scholarly journals Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts.

1991 ◽  
Vol 115 (1) ◽  
pp. 165-177 ◽  
Author(s):  
D S Hartman ◽  
N S Millar ◽  
T Claudio
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hybrid Cell ◽  
Muscle Cells ◽  
Specific Gene ◽  
Temperature Sensitive ◽  
Antigenic Modulation ◽  
Achr Clustering

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.

scholarly journals Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells.

1988 ◽  
Vol 107 (3) ◽  
pp. 1147-1156 ◽  
Author(s):  
L S Higgins ◽  
D K Berg
Keyword(s):  
Plasma Membrane ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Chromaffin Cells ◽  
Splanchnic Nerve ◽  
Temperature Sensitive ◽  
Adrenal Chromaffin Cells ◽  
Antigenic Modulation ◽  
Nicotinic Acetylcholine ◽  
Adrenal Chromaffin

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

1984 ◽  
Vol 4 (3) ◽  
pp. 475-483 ◽  
Author(s):  
C D Lobaton ◽  
A Moreno ◽  
D L Oxender
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Hybrid Cell ◽  
Chinese Hamster ◽  
Hybrid Cell Line ◽  
Temperature Sensitive ◽  
Acid Transport ◽  
Temperature Resistance ◽  
System L ◽  
Leucine Transport

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

Functional role of nicotinic acetylcholine receptors in apoptosis in HL-60 cell line

2003 ◽  
Vol 482 (1-3) ◽  
pp. 25-29 ◽  
Author(s):  
Delphine Gimonet ◽  
Regis Grailhe ◽  
Paul Coninx ◽  
Frank Antonicelli ◽  
Bernard Haye ◽  
...  
Keyword(s):  
Cell Line ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Functional Role ◽  
Nicotinic Acetylcholine

Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation

1988 ◽  
Vol 254 (3) ◽  
pp. C345-C364 ◽  
Author(s):  
R. J. Bloch ◽  
D. W. Pumplin
Keyword(s):  
Cell Surface ◽  
Muscle Cell ◽  
Acetylcholine Receptors ◽  
Membrane Skeleton ◽  
Postsynaptic Membrane ◽  
Molecular Assembly ◽  
Trophic Factors ◽  
Achr Cluster ◽  
Nerve Muscle ◽  
Achr Clustering

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane of newly innervated muscle fibers is one of the earliest events in the development of the vertebrate neuromuscular junction. Here, we describe two hypotheses that can account for AChR clustering in response to innervation. The "trophic factor" hypothesis proposes that the neuron releases a soluble factor that interacts with the muscle cell in a specific manner and that this interaction results in the local accumulation of AChR. The "contact and adhesion" hypothesis proposes that the binding of the nerve to the muscle cell surface is itself sufficient to induce AChR clustering, without the participation of soluble factors. We present a model for the molecular assembly of AChR clusters based on the contact and adhesion hypothesis. The model involves the sequential assembly of three distinct membrane domains. The first domain to form serves to attach microfilaments to the cytoplasmic surface of the muscle cell membrane at sites of muscle-nerve adhesion. The second domain to form is clathrin-coated membrane; it serves as a site of insertion of additional membrane elements, including AChR. Upon insertion of AChR into the cell surface, a membrane skeleton assembles by anchoring itself to the AChR. The skeleton, composed in part of actin and spectrin, binds and immobilizes significant numbers of AChR, thereby forming the third membrane domain of the AChR cluster. We make several predictions that should distinguish this model of AChR clustering from one that invokes soluble, trophic factors.

scholarly journals Temperature-Sensitive Expression of Drosophila Neuronal Nicotinic Acetylcholine Receptors

2002 ◽  
Vol 68 (5) ◽  
pp. 1812-1819 ◽  
Author(s):  
Stuart J. Lansdell ◽  
Bertram Schmitt ◽  
Heinrich Betz ◽  
David B. Sattelle ◽  
Neil S. Millar
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Temperature Sensitive ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Nicotinic Acetylcholine

scholarly journals High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

2017 ◽  
Vol 22 (6) ◽  
pp. 686-695 ◽  
Author(s):  
Lucas C. Armstrong ◽  
Glenn E. Kirsch ◽  
Nikolai B. Fedorov ◽  
Caiyun Wu ◽  
Yuri A. Kuryshev ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Line ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Brain Regions ◽  
Tobacco Product ◽  
Tobacco Products ◽  
Nicotinic Acetylcholine ◽  
Automated Patch Clamp ◽  
Subtype Selective

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity

1984 ◽  
Vol 4 (3) ◽  
pp. 475-483
Author(s):  
C D Lobaton ◽  
A Moreno ◽  
D L Oxender
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Hybrid Cell ◽  
Chinese Hamster ◽  
Hybrid Cell Line ◽  
Temperature Sensitive ◽  
Acid Transport ◽  
Temperature Resistance ◽  
System L ◽  
Leucine Transport

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

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