Phorbol ester binding to isolated muscle satellite cells compared to fetal myogenic cells from the rat.
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Biomedical subjects
Publications and source records attributed to J Gautron.
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A technique is described for isolating amphibian myogenic cells from the muscle of adult Xenopus laevis (Dauchin). Muscles were dissociated with 0.2% collagenase and 0.1% trypsin. The resulting cell suspensions were separated from the remaining myofibres by filtration through nylon grids. Most of the cells remaining in the filtrate suspension were satellite cells or fibroblasts. When plated in Petri dishes, satellite cells adhered to the substrate, became spindle-shaped and proliferated activity in a culture medium supplemented with fetal calf serum. Mitotic waves lasted 4 days and consequently cell density markedly increased. Satellite cells came into contact and began to fuse into myotubes on day 8 of culture. Horse serum, which replaced fetal calf serum in the medium on day 12, accelerated cell fusions which were almost complete on day 18. However, under these conditions, some mononucleated cells continued to undergo mitosis. Cell proliferation with a high rate of mitosis was prolonged by repeated trypsinization and replating in medium supplemented with fetal calf serum. When myofibres from dissociated muscles were cultured under the same conditions, they never fragmented or divided.
The effects of histamine on soluble calcium concentrations in gastrointestinal tract contents, acid secretion, urinary phosphorus excretion, plasma free-hydroxyproline and inorganic phosphorus levels were studied in laying hens during egg shell formation. Histamine induced hyperphosphataemia, hyperhydroxyprolinemia and decreased soluble gastrointestinal calcium at 3 hr after injection. Cimetidine inhibited all effects induced by histamine. This suggests that hyperphosphataemia arises from increased bone resorption provoked by decreased soluble intestinal calcium during egg shell formation.
In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.
After the metamorphosis of Tenebrio molitor (Coleoptera), Acetylcholinesterase (AChE) activity per adult brain was twofold the activity of the last larval instar. During metamorphosis, from the prepupa to the adult fluctuations occurred in brain AChE activity per protein amount. These fluctuations were superimposable to the molting hormone (ecdysterone) variations in hemolymph so that each rise of ecdysterone was accompanied by an AChE peak.
The biosynthesis of fibronectin and laminin was studied in satellite cells cultured from adult rat muscles before (day 4) and after fusion and formation of myotubes (day 14) using (35S) methionine as a tracer. The kinetics of incorporation into immunoprecipitable fibronectin and laminin were recorded at intervals from 1.5 to 24 hours of incubation with the tracer from the cells, the pericellular matrix and the culture medium. The rate of synthesis of fibronectin and laminin expressed as dpm/microgram DNA were constant from the mononucleated cell to the myotube state. Both glycoproteins were detected in the cells and in the pericellular matrix. When the results were expressed as the percentage of incorporation into total protein, major changes could be observed in the early phase of the kinetic studies in the cells and the pericellular matrix. Both showed an increase from the mononucleated myoblast to myotube, suggesting that an increasing fraction of total protein biosynthesis is directed towards these two extracellular matrix glycoproteins. At the same time, there was a decrease in the secretion into the medium of freshly synthesized radiolabeled fibronectin and laminin. Our results confirm the synthesis of varying ratios of both extracellular matrix macromolecules by undifferentiated mononucleated myogenic cells as well as myotubes.
The distribution pattern of fibronectin, laminin and type IV collagen in the striated muscle fiber of adult rat was studied using immunofluorescence staining and electron microscopy. The results indicate that fibronectin as well as laminin and type IV collagen precisely delineate each muscle fiber. Fibronectin is present on the sarcolemma extending from the cell membrane to the intercellular collagen fibers beyond the basal lamina lucida externa. This suggests a role for fibronectin in making contact between the cell membrane and the intercellular matrix.
Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.
A method is described allowing localization of acetylcholinesterase (AChE) by both light and electron microscopy. During the reaction lead thio-diacetyl is decomposed, and therefore precipitated as PbS in the presence of native -SH group produced by the hydrolysis of acetylthiocholine perchlorate. The reaction takes place at neutral pH, since improves the sensitivity of AChE localizations. Application of the method to parasympathetic neurons showed that AChE was mainly localized in the rough endoplasmic reticulum of the perikaryons. No reaction was visible in glial cells. AChE was also localized on the plasma membrane of parasympathetic neurons. In mouse embryo muscles AChE activity was seen to be high and was not yet restricted to the synaptic area. The well developed Schwann cells accompanying the neurites displayed constant AChE activity on their plasma membrane.
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Section of the preganglionic nerve of the superior cervical ganglion of the Rat produced a decrease followed by an increase of the acetylcholinesterasic activity. By cytochemical study it was possible to localize, from the seventh day after denervation, a strong acetylcholinesterase activity in the glial cells which lay close to the pericarya of the neurons.
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