Rat Leg Muscle Research Articles

31P Spin‐Lattice Relaxation Times and Resonance Linewidths of Rat Tissue in Vivo: Dependence upon the Static Magnetic Field Strength

Phosphorus-31 NMR spin-lattice relaxation times and resonance linewidths of rat leg muscle, brain, and liver metabolites in vivo have been examined at 1.9-, 4.7-, and 8.5-T static magnetic field strengths. The resonance linewidths expressed in ppm that have been measured are independent of field strength. The spin-lattice relaxation times of muscle and brain phosphorus metabolites decrease linearly with increasing field strength while those of liver are constant over the range of static fields examined.

Phospholipid methylation in skeletal muscle membranes

Phospholipid methylation is thought to modulate such vital cellular processes as calcium transport, receptor function, and membrane microviscosity. As these processes are fundamental to the function of muscle cells and are thought to be altered in disease states, we have characterized several features of phospholipid methylation reactions in skeletal muscle and have defined appropriate assay conditions. In rat leg muscle, methyltransferase activity was assayed radiometrically by measuring the incorporation of methyl groups from S-adenosyl-L-[methyl-3H]methionine into membrane phospholipids, the methylated derivatives of which were separated by thin-layer chromatography. Contrary to previous investigations of whole muscle, phospholipid methyltransferase activity was clearly present in skeletal muscle membranes, being highly localized in sarcoplasmic reticulum and present to a lesser extent in sarcolemma. Both the reaction products and the reaction kinetics were consistent with sequential methylation of phospholipids by two methyltransferase enzymes. S-adenosylhomocysteine and its analogues were potent inhibitors of phospholipid methylation in sarcoplasmic reticulum. The predominant localization of phospholipid methyltransferase activity in sarcoplasmic reticulum suggests that its functional role in skeletal muscle may be in calcium transport.

Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance.

Phosphorus nuclear magnetic resonance spectra at 145 MHz were obtained with a surface coil from the gastrocnemius-plantaris muscles of anesthetized rats. Phosphocreatine (PCr) and inorganic phosphate (Pi) contents and intracellular pH were measured before, during, and after periods of contractile activity induced by twitch or tetanic stimulation of the sciatic nerve. Reduced levels of PCr, increased levels of Pi, and intracellular acidification were achieved in a graded fashion with increased rates of twitch stimulation from 2 to 10 Hz without detectable changes in the ATP content. In all cases, the decrease in PCr was matched by a stoichiometric increase in Pi content. The time constant of resynthesis of PCr averaged 30 s, was five times faster than the restoration of intracellular pH to control levels, and was independent of the degree of intracellular acidosis at the beginning of recovery.

Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise.

Cellular damage caused by free radical reactions may play a role in the aging process. A bout of exercise can increase free radical concentration with damage to mitochondria in muscle (Davies et al., 1982). This study was undertaken to determine if muscle adapts to exercise training with an enhancement of enzymatic defenses against free radical damage. A program of running that induced two-fold increases in mitochondrial enzymes in leg muscles of rats resulted in no increase in catalase or cytoplasmic superoxide dismutase (SOD) activities. Mitochondrial SOD activity was increased 37% in fast-twitch red and slow-twitch red types of muscle and 14% in white muscle. Thus, despite an increase in mitochondrial SOD, the ratio of SOD to mitochondrial citrate cycle and respiratory chain enzymes was decreased. It seems unlikely that increased capacity for enzymatic scavenging of superoxide radical is a major protective adaptation against free radical damage in exercise-trained muscle.

A pulse sequence for simplifying hydrogen NMR spectra of biological tissues

1H NMR studies of many mammalian tissues are complicated by the presence of large lipid resonances that obscure much of the spectrum. In this communication a simple spin-echo homonuclear double-resonance difference sequence was used to separate the resonances of alanine, lactate, β-hydroxybutyrate, glutamate, and glutamine from the lipid resonances which overlap them in excised rat leg muscle and heart, and in an excised mouse liver which had been perfused with [3- 13C]alanine.

Glycogen resynthesis in leg muscles of rats during exercise.

This study was undertaken to determine whether glycogen resynthesis can occur in glycogen-depleted muscles in response to glucose feeding during prolonged exercise. Rats were exercised for 40 min with a treadmill running program designed to deplete muscle glycogen. One group was studied immediately after the glycogen-depletion exercise. A second group was given 1 g glucose by stomach tube and exercised for an additional 90 min at a running speed of 22 m/min on a treadmill set at an 8 degree incline; they were given additional 1-g glucose feedings after 30 and 60 min of running. The initial 40-min run resulted in liver glycogen depletion, large decreases in plasma glucose and insulin concentrations, and a marked lowering of muscle glycogen. The glucose feedings resulted in greater than twofold increases in the concentrations of glucose and insulin in plasma, and of glycogen in leg muscles, during the 90 min of running. No repletion of liver glycogen occurred. These results provide evidence that glycogen resynthesis can occur in glycogen-depleted muscle despite continued moderate intensity exercise if sufficient glucose is made available.

Tissue blood flow in control and cold-adapted hyperthyroid rats.

Chronic injections (once daily for 10-14 days) of triiodothyronine (T3) stimulated oxygen consumption by 50 and 15% in anaesthetized, control (24 degrees C), and cold-adapted (5 degrees C) rats, respectively, compared with euthyroid controls. Tissue blood flow, determined from the distribution of radioactive microspheres, was unaffected by T3 treatment in skeletal muscle, scrotum, brain, bone, skin, diaphragm, and brown adipose tissue (BAT) of rats housed at 24 degrees C, but was decreased in spleen (53% of control) and significantly increased in three white adipose tissue depots (average 267% increase) and liver (56%). Blood flow to epididymal fat and leg muscle of cold-adapted rats was increased by T3 treatment (100 and 138% increases, respectively), but other tissues were unaffected. Blood oxygen extraction and oxygen consumption in vivo by interscapular BAT was increased in hyperthyroid rats compared with euthyroid controls, but was reduced by T3 treatment in cold-adapted animals. These data show that BAT makes only a minor contribution (7%) to thyroid thermogenesis, but suggest that kidney, liver, gut, and particularly white adipose tissue may be involved.

Ca 2+-activated protease in denervated rat skeletal muscle measured by an immunoassay

Right hind leg muscles of adult male rats were denervated by transecting the sciatic nerve, while muscles of the left leg served as a control. On days 4, 9, and 18 after denervation, the rats were killed and the extensor digitorum longus and soleus muscles removed. Extracts were prepared which contained the Ca 2+-activated protease. The molecular weight of the protease in the extracts, measured immunologically on Western blots of SDS-polyacrylamide gels, was identical to that of purified standard protease and was unaffected by denervation. The amount of protease in each extract was measured by a solid phase immunoassay using monospecific IgG labeled with 125I, and the results were expressed as units of protease activity per milligram noncollagen protein. There were increases of 40 to 80% in the mean values obtained for both denervated muscles compared with controls. Because the protease is probably localized on the cell membrane, these modest increases are likely to be a consequence of a generalized over-development of muscle membranes which follows denervation.

The mononuclear cell population in rat leg muscle: its contribution to the lysosomal enzyme activities of whole muscle extracts.

The mononuclear cell fraction of rat hind-limb muscle was obtained by digestion with clostridial collagenase in the presence of calcium ions, filtration through nylon screens and washing to remove the enzyme. Final traces of contaminant myofibrillar debris were separated by isopycnic centrifugation in a Percoll density gradient. Whole muscle, washed cells and Percoll-fractionated cells were extracted in the presence of non-ionic detergent and the supernatants assayed for the lysosomal enzymes cathepsins B + L, N-acetyl-beta-glucosaminidase, beta-glucuronidase and protein. The enzyme levels were highest in the muscle from young rats, but the percentage of recovered activity in the mononuclear cell fraction was little altered by the age of the animal. The values obtained were: cathepsin B + L, 2.4-4.0%; N-acetyl-beta-glucuronidase, 4.3-7.6%; and beta-glucuronidase, 6.3-10.3%. Because of unavoidable losses in preparation these are minimal values and the actual levels of activity from the mononuclear cell fraction in muscle would be higher. The specific activity values of the cell lysates were raised after isopycnic centrifugation and were nearly constant over the age range 65-180 days. Substantially higher specific activity values were obtained for the cells from rats of 38 days. When grown in culture the mononuclear cell fractions were seen to contain mainly fibroblasts and myoblasts with only few leucocytes. The cultures reached confluence by the second week, at which time numerous myotubes had formed. In addition there were groups of large, circular cells with a prominent centrosphere. The origin of these latter cells is uncertain. It was concluded that although total lysosomal enzyme activity was higher in young rats there was little effect of age on the distribution of activity between muscle fibres and mononuclear cells in the muscle.

Labelling of cervival and thoracic spinal neurones following injections of horseradish peroxidase in the leg muscles of rats

Following injection of horseradish peroxidase (HRP) solution in the gastrocnemius muscle or the extensor digitorum longus muscle in neonatal and developing albino rats, some neurones in the cervical enlargement, mid-thoracic region and sacral region were labelled in addition to the region of the spinal cord from where the sciatic nerves emerged. The labelling was most probably the result of diffusion of HRP solution from the site of injection to nearby structures.

The components of the troponin complex and development in skeletal muscle

1. 1. Using the immunoperoxidase staining technique with monospecific antibodies to the polymorphic forms of the components of the troponin complex, two types of cells could be distinguished in rat leg muscles at 18 days' gestation. 2. 2. One kind of cell, designated presumptive type II, stained only with the fast skeletal muscle forms of troponin I, troponin T and troponin C. 3. 3. The remaining cells designated presumptive type I, stained with both the fast and slow skeletal muscle forms of the three troponin components. 4. 4. The amounts of the fast skeletal muscle forms of the troponin components progressively decreased in the presumptive type I cells until 10–11 days after birth these proteins could no longer be detected by the immunoperoxidase staining. At the same time the slow skeletal muscle forms of the troponin components steadily increased. These changes were complete about a day earlier in muscles containing a majority of type I cells. 5. 5. Differentiation into different muscle cell types during development could be distinguished earlier by staining with the troponin antibodies than by the classical histochemical methods. 6. 6. The results in general support the hypothesis that the genes responsible for synthesis of the components of the troponin complex are under some type of coordinated control.

Myotonia as a side effect of diuretic action.

1. Commonly used loop diuretics produce side effects in man which are similar to chemically induced myotonia. These diuretics have structural affinity with known myotonic agents. 2. We have observed EMG myotonia in vivo in leg muscles of rats treated with intravenous frusemide. 3. In the presence of several different diuretics, rat isolated diaphragm, soleus and extensor digitorum longus muscles as well as frog sartorius muscles produce typically myotonic contractions with relaxation times up to several seconds. 4. Intracellular recording of action potentials from diuretic-treated muscles reveals long lasting after-discharges following a brief electrical stimulus, again typical of chemically induced myotonia. 5. Having demonstrated a myotonic action of several diuretics we suggest a need for caution in using these drugs in persons with hereditary myotonia and a need to be aware of possible provocation of myotonia in subclinical cases. Myopathies and neuropathies which are known to result from chronic exposure to myotonic agents also need to be considered. 6. In our study, the diuretic, acetazolamide, unmasked subthreshold myotonia. This seems to be at variance with reports of its usefulness in the treatment of myotonia. 7. Diuretics should probably not be employed in the treatment of herbicide intoxication where their myotonic activity would be expected to add to the known myotonic activity of the herbicide.

Subunit structure of the acetylcholine receptor from denervated rat skeletal muscle

Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose. A control preparation containing only contaminants was made by performing a parallel purification in which alpha-bungarotoxin was added to the preparation prior to the cobrotoxin/Sepharose step. Comparison of the receptor was greater than 90% pure. Examination of the purified receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two major polypeptide chains with apparent weights of 45,000 and 51,000, along with minor components of 49,000, 56,000, 62,000, and 110,000. Polypeptides of the same molecular weight were found when purified acetylcholine receptor was radioiodinated and further purified by sucrose gradient sedimentation. Analysis of receptor purified from denervated muscles that had been incubated with [35S]methionine in organ culture showed that all of the identified polypeptide chains were radioactive, indicating that they had been synthesized de novo after denervation.

Differences in the cholinergic proteolipid isolated by affinity chromatography from normal and denervated rat leg muscle

The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents. The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid was used to desorb the specific fraction. From normal controls 36 μg of specific protein per g fresh tissue were obtained. In the denervated muscle there was no increase in this protein after three days, but it increased by 120% after six days. Binding studies carried out with 14C-acetylcholine, 3H-α-bungarotoxin and 14C-d-tubocurarine showed that only the specific fraction was able to bind the ligands. Three days after denervation the specific activity (nmoles/mg protein) for 14C-acetylcholine increased 400% and for 3H-α-bungarotoxin 100% over the control; on the contrary, there was no change in the binding of 14C-d-tubocurarine. These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle.

In vitro analysis of the general properties and junctional receptor characteristics of skeletal muscle membranes. Isolation, purification, and partial characterization of sarcolemmal fragments.

Muscle membranes were partially purified from rat leg muscles. Externally oriented membrane functions were used to monitor and characterize the resulting membrane fractions. Na(+)K(+)-stimulated Mg(++)-adenosinetriphosphatase, acetylcholinesterase, and cholinergic receptor activities are present and enriched in the density-gradient subfractions of crude sarcolemma when compared with the first pellet. The physical separation of the cholinesterase and receptor activities on the gradient subfractions is demonstrated. Receptor activity, determined by specific (125)I-labeled alpha-bungarotoxin binding, appears in fractions with densities similar to other plasma membranes (D(4) (20) 1.1015-1.1520). Acetylcholinesterase, on the other hand, is preferentially distributed in lighter density fractions (D(4) (20) 1.0507-1.0780) and parallels the gradient distribution of the ATPase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a high-molecular-weight glycoprotein sediments with the higher density fractions only. The data suggest a molecular dissection of the layers of the sarcolemma. The receptor is tentatively felt to be an integral component of the junctional plasma membrane. Acetylcholinesterase is felt to be superficially located on the ectolamina of the junctional sarcolemma, and may be woven within the matrix of the intersynaptic basement membrane.

Effect of Exercise on the Turnover of Cytochrome c in Skeletal Muscle

Abstract The concentration of cytochrome c has previously been shown to increase 2-fold in leg muscles of rats subjected to a program of prolonged exercise. In the present study it was found that the radioactivity of cytochrome c labeled with δ-amino[14C]levulinate decreased significantly more slowly in quadriceps muscles of rats subjected to a program of running than in muscles of sedentary animals. The t½ of muscle cytochrome c was 32 days in the sedentary animals and 48 days in the runners. The synthesis rate of cytochrome c was calculated to be approximately 0.630 nmole per muscle per day in the sedentary and 0.771 nmole per muscle per day in the exercised animals. Thus, a decrease in the rate of degradation plays the major role in accounting for the adaptive increase in the concentration of cytochrome c that occurs in muscle in response to exercise.

Protein synthesis in cultured muscle cells: Methylation of nascent proteins

Protein methylation was examined in primary cultures of rat leg muscle cells between 7 and 9 days of culture. Methyl[ 14C]- or [ 3H]-methionine was introduced into the culture medium and the cells were sampled for radioactive methylated protein residues. Incorporation of the total radioactivity was linear for at least 4 hr after introduction of the methionine label. When labeling was studied for periods between 10–30 min, the methylation of polyribosome-bound, presumably nascent, proteins was unaffected by addition of cycloheximide to the culture medium. The antibiotic, however, inhibited incorporation of methionine, and consequently increased the ratios of the incorporated methylated, to methionine residues and the ratio of ribosome-bound to free radioactivity. The methylated, polyribosome-bound proteins were decreased when puromycin was added to the culture medium. It is proposed that selective methylation of nascent proteins, such as myosin, can begin at the level of polyribosomes and be completed in the cytosol of muscle cells cultured in vitro.

Reversible Effects of Lysolecithin on Fusion of Cultured Rat Muscle Cells

Lysolecithin prevented the natural cell fusion in monolayer cultures of rat leg muscle cells when it was added in the range of 120 to 160 μg/ml of complete growth medium. The cells rapidly recovered the ability to fuse after withdrawal of the lysolecithin medium and replacement by the regular growth medium. Lysolecithin prevented myogenic cell fusion within a narrow concentration range in the presence of sufficient concentration of calcium and pH values between 7.4 and 7.8. Comparisons between effects of lysolecithin, high cyclic-AMP and low calcium-containing media on cultures of muscle cells were also made. The elaboration of long, thin processes from the bipolar ends of myogenic cells was a feature particularly noticeable with lysolecithin and cyclic-AMP-containing culture plates. The possible influences of other, non-specified, natural surfactants, reacting with lipoprotein micelles of the plasmalemma and subsequently affecting the orientation and arrangement of cell boundaries during differentiation of various cell types, are discussed.

Acetylcholine receptors. Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity.

Using (125)iodine-labeled alpha-bungarotoxin (alpha-BGT-(125)I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/microm(2) at 14 days, subsequently decreasing to 529 receptors/microm(2) at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by alpha-BGT-(125)I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [ 14C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [ 32P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [ 14C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [ 32P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [ 32P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [ 32P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.