Concentration Of Acetylcholine Receptors Research Articles

Development of abnormalities at the neuromuscular junction in the SOD1-G93A mouse model of ALS: dysfunction then disruption of postsynaptic structure precede overt motor symptoms.

The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.

Detection of Acetylcholine Receptor Modulating Antibodies by Flow Cytometry

To determine the clinical utility and performance characteristics of a laboratory-adapted flow cytometric method for the detection of acetylcholine receptor (AChR) modulating antibodies in myasthenia gravis (MG). Serum samples from 120 healthy donors and 100 patients with suspected MG were assessed for the ability to reduce surface AChR concentrations (antigenic modulation) in RD (TE671) or DB40 human muscle cell lines by flow cytometry. Reference ranges were established by receiver operating characteristic curve analysis, and results were then compared with those of the current radioimmunoassay (RIA). Flow cytometric results from the RD cell line had an interpretive threshold of 46% modulation or greater and correlated best (98% sensitivity, 99% specificity) with those of the current RIA. The new flow cytometric method using the RD cell platform provided higher quality clinical results, a more robust and efficient assay format, a significant cost savings, and less environmental burden.

Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis

Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The AChR is clustered and anchored in the postsynaptic membrane of the neuromuscular junction (NMJ) by a cytoplasmic protein called rapsyn. We previously showed that resistance to experimental autoimmune myasthenia gravis (EAMG) in aged rats correlates with increased rapsyn concentration at the NMJ. It is possible, therefore, that endogenous rapsyn expression may be an important determinant of AChR loss and neuromuscular transmission failure in the human disease, and that upregulation of rapsyn expression could be used therapeutically. To examine first a potential therapeutic application of rapsyn upregulation, we induced acute EAMG in young rats by passive transfer of AChR antibody, mAb 35, and used in vivo electroporation to over-express rapsyn unilaterally in one tibialis anterior. We looked at the compound muscle action potentials (CMAPs) in the tibialis anterior, at rapsyn and AChR expression by quantitative radioimmunoassay and immunofluorescence, and at the morphology of the NMJs, comparing the electroporated and untreated muscles, as well as the control and EAMG rats. In control rats, transfected muscle fibres had extrasynaptic rapsyn aggregates, as well as slightly increased rapsyn and AChR concentrations at the NMJ. In EAMG rats, despite deposits of the membrane attack complex, the rapsyn-overexpressing muscles showed no decrement in the CMAPs, no loss of AChR, and the majority had normal postsynaptic folds, whereas endplates of untreated muscles showed typical AChR loss and morphological damage. These data suggest not only that increasing rapsyn expression could be a potential treatment for selected muscles of myasthenia gravis patients, but also lend support to the hypothesis that individual differences in innate rapsyn expression could be a factor in determining disease severity.

Neuregulins: primary or secondary signals for the control of synapse-specific gene expression.

The selective transcription of acetylcholine receptor (AChR) subunit genes in synaptic myonuclei leads to the accumulation of AChR subunit mRNAs at the neuromuscular junction (NMJ). This mechanism contributes to the concentration of AChRs at the postsynaptic sarcolemma, and its physiological significance is underscored by the cases of human congenital myasthenias caused by mutation in a cis-regulatory element of the AChRepsilon-subunit promoter, which is necessary for its synaptic expression. The signal(s) that drives synapse-specific expression is unknown but neuregulin-1 (Nrg-1), a group of growth-factor-like polypeptides encoded by the nrg-1 gene, has been a favorite candidate. Nrg-1 was originally thought as a nerve-derived factor, acting in parallel to pathways controlling AChR clustering at the synapse ( i.e. agrin signaling). However, recent work suggests that Nrg-1 may actually be a muscle-derived signal that is concentrated at the NMJ, together with its receptors, by agrin and that acts as a secondary, downstream signal to enhance synapse-specific AChR transcription. Here, I review studies for and against Nrg-1 as a secondary signal driving synapse-specific expression at the NMJ. In addition, I briefly present new evidence that raise the possibility that Nrgs encoded by the ngr-1 -related gene nrg-2 might have a role controlling AChR expression.

Building the vertebrate neuromuscular synapse.

Neuromuscular synapses form following a series of complex interactions between motor neurons, muscle fibers, and Schwann cells that culminate in the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal (Son and Thompson, 1995; Burden, 1998; Sanes and Lichtman, 2001; Schaeffer et al., 2001). In adult muscle, motor axon terminals are situated in shallow depressions of the muscle cell membrane, which is invaginated further into deep and regular folds, termed postjunctional folds. Acetylcholine receptors (AChRs) and additional proteins are concentrated at the crests of these postjunctional folds, while other proteins, including sodium channels, are enriched in the troughs of the postjunctional folds. The genes encoding many of these muscle-derived synaptic proteins are transcribed preferentially in the myofiber nuclei that are situated near the synaptic site, resulting in an accumulation of the encoded mRNAs near synaptic nuclei. The nerve terminal is likewise spatially organized, and its substructural organization reflects that of the postsynaptic membrane. Synaptic vesicles are clustered adjacent to poorly characterized specializations of the presynaptic membrane, termed active zones, which are the sites of synaptic vesicle fusion. Active zones are organized at regular intervals and are aligned precisely with the mouths of the postjunctional folds. This precise registration of active zones and postjunctional folds insures that acetylcholine encounters a high concentration of AChRs within microseconds after release, thereby facilitating synaptic transmission. This precise organization of molecules in presynaptic and postsynaptic membranes belies the concept that the neuromuscular synapse is a simple synapse. Rather, the substructure of presynaptic and postsynaptic membranes suggests that complex mechanisms, requiring spatially restricted signaling between presynaptic and postsynaptic cells, are required to assemble the synapse and to coordinate presynaptic and postsynaptic differentiation.

The Role of Cholinergic Transmission in Outer Hair Cell Functioning Evaluated by Distortion Product Otoacoustic Emissions in Myasthenic Patients

Outer hair cells (OHCs) are the source of otoacoustic emissions, following a tropomyosin-miosin-dependent contraction, which are regulated by the olivocochlear bundle via the release of acetylcholine (ACh). ACh acts on ACh receptors (AChR) located on the OHC post-synaptic membrane. In myasthenia gravis (M.G.) neuromuscular transmission is reduced due to the action of AChR autoantibodies. It has previously been shown that M.G. induces a reduction in transient evoked otoacoustic emissions (TEOAEs), which is reversed after administration of a cholinesterase (AChE) inhibitor. Distortion product otoacoustic emissions (DPOAEs) were recorded before and 60 min after oral administration of 60 mg pyridostigmine bromide in 25 patients with normal hearing affected by M.G. The results were compared with those from 25 age-matched normal controls. Mean values of DPOAE amplitude in myasthenic patients were significantly (p<0.05) lower at all frequencies before drug administration. All patients showed an overall significant (p<0.05) increase in DPOAE amplitude after drug administration, although without reaching the control values. Such a recovery was more evident and highly significant (p<0.01) for middle and high frequencies and could be explained by a higher concentration of ACh receptors in the basal and middle cochlear turns. These data seem to confirm the role of ACh in the neurotransmission of the auditory efferent system and may represent a new in vivo model for the investigation of the physiology of this system.

Different functions of fetal and adult AChR subtypes for the formation and maintenance of neuromuscular synapses revealed in epsilon-subunit-deficient mice.

Mice deficient in epsilon-subunits of the acetylcholine receptor (AChR) channel die prematurely due to severe AChR deficiency that leads to the progressive reduction in AChR density at the neuromuscular endplate [Witzemann, V., Schwarz, H., Koenen, M., Berberich, C., Villarroel, A., Wernig, A., Brenner, H.R. & Sakmann, B. (1996) Proc. Natl Acad. Sci. USA, 93, 13286-13291]. The mice may serve as a model for studying AChR-related myasthenic diseases. The postnatal development of the subsynaptic apparatus takes place in the absence of the adult type, epsilon-subunit-containing receptors which normally replace the fetal gamma-subunit-containing receptors. During later development the secondary folds of the postsynaptic membrane disappear concomitant with the decrease in AChR density, so that the flattened-out membrane with its remaining nicotinic receptors is in close proximity to the subsynaptic cytoplasmatic compartment and the subsynaptic myonuclei. The decrease in AChR concentration is correlated with a decrease of postsynaptic rapsyn, but has less effect on agrin, a neuronally released aggregating factor for AChRs. Thus, despite the presence of agrin at the synapse, AChR expression is not maintained at the level required to stabilize normal synaptic structure comprising secondary postsynaptic membrane folds. Collectively the results suggest that the postnatal switch from the global, activity-sensitive gamma-subunit gene transcription to the synapse-specific, activity-independent epsilon-subunit gene transcription is not required for the formation and differentiation of synapses but is essential for the maintenance of the highly organized structure of the neuromuscular endplate.

Uncoupling of in vivo torque production from EMG in mouse muscles injured by eccentric contractions.

1. The main objective of this study was to determine whether eccentric contraction-induced muscle injury causes impaired plasmalemmal action potential conduction, which could explain the injury-induced excitation-contraction coupling failure. Mice were chronically implanted with stimulating electrodes on the left common peroneal nerve and with electromyographic (EMG) electrodes on the left tibialis anterior (TA) muscle. The left anterior crural muscles of anaesthetized mice were stimulated to perform 150 eccentric (ECC) (n = 12 mice) or 150 concentric (CON) (n = 11 mice) contractions. Isometric torque, EMG root mean square (RMS) and M-wave mean and median frequencies were measured before, immediately after, and at 1, 3, 5 and 14 days after the protocols. In parallel experiments, nicotinic acetylcholine receptor (AChR) concentration was measured in TA muscles to determine whether the excitation failure elicited a denervation-like response. 2. Immediately after the ECC protocol, torque was reduced by 47-89 %, while RMS was reduced by 9-21 %; the RMS decrement was not different from that observed for the CON protocol, which did not elicit large torque deficits. One day later, both ECC and CON RMS had returned to baseline values and did not change over the next 2 weeks. However, torque production by the ECC group showed a slow recovery over that time and was still depressed by 12-30 % after 2 weeks. M-wave mean and median frequencies were not affected by performance of either protocol. 3. AChR concentration was elevated by 79 and 368 % at 3 and 5 days, respectively, after the ECC protocol; AChR concentration had returned to control levels 2 weeks after the protocol. At the time of peak AChR concentration in the ECC protocol muscles (i.e. 5 days), AChR concentration in CON protocol muscles was not different from the control level. 4. In conclusion, these data demonstrate no major role for impaired plasmalemmal action potential conduction in the excitation-contraction coupling failure induced by eccentric contractions. Additionally, a muscle injured by eccentric contractions shows a response in AChR concentration similar to a transiently denervated muscle.

Neuromuscular junction physiology in myasthenia gravis

Article abstract-The mammalian neuromuscular junction is a chemical synapse that uses acetylcholine as transmitter. Acetylcholine (ACh) is stored in the nerve terminal and is released from small membrane-bound vesicles that fuse with the nerve terminal membrane after depolarization of the nerve terminal. Calcium entry via P/Q-type calcium channels is necessary for transmitter release. The postsynaptic membrane of the muscle fiber has several unique properties that facilitate neuromuscular transmission. The postsynaptic membrane area is greatly expanded by synaptic folds. Acetylcholine receptors (AChR) are concentrated at the peaks of synaptic folds and sodium channels are concentrated in the troughs of the synaptic folds. The safety factor for neuromuscular transmission results from a combination of factors: the amount of ACh released, the high concentration of AChR, and the high concentration of sodium channels. AChR are firmly anchored in the membrane by a complex of proteins. The autoimmune attack in myasthenia gravis (MG) directed at acetylcholine receptors results in loss of postsynaptic membrane, AChR, and sodium channels. This article describes the different types of neuromuscular junctions on extraocular muscle fibers and their possible relevance to the susceptibility of extraocular muscle to MG. <b>NEUROLOGY 1997;48(Suppl 5): </b> S8-S17

A transfected human muscle cell line expressing the adult subtype of the human muscle acetylcholine receptor for diagnostic assays in myasthenia gravis.

Immunoprecipitation of human acetylcholine receptor (AChR) is used in the diagnostic assay for myasthenia gravis (MG). We compared human AChR derived from TE671 cells, which express fetal-type AChR, with AChR from TE671-epsilon cells, which we have engineered to express adult-type AChR. Some low-titer MG sera distinguished strikingly between the two subtypes. Four out of seven MG sera that had equivocal titers in standard assays gave positive titers with TE671-epsilon AChR, whereas only one out of seven gave a positive titer with TE671 cells. The new cell line provides a greater concentration of adult AChR than can be obtained from normal human muscle and increases the sensitivity of the diagnostic assay.

D-Tubocurarine Accentuates the Burn-Induced Upregulation of Nicotinic Acetylcholine Receptors at the Muscle Membrane

BACKGROUND Increases in acetylcholine receptors (AChRs) at the muscle membrane, induced by burn injury, have been associated with a hyperkalemic response to succinylcholine and resistance to d-tubocurarine-like drugs. Muscle relaxants often are administered to burn-injured patients in the intensive care unit to facilitate mechanical ventilation. This study in rats tested whether continuous administration of d-tubocurarine in subparalytic doses exaggerates the upregulation of AChRs induced by burn trauma. Subparalytic doses were used to avoid the confounding effects of immobilization. METHODS Three days after an approximate 50% body surface area burn or sham injury, the animals received an infusion of 3.03 +/- 0.05 micrograms/h of d-tubocurarine or equal volume of saline directly to the left gastrocnemius muscle via catheter connected to a subcutaneously implanted osmotic pump. After 7 days of d-tubocurarine or saline infusion, the AChRs were quantitated using 125I-alpha-bungarotoxin. The AChRs on the d-tubocurarine or saline-infused left gastrocnemius were compared to the contralateral gastrocnemius in the same group. The right or left gastrocnemius AChRs were compared to the ipsilateral muscles between groups. These intra- and intergroup comparisons allowed the delineation of the effects of catheter irritation, burns, or d-tubocurarine on AChRs. RESULTS Daily examination of the withdrawal response to toe-pinch revealed no evidence of paralysis. Weight loss in the burn-injury animals receiving d-tubocurarine or saline was similar, confirming that the infusion of d-tubocurarine did not impair the mobility of the animals to move and feed. The plasma d-tubocurarine concentration after 7 days of infusion was 26.0 +/- 12 ng/ml (mean +/- SE). Regardless of burn or sham injury or of d-tubocurarine or saline infusion, the concentration of AChRs on the left was consistently greater than in the contralateral right gastrocnemius muscles within the same group, indicating that manipulation of the area alone can result in upregulation of AChRs. The AChRs in the right gastrocnemius of burn-injured animals were greater than those in the same muscle of sham-injured animals, regardless of saline (7.24 +/- 0.9 vs. 5.7 +/- 0.5 fmoles/mg protein, P = 0.06) or d-tubocurarine (7.3 +/- 0.4 vs. 5.7 +/- 0.5, P < 0.05) infusion to the burn-injury groups. AChRs in the left gastrocnemius of burn-injury animals receiving d-tubocurarine were significantly greater than those in burn- or sham-injury animals receiving saline (13.9 +/- 1.1 vs. 9.8 +/- 1.2 and 7.1 +/- 0.5 fmoles/mg protein, respectively, P < 0.05). CONCLUSIONS Burn-induced upregulation of AChRs is accentuated by infusion of subparalytic doses of d-tubocurarine. Concomitant administration of d-tubocurarine to burn-injured patients may result in further exaggeration of the aberrant responses to neuromuscular relaxants.

Prolonged d-tubocurarine infusion and/or immobilization cause upregulation of acetylcholine receptors and hyperkalemia to succinylcholine in rats.

Hyperkalemic cardiac arrest after the administration of succinylcholine (SCh) to critically ill intensive care patients has been attributed to changes in the acetylcholine receptors (AChRs) at the muscle membrane. The current study attempts to characterize the contributory roles of chronic administration of nondepolarizing muscle relaxants typified by d-tubocurarine (dTC) and/or of immobilization on AChR upregulation and the relationship of these AChR changes to SCh-induced hyperkalemia. Rats received chronic subparalytic infusion of saline or dTC for 28 days via subcutaneous osmotic pumps inserted while they were under anesthesia. Approximately half of the saline- or dTC-treated rats underwent bilateral hind-limb immobilization with plaster casts for the same duration as the infusion. After 4 weeks, the osmotic pumps were removed, and 24-48 h later, the blood potassium concentrations were measured at baseline and at 1, 3, 5, 7, and 10 min after SCh (3 mg/kg). At the end of this period, the gastrocnemius muscle was excised for quantitation of AChR number using (125)I-alpha-bungarotoxin. At 28 days, the weight gain in mobile animals receiving saline or dTC infusion did not differ, nor did that in immobilized animals receiving saline or dTC infusion, confirming that infusion of dTC did not unduly affect the ability of the animals to feed. The maximal potassium change after SCh occurred at 5 min. Potassium responses to SCh changed (mean +/- SE): (1) from 3.9 +/- 0.04 to 4.5 +/- 0.1 mEq/1 in the mobile saline- treated control group, where the AChR concentration was 18.4 +/- 2 fmol/mg protein; (2) from 3.9 +/- 0.03 to 5.1 +/- 0.1 in the mobile dTC-infused group (AChRs = 48.6 +/- 7); (3) from 3.8 +/- 0.1 to 5.5 +/- 0.3 in the immobilized saline- treated group (AChRs = 107.4 +/- 14); and (4) from 3.8 +/- 0.1 to 6.3 +/- 0.2 in the immobilized-dTC-treated group (AChRs = 183.5 +/- 23). There was a significant positive correlation between maximal change in blood potassium concentration and the respective AChR concentration in the gastrocnemius of the same animal (r = 0.81, P<0.01). Subtherapeutic (subparalytic) doses of chronic infusion of dTC (with no immobilization) or immobilization alone (with no dTC) independently increased number of AChRs. The infusion of dTC with immobilization caused the greatest upregulation of AChRs. The magnitude of the increase in blood potassium to SCh was directly dependent on AChR number. This study shows direct evidence and confirms previous speculation that AChR number plays an important role in the magnitude of the hyperkalemic response to SCh. Presuming this represents an appropriate model for patients who are immobilized and/or receiving nondepolarizing muscle relaxants for prolonged periods, exaggerated blood potassium responses to SCh are possible when either or both of these perturbations are present in patients.

D-Tubocurarine accentuates the burn-induced upregulation of nicotinic acetylcholine receptors at the muscle membrane.

Increases in acetylcholine receptors (AChRs) at the muscle membrane, induced by burn injury, have been associated with a hyperkalemic response to succinylcholine and resistance to d-tubocurarine-like drugs. Muscle relaxants often are administered to burn-injured patients in the intensive care unit to facilitate mechanical ventilation. This study in rats tested whether continuous administration of d-tubocurarine in subparalytic doses exaggerates the upregulation of AChRs induced by burn trauma. Subparalytic doses were used to avoid the confounding effects of immobilization. Three days after an approximate 50% body surface area burn or sham injury, the animals received an infusion of 3.03 +/- 0.05 micrograms/h of d-tubocurarine or equal volume of saline directly to the left gastrocnemius muscle via catheter connected to a subcutaneously implanted osmotic pump. After 7 days of d-tubocurarine or saline infusion, the AChRs were quantitated using 125I-alpha-bungarotoxin. The AChRs on the d-tubocurarine or saline-infused left gastrocnemius were compared to the contralateral gastrocnemius in the same group. The right or left gastrocnemius AChRs were compared to the ipsilateral muscles between groups. These intra- and intergroup comparisons allowed the delineation of the effects of catheter irritation, burns, or d-tubocurarine on AChRs. Daily examination of the withdrawal response to toe-pinch revealed no evidence of paralysis. Weight loss in the burn-injury animals receiving d-tubocurarine or saline was similar, confirming that the infusion of d-tubocurarine did not impair the mobility of the animals to move and feed. The plasma d-tubocurarine concentration after 7 days of infusion was 26.0 +/- 12 ng/ml (mean +/- SE). Regardless of burn or sham injury or of d-tubocurarine or saline infusion, the concentration of AChRs on the left was consistently greater than in the contralateral right gastrocnemius muscles within the same group, indicating that manipulation of the area alone can result in upregulation of AChRs. The AChRs in the right gastrocnemius of burn-injured animals were greater than those in the same muscle of sham-injured animals, regardless of saline (7.24 +/- 0.9 vs. 5.7 +/- 0.5 fmoles/mg protein, P = 0.06) or d-tubocurarine (7.3 +/- 0.4 vs. 5.7 +/- 0.5, P < 0.05) infusion to the burn-injury groups. AChRs in the left gastrocnemius of burn-injury animals receiving d-tubocurarine were significantly greater than those in burn- or sham-injury animals receiving saline (13.9 +/- 1.1 vs. 9.8 +/- 1.2 and 7.1 +/- 0.5 fmoles/mg protein, respectively, P < 0.05). Burn-induced upregulation of AChRs is accentuated by infusion of subparalytic doses of d-tubocurarine. Concomitant administration of d-tubocurarine to burn-injured patients may result in further exaggeration of the aberrant responses to neuromuscular relaxants.

Neurotoxins in the study of neural regulation of membrane proteins in skeletal muscle

The discovery and purification of several neurotoxins, including α-bungarotoxin and tetrodotoxin has provided very high-affinity ligands which have proved to be central to the elucidation of the neural control of skeletal muscle membrane proteins and to the purification and characterization of the nicotinic acetylcholine receptor (AChR) and the Na + channel, respectively. This review describes the use of neurotoxins for quantification and localization of receptors and ion channels in normal and denervated skeletal muscles with particular emphasis on the appropriateness of the muscle preparation and ligand used in the studies. It is now clear that the nerve controls the synthesis and spatial distribution of AChRs and Na + channels by regulating gene expression in extrajunctional and subjunctional nuclei. The down-regulation of extrajunctional AChRs is primarily mediated by neuromuscular activity and the concentration of AChRs and Na + channels in specific membrane domains at the neuromuscular junction is controlled by a number of neurotrophic substances at the neuromuscular junction. These include agrin, ARIA, and CGRP.

The effect of tetanus toxin on in vitro synaptogenesis

Cultures of spinal cord neurons and cocultures of rat embryo neurons and muscle cells have been studied in the presence of tetanus toxin (TT) at a concentration of 40 μg/ml of medium. TT strongly stimulated neurite outgrowth, notably branching from the cell bodies. In addition it induced a marked, overall increase in acetylcholine receptor (AChR), but inhibited focalisation of AChR and acetylcholinesterase (AChE) at the synaptic sites. TT seems to act on neurite emergence, on the neuronal factor(s) controlling AChE and AChR concentrations, and on the factor(s) modulating degradation and/or synthesis of AChR.

Acetylcholine receptors from normal human muscle: Concentration, purification, and use in radioreceptor assays for autoantibodies

We measured the concentration of acetylcholine receptor (AChR) in various normal human muscles, obtained at autopsy, in order to find a more reliable and convenient source than the amputated leg muscle from diabetic patients for the isolation of AChR. We found that human calf muscle contains the highest concentration of AChR, approaching the concentration of receptor required for the radioreceptor assay for autoantibodies in myasthenia gravis (MG). Because the amount of contaminant proteins in the receptor preparations affect the sensitivity and precision of the assay, various chromatographic techniques were tested to improve the purity of the receptor preparation. We found that both G-100 Sephadex and DEAE Sephacel chromatographies were effective in removing contaminant proteins. DEAE Sephacel chromatography is particularly useful because the procedure provides higher recovery of receptor. Against the same pool of autoantibodies from patients with MG, AChRs from diabetic and normal leg muscle exhibit similar affinities. We conclude that AChR from normal human calf muscle can be used in radioreceptor assays for measuring autoantibodies in patients with myasthenia gravis.

Effect of local anaesthetics on steroid-nicotinic acetylcholine receptor interactions in native membranes of Torpedo marmorata electric organ

Interactions between steroids and the nicotinic acetylcholine receptor (AChR) have been studied in native membrane vesicles from Torpedo marmorata electric organ by electron spin resonance (ESR) and fluorescence techniques. ESR spectra of spin-labelled cholestane (CSL) revealed that this steroid probe was incorporated into the AChR-rich membrane vesicles in regions which were to a certain extent enriched preferentially in the steroid, both in the presence and in the absence of local anaesthetics. Since the nitroxide group present in CSL is also a paramagnetic quencher of the intrinsic protein fluorescence, this property was used to characterize the AChR-steroid interactions. The quenching induced by CSL was sensitive both to AChR concentration and to the action of cholinergic agonists. In competition experiments, the ability of CSL to quench the AChR intrinsic fluorescence was markedly inhibited by benzocaine, tetracaine and QX-222 (a quaternary trimethylammonium derivative of lidocaine), and was totally inhibited by procaine. The effectiveness of local anaesthetics in inhibiting CSL-induced quenching followed the order: procaine ⪢ benzocaine ⪆ tetracaine > QX-222 . This inhibition effect was shown not to be charge-dependent. The data can be interpreted in terms of a model requiring specific association sites for local anaesthetics on the hydrophobic surface of the AChR which at least partially overlap with those for steroids.

Intracellular and surface acetylcholine receptors during the normal development of a frog skeletal muscle

125I-alpha-bungarotoxin (125I-alpha BT) was used to measure the pool sizes of surface and intracellular acetylcholine receptors (AChRs) in the myotomal muscle of Xenopus laevis over a developmental period (stages 23-48; 1.03-7.5 d) which ranged from initial to mature stages of neuromuscular synaptogenesis. The surface pool increased progressively throughout development. The intracellular pool increased more slowly and also underwent a transient decrease. Linear regression indicated that AChRs begin to appear intracellularly and in the surface membrane at embryonic ages of 13.2 and 18.5 hr, respectively. The findings also suggest that newly synthesized AChRs contribute much more to the intracellular pool than do AChRs internalized from the surface membrane and that the rates of supply and/or intracellular resident times of these 2 sources of intracellular AChRs change during the course of normal development. Carbamylcholine, even at concentrations 10-fold greater than needed to block completely 125I-alpha BT binding to surface AChRs, blocked specific intracellular binding by only 80%. Considered in the light of previous studies on cell cultures, these results suggest that 20% of the intracellular sites are on alpha-subunits not yet assembled into pentameric AChRs. Light microscope radioautography revealed an essentially uniform distribution of intracellular AChRs along the length of the muscle cells. It is concluded that during the normal development of Xenopus myotomal muscle the accumulation and maintenance of AChRs in the postsynaptic membrane occurs in the absence of any preferential concentration of intracellular AChRs in the subsynaptic region.