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T R Podleski

Publications and source records attributed to T R Podleski.

At least 19 recordsLinked to original sources
PubMed1993

Secreted collagen induced by ascorbic acid in L5 cloned muscle cultures does not affect acetylcholine receptor expression.

Previous studies have shown that ascorbic acid increases both the total surface acetylcholine receptor (AChR) expression and the mRNA for the alpha-subunit of this receptor in myotubes of cloned L5 muscle cultures. Since ascorbic acid increases collagen synthesis in fibroblasts, we studied the effect of ascorbic acid on collagen secretion in L5 muscle cells and investigated the possibility that the effects of ascorbic acid on collagen and AChR are related. We report that L5 muscle cells secrete collagen types I, III, and V, with collagen type I being the most abundant species, and that accumulation of secreted collagens increased in the medium approximately two- to ninefold within 3 h of ascorbic acid treatment. The increase in surface AChRs, on the other hand, developed more slowly, and was detected only about 20-24 h after ascorbic acid treatment. A short (5 h) treatment with ascorbic acid is, however, sufficient to trigger an increase in AChRs 24 h later. Since ascorbic acid caused a rapid increase in collagen secretion, whereas the effect on total surface AChRs occurs more slowly, we tested the possibility that an increase in secreted collagen might be necessary for the increase in AChRs. However, when the L5 cultures were treated with bacterial collagenase, the ascorbic acid-induced increase in secreted collagen was abolished but its inductive effect on AChRs was unchanged. The increase in secreted collagen is therefore not necessary for the increase in AChRs to occur.

Animals↗
PubMed1991

Acetylcholine receptor regulation in L5 muscle cells is independent of increases in collagen secretion induced by ascorbic acid.

Ascorbic acid is the active component of fetal brain extract that induces increased acetylcholine receptor (AChR) expression in L5 rat clonal muscle cell cultures. The induction of AChR expression, as determined by 125I-alpha-bungarotoxin binding, occurs with a delay of 20-25 h. We report that the delayed increase in AChR can be triggered by a 5-h exposure to ascorbic acid. These studies suggest that intermediary processes may be involved. Ascorbic acid treatment also causes a threefold increase in collagen secretion in L5 cultures by 3 h. The rapid increase in collagen secretion and the delayed induction of surface AChR suggested that there may be a link between these two responses. However, although bacterial collagenase eliminates secreted collagen, it had no effect on the increase in surface AChR. Thus, the ascorbic acid effect on elevating AChR expression is independent of its effect on collagen secretion.

Animals↗
PubMed1989

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

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.

Animals↗
PubMed1988

Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata).

We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors (MAb 35 of Tzardos et al., J. Biol. Chem., 250: 8635-8645, '81; and MAb 270 of Whiting and Lindstrom: J. Neurosci. 6: 3061-3069, '86). Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei. Lobus parolfactorius and nucleus vestibularis medialis were labelled by only MAb 270, whereas only MAb 35 labelled nucleus laminaris and the medial and lateral pontine nuclei. These data extend previous reports of cholinergic participation in the song system (Ryan and Arnold: J. Comp. Neurol. 202: 211-219, '81) to suggest that the zebra finch song system may contain several closely related nicotinic receptors. In several brain nuclei it appeared that certain anatomical portions of a nucleus or a certain class of neurons were specifically labelled. Furthermore, in certain cases, the labelling appeared to be clustered around Nissl-stained cell nuclei, thus suggesting that the receptors are concentrated on or in somata.

Animals↗
PubMed1988

Acetylcholine receptor clustering and triton solubility: neural effect.

Previous studies by Prives et al. (1980, 1982a and b) have shown that acetylcholine receptors (AchRs) are extracted from muscle cells in vitro by Triton X-100 at different rates, and that clustered receptors extract most slowly. The present study was aimed at comparing the relative extractability of receptors in clusters with those in intercluster regions and the role of neural factors in regulating this extractability. Using primary rat muscle cells in vitro we confirmed that receptor extraction with Triton X-100 does not fit a single exponential but has more than one rate, and that in control cells clustered receptors extract more slowly than do receptors in intercluster regions. The major new observation in this study was that neural extract lowered the overall Triton extraction rate of intercluster receptors to that of clustered receptors. Additional new observations include the findings that (1) both clustered and intercluster receptors show multiphasic extraction rates; (2) stabilization of AchRs against Triton extraction increases with time in the surface membrane; (3) the effect of neural extract on Triton extractability of AChR is dependent on factors that control RNA synthesis, cytoskeletal elements, and collagen; (4) fixation and/or buffer washes accelerate receptor extraction only in cells that are treated with Triton, but not in control cells; (5) in control cells (not exposed to neural factors) Triton X-100 causes new clusters to form. From experiments using Con A we suggest that the Triton-induced new clusters may not be formed by a redistribution of receptors but are, most likely, due to the presence of groups of intercluster receptors with extraction rates lower than those of surrounding receptors.

Aminopropionitrile↗
PubMed1986

Effects of pineal factors on the action potentials of sympathetic neurons.

Neurons from rat superior cervical ganglia were grown in coculture with pineal cells. Action potentials of neurons in cocultures were 25% longer and were 25% greater in amplitude than those recorded from neurons grown in the presence of ganglionic nonneuronal cells alone. Neurons showed an increase in action potential duration with increasing time in culture. This may have been related to the recovery of nonneuronal cell populations after an initial exposure to the antimitotic agent Ara-C. In cultures not initially exposed to Ara-C, a subsequent exposure after 7 days in culture resulted in a shortening of the action-potential duration. Neuronal cultures were exposed to gel slabs containing the pineal indolamines, serotonin, N-acetylserotonin, and melatonin. Serotonin and N-acetylserotonin showed no effect on the neuronal action potentials at the concentrations tested. Melatonin caused an increase in action-potential duration that was associated not with an increase in action-potential amplitude, but with a decrease in action-potential rise rates. The effects of long-term exposure in melatonin appeared to be reversible in some cells but not in all. Short-term effects of melatonin were observed in older cultures and in younger cultures after the cells were stimulated repeatedly. Older cultures also had higher levels of spontaneous activity. The dependence of the short-term effects of melatonin on electrical activity may suggest a role for melatonin as a neuromodulator.

Action Potentials↗
PubMed1986

Selective effects of ascorbic acid on acetylcholine receptor number and distribution.

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 (t1/2 = 19 h) was not significantly different from that in untreated controls of 0.050 +/- 0.001 h-1 (t1/2 = 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 microM 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.

Animals↗
PubMed1985

Differential responses of L5 and rat primary muscle cells to factors in rat brain extract.

Crude brain extract (100,000 g supernate from newborn or fetal rat brain homogenate) was studied for its effects on the number and distribution of acetylcholine receptors (AChRs) on myotubes of the L5 cloned myogenic cell line and compared to that of rat primary cultures. Gamma counting, light autoradiography and scanning electron microscopic autoradiography were used. We found that the L5 cells responded to the brain extract with an increase in the average AChR site density (2-5-fold) and with an increase in AChR clustering. Clustering was manifested by both an increase in the number of AChR clusters and in the ratio of receptor site density within clusters relative to that between clusters. The increase in average AChR site density was shown to be due to an increase in the rate of AChR insertion into the surface membrane with little change in the rate of receptor degradation. As also previously reported, the rat myotubes had a similar clustering response but only a very slight (approximately 1.2-fold) increase in average AChR site density. The surface area of myotubes was also increased slightly (approximately 1.2-1.3-fold) by the brain extract. Autoradiography viewed by scanning EM was found to be very useful in illustrating the shape and distribution of the receptor clusters. After the brain extract was fractionated on Sephadex G-200, the fractions with greatest clustering activity could be separated from those causing predominantly an increase in receptor site density. Increased receptor site density was primarily produced by the low molecular weight fractions (less than 12 kD), whereas the strongest (but not exclusive) effect on clustering was produced by the high molecular weight fractions (greater than 140 kD). Furthermore, the two cell types assayed had different sensitivity to the different factors. L5 cells responded to both the high and low molecular weight factors while rat primary cells are sensitive primarily to the high molecular weight factors.

Animals↗
PubMed1982

Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions.

We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.

Animals↗
PubMed1981

Physical and chemical characterization of the major lactose-blockable lectin activity from fetal calf skeletal muscle.

The lactose-blockable lectin activity from fetal calf skeletal muscle has been purified to apparent homogeneity. The purification entails differential centrifugation, ammonium sulfate precipitation, asialofetuin affinity chromatography with a lactose gradient and ion-exchange chromatography on DEAE-cellulose. In the last step, the activity is resolved into a major and minor species, designated ion-exchange-purified lectins I and II, respectively. Both lectin activities are reversibly inhibited by lactose and appear as single bands with identical mobilities on SDS-polyacrylamide gel electrophoresis. Lectin II was not obtained in sufficient quantities for further characterization. Lectin I is characterized by a functional requirement for reducing agents and sensitivity to N-ethylmaleimide, which suggests a role for an essential thiol in its activity. Subunit molecular weight determinations by SDS-polyacrylamide gel electrophoresis (12 000 +/- 1 000) and by gel filtration in 6 M guanidine . HCl (13 000 +/- 1 000), when compared with that obtained under native conditions on Bio-Rad P-60 gels (27 000 +/- 2 000), suggest a true Mr of 25 000 +/- 3 000 for the dimeric molecule. Amino acid composition data, when fitted to this molecular weight, lead to the tentative conclusion that the intact dimer is composed of two very similar but compositionally non-identical chains, designated by alpha and beta. While the only detectable N-terminal amino acid is tryptophan, the isoelectric focusing pattern of lectin I supports this heterodimeric structure. In addition, a lactose-sensitive hemagglutinating activity which can be separated from the lactose-blockade activity by affinity chromatography was also observed.

Amino Acids↗
PubMed1980

Distribution and activity of endogenous lectin during myogenesis as measured with antilectin antibody.

Antibodies to electrolectin, a lectin endogenous to embryonic skeletal muscle, have been used to study the distribution of electrolectin during myogenesis in L6 cells and rat primary muscle cultures. Antibody binding is highest to mononucleated cells and is low to myotubes in both systems. Binding is much lower to fibroblasts in the primary cultures. Binding appears to be on the surface of these cells, although evidence is presented for there being binding on the inside of cells as well. When observed on myotubes, binding is generally associated with highly stained patches and in some instances is near regions where fusion may be occurring, In L6 cells, binding sites can be exposed by treating mononucleated cells with trypsin. These results are discussed in terms of their possible role in myogenesis and synaptogenesis.

Animals↗
PubMed1978

Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells.

The effect of rat spinal cord explants and cell-free nerve extract on acetylcholine receptor site density and distribution was studied using (125)I- and rhodamine-labeled alpha-bungarotoxin on L(6), a cloned rat muscle cell line. Control L(6) myotubes have a low and uniform distribution of acetylcholine receptors (20 +/- 3 sites per mum(2) in the present study). The addition of spinal cord explants caused an increase in average receptor site density of about 6 times on myotubes within 2 mm of the explant, while a smaller increase of 3 times was observed at distances greater than 5 mm. The formation of high-density patches of receptors was also stimulated. These observations suggested that a diffusible substance originating from the explant was responsible for these changes. Cell-free homogenates of the central nervous system were prepared and found to produce the same effects. The effect of the homogenate was not strongly dependent on the age of the fetus from which the tissue was isolated, and fetal liver had little or no effect. The active component(s) appears to be a protein(s) with a molecular weight of about 100,000. Because the nerve homogenates make the L(6) cells resemble primary muscle cultures, we suggest that a common factor is responsible for regulating the acetylcholine receptor in the two types of muscle culture. The normally acetylcholine receptor-poor L(6) cells may provide a more sensitive assay for these factors than do primary muscle cultures.

Acetylcholine↗
PubMed1977

Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity.

1. A linear relation, with a slope of 0-9 +/- 0-2 on a log-log plot, was obtained between acetylcholine (ACh) sensitivity and alpha-bungarotoxin (alpha-BTX) binding site density in developing L6 and rat primary myotubes. ACh sensitivity was defined as g/Qn where g is conductance, Q is ACh charge and n is the Hill coefficient. Experimentally we found n approximately 1-7 for our myotubes, which is similar in value to that reported for adult systems. 2. The linear relationship is compatible with an organization whereby each ion channel is always complexed with a fixed number of ACh receptors such that the dose-response characteristics of each such complex are independent of average ACh receptor density. 3. Light microscope autoradiography showed that the alpha-bungarotoxin binding sites on L6 myotubes are uniformly distributed over the surface, while primary rat myotubes exhibit gradients and hot spots. Electron microscope autoradiography indicated that about 70% of the [125I]alpha-bungarotoxin label was on the surface of the myotubes. The alpha-bungarotoxin site density, after subtracting myoblast background, varied from 5 to 400 sites/micrometer2 on different L6 myotubes, and from 54 to 900 sites/micrometer2 on primary rat myotubes, with occasional hot spots of 3000-4000 sites/micrometer2. The conductance sensitivities varied from 10(-4) to 2 X 10(-2) Momega-1/nC1-7.

Acetylcholine↗