Acetylcholine Receptor Density Research Articles

Acetylcholine receptors at mature and aged mouse neuromuscular junctions

The density and distribution of junctional and perijunctional ACh receptors (AChR) were studied in young (8-12 months) and old (24-25 months) C57 mice to determine: (1) if increased amplitude of spontaneous postsynaptic potentials previously reported in old C57 muscle was due to increased junctional AChR; (2) if increased extrajunctional AChR would be found in association with previously reported nerve terminal complexity; and (3) if extrajunctional AChR was present as in disused or denervated muscle. Microdissection of individual muscle fibers combined with I125-alpha-bungarotoxin labeling, gamma counting, measurement of surface area, cholinesterase stains, and autoradiography were used to obtain the results. In both young and old mice there was a sharp gradient in AChR between the end-plate and the perijunctional region. End-plate AChR densities and total AChR per end-plate were the same at old and young end-plates, as were perijunctional values. Thus, neither end-plate nor extrajunctional AChR density changes with age. An increased mepp amplitude reported previously in old CB57 animals must be due to other factors. The perijunctional AChR in old mice show no changes characteristic of disuse or denervation, or those which might give rise to the observed nerve terminal complexity.

A human embryonic lung fibroblast with a high density of muscarinic acetylcholine receptors.

Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and N-methylscopolamine showed that the human embryonic lung fibroblast CCL137 possesses approximately 2 X 10(5) muscarinic receptors/cell, i.e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.

Nicotinic acetylcholine receptors in the rat stomach: II. Alterations after vagotomy

Nicotinic and muscarinic acetylcholine receptors were studied in the stomach of vagotomized rats, with ligand binding techniques, and with the concomitant measurement of choline acetyltransferase (ChAT). The maximum binding capacity (B max) of the high affinity sites of (−)-[ 3H]nicotine binding was significantly increased in all the regions of the stomach of subdiaphragmatically vagotomized animals while the values for the low affinity sites remained the same as those in sham-operated animals. On the other hand, [ 3H]quinuclidinyl benzilate binding to the stomach was unchanged after vagotomy. ChAT activity was significantly reduced in the vagotomized rats. These results suggest that surgical vagal denervation causes an increase in the density of nicotinic acetylcholine receptors presumably located on the parasympathetic ganglion cells. There is also a reduction of ChAT activity, probably due to a degenerative loss of preganglionic cholinergic neurons. Preganglionic denervation (decentralization) has little influence on the muscarinic receptors of postsynaptic effector organs.

Acetylcholine receptor density and binding in murine dystrophy

The concentration and binding characteristics of acetylcholine receptors (AChR) in sarcolemmal fractions from normal and dystrophic mice have been measured. Unlike the results following denervation, AChR concentration (per g wet weight) is unchanged and AChR affinity for two different α-neurotoxins is also unchanged. In contrast sarcolemmal ATPase is significantly reduced in dystrophic tissues. These data reaffirm that denervation is a poor model of dystrophy.

Development of the electromotor system of Torpedo marmorata: distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization.

A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptor-rich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electron-microscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.

Correlation of clustering peak density and total amount of acetylcholine receptor on cultured mouse myotubes

The development of acetylcholine receptor (AChR) and its clustering were studied quantitatively on mouse myotubes in nerve-muscle co-cultures. AChR was visualized by fluorescence-labeled antibodies (F-Ab) against crude AChR or fluorescence-labeled α-bungarotoxin (F-α-BuTX). The F-Ab stain was observed throughout the entire surface of the myotube at day 8 and appeared clustered at day 13. Both peak density and total amount of fluorescence in F-Ab stained myotubes were plotted against days in culture. Both fluorescence indices markedly increased from days 8 to 13 of culture were greater in extent in myotubes incubated in the presence of spinal cord explant, as compared with its absence. Similar results were observed in myotubes stained with F-α-BuTX. d-Tubocurarine (d-TC, 0.1 mg/ml) and native α-BuTX (1 μg/ml) clearly inhibited both the total amount of fluorescence and the development of peak fluorescence density in the F-Ab stained myotubes. But the inhibition by d-TC appeared at the later day in culture than α-BuTX did. Low temperature (28°c) and cholesterol (1 μg/ml) treatment inhibited peak fluorescence density without affecting total amount of fluorescence. These results show that the development of ACh can be characterized both by clustering peak density (indicating the lateral mobility of AChR) and by total amount of fluorescence.

Mechanisms of toxicity and tolerance to diisopropylphosphorofluoridate at the neuromuscular junction of the rat

Diisopropylphosphorofluoridate (DFP), an irreversible inhibitor of acetylcholinesterase (AChE) activity, when given as an acute dose (1.5 mg/kg, sc) caused fasciculations and induced necrosis in rat skeletal muscle fibers. No adaptation was seen to daily dosing of DFP (1.5 mg/kg, sc) since all rats died after the second or third injection. Daily dosing of DFP in a concentration (0.5 mg/kg, sc) that as a single dose did not cause symptoms, produced onset of fasciculations on the third day associated with a reduced number of muscle fiber lesions. Further administration of DFP (14 days) caused disappearance of fasciculations and loss of sensitivity to the necrotizing actions in all muscles tested (diaphragm, soleus, and extensor digitorum longus). Activity of all molecular forms of AChE was reduced to 20–24% of control when symptoms of cholinergic hyperactivity appeared. Continuous injections of DFP (0.5 mg/kg/day, sc) up to 14 days did not cause greater inhibition of AChE activity. Instead, recovery of enzyme activity, especially of the 4S and 10S forms, was seen. During this period choline acetyltransferase activity (ChAT) was increased in muscle (intramuscular nerves) while the postsynaptic nicotinic acetylcholine receptor (nAChR) density ( B max) was decreased to 44% without a change in the affinity constant ( K d). It is concluded that neuromuscular adaptation to DFP is caused by recovery of AChE activity due to de novo synthesis and reduction in the number of nAChR.

Localization of acetylcholine receptors and cholinesterase on nerve- contacted and noncontacted muscle cells grown in the presence of agents that block action potentials

The possible role of nerve activity in triggering changes in the localization of acetylcholine receptors (AChRs) and cholinesterase (ChE) on nerve-contacted Xenopus muscle cells has been assessed. The localization of these molecules was examined on nerve-contacted and noncontacted muscle cells in cultures of spinal cord and myotomal muscle derived from Xenopus embryos. Sites of high AChR density were revealed by staining with fluorescent alpha-bungarotoxin and sites of ChE localization were revealed histochemically. Localization of AChRs and ChE at sites of nerve-muscle contact occurred when the culture medium contained 1.2 micron tetrodotoxin (TTX), 1.2 micron TTX, 10 mM magnesium, and no calcium salts, 1.2 micron TTX and 2 mM manganese, or 106 mM potassium methyl sulfate instead of sodium chloride. The nerve-contacted muscle cells in each of these modified culture media also exhibited a reduced incidence of AChR and ChE patches away from the site of contact. It is concluded that the neural factor(s) that triggers the local and remote changes in AChR and ChE distribution can be supplied to the neurites and externalized in the absence of nerve impulses, and that the nerve and muscle cells can interact even when they are largely depolarized.

Variability of quantal events in control solution and after cholinesterase blockade in frog

Amplitudes of miniature end-plate currents ( A MEPCs) as well as their time constants of decay ( T MEPCs) contribute to the amplitudes of miniature end-plate potentials and hence determine their mean values and variability. Larger mean amplitudes of miniature end-plate potentials observed after cholinesterase blockade result from both greater mean A MEPCs and greater mean T MEPCs. However, enhanced variability of amplitudes of miniature end-plate potentials is a result mainly of greater variability of T MEPCs. This occurs almost exclusively because the amplitude dependence of T MEPCs becomes markedly enhanced. Although the scatter of T MEPCs about T MEPCs vs A MEPCs curve is also larger, it does not contribute much to the increased variability of T MEPCs. The marked amplitude dependence of T MEPCs, indicates that repetitive binding strongly depends on the quantal size—the number of acetylcholine molecules released per quantum, and suggests that if a quantum is the result of a release of several vesicles, then they should be released very close to each other. Marked amplitude dependence of T MEPCs also shows that, with cholinesterase inhibited, the quantal size can be estimated not only from A MEPCs, but also from T MEPCs. Greater scatter of A MEPCs, from the T MEPC vs A MEPC curve is of more obscure origin and probably results because repetitive binding occurs over enlarged “critical areas” with more variable density of acetylcholine receptors.

Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

The ultrastructure of cultured rat myotubes was examined at stages in the initial assembly of acetylcholine receptor (AChR) aggregates in order to elucidate the role of cell-surface specializations in aggregate formation. Within 4-6 hr, embryonic brain extract (EBX) induces the formation of sites of AChR density elevated 5-9 X above that of surrounding regions, and the appearance of these aggregates is preceded by the formation of clouds of punctate microaggregates (Olek et al., 1983). A video image-intensification system was used to monitor this redistribution of fluorescently labeled AChR, and sites of aggregation were mapped on identified myotubes. After processing the cultures for electron microscopy, thin sections were taken through identified aggregate sites at various stages in assembly. Specializations, including a basal lamina, mound-shaped plasma membrane contours with occasional deep infoldings, and a subjacent dense cytoskeletal specialization, which tended to exclude other cytoplasmic organelles, were associated with newly formed aggregates found 4-6 hr after adding EBX to the cultures. Analysis of random thin sections through EBX-treated and untreated myotubes showed that the extent of specializations of the basal lamina and cytoplasm was approximately threefold greater in cells exposed to EBX for 4 hr, suggesting a concurrent, and possibly interdependent, organization of such specializations with AChR aggregate assembly. Examination of sections through clouds of microaggregates, which formed within 90 min, revealed mound-shaped plasma membrane contours and underlying cytoplasm depleted of organelles but relatively little basal lamina and submembrane cytoskeletal density. These results suggest that the initial stage of AChR aggregate assembly involves relatively subtle changes in the structure of the cell cortex and that the evolution of microaggregates to aggregates may require the formation of additional cytoskeletal and extracellular matrix structures.

Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes.

Two populations of acetylcholine receptors (AChRs) are present in cultured myotubes. One forms large aggregates or clusters and the other has a much lower density of AChRs, which are diffusely distributed. Both clustered and diffuse AChRs are inserted and removed (internalized) from the sarcolemma. To determine the insertion and removal rates of AChRs in these two plasma membrane domains, we used a double label technique to distinguish and quantitate newly inserted and "old" AChRs. Application of our method revealed that the rate of AChR internalization is the same at the clustered and diffuse regions of the plasma membrane, whereas the rate of insertion is threefold greater at the clusters than elsewhere in the plasma membrane. Thus, the increase in AChR number at the clusters is not due to an increase in their half-life, but to an increase in their rate of insertion.

Changes in cholinergic parameters associated with failure of conotruncal septation in embryonic chick hearts after neural crest ablation.

Cells from the neural crest over occipital somites migrate to the heart, where they give rise to parasympathetic postganglionic neurons as well as ectomesenchymal elements which contribute to conotruncal septation. With a microcautery needle, the neural crest over occipital somites was ablated bilaterally in chicken embryos at an early stage of development. Histological examination on incubation day 15 revealed conotruncal malformations, involving malformation or absence of the conotruncal septum in all embryos. Two peaks of embryo mortality were observed. One peak (incubation days 6-8) occurred at the same time as conotruncal septal closure; the second peak (incubation days 11-13) was concurrent with the onset of functional parasympathetic innervation. A disruption of parasympathetic innervation was indicated by: (1) a decrease in acetylcholinesterase staining, (2) a decrease (27%) in the number of ganglion cells in the conotruncus, (3) decreases in the acetylcholine content of atrium (31%) and ventricle (39%), and (4) a decrease (21%) in muscarinic acetylcholine receptor density on incubation day 15. Radiolabeled ligand-binding studies revealed no change in the affinity of cardiac muscarinic receptors for [3H]methylscopolamine (KD = 0.17-0.21 nM). Agonist-binding affinity and sensitivity to guanine nucleotides were similarly unaffected. The reasons for the limited extent of the parasympathetic lesion are unclear, but may involve recruitment of precursor cells from other regions of the neural crest, partial regeneration of the neural crest following surgical removal, or an alteration in the contribution of incoming sympathetic or preganglionic parasympathetic elements. No such plasticity was associated with neural crest contributions to the structural development of the conotruncus. Malformations were observed in all lesioned embryos.

Congenital canine myasthenia gravis: I. Deficient junctional acetylcholine receptors.

The neuromuscular junction was studied during growth in two breeds of dog with a congenital familial form of myasthenia gravis (CMG): the Jack Russell terrier and the springer spaniel. Light microscopy revealed no difference in endplate size or nerve terminal morphology in age-matched CMG dogs and unaffected littermates. At all ages there was in individual muscle fibers of CMG dogs an approximately 75% lower postsynaptic membrane density of acetylcholine receptors (AChR). There was no evidence that this abnormality had an autoimmune basis. Measurements of miniature endplate potential amplitude in relation to fiber diameter revealed that the low density of AChR in the postsynaptic membrane of CMG dogs remained constant and did not change with the marked progression of muscle weakness during growth. Antigenic determinants of AChR were deficient in CMG muscle to the same extent as alpha-bungarotoxin (alpha BT) binding sites. Thus, a low density of AChR in the muscle's postsynaptic membrane appears to be the principal abnormality of CMG. The data suggest that the normal increase in number of acetylcholine (ACh) quanta released by nerve impulse may not fully compensate for the reduction in depolarization produced by a single quantum as the muscle fiber diameter increases. This could cause progression of weakness during growth. In addition to being a useful animal model of a rare form of CMG that resembles one form of human CMG, canine CMG offers a unique model for investigating events of synaptogenesis at the neuromuscular junction.

Manganese ions inhibit acetylcholine receptor synthesis in cultured mouse soleus muscles

Thin bundles dissected from mouse soleus muscles were kept in culture media containing 10% calf serum. After 3 days of culture at 37°C the muscles were tested for acetylcholine (ACh) sensitivity by measuring the force of the contracture produced by 10 μM ACh. The ACh-receptor density was measured by the [ 125I]α-bungarotoxin method. When the [Ca 2+] of the culture medium was 0.02 mM (instead of the normal 2 mM), ACh sensitivity and ACh-receptor density were not different from control. Replacement of 99% Ca 2+ by Sr 2+ or by Mg 2+ was also without effect. The muscles did not survive in solutions containing Ni ions. ACh sensitivity and ACh-receptor density were at levels characteristic of non-cultured muscles when the culture medium contained 0.4–1.8 mM Mn 2+. Since the incorporation of ACh receptors into the membrane was not affected by Mn ions these results indicate that Mn ions inhibit the synthesis of ACh receptors.

Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes.

Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.

Association of laminin and other basement membrane components with regions of high acetylcholine receptor density on cultured myotubes

The distribution of immunoreactivity to basement membrane components in cultures of rat skeletal myotubes was compared to acetylcholine receptor distribution by fluorescence microscopy. Laminin occurred in patches on the myotube surface, and most laminin patches coincided or overlapped with acetylcholine receptor aggregates. Almost all receptor aggregates coincided with laminin patches. Most of the heparan sulfate proteoglycan and fibronectin was associated with non-muscle cells, but some patches coincided with receptor aggregates on myotubes. In cultures treated with l-ascorbate, collagen types IV and V covered much of the myotube surface and receptor aggregates often coincided with intense collagen patches. When receptor aggregation was induced by treatment of cultures with soluble neural factors, the newly formed receptor aggregates coincided with laminin patches.The results suggest that each of the basement membrane components studied has a distinct distribution pattern in primary rat muscle cell cultures. The striking association of laminin immunoreactivity with receptor aggregates, together with the enhancement of receptor aggregation by laminin reported previously (Vogel et al., J. Neurosci. 3, 1058–1068, 1983) suggests that laminin could have a role in the organization of acetylcholine receptors on developing muscle fibers.

Regulation of acetylcholine receptor density in membranes of denervated mouse muscles.

The ACh sensitivity of denervated muscles of rats stimulated in situ or in culture for several days decreases to low levels characteristic of normal muscles. Possible causes are the electrical activity, depolarization per se, contraction and intracellular [Ca2+] changes. To test the last three hypotheses, isolated bundles of denervated mouse soleus muscles were placed in narrow chambers (internal diameter 2 mm) which were periodically perfused with standard mixtures of Minimal Essential Medium (MEM) and calf serum. At 10-15-min intervals the chambers were filled for 15-43 s with a Ringer solution containing 55 microM ACh or 8-10 mM caffeine or with a solution containing 200 mM KCl. After 3-7 days the muscles were tested for ACh sensitivity by comparing the force developed in response to 110 microM ACh and that to 400 mM K methanesulphonate. In addition, the ACh receptor density was measured with 125I alpha-bungarotoxin. The results showed no difference in ACh sensitivity or receptor density between treated and untreated muscles. Small differences produced by caffeine were probably caused by muscle damage. Ca influx, but not efflux, was strongly elevated during exposure to ACh. It was concluded that neither depolarization per se nor contractile activity or the associated calcium movements have any effect on ACh receptors.

Developmental changes in the distribution of acetylcholine receptors in the myotomes of Xenopus laevis.

The acquisition and distribution of nerve fibres and of acetylcholine (ACh) receptors were examined in the myotomes of Xenopus laevis during normal development. This muscle is well-suited for investigating temporal relationships during neuromuscular synaptogenesis because the age of the Xenopus embryo at the onset of innervation can be assessed with an accuracy of about one hour. Myotomal nerve fibres were visualized after staining them with nitroblue tetrazolium and ACh receptors were examined after exposure to alpha-bungarotoxin labelled with 125I or fluorescent dye. Nerve fibres were seen in the myotomes of some embryos as early as stage 19 (20 . 75 hr) and in virtually all embryos by stage 24 (26 . 25 hr). From the outset they were located mainly at the ends of the myotomes, but some myotomes also exhibited nerve fibres in more central regions. ACh receptors were already present in myotomes by stage 19 (20 . 75 hr) and initially had a widespread, uniform distribution. The density of extrajunctional ACh receptors increased until stage 36 (50 hr) and then declined less than 3-fold over the next 10 days of development. Discrete patches of high ACh receptor density began to appear at the ends of the myotomes at stage 22 (24 hr) and were seen in almost all embryos by stage 26 (29 . 5 hr). ACh receptor patches were also seen in central regions of some myotomes and these were usually aligned in patterns which resembled the course of nerve fibres. The present findings suggest that myotomal muscle cells in Xenopus embryos begin to acquire ACh receptors shortly before the arrival of nerve fibres and that discrete patches of ACh receptors begin to form at presumptive synaptic sites on the average about 3 hr after the arrival of the nerve fibres. The latter delay is considerably shorter than that in developing rat muscle. The temporal and spatial relationships between nerve fibres and the development of ACh receptor patches in Xenopus myotomes in vivo are consistent with findings in Xenopus cell cultures which indicate that nerve fibres can rapidly induce ACh receptor localization at sites of nerve--muscle contact.

Absence of [125I] alpha-bungarotoxin binding to motor nerve terminals of frog, lizard and mouse muscle

The existence of nicotinic acetylcholine receptors (AChRs) on the motor nerve terminals of vertebrates has long been controversial. We have re-examined this issue by electron microscope autoradiography with [125I] alpha-bungarotoxin, following separation of nerve terminals from muscle fibers by collagenase and protease treatment. We found no label over nerve terminal membranes other than that due to background, and we calculate upper limits of less than 0.1% of the postsynaptic AChR density for nerve terminals in frogs, lizards, and mice. We conclude that there are essentially no presynaptic acetylcholine receptors that bind alpha-bungarotoxin at vertebrate neuromuscular junctions.