α-BGT Binding Research Articles

Heterogeneity of antibodies directed against the α-bungarotoxin binding site on human acetylcholine receptor and severity of myasthenia gravis

A rapid and sensitive radioimmunological method is described, using decamethonium (DC), which revealed antibodies which blocked α-bungarotoxin (α-Bgt) binding to human acetylcholine receptor (AChR) in 98% of myasthenia gravis (MG) patients' sera tested. These sera had anti-AChR antibody titres by the conventional assay. The titre of blocking antibodies (1 to 110 nM) could be measured and was found to produce from 1 to 54% inhibition of α-Bgt binding. No relationship was found between these titres and anti-AChR antibody titres. MG sera were divided into 2 major groups on the basis of their blocking effects, with and without DC, but there was no correlation between these and the clinical status, as defined by Osserman's classification. However, no sera from asymptomatic or ocular MG patients had the dual capacities of blocking α-Bgt binding, directly and in the presence of DC.

Contrasting effect of a brain supernatant extract on neuronal vs neuromuscular α-bungarotoxin receptors

The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [ 125I]α-bungarotoxin (α-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited α-BGT binding to brain membranes in a dose-dependent fashion, ( Brain Research, 245 (1982) 57–67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled α-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [ 125I]α-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic α-bungarotoxin binding sites.

Modulation of the nicotinic α-bungarotoxin site in chromaffin cells in culture by a factor(s) endogenous to neuronal tissue

An endogenous factor(s) which affects the in vitro binding of α-bungarotoxin (α-BGT) to rat brain membranes has previously been found in brain supernatant. This fraction, as well as a partially purified preparation of this material from bovine brain, is here shown to affect the binding of α-BGT to chromaffin cell membranes. To study possible long term effects, the supernatant extract was added to adrenal medullary chromaffin cells in culture. The cells were incubated for several days and at the end of this time, the medium bathing the cells, which contained the endogenous factor(s), was removed and α-BGT binding to the cells measured. Binding to control cultures had shown that α-BGT bound to the chromaffin cells in a saturable manner, with high affinity (K d = 1.5 nM) and the specificity of a nicotinic receptor ligand. After incubation of the cells with supernatant factor, a marked decline in the number of a α-BGT binding sites was observed with no change in affinity. This does not appear to be due to a detrimental effect on the cells as cell number did not appear to be decreased in the cultures preincubated with the supernatant extract and the DNA and protein content were similar in the control and treated cultures. The possibility that there was some non-specific detrimental effect to the chromaffin cell membrane was considered; however, the stimulated release of noradrenaline from the cells was not affected by treatment of the cultures in the presence of the supernatant fractions. In addition, tyrosine hydroxylase activity was significantly increased in the treated cultures. d-Tubocurarine, an antagonist at the acetylcholine receptor, caused an increase in α-BGT binding after 7 days of treatment, while the agonist nicotine and choline had no effect. These results suggest that in brain supernatant there may exist an endogenous factor(s), which may function in the regulation of the nicotinic-like α-BGT receptors in neuronal cell.

Localization and function of cat carotid body nicotinic receptors

Acetylcholine and nicotinic agents excite cat carotid body chemoreceptors and modify their response to natural stimuli. The present experiments utilized [ 125I]α-bungarotoxin ([ 125I]α-BGT) to localize within the chemosensory tissue the possible sites of action of exogenous and endogenous nicotinic cholinergic substances. In vitvo equilibrium binding studies of intact carotid bodies determined a K d of 5.57 nM and a B max of 9.21 pmol/g of tissue. Chronic section (12–15 days) of the carotid sinus nerve (CSN) did not change the amount of displaceable toxin binding. In contrast, the specific binding was reduced by 46% following removal of the superior cervical ganglion. Light microscope autoradiography of normal, CSN-denervated and sympathectomized carotid bodies revealed displaceable binding sites concentrated in lobules of type I and type II cells. Treatment of carotid bodies with 50 nM α-BGT in vitro reduced by 50% the release of [ 3H]dopamine (synthesized from [ 3H]tyrosine) caused by hypoxia or nicotine, and also significantly reduced the stimulus-evoked discharges recorded from the CSN. The data suggest (1) an absence of α-BGT binding sites on the afferent terminals of the CSN and (2) that nicotinic receptors located within parenchymal cell lobules may modulate the release of catecholamines from these cells.

Effects of phencyclidine and analog drugs on acetylcholine receptor of cultured muscle cells

Myotubes grown in culture provided a convenient experimental system for the study of the effects of phencyclidine (PCP) and analog drugs on both acetylcholine receptor (AChR) function and on its binding properties. The extent of PCP retention by these cells was studied on the same preparations. PCP, N-ethyl-1-phenylcyclohexylamine (PCE), PCP methiodide (PCPMeI), 1-[1-(3-aminophenyl)-cyclohexyl] piperidine (NH 2PCP) and 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) were found to inhibit carbamylcholine (CbCh)-induced 22Na and 45Ca ion fluxes with 50% inhibition (I 50) at 2–6 μM drug concentration. The I 50 for CbCh-induced 42K + efflux was 8–20 μM. Ketamine was less efficient with an I 50 of 100 μM. Binding of [ 125I] α-bungarotoxin ([ 125I]α-BGT) was not affected at drug concentrations that cause 100% inhibition of ion fluxes. Retention of [ 3H]PCP by the myotubes was a saturable process with half-maximal saturation at ≈20 μM PCP. It was inhibited by PCP and several tertiary analogs, with an I 50 of ≈ 20μM. PCPMeI was much less effective, with an I 50 of 1 mM. PCPMeI was, however, as potent as PCP in its inhibition of the AChR function although the amount retained by the cells was 50-fold lower than that of PCP. These results are consistent with the theory that PCP and analog drugs affect AChR at a site other than the α-BGT binding site, possibly at the ionic channel of the nicotinic receptor.

Quantitative light microscopic autoradiography for .ALPHA.-bungarotoxin binding capacity in the mouse brain.

α-Bungarotoxin (α-BGT) binding capacity on slide-moullted brain sections which had been fixed with 4% paraformaldehyde (PFA) or periodate-lysine-paraformaldehyde (PLP) was determined by quantitative light microscopic autoradiography using radioiodinated toxin. The results are consistent with our previous data obtained by a biochemical method on tissue homogenate. This is a new methodology for estimation of α-BGT binding capacity in brain regions of a limited area.

A new mechanism revealed for the action of antibodies on acetylcholine receptor function

Rabbits and sheep were inoculated with acetylcholine (ACh) receptor protein from Torpedo electric organ. Immunoglobulins (IgGs) were purified from the sera, and their effects on binding of ligands to Torpedo membranes as well as ACh receptor-induced 22Na + flux in these membranes were determined. Immune rabbit IgGs inhibited binding of [ 3H]ACh and [ 125I]α-bungarotoxin to the ACh receptor sites but not the binding of [ 3H]perhydrohistrionicotoxin ([ 3H]H 12-HTX) to the receptor's ionic channel site. On the other hand, immune sheep IgGs did not inhibit binding of any of these three ligands. Yet, the two IgGs from rabbit and sheep inhibited the ACh receptor-induced 22Na + flux. The initial rate of binding of [ 3H]H 12-HTX to the membranes was potentiated by carbamylcholine, which reflected the allosteric effect of the receptor site on the ionic channel site. Both rabbit and sheep IgGs inhibited this potentiation. It is suggested that these immune sheep sera contain IgGs which inhibit ACh receptor function by means of interfering with the agonist-induced conformational changes in the receptor-channel complex. Immune rabbit IgGs inhibit receptor function possibly by a similar mechanism but, in addition, by inhibiting [ 3H]ACh binding to the receptor. Antibodies such as those found in the sheep sera would inhibit receptor function by blocking its conformational changes, while leaving the α-BGT binding site unoccupied.

Whole brain and regional [125I]-α-bungarotoxin binding in developing rat

These experiments were conducted in order to determine if the total number of binding sites for [125I]-α-bungarotoxin ([125I]-α-BGT) in rat brain increases and then decreases during postnatal development as predicted by comparison with skeletal muscle, and, if so, to determine at approximately what age the peak in binding occurs in the brain as a whole. A further purpose was to investigate the time-course of development of the [125I]-α-BGT binding sites in several brain regions.Specific binding for [125I]-α-BGT was studied using the pellets from a 20 min, 14,000 × g centrifugation of rat brain homogenates from 4 or 5 postnatal ages. At least three binding assays were done per region and per age, on cerebral cortex, cerebellum, caudate-putamen, posterior hippocampus, pons-medulla and whole brain. In most regions, the [125I]-α-BGT specific binding is measurable, but is low at day one, peaks at about 12–20 days and declines by adulthood. With a few exceptions, these data hold true whether binding is expressed as specific binding per mg protein, specific binding per gram wet tissue, or total specific binding per brain region. The absolute number of specifically bound [125I]-α-BGT molecules is undistorted by simultaneous or non-linear growth of cells uninvolved with α-BGT binding and, thus, is the measurement most useful in determining developmental changes. Whole brain has the same age-related pattern as in the majority of the brain regions, i.e., compared to 19–20 days, the adult brain actually has fewer total binding sites.

Effects of antibodies to Torpedo acetylcholine receptor on the acetylcholine receptor-lonic channel complex of Torpedo electroplax and rabbit intercostal muscle

An electrophysiological, biochemical, and ultrastructural study of the acetylcholine (ACh) receptor-ionic channel complex was undertaken in electric organ membranes from the electric ray, Torpedo ocellata, and intercostal muscle of the rabbit. Rabbits immunized with receptor purified from Torpedo ocellata were paralyzed as is characteristic of experimental autoimmune myasthenia gravis. Incubation of Torpedo membranes with antisera from six paralyzed rabbits, but not control rabbits, resulted in inhibition of [ 3H]ACh and [ 125I]α-BGT binding by 19 to 95% and 43 to 86%, respectively, and did not significantly affect the binding of [ 3H]H 12-HTX. Anti-ACh-receptor antibodies bound to Torpedo microsacs were identified in electron micrographs by specific binding of ferritin-labeled goat anti-rabbit antibodies. Treatment of Torpedo microsacs with ACh-receptor antisera from rabbits inhibited their carbamylcholine-induced 22Na efflux, which would suggest direct effect of the antibodies on Torpedo ACh-receptor function. However, addition of the same anti- Torpedo-ACh-receptor antisera for 240 min to inter-costal muscles of rabbits produced only a small reduction in the amplitude of the response to microiontophoretically applied ACh, miniature end-plate potentials, and end-plate potentials. It is suggested that the muscle weakness observed in rabbits immunized with Torpedo ACh-receptor protein may be due to time-requiring action on receptors such as their accelerated degradation, and the amount of antimuscle receptor in rabbit antisera against Torpedo receptor may be too small.

Acetycholine receptor production and incorporation into membranes of developing muscle fibers

Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing 125I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm 2 per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm 2 per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors.