scholarly journals Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

1992 ◽  
Vol 118 (5) ◽  
pp. 1201-1212 ◽  
Author(s):  
K W Tsim ◽  
I Greenberg ◽  
M Rimer ◽  
W R Randall ◽  
M M Salpeter
Keyword(s):  
In Situ Hybridization ◽  
Acetylcholine Receptor ◽  
Gaussian Distribution ◽  
Muscle Cells ◽  
Skewed Distribution ◽  
Alpha Subunit ◽  
Subunit Mrna ◽  
Nuclear Label ◽  
The Mean

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

scholarly journals Regulation of acetylcholine receptor synthesis at the level of translation in rat primary muscle cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1817-1822 ◽  
Author(s):  
O Horovitz ◽  
V Spitsberg ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Alpha Subunit ◽  
Brain Extract ◽  
Primary Cells ◽  
Active Components ◽  
Subunit Mrna ◽  
Threefold Increase

Previous studies have shown that rat primary muscle cells do not respond to crude rat brain extract or one of its active components, ascorbic acid, with a significant increase in surface acetylcholine receptor (AChR) number. We report here that, although little or no response is seen on the cell surface, rat primary muscle cells do respond to both crude brain extract and to ascorbic acid with an approximately threefold increase in AChR alpha-subunit mRNA. The response of the mRNA is similar to that seen in the cloned L5 cells. However, while in L5 cells the increase in alpha-subunit mRNA is further translated into increased levels of alpha-subunit protein, there is no such increase in alpha-subunit synthesis in the primary cells. This study thus shows a regulation of surface AChR synthesis in rat primary cells at the level of alpha-subunit translation. This level of regulation is different from that involving subunit transcription or subunit assembly reported by others.

In situ hybridization localization of the Na+ channel β1 subunit mRNA in rat CNS neurons

1994 ◽  
Vol 176 (1) ◽  
pp. 119-122 ◽  
Author(s):  
Youngsuk Oh ◽  
Shunsuke Sashihara ◽  
Stephen G. Waxman
Keyword(s):  
In Situ Hybridization ◽  
Na Channel ◽  
Subunit Mrna ◽  
Cns Neurons ◽  
Rat Cns

scholarly journals Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.

1989 ◽  
Vol 108 (3) ◽  
pp. 1025-1037 ◽  
Author(s):  
B Fontaine ◽  
J P Changeux
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Muscle Development ◽  
Neuromuscular Junctions ◽  
Primary Cultures ◽  
Motor Endplate ◽  
Alpha Subunit ◽  
Subunit Gene ◽  
Nicotinic Acetylcholine

In 15-d-old chick latissimi dorsi muscles, the nicotinic acetylcholine receptor (AChR) alpha-subunit mRNA is densely accumulated at the level of subsynaptic nuclei of the motor endplate (Fontaine et al., 1988). In this paper, using in situ hybridization with genomic probes, we further show that the expression of the AChR alpha-subunit gene in the embryo, revealed by the accumulation of mature mRNAs, starts in myotomal cells and persists during the first stages of muscle development in a majority of muscle nuclei. Subsequently, the distribution of AChR alpha-subunit mRNAs becomes restricted to the newly formed motor endplates as neuromuscular junctions develop. To assess the transcriptional activity of individual nuclei in developing muscles, a strictly intronic fragment of the AChR alpha-subunit gene was used to probe in situ the level of unspliced transcripts. AChR alpha-subunit unspliced transcripts accumulate around a large number of sarcoplasmic nuclei at embryonic day 11, but can no longer be detected at their level after embryonic day 16 in the embryo. A similar decrease in the accumulation of AChR alpha-subunit transcripts is observed between day 4 and day 6 in primary cultures of muscle cells. On the other hand, in vivo denervation and in vitro blocking of muscle electrical activity by the sodium channel blocker tetrodotoxin results in an increase in the labeling of muscle nuclei. Yet, only 6% of the muscle nuclei appear labeled by the strictly intronic probes after denervation. The possible significance of such heterogeneity of muscle nuclei during motor endplate formation in AChR gene expression is discussed.

scholarly journals BiP forms stable complexes with unassembled subunits of the acetylcholine receptor in transfected COS cells and in C2 muscle cells

1992 ◽  
Vol 117 (4) ◽  
pp. 841-847 ◽  
Author(s):  
JR Forsayeth ◽  
Y Gu ◽  
ZW Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Stable Complex ◽  
Cos Cells ◽  
Toxin Binding ◽  
Immunoglobulin Binding Protein ◽  
Alpha Bungarotoxin ◽  
Immunoglobulin Binding

We have investigated the role of the immunoglobulin-binding protein (BiP) in the folding and assembly of subunits of the acetylcholine receptor (AChR) in COS cells and in C2 muscle cells. Immunoprecipitation in COS cells showed that alpha, beta, and delta subunits are associated with BiP. In the case of the alpha subunit, which first folds to acquire toxin-binding activity and is then assembled with the other subunits to form the AChR, BiP was associated only with a form that is unassembled and does not bind alpha-bungarotoxin. Similar results were found in C2 cells. Although the alpha and beta subunits of the AChR are minor membrane proteins in C2 cells, they were prominent among the proteins immunoprecipitated by antibodies to BiP, suggesting that BiP could play a role in their maturation or folding. In pulse-chase experiments in C2 cells, however, labeled alpha subunit formed a stable complex with BiP that was first detected after most of the alpha subunit had acquired toxin-binding activity and whose amount continued to increase for several hours. These kinetics are not compatible with a role for the BiP complex in the folding or assembly pathway of the AChR, and suggest that BiP is associated with a misfolded form of the subunit that is slowly degraded.

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