Endplate Acetylcholine Receptor Research Articles

Acetylcholine receptor availability and transmission efficacy

Recovery of cholinergic transmission after in vivo blockade with α-bungarotoxin (α-BTX), and the relationship of recovery to availability of unbound acetylcholine receptors (AChR) were studied in rat diaphragm. When 83% of endplate acetylcholine receptor binding sites were blocked, transmission was absent. A barely detectable recovery of the blocked receptors (25 h after exposure to α-bungarotoxin) restored transmission. In fact, 25% of the endplate receptor ACh binding sites were just sufficient for action potential generation. As discussed, slow turnover ( t 1 2 = 11 days) of junctional receptors would be sufficient to provide the observed recovery of transmission. Good agreement was observed between the minimum fraction of total receptor sites required for transmission and the computed fraction of maximum quantal release of acetylcholine required to reach threshold. In addition, the data are consistent with the hypothesis that at the neuromuscular junction, AChR exists in considerable excess.

Acetylcholine receptors in human thymic myoid cells in situ: an immunohistological study.

Myoid cells were studied by double immunofluorescence in sections of thymus from 47 patients with myasthenia gravis and 15 control subjects, using polyclonal sheep anti-troponin T and monoclonal antibodies to troponin I, striated muscle myosin, and acetylcholine receptor (AChR). The myoid cells were rare and located mainly in the medulla, and most were clearly positive for AChR; labeling was similar with four individual monoclonal antibodies specific for extrajunctional AChR and five that also recognize endplate AChR. They were mostly keratin-positive and consistently HLA-DR-negative. In the myasthenia gravis samples, the myoid cells were similar but largely confined to medullary epithelial areas; AChR labeling was slightly weaker, but otherwise they did not differ noticeably from those of control subjects. A preliminary finding was of even rarer AChR-positive/HLA-DR-positive antigen-presenting (possibly) cells seen in 9 of 9 myasthenia gravis samples and in none of 9 control samples. Although myoid-cell AChR appears antigenically similar to extrajunctional muscle AChR, and must therefore express the epitopes that myasthenics' antibodies recognize, these cells do not appear to be foci of immunological stimulation in myasthenia gravis.

Concentration-dependent effects of neostigmine on the endplate acetylcholine receptor channel complex

The concentration-dependent actions of neostigmine, a carbamate anticholinesterase agent, were studied on the acetylcholine receptor channel complex in voltage-clamped twitch fibers of costocutaneous muscles of garter snakes. Low concentrations of neostigmine (10(-6) or 10(-5) M) increased miniature endplate current (MEPC) amplitude and the time constant of MEPC decay without changing the relationship between the MEPC decay time constant and membrane potential. Acetylcholine- or carbachol-induced endplate current fluctuation spectra were well fitted by a single Lorentzian curve with a characteristic frequency and single-channel conductance unaltered by low concentrations of neostigmine. Concentrations of neostigmine greater than 5 X 10(-5) M decreased MEPC amplitude and split the decay of MEPCs into two components, one faster and one slower than the control rate. These effects were both voltage and concentration dependent. Spectra of current fluctuations recorded in concentrations greater than or equal to 5 X 10(-5) M neostigmine required two time constants, one faster and one slower than the control. Two component spectra were also obtained with carbachol-induced current fluctuation spectra, indicating that these effects of neostigmine were direct and not a consequence of acetylcholinesterase inhibition. Similar results were also obtained in muscles pretreated with collagenase to remove junctional acetylcholinesterase. The fast and slow time constants obtained from current fluctuation spectra decreased and increased, respectively, with either increases in the concentration of neostigmine or membrane hyperpolarization when analyzed in the same fiber. The effects of neostigmine on channel lifetime were reversible with washing. These results indicate that the effects of neostigmine are concentration dependent. Concentrations greater than 2.5 X 10(-5) M exhibit direct effects on the endplate receptor channel complex which are unrelated to acetylcholinesterase inhibition. These actions include: a prolongation of the gating kinetics of the endplate receptor channel complex, the production of an altered state of the receptor channel complex evidenced by a high frequency component to current fluctuation spectra, and a direct action to block the acetylcholine receptor.

The effects of myasthenic immunoglobulins on neuromuscular transmission and thymus histology in mice.

An autoimmune reaction to the endplate acetylcholine receptor (AChR) is thought to be responsible for the muscular weakness of myasthenia gravis (MG). However, the significance of antithymic antibodies and the thymic AChR-like protein is still uncertain. We transferred immunoglobulins (Igs) from patients with MG to mice. Thymitis was observed in 12 of 14 mice, and miniature endplate potential amplitudes were reduced in 8. Control mice showed none of these abnormalities. Immunofluorescence examination failed to reveal the binding of human IgG, IgM, or IgA to the thymic tissue. Our findings support the hypothesis that antithymic antibodies may alter thymic histology.

Acetylcholine receptor turnover in mice with passively transferred myasthenia gravis. II. Receptor synthesis.

The in vivo synthesis of diaphragm endplate acetylcholine receptors was estimated in mice treated daily with IgG from eight myasthenia gravis patients. Myasthenia gravis IgG preparations which had previously been shown to increase the rate of receptor degradation also increased the rate of receptor synthesis, suggesting the existence of a compensatory mechanism serving to stabilise the number of acetylcholine receptors.

Acetylcholine receptor turnover in mice with passively transferred myasthenia gravis. I. Receptor degradation.

The in vivo degradation of endplate acetylcholine receptors was investigated in mice treated daily with IgG from 10 myasthenia gravis patients. Four preparations increased the rate of degradation. The number of endplate acetylcholine receptors after 7 days of IgG treatment was greater than that predicted on the basis of the increased degradation rate, suggesting a compensatory increase in receptor synthesis.

Denervation increases the degradation rate of acetylcholine receptors at end‐plates in vivo and in vitro.

We have studied the effect of denervation on the degradation of the existing junctional acetylcholine (ACh) receptors at end-plates in rat muscles. ACh receptors were labelled by injecting animals with iodinated alpha-bungarotoxin (I-alpha BT); 1 day later the left hemidiaphragm was denervated. The degradation of bound I-alpha BT in normal and denervated muscles was examined in organ culture, beginning at various times after denervation in vivo. The original, pre-labelled end-plate ACh receptors are degraded more rapidly after denervation. The rate of degradation begins to increase shortly after the nerve is cut and reaches a maximum value at about 9 days of denervation. Muscles denervated only on transfer to organ culture also show an increase in the degradation rate of bound I-alpha BT with increasing time of denervation (time in culture). In normal diaphragm muscles, the initial rate of degradation of functional ACh receptors, after correcting for non-degradative loss of I-alpha BT, is 0.0018 h-1 (t1/2 = 383 h). The maximal rate at denervated end-plates is 0.0073 h-1 (t1/2 = 94 h). For soleus, sternomastoid, plantaris and intercostal innervated muscles the apparent rate of ACh receptor degradation either in vitro or in vivo ranged from 0.0005 h-1 to 0.002 h-1. The rate of loss of bound I-alpha BT in vivo is more rapid at denervated end-plates than at innervated end-plates. For diaphragm muscles, the rates of I-alpha BT degradation measured in organ culture are able to describe the relative rates of loss of I-alpha BT from innervated and denervated muscles in vivo. At short times after labelling, a fraction (10-20%) of the I-alpha BT bound to innervated muscles is degraded more rapidly than the remaining toxin. The possibility that these I-alpha BT binding sites are degraded at the rate characteristic of extrajunctional receptors on denervated muscle fibres is discussed.

Ultrastructural aspects of acetylcholine receptor turnover at the normal end-plate and in autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) deficiency at the myasthenic end-plate has been attributed to complement-mediated lysis of the junctional folds and to increased fractional degradation rate of AChR cross-linked by myasthenic immunoglobulin. This paper addresses the manner in which AChR is internalized and degraded at the normal end-plate and provides morphologic evidence for accelerated AChR degradation at the end-plate of rats with experimental autoimmune myasthenia gravis (EAMG). We sequentially traced the fate of end-plate AChR labeled in vivo with intramuscularly-injected peroxidase-alpha-bungarotoxin (PBGT) in control rats and rats with chronic EAMG. At both control and EAMG end-plates, AChR is internalized by endocytosis. The endocytosed vesicles containing AChR are transferred into the lysosomal compartment which extends from the junctional folds into the junctional sarcoplasm. Regardless of whether the initial intensity of the reaction for AChR at the EAMG end-plate appeared normal or reduced. AChR disappeared more rapidly from the EAMG than from the control end-plates. Despite the accelerated fractional turnover rate of end-plate AChR in EAMG, the postsynaptic membrane surface which could be labeled with PBGT for AChR remained unchanged over a 120-hour period. These data suggest that end-plate AChR is at a steady state in chronic EAMG.

Voltage clamp analysis of acetylcholine receptor desensitization in isolated mollusc neurones.

1. Desensitization produced by acetylcholine (ACh) in completely isolated Limnaea stagnalis neurones with chloride-selective membrane channels was studied using a voltage-clamp technique. 2. A difference in the time course of the neurone responses to ACh, depending on whether the measured parameter was voltage or current, was observed and explained on the basis of an equivalent electric scheme of the neurone soma membrane. 3. Desensitization onset was shown not to depend on membrane potential in the range of -30 to -120 mV. 4. Variation of external Ca2+, Na+ and Cl- concentrations over a wide range had little influence on the onset of desensitization and recovery from it. 5. An obvious difference is shown to exist between features of desensitization in mollusc neurone and frog muscle end-plate ACh receptors.

Binding of d-tubocurarine and α-bungarotoxin in normal and denervated mouse muscles

The action of native and mono- 3H-acetylated-α-bungarotoxin ( 3H-BuTX) was studied at the acetylcholine (ACh) receptors of innervated and of chronically denervated diaphragm and soleus muscles of the mouse. The 3H-BuTX was similar in potency to the native toxin, and both toxins produced complete blockade of endplate potentials of innervated muscles and of ACh sensitivity of chronically denervated muscles. When the innervated muscles were washed for 4–7 hr after exposure to either toxin (at 1–5 μg/ml), recovery to an endplate potential value of 0.5–1 mV was recorded in most of the endplate regions of the surface fibers. Parallel experiments on the chronically denervated muscle after a 4–7 hr wash showed a much larger fraction of reversibility; i.e., while in control denervated preparations the ACh sensitivity was 50–75 mV/nC, after the toxin treatment and washing the values were 5–10 mV/nC. When d-tubocurarine ( d-TC, 28 × 10 −6 m) was present to protect against the blockade of the toxin at the endplates, a complete recovery of neuromuscular transmission could always be obtained upon washing. In the chronically denervated muscles, however, much less protection from the 3H-BuTX blockade was observed. Uptake of 3H-BuTX at the endplates was measured by radioactivity analyses; these revealed that only about 60% of the endplate ACh receptors are protected at saturating d-TC levels. Kinetic analyses of the uptake confirmed this. The observations can be interpreted in terms of two types of sites at the endplate; the two are equally reactive with α-bungarotoxin, but only one of them binds d-TC firmly. Similar observations were made with d-TC protection in the denervated muscles; the results showed much lower affinity of d-TC in these muscles. The changes in d-TC effectiveness in the receptors after denervation are all parallel, when measured in several muscles by three approaches—blockade of ACh sensitivity, prevention of α-bungarotoxin blockade of ACh sensitivity, and inhibition of uptake of 3H-BuTX. We conclude that the ACh receptor molecules themselves, when induced by denervation, are different from normal receptors in that they interact much less strongly with d-TC.

Blockade by lobeline of potassium exchange in skeletal muscle. Relationship to receptor desensitization at the endplate.

Lobeline (0.05 to 2.5 mM) caused depolarization of muscle fibers in frog sartorius muscle bathed in chloride-free solution. When the extent of depolarization was plotted against the log of the concentration of lobeline, the relationship was described by a straight line with a slope of 57 mV for a 10-fold change in the concentration of lobeline (0.2 to 2.0 mM). Like lobeline, butacaine (0.1 to 0.5 mM) caused muscle depolarization with a 57 mV change in membrane potential for a 10-fold change in the concentration of the local anesthetic. Lobeline (0.05 to 0.15 mM) depressed the depolarization of the muscle fibers caused by the elevation of (K+]0 over a range of 2.5 to 50 mM. 42K-exchange in resting muscle was also depressed by lobeline (0.05 to 0.25 mM). The blockade by lobeline of 42K-efflux followed a typical dose-response relationship with 0.06 mM lobeline required to cause a 50% decrease of 42K-efflux. These findings show that lobeline in concentrations not much greater than those required to block neuromuscular transmission, has a direct action on ion exchange in skeletal muscle. These actions on electrogenic mechanisms may contribute to the anti-nicotinic action of lobeline and to the desensitization of endplate acetylcholine receptors caused by lobeline.

Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes

Incorporation of a fraction isolated from rat diaphragm muscle that contained the specific endplate cholinergic receptor into phospholipid bilayer membranes resulted in the production of an acetylcholine-stimulated conductance increment of large magnitude. The acetylcholine-stimulated conductance shows several characteristics of the in vivo post-synaptic excitable system.

Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes

Incorporation of a fraction isolated from rat diaphragm muscle that contained the specific endplate cholinergic receptor into phospholipid bilayer membranes resulted in the production of an acetylcholine-stimulated conductance increment of large magnitude. The acetylcholine-stimulated conductance shows several characteristics of the in vivo post-synaptic excitable system.

Acetylcholine receptor and ion conductance modulator sites at the murine neuromuscular junction: evidence from specific toxin reactions.

The perhydro derivative of histrionicotoxin reversibly blocks the excitatory ionic transduction system in the synaptic and sarcolemmal membranes of mammalian skeletal muscle cells. The efficacy of perhydrohistrionicotoxin as an antagonist at the post-synaptic membrane is increased by the transient presence of acetylcholine in the endplate of innervated muscles and at extrajunctional receptors in denervated muscles. alpha-Bungarotoxin and [(3)H]monoacetyl-alpha-bungarotoxin block the endplate acetylcholine receptors, each binding to the same extent. The effect of bungarotoxin is partially reversible. These electrophysiological results, together with the effects of perhydrohistrionicotoxin and/or d-tubocurarine on the binding of [(3)H]monoacetyl-alpha-bungarotoxin at endplates of murine diaphragm muscle and on the bungarotoxin-elicited irreversible blockade of neuromuscular transmission, suggest that at least two types of sites participate in the synaptic excitation by acetylcholine. One site, competitively blocked by bungarotoxin and by curare, is presumably the acetylcholine receptor. Binding of bungarotoxin at this site is responsible for an irreversible blockade of neuromuscular transmission. The second site, competitively blocked by bungarotoxin and perhydrohistrionicotoxin, is proposed to be part of the cholinergic ion conductance modulator. Binding of bungarotoxin to this site does not result in an irreversible blockade.