Abstract. 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 Ls. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR ct-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the B-, 7-, and &subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, ot-actin, and creatine kinase. By in situ hybridization, we further show that the increase in a-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei, mRNA levels for the ot-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 oL-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 a-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR a-subunit mRNA.

Extracts of fetal calf brain cause a 3-to 5-fold increase in acetylcholine receptors (AcChoR) on cultured myogenic L5 cells. Purification of the substance causing the major portion of this receptor increase has been completed. Ultraviolet spectral characteristics, nuclear magnetic resonance, mass spectra, and AcChoR induction by the active factor are the same as those of commercially available ascorbic acid. The biological activity of ascorbic acid is not mimicked by reducing agents with or without sullhydryl groups. Compounds related to ascorbic acid were tested for their ability to induce AcChoR increases on L5 cells. D-Isoascorbic acid is the only substance with identical biological activity to ascorbic acid. Dehydroascorbic acid and ascorbic acid 2-0-sulfate also induce AcChoR increases but with lower specific activity. These data show that ascorbic acid can play a role in regulating AcChoR expression in myogenic tissue, and the presence of ascorbic acid in the purified fraction from fetal calf brain accounts for its ability to increase AcChoR in L5 cells.A major effect of coculturing rat spinal cord explants with L6 myogenic cells is an increase in the average site density of acetylcholine receptors (AcChoR) on the differentiated myotubes. The increase in receptor site density on myotubes adjacent to the explant is greater than that occurring on myotubes a few millimeters away. This graded distribution has been taken as an indication of the release, from the explant, of a diffusible substance causing the receptor increase (1). Similar results have been obtained in cocultures derived from chicken embryos (2). In addition to the increase in receptor site density, there is also an increase in the number of AcChoR clusters in these cultures (1, 2).Since it appears that the effects on AcChoR clustering and site density may be caused by different substances (3), in the present study we directed our attention toward the overall increase in AcChoR. This effect is of interest because changes in average AcChoR site density also occur during muscle development in vivo (4). Shortly after the fusion of mononucleated myoblasts, AcChoR levels increase dramatically (5, 6), and then decrease following innervation (7,8). Denervation of adult skeletal muscle results in a return to preinnervation site densities (9-11). The purification and identification of a soluble factor capable of inducing AcChoR site density increases offers an approach to the study of in vivo AcChoR site density regulation and, possibly, the control of AcChoR synthesis as well.The effects of nerve explants on AcChoR site density in myogenic tissue can be mimicked by soluble extracts of fetal rat, chicken, or calf brain (1,(12)(13)(14). We have used the increase in total AcChoR on L5 cells caused by fetal calf brain extracts (14) as an assay for the purification of the active factor. The L5 (46) cell line has similar characteristics to the L6 cell line isolated by Yaffe (15). We report here the identification of the material from fetal calf brain ...

Abstract. Ascorbic acid in soluble extracts of neural tissue can account for the increase in surface acetylcholine receptors (AChR's) seen on L5 myogenic cells treated with crude brain extract (Knaack, D., and T.R. Podleski, 1985, Proc. Natl. Acad. Sci. USA., 82:575-579). The present study further elucidates the nature of the response of L5 cells to ascorbic acid. Light autoradiography showed that ascorbic acid treatment affects both the number and distribution of surface AChR's. Ascorbic acid, like crude brain extracts, caused a three-to fourfold increase in average AChR site density. However, the number of AChR clusters induced by ascorbic acid was only one-fifth that observed with crude brain extract.The rate constant for degradation of AChR in ascorbic acid-treated cells of 0.037 __. 0.006 h -1 (t,/2 = 19 h) was not significantly different from that in untreated controls of 0.050 ___ 0.001 h -1 (t,/2 = 14 h). The increase in AChR site density is primarily due to a 2.8-fold increase in the average rate of AChR incorporation. Ascorbic acid also stimulates thymidine incorporation and increases the total number of nuclei per culture. However, cellular proliferation is not responsible for the increase in AChR's since 10 #M cytosine arabinofuranoside blocks the mitogenic effect without affecting the AChR increase. The specificity of ascorbic acid on AChR expression was established by showing that (a) ascorbic acid produced only a slight increase in total protein, which can be accounted for by the mitogenic effect, and (b) the normal increase seen in creatine kinase activity during muscle differentiation was not altered by the addition of ascorbic acid.We conclude that the action of ascorbic acid on AChR number cannot be explained by changes in cell growth, survival, differentiation, or protein synthesis. Therefore, in addition to a minor stimulation of AChR clustering, ascorbic acid specifically affects some aspect of the AChR biosynthetic pathway.W HEN spinal cord explants are co-cultured with myogenic cells, the expression of acetylcholine receptors (AChR's)' on myotubes is affected primarily in two ways. The overall receptor site density is increased and discrete receptor clusters are formed (9, 34). Several studies have suggested that the spinal cord explants could affect AChR number and distribution via the release of soluble factors (2, 7, 17, 34, 4 l).Studies using partially purified brain and nerve-derived substances indicate that more than one factor may affect AChR expression. Formation of receptor clusters has been observed in a number of systems and has been associated with components having a range of molecular weights (2,17,28,34). But according to Neugebauer et al. (3 l), clustering activity is greatest in the high molecular weight range. On the other hand, significant increases in the overall AChR site density have only been demonstrated on primary chick skeletal muscle (17) and the L5 and L6 cloned cell lines (21,23,31,34), and are produced by substance(s) with apparent molecular weight(s) of < ...

A number of pancreatic beta-tumor cell (beta TC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a beta TC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for > 55 passages (over a year) in culture. One of these clonal lines, denoted beta TC6-F7, was characterized in detail. beta TC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal beta-cells. These results demonstrate that transformed beta-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous beta-cell lines for transplantation therapy of diabetes.

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