Showing papers by "Jon Lindstrom published in 1985"

Immunobiology of Myasthenia Gravis, Experimental Autoimmune Myasthenia Gravis, and Lambert-Eaton Syndrome

Abstract: The muscular weakness and excessive fatigability that characterize the disease now known as myasthenia gravis (MG) were initially described by Thomas Willis in 1672 (1). In 1973 it was shown that immunization of rabbits with acetylcholine receptors purified from fish electric organs caused similar symptoms (2). During the following decade, researchers demonstrated that the symptoms of both MG and experimental autoim­ mune myasthenia gravis (EAMG) are caused by an antibody-mediated autoimmune response to acetylcholine receptors (3-56). Although we now have a reasonable understanding of the complex mechanisms by which this autoimmune response impairs neuromuscular transmission, we still do not know either what initiates and regulates this response in MG or the significance of the thymoma frequently associated with it. Although muscular weakness can be partially alleviated by treatment with inhibitors of acetylcholinesterase (which increase the concentration and duration of acetylcholine to compensate for the loss of functional receptors) and although thymectomy (7, 8), immunosuppressive drugs (9, 10), and plasmapheresis (11, 12) are beneficial therapies, there is no cure for MG. The discovery of EAMG was a by-product of the initial purification of acetylcholine receptors. Nicotinic acetylcholine receptors are now by far

Mutation at I-A beta chain prevents experimental autoimmune myasthenia gravis.

Abstract: Immune response (Ir) gene(s) at the I-A subregion of the mouse H-2 complex influence susceptibility to experimental autoimmune myasthenia gravis (EAMG). To determine the importance of the Ir gene product, the Ia antigens, in EAMG pathogenesis, we studied the degree of EAMG susceptibility of an I-A mutant strain, the B6.C-H-2 bm12 (bm12), and its parent B6/Kh. According to the cellular, humoral, biochemical, and clinical manifestations of EAMG, the I-A mutation converted an EAMG susceptible strain (B6/Kh) into a relatively resistant strain (bm12). The relative resistance to EAMG induction in bm12 may be due to the lack of Ia.8 and/or la.39 determinants and/or quantitative expression of la antigens.

Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits

Abstract: Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the alpha subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, alpha subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide alpha 159-169 was less by a factor of 10(4), suggesting that the mAbs bind primarily to the sequence alpha 152-159. Cholinergic ligands did not inhibit mAb binding. No crossreaction was observed with the receptor in native membranes, but the mAbs could bind to receptor reconstituted into liposomes in which 50% of the receptors have their cytoplasmic surface oriented outside. When native membranes were permeabilized with saponin, mAbs directed against cytoplasmic determinants of the receptor could bind to them, but mAbs 236 and 237 could not. However, after treatments that removed peripheral proteins from the cytoplasmic surface, binding of both mAbs was observed. Further evidence for the cytoplasmic localization of this sequence was provided by observation of partial competition for binding between mAbs 236 and 237 and mAbs previously demonstrated to bind to the cytoplasmic surface of the receptor. To account for these findings, a model for the organization of the polypeptide chains in receptor subunits is proposed that has a total of seven transmembrane domains in each subunit, two of which are amphipathic and one of which is not alpha-helical.

Characterization of a component in chick ciliary ganglia that cross- reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor

Abstract: Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the “main immunogenic region” of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross- reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles.

Abstract: We tested the effects of 62 monoclonal antibodies (mAbs) to acetylcholine receptors from Torpedo californica on the function of receptor reconstituted into lipid vesicles. Two of these mAbs, mAbs 148 and 168, inhibited carbamylcholine-induced 22Na+ uptake into vesicles. The rate of 125I-labeled alpha-bungarotoxin (125I-alpha BGT) binding to the reconstituted liposomes was also reduced, although 125I-alpha BGT binding at equilibrium was not affected. Agonist-induced desensitization of the receptor was also affected by these mAbs. mAb 148 binds to the beta subunit of receptor, and mAb 168 binds to the gamma subunit. Both mAbs bind to the cytoplasmic surface of the receptor; correspondingly, both block function when added before reconstitution, and both were found to have no effect on function when added to preformed vesicles. Their effects were not due to interference with the reconstitution process. Both mAbs were capable of cross-linking receptors. In contrast to the bivalent mAbs, monovalent Fab fragments of these two mAbs had little effect on receptor function, which suggests that the effects of the bivalent mAbs resulted primarily from cross-linking receptors.

The occurrence of long openings in the purified cholinergic receptor channel increases with acetylcholine concentration.

Abstract: To establish the relationship between cholinergic ligand occupancy and channel open states, we recorded single-channel currents activated by different acetylcholine (ACh) concentrations from Torpedo californica ACh receptors reconstituted in lipid bilayers. Inspection of single- channel records shows that the frequency of occurrence of long openings increases with ACh concentration. Analysis of the probability distribution of open dwell times indicates that the ACh receptor channel has two distinct channel open states, short- and long-lived. The frequency of occurrence of the long openings over the short increased with ACh concentration, whereas the corresponding time constants were virtually unaltered. The extent of agonist occupancy at the ACh-binding sites in the purified cholinergic receptor appears, therefore, to correlate with an increased probability of the long-lived open state. These results are consistent with the notion that the two open-channel states arise from different extents of ligand occupancy at the receptor molecule.

The Molecular Basis of Neurotransmission: Structure and Function of the Nicotinic Acetylcholine Receptor

Abstract: Acetylcholine was identified as the first neurotransmitter as a result of elegant experiments by Otto Loewi in 1921 who demonstrated that the vagus nerve liberates a substance that has an inhibitory effect on the rate of the heartbeat of an isolated frog heart. Loewi showed that this “vagus substance” could be transferred from the fluid filling the heart onto another heart and there reproduce the same inhibitory effect. He coined the term “humoral transmission” to describe this activity (Loewi, 1921). Subsequent experiments identified the “vagus substance” as acetylcholine. It became clear that acetylcholine, in addition to its influence on the heart, exerted a variety of pharmacologically distinct effects, which were classified by Sir Henry Dale (1934) as “muscarinic” and “nicotinic” actions, because some were mimicked best by muscarine and others by nicotine. Acetylcholine receptors with muscarinic ligand-binding properties are characterized by prolonged responses of slow onset which are mediated through nucleotide cyclases (for short reviews see Sokolovsky and Bartfai, 1981; Hartzell, 1982), whereas acetylcholine receptors with nicotinic-binding properties are characterized by rapid responses in which ligand binding regulates the opening and closing of a cation-specific channel through a conformational alteration in the molecule. Acetylcholine receptors at the neuromuscular junctions of striated muscle and at the synapses of fish electric organs (which are phylogenetically related to muscle tissue) are the best-studied nicotinic acetylcholine receptors and the subjects of this review.

Myasthenia gravis--current concepts.

Abstract: An edited summary of an Interdepartmental Conference arranged by the Department of Medicine of the UCLA School of Medicine, Los Angeles. The Director of Conferences is William M. Pardridge, MD, Associate Professor of Medicine. Current findings indicate that autoimmune myasthenia gravis is an acquired immune complex disorder of neuromuscular transmission in voluntary striated muscle. There is a break in immunologic tolerance leading to blocking and degradation of acetylcholine receptors, together with widening of the synaptic cleft associated with partial destruction, simplification and shortening of the postjunctional membrane. Thymic hyperplasia and thymoma may be present. A decremental response to nerve-muscle stimulation, blocking and jitter on single-fiber electromyography and circulating antibodies to acetylcholine receptor are detectable in most patients with generalized weakness. Although the cause of this abnormal immunologic mechanism remains to be discovered, anticholinesterases, corticosteroids, immunosuppressants, plasmapheresis or thymectomy (individually or in combination) provide control and better prognosis in most patients.

Production of Rat × Mouse Hybridomas for the Study of the Nicotinic Acetylcholine Receptor

Abstract: In this chapter we will discuss methods used in our laboratory for the production of monoclonal antibodies (MAbs) to acetylcholine receptors (AChRs) and their use in obtaining information on AChR structure, function, and synthesis, the autoimmune response to AChR, and the identification of putative AChRs in neurons. Space limitations preclude extensive discussion and referencing of work done in other laboratories in this field.