Embryonic Chick Pectoral Muscle Research Articles

The Activity of an Endoplasmic Reticulum-localized Pool of Acetylcholinesterase Is Modulated by Heat Shock

Primary cultures prepared from embryonic chick pectoral muscle were subjected to heat shock, and the effect on acetylcholinesterase activity in the cultures was examined. A rapid recovery in enzyme activity was observed soon after an initial heat shock-induced drop and was shown to be independent of de novo synthesis of protein, since it could occur in the presence of an inhibitor of protein synthesis. Lectin binding and sucrose gradient centrifugation studies suggested that molecular monomers and dimers found in the endoplasmic reticulum are involved in the observed recovery of acetylcholinesterase activity. Enhanced activation of a pre-existing pool of inactive enzyme was clearly not the main agent of the recovery in enzymic activity. Recovery relied principally on restoration of the activity of previously active, heat-denatured acetylcholinesterase molecules found in the endoplasmic reticulum. Possible agents involved in the recovery of enzymatic activity might be heat shock proteins acting as molecular chaperones.

Transport of beta-alanine and biosynthesis of carnosine by skeletal muscle cells in primary culture.

Uptake of beta-alanine and synthesis of carnosine (beta-alanyl-histidine) could be demonstrated in primary cell cultures derived from embryonic chick pectoral muscle. Concomitant with the morphological changes, cessation of cell division and the induction of creatine kinase, a rapid increase in the rate of beta-alanine uptake and also in the rate of carnosine synthesis could be observed. The uptake of beta-alanine is sodium and chloride dependent and obeys Michaelis-Menten kinetics with Km values of about 40 microM that are essentially identical for myoblasts and myotubes. In contrast, Vmax increases considerably during differentiation. The beta-alanine transport system is highly specific for beta-amino acids and exhibits a substantial anion dependency (Cl- > J- > CSN- > SO(4)2-). Stoichiometric studies suggest that the transport of one beta-alanine molecule involves two sodium ions and one chloride ion. This ratio is not altered by the process of cell differentiation.

Biosynthesis of carnosine and related peptides by skeletal muscle cells in primary culture.

Synthesis of carnosine (beta-alanyl-L-histidine) and related dipeptides could be demonstrated in primary muscle cell cultures derived from embryonic chick pectoral muscle. After incubation with radiolabeled beta-alanine or gamma-aminobutyric acid, the radiolabeled dipeptides were isolated from the cell extracts and also in small amounts from the culture medium. The kinetics of dipeptide formation indicated that anserine (beta-alanyl-1-methylhistidine) is not formed directly by these cells but as a secondary product via the methylation of carnosine. Coinciding with the morphological differentiation of the mononucleated myoblast to form multi-nucleated myotubes, a rapid increase in beta-alanine uptake and also in dipeptide synthesis could be observed. These results demonstrate that carnosine and related peptides are not merely deposited in skeletal muscles but that they are actively synthesized by muscle cells in culture.

Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development.

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.

Denervation-induced alterations of acetylcholinesterase in denervated and nondenervated muscle

The influence of denervation on acetylcholinesterase (AchE) molecular forms in rat skeletal muscle for durations up to 30 days is examined in denervated anterior tibialis, the innervated contralateral muscle, and diaphragm. Denervated rats at a common age of 8.5 weeks are compared with age-matched, nondenervated animals. The results indicate that time-dependent losses of AchE in denervated muscle occur more rapidly than loss of muscle mass and are not uniform among the different molecular forms. Loss of the 4 S and 16 S forms is rapid and essentially complete within 3.5 days of denervation, while during this same period the 10.5 S form undergoes a transient twofold increase and its presence in denervated muscle is never abolished. Within 30 days of denervation, all forms of AchE including the 16 S species reappear. A salient finding of these studies is that the effects of denervation are evident also in anatomically remote, innervated muscle such as anterior tibialis of the contralateral limb and in diaphragm. These alterations appear as pronounced reductions in 4 S AchE and increases in 10.5 S AchE; the asymmetric collagen-tailed 16 S form is unaltered. Treatment of primary cultures of embryonic chick pectoral muscle with sera from denervated but not nondenervated rat causes reductions in AchE. These results indicate that the appearance and retention of AchE, in particular the 16 S form, occur in the absence of functional innervation. The effects of denervation on AchE metabolism in remote, innervated tissue are consistent with the action of a diffusible factor released from severed nerve or muscle, or both.

Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures.

The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15% of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K+] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K+] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K+] medium was reversible. When cultures maintained in high [K+] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC.

Phorbol ester inhibits myoblast fusion and activates β-adrenergic receptor coupled adenylate cyclase

Primary cultures of myoblasts, derived from embryonic chick pectoral muscle, were treated with phorbol ester (TPA) for 8–96 h. TPA treatment blocked the fusion of myoblasts along with the expression of the MM form of creatine kinase. Interestingly, TPA treatment markedly increased the activity of β-adrenergic receptor coupled adenylate cyclase (AC) activity. The study suggests that TPA treatment augments the functional interaction between a coupling N s protein and catalytic unit of AC. The likely significance of these results is briefly presented.

Molecular forms of acetylcholinesterase in chick embryonic fast muscle: developmental changes and effects of DFP treatment.

Multiple molecular forms of acetylcholinesterase (AChE) from embryonic chick pectoral muscle were examined by sucrose density sedimentation. Embryonic muscle in ovo possessed three molecular forms with apparent sedimentation coefficient of 7S, 11S, and 20S. The proportion of the 20S form increased 4-fold between days 11 and 18 in ovo and there was a decrease in the proportion of the 7S and 11S forms. Cultured embryonic muscle contained only 7S and 11S AChE despite the presence of the 20S form in the tissue from which the cultures were derived. Brief treatment with diisopropylphosphorofluoridate (DFP) completely inhibited the enzyme activity; the newly synthesized AChE first appeared as a 7S form. Polyacrylamide gel electrophoresis of the peak fractions from the sucrose gradients revealed that the 7S AChE was composed of two electrophoretic forms and gave evidence that the 20S AChE was too large to enter the running gel. The results are consistent with the ideas that the distribution of AChE molecular forms is regulated during embryonic development and that AChE is first synthesized as a low molecular weight form following inhibition with organophosphate compounds.

Heparin-inhibitable lectin. Purification from chicken liver and embryonic chicken muscle.

The heparin-inhibitable lectin activity from adult chicken liver and embryonic chick pectoral muscle has been purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Specific activity of this abundant material was increased about 70-fold with about 28% recovery of activity. The purified lectin, which behaved as a large aggregate, could be dissociated into two proteins with apparent molecular weights of 13,000 and 16,000 upon electrophoresis in sodium dodecyl sulfate. 125I-tryptic peptide maps of these proteins showed that they were distinct from each other and also from lectins of similar molecular weight previously purified from chicken. Agglutination activity of the lectin, tested with specially prepared rabbit erythrocytes, was very sensitive to heparin which inhibited 50% at concentrations of approximately 10(-5) mM. Dermatan sulfate and heparan sulfate were also potent inhibitors, whereas other glycosaminoglycans were not. The most potent monosaccharide hapten inhibitor was N-acetyl-D-galactosamine. Because of its properties, we operationally refer to this material as chicken heparin-lectin, recognizing that this does not necessarily indicate that it functionally interacts with heparin in vivo.

Purified lectin from skeletal muscle inhibits myotube formation in vitro.

A lactose-extractable lectin obtained from 14--16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture. When applied to muscle cultures at 0.09 micrograms/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation. Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4 degrees C. Incubation in L-15 solution at 37 degrees C with or without 2 mM dithiothreitol resulted in inactivation in 2--3 h. To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca+2] medium to inhibit fusion, and then [Ca+2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93%. Continued incubation at 37 degrees C. without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.

Developmentally regulated lectin from embryonic chick pectoral muscle. Purification by affinity chromatography.

A lectin, whose specific activity in soluble extracts of embryonic chick pectoral muscle increases strikingly between 8 and 16 days of development, has been purified by affinity chromatography on derivatized Sepharose 4B coupled to p-aminophenyl-beta-D-lactoside. After affinity chromatography the lectin is pure except for minor contamination with another protein possibly representing a second muscle lectin. The latter can be completely removed by preparative isoelectric focusing. The purified lectin has an apparent molecular weight of 30,000 and an apparent subunit molecular weight of 15,000. Its isoelectric point is 4.0. The most potent saccharide inhibitors tested were thiodigalactoside and lactose. An antibody has been raised to the pure lectin. Studies with this antibody indicate that the lectin is present both on the surface of and within myoblasts.

Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line

Soluble extracts of embryonic chick pectoral muscle and myoblast clone L6 agglutinated trypsin treated glutaraldehyde fixed rabbit erythrocytes. Agglutination activity was blocked by thiodigalactoside, lactose and related saccharides but not by many other saccharides. Agglutination activity of chick pectoral muscle extracts increased at least one order of magnitude between 8 and 16 days of chick embryo development, as the pectoral muscle differentiated. With L6 myoblasts there was a three-fold increase in activity of the extracts as the myoblasts fused to form multinucleated myotubes.