified in complete Freund's adjuvant resulted in the production of precipitating antibody to acetylcholine receptor. After the second injection of antigen, the animals developed the flaccid paralysis and abnormal electromyographs characteristic of neuromuscular blockade. Treatment with the anticholinesterases edrophonium or neostigmine dramatically alleviated the paralysis and the fatigue seen in electromyography.
Animals↗
Studies on purified eel acetylcholine receptor and anti-acetylcholine receptor antibody.
Rabbit antiserum against purified Electrophorus electricus acetylcholine receptor is studied using an immunoprecipitin assay to measure either antibody titer or concentration of toxin-binding sites in solubilized receptor preparations. This antiserum, unlike control serum, blocks the electrophysiological response of the electroplax to carbamylcholine. Toxin (alpha-neurotoxin, Naja naja) and several cholinergic ligands produce partial inhibition of the reaction of antiserum with purified acetylcholine receptor. Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin. In addition, evidence is presented that antireceptor antiserum will cause precipitation of more toxin-binding sites present in an initial extract than in purified receptor preparations.
Acetates↗
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.
Acetylcholine↗
Photo-affinity labeling of specific acetylcholine-binding sites on membranes.
Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites.
Acetylcholine↗
Microcirculation in mucous membranes of respiratory airways.
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Animals↗
Specificities of antibodies to acetylcholine receptors.
Antisera to acetylcholine receptor purified from the electric organs of Torpedo and Electrophorus were highly species specific, but showed limited cross-reaction with receptors from all mammalian species tested. Antisera to receptor purified from rat muscle cross-reacted extensively with receptor from the muscles of other mammals. Antibodies to receptor from Electrophorus cross-reacting with receptor from Torpedo were affinity purified. Subsequently these showed increased cross-reaction with receptor from mammalian muscle. Sera from patients with myasthenia gravis reacted best with receptor from human muscle, but such sera also cross-reacted with receptor from other mammals, especially squirrel monkeys. These sera cross-reacted slightly with receptor from Torpedo, but not detectably with receptor from Electrophorus,
Animals↗
Antigenic modulation and receptor loss in experimental autoimmune myasthenia gravis.
Immunization of groups of rats with 0.1- 100 microgram of acetylcholine receptor (AChR) purified from the electric organ of Torpedo californica resulted in dose-dependent (1) loss of acetylcholine receptor from the rats' muscles, (2) binding of antibodies to many of the receptors remaining in muscle and (3) production of antibodies in serum capable of cross-reacting with receptor solubilized from rat muscle. Addition of antibodies from rats immunized with electric organ acetylcholine receptors to muscle cells in culture caused loss of receptor by accelerating the rate of receptor degradation. Monovalent antibody fragments did not accelerate degradation unless antiantibody was added to cross-link the monovalent antibody fragments bound to receptors. This indicates that cross-linking of receptors by antibody molecules triggers accelerated receptor degradation, leading to receptor loss. The rate of increase in receptor destruction due to antigenic modulation observed in vitro appears sufficient to account for the extent of receptor loss observed in vivo. Endocytosis of antibody cross-linked receptors may be a rate-limiting step common to antigenic modulation in vitro and in vivo.
Antigen-Antibody Reactions↗
Expression of functional acetylcholine receptor from cloned cDNAs.
The cloned cDNAs encoding the four subunits of the Torpedo californica acetylcholine receptor, each carried by a simian virus 40 vector, direct the synthesis of the functional receptor in a combined expression system consisting of COS monkey cells and Xenopus oocytes. Our results suggest that all four subunits are required to elicit a normal nicotinic response to acetylcholine, whereas only the alpha-subunit is indispensable for alpha-bungarotoxin binding activity.
Animals↗
Immunochemical tests of acetylcholine receptor subunit models.
Acetylcholine receptors of fish electric organs and mammalian skeletal muscle comprise four structurally homologous glycoprotein subunits in the mole ratio alpha 2 beta gamma delta (refs 1-4). All four subunits have leader sequences and are exposed on both sides of the membrane. From amino acid sequencing, three groups have predicted that each subunit has four hydrophobic alpha-helical transmembranous domains. Because the N-terminus of each subunit is thought to remain on the extracellular surface after cleavage of the leader sequence, this model predicts that the N- and C- termini are both on the extracellular side. An alternative model proposed by two other groups predicts that there is, in addition, a fifth amphipathic transmembranous domain which would place the C-terminus on the cytoplasmic side. Here, using anti-subunit sera and monoclonal antibodies and their reaction with synthetic subunit peptides, we demonstrate that the C-terminus is in fact on the cytoplasmic surface. We also show that, contrary to other predictions, the most hydrophilic sequence on the extracellular domain of alpha-subunits is not the main immunogenic region.
Amino Acid Sequence↗
Acquired myasthenia gravis. Selective involvement of esophageal, pharyngeal, and facial muscles.
Serum samples from 152 dogs with a clinical diagnosis of idiopathic megaesophagus without detectable generalized muscle weakness were tested for the presence of antibodies to acetylcholine receptors by immunoprecipitation radioimmunoassay. Positive serum antibody titers (mean, 3.1 nmoL/L; range, 0.77-30 nmoL/L; reference values less than 0.6 nmoL/L) were found in 40 dogs (26%), with German Shepherd dogs (8/25, 32%) and Golden Retrievers (7/20, 35%) having a greater percentage of positive submissions. By immunocytochemical methods, localization of immune complexes at the neuromuscular junction after incubation of serum with normal canine muscle was documented in an additional 17 cases (11% of all samples submitted) that did not have increased antibody titers to acetylcholine receptors. Of the 40 seropositive dogs, 17 (48%) had a clinical improvement or remission of clinical signs associated with decreasing AChR antibody titers. Idiopathic megaesophagus has been associated with a poor prognosis; however, this study demonstrates that a large percentage of the dogs have myasthenia gravis and that with supportive treatment, the clinical signs may improve or resolve.
Animals↗