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Biomedical subjects

Z W Hall

Publications and source records attributed to Z W Hall.

At least 73 records · Page 4Linked to original sources
PubMed1987

Genetic variants of C2 muscle cells that are defective in synthesis of the alpha-subunit of the acetylcholine receptor.

We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.

Animals↗
PubMed1987

Isolated muscle cells as a physiological model.

Summary of a symposium presented by the American Physiological Society (Cell and General Physiology Section and Muscle Group) at the 70th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Missouri, April 15, 1986, chaired by M. Lieberman and F. Fay. This symposium reflects a growing interest in seeking new technologies to study the basic physiological and biophysical properties of cardiac, smooth, and skeletal muscle cells. Recognizing that technical and analytical problems associated with multicellular preparations limit the physiological significance of many experiments, investigators have increasingly focused on efforts to isolate single, functional embryonic, and adult muscle cells. Progress in obtaining physiologically relevant preparations has been both rapid and significant even though problems regarding cell purification and viability are not fully resolved. The symposium draws attention to a broad, though incomplete, range of studies using isolated or cultured muscle cells. Based on the following reports, investigators should be convinced that a variety of experiments can be designed with preparations of isolated cells and those in tissue culture to resolve questions about fundamental physiological properties of muscle cells.

Animals↗
PubMed1985

Developmental regulation of laminin accumulation in the extracellular matrix of a mouse muscle cell line.

We have investigated the synthesis, accumulation, and secretion of laminin, an extracellular matrix glycoprotein, during differentiation of the C2 mouse skeletal muscle cell line in culture. Myoblasts actively synthesized laminin, as measured by incorporation of [35S]methionine and by a dot-immunobinding assay. In myoblast cultures laminin accumulated in an intracellular compartment and could be extracted with a physiological salt solution containing the detergent Triton X-100. After the culture medium was replaced to promote differentiation of myoblasts to myotubes, laminin synthesis was increased, and laminin began to accumulate in the medium in soluble form. During differentiation, laminin also accumulated in an insoluble cell-associated fraction that required guanidinium chloride for extraction. Indirect immunofluorescence and immunobinding assays showed that myotubes but not myoblasts contained laminin on their external surface. The time course of increase in surface laminin paralleled that of the accumulation of insoluble laminin. These results suggest that the insoluble fraction represents laminin bound to the extracellular matrix at the cell surface. Our experiments demonstrate, contrary to previous observations, that myotube cultures synthesize and accumulate laminin, and further, that the differentiation of proliferating myoblasts to multinucleated myotubes is accompanied by increased laminin synthesis, by secretion of laminin into the medium, and by the deposition of laminin into an extracellular matrix on the myotube surface.

Animals↗
PubMed1985

Use of a replica technique to isolate muscle cell lines defective in expressing the acetylcholine receptor.

We have isolated genetic variants of the C2 muscle cell line that are defective in expressing the acetylcholine receptor (AcChoR). Because the AcChoR is expressed only after C2 myoblasts have fused to form myotubes, we employed a replica technique to detect the variants. This technique yields two copies of each clone, one of which can be used for screening and the other, as a source of dividing cells. In a screening of about 10,000 clones derived from mutagenized cells, we found 2 that fused normally and expressed normal levels of acetylcholinesterase but had reduced amounts of AcChoR on their surface. One of these also had a reduced level of intracellular AcChoR, but, in the other, the amount of intracellular AcChoR was 5-fold higher than normal. Several variants were found that failed to fuse and had reduced levels of both AcChoR and acetylcholinesterase. Though we relied on 125I-labeled alpha-bungarotoxin to distinguish wild-type from deficient clones, we found that an antiserum to the AcChoR, followed by a biotinylated second antibody and a horseradish peroxidase-avidin complex, could also be used. Therefore, it should be possible to obtain muscle cell variants defective in the expression of a variety of proteins for which specific antibodies are available.

Animals↗
PubMed1985

Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.

Amino Acid Sequence↗
PubMed1985

Myasthenic serum selectively blocks acetylcholine receptors with long channel open times at developing rat endplates.

We have examined the physiological effects of antibodies from a highly specific myasthenic serum on acetylcholine receptors at developing rat endplates. The antibodies reduced the amplitude of miniature endplate potentials by 60% in 3- to 6-day-old animals but had no effect after day 14. Between days 7 and 12 the antibodies had an intermediate effect. This is the same period during which acetylcholine receptors with long channel open times (slow channels) disappear and receptors with short open times (fast channels) increase in number. Therefore, we examined the effect of the antibodies at endplates with a mixture of channel types more carefully. At all times tested, both noise analysis and analysis of miniature endplate currents showed that the antibodies reduced slow channel activity selectively. Single-channel recordings indicated that acetylcholine receptors that remained active after antibody treatment had normal gating properties. Thus, the antibodies appeared to silence slow channels selectively.

Animals↗
PubMed1985

Atypical distribution of asymmetric acetylcholinesterase in mutant PC12 pheochromocytoma cells lacking a cell surface heparan sulfate proteoglycan.

We studied the distribution of the molecular forms of acetylcholinesterase (AChE) in a stable variant (F3) of the rat pheochromocytoma cell line, PC12, that lacks a heparan sulfate proteoglycan on the cell surface. After treatment with nerve growth factor F3 cells synthesize less 4S enzyme, and more 10S and 16S enzyme than normal PC12 cells. This distribution is similar to that seen in normal cells after incubation with beta-D-xylosides, molecules that interfere with proteoglycan assembly. Using collagenase treatment and membrane-permeable and -impermeable inhibitors of AChE, we determined the cellular location of the AChE forms. Although in normal cells greater than 90% of the 16S AChE is on the cell surface, approximately 60% is present in an internal pool in the variant. Following irreversible inhibition of all forms of AChE in the variant, the newly synthesized 16S AChE appears in the internal pool after a 1-h lag, but is not detected on the cell surface until after 2.5 h. Our results thus show that 16S AChE is assembled internally within neuronal cells and that alterations in the synthesis and distribution of proteoglycans affect the total amount and cellular localization of the 16S AChE form.

Acetylcholinesterase↗
PubMed1985

A postnatal change in the immunological properties of the acetylcholine receptor at rat muscle endplates.

We have used a myasthenic serum that in adult rat muscle is specific for acetylcholine receptors (AChRs) in the extra-junctional membrane to characterize the AChRs at developing endplates. Immunocytochemical experiments show that AChRs at endplates in the rat diaphragm bind the myasthenic antibodies during the first week after birth but lose their reactivity during the second and third postnatal weeks. AChRs at endplates in adult rat diaphragm do not bind the antibodies even after denervation; in contrast, AChRs at endplates in an adult chicken muscle (anterior latissimus dorsi) are recognized by the antibodies. The loss of immunological reactivity thus may be correlated with a change in the channel properties of the AChR and with the appearance of synaptic folds, two postnatal developmental changes that occur at the endplates of rats, but not of chickens.

Animals↗
PubMed1985

The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. I. Immunological distinction between the two toxin-binding sites of the receptor.

We have examined the effect of a serum from a patient with myasthenia gravis on the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptors (AChRs) of a mouse muscle cell line, C2. After a 2-hr incubation, antibodies in the serum reduced toxin binding to C2 myotubes to a maximal extent of approximately 50%. The degradation of surface AChRs could account for the loss of only 5% of sites during the incubation; the remainder, therefore, must have been lost by blockage of binding. To investigate whether the antibodies blocked specifically one of the two toxin-binding sites that each AChR possesses, we used an analysis based on that of Sine, S. W., and P. Taylor, [1981) J. Biol. Chem. 255: 10144-10156). Although the two sites could not be distinguished by their rates of binding of alpha-BuTx, d-tubocurarine (dTC) inhibition of the initial rate of toxin binding revealed that the sites had affinities for dTC that differed by approximately 30-fold. Incubation with the myasthenic antibodies reduced the number of high affinity dTC sites, without affecting those of low affinity. We conclude that the two toxin-binding sites of the AChR are immunologically distinct.

Animals↗
PubMed1984

Acetylcholine receptors from Torpedo californica membrane vesicles are metabolized after fusion with cultured mammalian muscle cells.

Polyethylene glycol was used to fuse Torpedo californica membrane vesicles containing acetylcholine receptors to monolayers of mammalian muscle cells in culture. Ten to 15% of the transferred Torpedo receptors had an outwardly-directed alpha-bungarotoxin binding site and were degraded by the host cells with an apparent halftime of 30-40 h. The apparent half-time of degradation of host cell AChRs in contrast, was 8-12 h. Treatment of the cells with NaCN or puromycin reduced the rate of degradation of both endogenous and Torpedo acetylcholine receptors. The remaining Torpedo receptors that were associated with the host cells were not accessible to alpha-bungarotoxin, but were in membrane vesicles that were sloughed off into the medium. These data suggest that a portion of the transferred receptors are integrated into the host cell membranes.

Animals↗
PubMed1984

Acetylcholinesterase is functional in embryonic rat muscle before its accumulation at the sites of nerve-muscle contact.

We have examined the expression, the location, and the physiological activity of acetylcholinesterase (AChE) in developing intercostal muscles in the rat. Although focal accumulations of AChE at developing end plates do not appear until Embryonic Day (ED) 16-17, 16 S AChE is present at ED 14. Experiments with permeable and impermeable inhibitors established that prior to focal accumulation most of the 16 S enzyme is on the surface of muscle fibers, where it constitutes the major species. Intracellular recording from developing muscle fibers showed that as early as ED 14, AChE inhibitors prolonged evoked end-plate potentials. We conclude that prior to its focal accumulation, AChE is present on the surface of muscle fibers and is physiologically active. Histochemical staining of the focally accumulated enzyme demonstrated that the enzyme is concentrated both intracellularly and extracellularly at the sites of developing nerve-muscle contacts.

Acetylcholinesterase↗
PubMed1984

Redistribution of acetylcholine receptors on developing rat myotubes.

The mechanism of formation of acetylcholine receptor (AChR) clusters at developing mammalian endplates was investigated in vitro, using intercostal muscles from embryonic rats. The muscles were explanted in organ culture with the spinal cord attached, as described previously (Ziskind-Conhaim, L., and M. J. Dennis (1981) Dev. Biol. 85: 243-251). AChRs on the myofibers were labeled with [125I]-alpha-bungarotoxin shortly before clusters appeared and subsequently were cultured in unlabeled toxin for 1 day. Autoradiography of the cultured fibers demonstrated the presence of labeled clusters of AChRs indicating that the AChRs in the newly formed clusters arise from AChRs that had previously been uniformly distributed on the muscle surface.

Animals↗
PubMed1983

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line.

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.

Acetylcholinesterase↗
PubMed1983

Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin.

We have used immunocytochemical methods to investigate the cytoskeletal constituents of the vertebrate neuromuscular junction. Specific, affinity-purified antibodies to three cytoskeletal proteins, vinculin, alpha-actinin, and filamin, bound to neuromuscular junctions in sections of normal rat, mouse, chick, and Xenopus muscles. All three antibodies bound to the synaptic regions of denervated rat muscle fibers, indicating that the proteins recognized by these antibodies are associated with postsynaptic structures. The three proteins are present at the neuromuscular junction in muscle fibers of embryonic and neonatal animals, and therefore, may play an important role in its differentiation.

Actinin↗
PubMed1982

Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells.

Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16S form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16S enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16S acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16S form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholinesterase activity on the cell surface. Collagenase treatment, which removes only the 16S form, abolishes this staining pattern, indicating that the patches consist of the 16S enzyme. We conclude that the 16S enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix.

Acetylcholinesterase↗