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Biomedical subjects

A Shainberg

Publications and source records attributed to A Shainberg.

At least 73 records · Page 4Linked to original sources
PubMed1985

Inhibition of myogenesis by trifluoperazine and compound 48/80.

When trifluoperazine (TFP), a calmodulin antagonist, was given to chick or rat myoblasts in cultures, formation of multinucleated myotubes was inhibited. The inhibition of cell fusion by TFP in rat cultures prevents the normal increase in the amount of acetylcholine receptors (AChR) and creatine kinase (CK), while the levels of these proteins in chick muscle cultures are hardly affected. Another calmodulin antagonist, compound 48/80, inhibits fusion at doses that correspond closely to its antagonistic effects on calmodulin. Thus, our results suggest a possible role for calmodulin in the regulation of myoblast fusion, but not on the appearance of muscle proteins.

Animals↗
PubMed1985

Differentiation of myoblasts with nerve cells on microcarriers in culture.

Differentiation of embryonic rat and chick myoblasts was investigated using a tridimensional support made of positively charged, uncoated DEAE-cellulose microcarriers (MC). Following rapid cell attachment, the MC interconnected to form large cell-MC conglomerates which remained floating in the nutrient medium. Cells within the conglomerates fused to form myotubes which synthesized muscle-specific proteins such as: creatine kinase, acetylcholinesterase, acetylcholine receptors, and contracted in response to electrical stimulation. Myotubes, at different stages of differentiation, showed characteristic morphology (as observed by transmission and scanning electron-microscopies). Upon addition of dissociated spinal cord cells to these muscle-MC cultures, intensive sprouting of nerve fibres took place. After a few days an extensive network of nerve fibres was formed on the top of muscle myotubes and nerve-muscle contacts were established.

Animals↗
PubMed1984

Inhibition of acetylcholine receptor synthesis by conditioned medium of electrically stimulated muscle cultures.

In previous studies, it has been reported that electrical stimulation of muscle cultures is followed by inhibition of acetylcholine receptor (AChR) synthesis. In the study reported here, we show that even media taken from electrically stimulated muscle cultures are capable of inhibiting receptor synthesis. This inhibition is specific, since the levels of acetylcholinesterase and creatine kinase are not reduced. The electrically stimulated myotubes probably synthesize and release to the medium a protein(s) responsible for reducing AChR synthesis.

Acetylcholinesterase↗
PubMed1984

Influence of thyroid hormone on some electrophysiological properties of developing rat skeletal muscle cells in culture.

Effects of thyroxine (T4) were examined on some electrophysiological properties of developing rat myotubes in culture. Thyroxine caused an increase in transmembrane resting potential (Em) of skeletal myotubes. Ouabain decreases Em in both control and T4-treated cells within 5-15 min of addition to the culture. Moreover, the effect of T4 is nearly eliminated by short-term ouabain treatment and by reduced extracellular K+ concentration. The change in Em in response to a 10-fold increase in extracellular K+ concentration is not altered by T4-treatment despite the higher Em of T4-treated cells. Studies on developmental effects of T4 showed that Em of T4-treated cells is higher than controls by as early as day 5. By this time, ouabain decreases Em of T4-treated cells but not that of untreated controls. Treatment with T4 also causes an increase in frequency of spontaneously-occurring action potentials generated by cultured myotubes. Chronic treatment with tetrodotoxin blocks this effect without affecting the T4-induced increase in Em. The findings demonstrate that activity of electrogenic Na,K-ATPase is increased by T4-treatment and that this effect is primarily responsible for the elevated Em in T4-treated cells. In addition, it appears that T4 causes the activity of this enzyme to be manifest earlier in development than might otherwise occur in cultured muscle. Finally, stimulation of electrogenic pump activity is not secondary to increased influx of Na+ ions associated with increased electrical activity and probably results from synthesis of new enzyme units in both developing and mature myotubes.

Animals↗
PubMed1984

Inhibition of acetylcholine receptor synthesis by thyroid hormones.

Studies were made on the effect of thyroid hormones on the level of acetylcholine receptors (AChR) in cultured rat skeletal muscle. Treatment of differentiated myotubes in vitro with thyroxine (T4; 2 X 10(-7) mol/l) for 2-3 days caused a marked decrease in the amount of AChR (P less than 0.05) and an increase in activity of Na+-K+-ATPase (P less than 0.05). There was no significant effect of hormone treatment on other muscle proteins, such as creatine kinase and acetylcholinesterase. Measurements of the turnover rate of AChR in T4-treated myotubes showed only a very slight effect of T4 on the rate of AChR degradation. To study the mechanism by which the hormone exerts its effect, muscle cells were labelled with radioactive amino acid and the rate of its incorporation into AChR protein was measured. The AChR was then isolated using anti-AChR antibodies. The specific activity of labelled AChR was lower in hormone-treated cells. These experiments suggest that the decreased level of AChR in response to thyroid hormone treatment is due to a partial suppression of receptor synthesis.

Acetylcholinesterase↗
PubMed1983

Evidence for a functional role of acetylcholinesterase in cultured chick myotubes.

This study was undertaken in order to assess the functional role of acetylcholinesterase (AChE) in cultures of chick skeletal muscle cells. Cultures of skeletal myotubes were prepared by mechanical dissociation of limb muscle removed from 11-day-old chick embryos and plating at a concentration of 0.8 X 10(6) cells/ml. Cultures incubated for 4-10 days were used for electrophysiological studies with intracellular microelectrodes. Individual myotubes differed with respect to the time course of repolarization following depolarization by acetylcholine (ACh), some cells repolarizing within 2-3 min and others only after 8-10 min. Physostigmine (10(-8)-10(-6) M) prolonged or sometimes completely prevented repolarization following ACh-induced depolarization. These results demonstrate that hydrolysis of ACh by AChE in cultured chick skeletal myotubes plays an important role in the repolarization of these cells following ACh-induced depolarization.

Acetylcholine↗
PubMed1983

Alterations in purine nucleotide metabolism during muscle differentiation in vitro.

Pathways of purine nucleotide metabolism affecting the availability of ATP in the muscle tissue were studied in differentiating rat muscle cultures. The rate of de novo purine nucleotide synthesis and of AMP deamination were found to increase markedly with cell differentiation, but the rate of IMP dephosphorylation was similarly low in both myoblasts and contracting fibers. The above differentiation-associated alterations in purine nucleotide metabolism conform with the greater need for ATP as a source of energy in the contracting myotubes.

AMP Deaminase↗
PubMed1983

Myogenesis on microcarrier cultures.

The capacity of embryonic chick myoblasts to grow in vitro on DEAE-cellulose microcarriers (MC) has been investigated biochemically and morphologically. The cells attached to the MC, replicated and fused to form elongated myotubes. These myotubes synthesized muscle-specific proteins, such as creatine kinase (CK) and acetylcholine receptors (AChR), and they contracted spontaneously. Some of the advantages of this technique are: a) Tridimensional development of myotubes on MC with orientation of fibers parallel to each other; b) Muscle cells can be cultured on MC for long periods (months); c) Easy harvesting of samples at any time during cultivation; d) DEAE-cellulose MC are commercially available, inexpensive and easy to handle.

Animals↗
PubMed1982

Characterization of purine nucleotide metabolism in primary rat muscle cultures.

The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in primary rat muscle cultures. The cultures were found to produce purine nucleotides, by de novo and salvage pathways, both exhibiting dependence on cellular availability of substrate 5-phosphoribosyl-1-pyrophosphate (PPRibP). Depletion of cellular PPRibP decelerated the rate of purine synthesis, whereas increasing PPRibP generation by high Pi concentration in the incubation medium, accelerated purine synthesis. Ribose accelerated purine synthesis, indicating that ribose 5-phosphate availability in the cultured muscle is limiting for PPRibP synthesis. The study in the muscle cultures of the metabolic fate if IMP formed from [14C]formate and that of nucleotides formed from labeled purine bases, revealed that the main flow in the nucleotide interconversions pathways is from AMP to IMP. The flow from IMP to GMP and to AMP appeared to be of a lesser magnitude and virtually no flow could be detected from GMP to IMP. The greatest proportion of radioactivity of purine nucleotides following synthesis by either de novo or salvage pathways, accumulated in IMP, reflecting the relative rates of flows between the various nucleotides and probably also a relatively low, or inhibited activity of the IMP nucleotidase. The results suggest that primary muscle cultures are a plausible model for the study of the role of purine metabolism in muscle work.

Adenine↗
PubMed1982

Effects of thyroxine on transmembrane resting potentials of skeletal muscle cells in culture.

The effect of thyroxine (T4) was studied on the transmembrane resting potential (Em) of rat skeletal muscle cells in culture. In agreement with previous studies, we found the Em of developing muscle cells to increase with age from Day 3 to Day 7 or 8, at which time a plateau is reached. The values of Em we obtained (-65 to -75 mV) were 15-20 mV higher than those (-50 to -55 mV) reported by others. Treatment of mature myotubes with T4 resulted in a significant increase in resting Em. There was only a slight difference between T4-treated and control cells with regard to the relation between extracellular K+ concentration and resting Em. Within 20-30 min of exposure to ouabain (10(-4)--10(-3)M) resting Em of both T4-treated and control cells had fallen to about the same value. This effect was reversible. Thus, T4 stimulates the activity of an electrogenic Na/K-ATPase in the membrane of cultured skeletal muscle cells.

Animals↗
PubMed1980

Role of calcium in the regulation of acetylcholine receptor synthese in cultured muscle cells*.

Embroyonic muscles differentiated in vitro were used to study the effects of intracellular Ca2+ ([Ca2+1]i) variations on the amount of acetylcholine receptors ([AChR]) in the cell membrane. 2. Increased Ca2+ concentration in the growth medium ([Ca2+]o) caused a marked elevation of AChR levels, apparently through de novo synthesis. 3. Agents known to increase [Ca2+]i and its accumulation in the sarcoplasmic reticulum (SR), such as ionophore A23187, sodium dantrolene (DaNa), or high [Mg2+]o all enhanced alpha-bungarotoxin (alpha-BGT) binding after 48 h of treatment. 4. Electrical stimulation or caffeine, both affectors of SR calcium release, brought about a decrease in [AChR] probably by suppressing its synthesis. 5. The effects of simultaneous treatment with two AChR-inducing agents, namely, high [Ca2+]o in the presence of tetrodotoxin (TTX) or high [Mg2+]o were not additive, thus suggesting action via a common saturable mediator. 6. Intermediate AChR levels obtained following simultaneous treatments with opposing effects, e.g., electrical stimulation in the presence of high [Ca2+]o or DaNa, suggest contradictory actions on a common mediator. 7. All these observations indicate a strong correlation between SR calcium levels and [AChR] on myotubes; while calcium accumulation in the Sr was followed by increased AChR synthesis, calcium release was accompanied by suppression of receptor synthesis.

Acetylcholine↗
PubMed1976

Induction of acetylcholine receptors in muscle cultures.

Acetylcholine receptors in muscle cells differentiated in vitro were monitored by using 125 I-alpha-bungarotoxin. The number of cholinergic receptors was increased 4-8 fold in 2 days due to inhibition of spontaneous contraction of the muscle fibers. The inhibition of this activity, whether mediated through tetrodotoxin, lidocaine or D-600, did not affect the biochemical differentiation of muscle, as represented by creatine-phosphokinase and acetylcholinesterase activity. "Induction" of receptors by tetrodotoxin was inhibited by cycloheximide, actinomycin-D, or 5-bromotubericidine. Dystrophic muscle responded in vitro to inhibition of contraction similar to normal tissue.

Acetylcholine↗
PubMed1975

The hypothalamo-neurohypophysial complex in organ culture: morphologic and biochemical characteristics.

Organ cultures of the guinea pig hypothalamo-neurohypophysial complex could be maintained for periods as long as 3 weeks. Morphological studies using conventional light microscopy as well as electron microscopy indicate that neurosecretory cells remain viable during this time. Biochemical studies show that the capability of the cultures to synthesize a normal spectrum of cellular RNA species is impaired during the initial day in culture, but that this capability is restored after 4-5 days. Similarly, protein synthesis proceeds at low levels during initial days of culture, but increases after 5 days. These phenomena appear to be independent of changes in the specific radioactivity of precursor pools and were also observed when outgrowth of non-neuronal cells is inhibited with fluorodeoxyuridine. The content of vasopressin, a product of a specific class of neurosecretory neurons, was found to decrease in the posterior pituitary during 7 - 10 days in culture by 50-70 percent; the levels then plateaued and were maintained for up to 3 weeks. The hypothalamic content of the hormone was relatively constant throughout the culture period. Of most importance was the demonstration that the organ cultures were capable of vasopressin biosynthesis. This capability paralleled the biosynthetic activity of RNA and gross protein metabolism in that there was an initial refractory period of several days duration.

Animals↗
PubMed1975

The hypothalamo-neurohypophysial complex in organ culture: effects of metabolic inhibitors, biologic and pharmacologic agents.

Organ cultures of the guinea pig hypothalamo-neurohypophysial complex synthesize the octapeptide hormone, vasopressin, a specific product of the neurosecretory cells of the supraoptic nucleus. Inhibitors of both protein and RNA synthesis (cycloheximide and bromotubercidin respectively) were found to block vasopressin biosynthesis. In the presence of bromotubercidin, the apparent half-time of decline in the rate of hormone biosynthesis was about 28 h. Colchicine inhibited the distal transport of vasopressin into the posterior pituitary. Ultrastructural studies on colchicine-treated cultures indicated the neuronal stalks were intact and that neurotubules were still present. The narcotic drug, levorphanol at 10-7 M and 10-9 M was found to inhibit RNA synthesis by 20 percent. At these concentrations it had no demonstrable effect on vasopressin synthesis. Cultures established from animals that had been rendered tolerant to narcotics also had no observable alterations in vasopressin biosynthesis, although the initial pituitary vasopressin content of these cultures was reduced by about 35 percent. Various pharmacologic and biologic compounds were tested for their effects on vasopressin biosynthesis in organ cultures. Dibutyryl cyclic AMP, estradiol-17beta, nicotine, nerve growth factor (NGF), and pineal extract all had no effects under the present experimental regimen. Medium conditioned by the presence of fetal hypothalami of 40-55 days gestation produced a 2-4 fold increase in vasopressin biosynthesis in cultures established from adult animals. Medium conditioned by fetal cerebral cortex, liver, or hypothalamic tissue from fetuses of less than 33 days gestation did not have this stimulatory effect.

Antibiotics, Antineoplastic↗
PubMed1973

Choline acetyltransferase activity is increased in combined cultures of spinal cord and muscle cells from mice.

The activity of choline acetyltransferase was more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord cells alone. This increase was associated with the formation of functional neuromuscular junctions in culture. Counts of silver-stained cells and determinations of other enzyme activities indicated that the increased choline acetyltransferase activity was not due to nonspecific neuronal survival but reflected greater activity in the surviving neurons. Hence, muscle had a marked, highly specific trophic effect on the cholinergic neurons that innervated it.

Acetyltransferases↗