The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation.

Rates of protein synthesis were significantly lower in the cut soleus and extensor digitorum longus muscles than in their uncut counterparts. Rates of protein degradation were significantly higher in cut soleus muscles, but not in cut extensor digitorum longus muscles as compared with their uncut controls. Concentrations of ATP and phosphocreatine were significantly lower in cut soleus and extensor digitorum longus muscles after incubation in vitro in contrast with respective control uncut muscles. These data indicate that cutting of muscle fibres alters rates of protein synthesis and degradation, in addition to altering concentrations of high-energy phosphates. Since these findings stressed the importance of using intact muscles to study protein metabolism, additional studies were made on intact muscles in vitro. Stretched soleus muscles had higher concentrations of high-energy phosphates at the end of an incubation period than did unstretched muscles. However, the length of the soleus, extensor digitorum longus and diaphragm muscles during incubation did not affect rates of protein degradation.U     

Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N^G-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells. muscle regeneration     

Changes in Acid-soluble Nucleotides in Cut-injured Sweet Potato Root Tissue

ATP and ADP increased in cut-injured sweet potato root tissue during the 3 to 6-hr incubation period, and showed the maximum for the 9 to 18-hr, and 6 to 9-hr incubation periods, respectively, then decreased. ATP was present in the highest amount among ATP, ADP and AMP throughout the 72-hr incubation period, while AMP was in the lowest. Total acid-soluble nucleotides increased gradually, and showed the peak content at the 12-hr incubation period, and decreased thereafter. Adenine mononucleotides such as ATP, ADP and AMP occupied about 40 to 65% of total acid-soluble nucleotides.     

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.     

Protein synthesis in isolated rabbit forelimb muscles. The possible role of metabolites of arachidonic acid in the response to intermittent stretching.

Protein synthesis was measured in isolated intact rabbit muscles by the incorporation of [3H]phenylalanine added at a high concentration (2.5 mM) to the incubation medium. Intermittent mechanical stretching substantially increased the rate of protein synthesis relative to that in control muscles incubated under a constant tension. Indomethacin and meclofenamic acid, inhibitors of the enzyme cyclo-oxygenase, which converts free arachidonic acid into the prostaglandins, prostacyclins and thromboxanes, decreased the rate of protein synthesis in intermittently stretched muscles, but had no effect on synthesis rates in the unstimulated controls. Arachidonic acid at concentrations of 0.2 and 1.0 microM gave a highly significant increase in the rate of protein synthesis in muscles incubated under a constant tension. The ability of arachidonic acid to increase protein-synthesis rates was abolished by the addition of indomethacin. Activation of protein synthesis by intermittent stretching persisted for 10-20 min after the stretch stimulation had ceased. Indomethacin, added either during the initial incubation with intermittent stretching or during the subsequent period when protein synthesis was measured after stimulation had ceased, decreased protein-synthesis rates. This decrease was similar whether indomethacin was present during the initial, final or entire incubation period. In experiments analogous with those in (4) above, when Ca2+ was withheld and EGTA added for the entire incubation, rates of protein synthesis were again decreased. The rates of protein synthesis observed when Ca2+ was present during either an initial stimulation phase or a final, unstimulated, measurement phase were similar, and were intermediate between control rates and those in muscles incubated without Ca2+ for the whole experiment. Two prostaglandins, F2 alpha (2.8 microM) and A1 (28 microM), increased rates of protein synthesis in unstimulated muscles, but prostaglandins E2 and D2 and the leukotrienes C4 and D4 failed to do so. It is concluded that the stretch-stimulated increase in protein synthesis may be caused by activation of membrane phospholipases, release of arachidonic acid and a consequent increase in prostaglandin synthesis.     

Age-related apoptotic responses to stretch-induced hypertrophy in quail slow-tonic skeletal muscle

In the present study, we examined the responses of apoptosis and apoptotic regulatory factors to muscle hypertrophy induced by stretch overload in quail slow-tonic muscles. The wings from one side of young and aged Japanese quails were loaded by attaching a tube weight corresponding to 12% of the bird's body weight for 7 or 21 days. Muscle from the contralateral side served as the intraanimal control. Relative to the intraanimal contralateral control side, the muscle wet weight increased by 96% in young birds, whereas the muscle weight gain in aged birds was not significant after 7 days of loading. After 21 days of loading, muscle weight significantly increased by 179% and 102% in young and aged birds, respectively. Heat shock protein (HSP)72 and HSP27 protein contents in the loaded sides were higher than on the control sides exclusively in young birds after 7 days of loading. Compared with the contralateral control muscle, the extent of apoptotic DNA fragmentation and the total cytosolic apoptosis-inducing factor protein content were reduced in all loaded muscles except for the 7-day-loaded muscles from the aged birds. Bax protein content was diminished in the loaded muscle relative to the control side from all groups, whereas Bcl-2 protein content was reduced in the young and aged muscles after 21 days of loading. The total cytosolic cytochrome c protein content was decreased and the X chromosome-linked inhibitor of apoptosis protein content was elevated in 7- and 21-day-loaded muscles relative to the intraanimal control muscle from young birds. Furthermore, after 7 days of loading the muscles of aged birds, H₂O₂ content and the total cytosolic protein content of second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point were elevated compared with the intraanimal control side. These data suggest that stretch overload-induced muscle hypertrophy is associated with changes in apoptosis in slow-tonic skeletal muscle. Moreover, discrepant apoptotic responses to muscle overload in young and aged muscles may account in part for the age-related decline in the capability for muscle hypertrophy. aging; sarcopenia; Bcl-2; Bax; heat shock proteins; apoptosis-inducing factor     

The effect of intermittent changes in tension on protein and collagen synthesis in isolated rabbit muscles.

Isolated intact rabbit muscles were incubated in a medium containing radioactive proline. The rates of synthesis of collagen and total muscle protein after incubation with a constant tension or intermittent mechanical stretching were compared with the rates in vivo. Muscles incubated under a constant tension synthesized protein at 22% of the rate observed in vivo; intermittent mechanical stretching resulted in an increase of 73% in the rate of protein synthesis, to 38% of that found in vivo. Collagen synthesis was affected in the same way as total protein synthesis by both types of incubation, therefore the relative rates of collagen and total protein synthesis were unchanged. ATP concentration in the isolate muscles and the uptake of glucose from the medium were increased by intermittent mechanical stretching. Incubating the muscles with a gas phase containing 5% O2 decreased the rate of protein synthesis, abolished the effect of intermittent mechanical stretching, lowered the concentration of ATP and increased the lactate concentration. The rate of protein synthesis in muscles maintained with a constant or intermittently applied tension was not affected by a previous period of incubation with the other type of stimulus.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

Two fast-type fibers in claw closer and abdominal deep muscles of the Australian freshwater crustacean, Cherax destructor, differ in Ca2+ sensitivity and troponin-I isoforms

One type of fast fiber and two types of slow (slow-twitch, S1 and slow-tonic, S2) fibers are found in decapod crustacean skeletal muscles that differ in contractile properties and myofibrillar protein isoform compositions. In this study the structural characteristics, protein isoform compositions, and Ca2+-activation properties of fast fibers in the claw closer (F1) and abdominal deep flexor (F2) muscles of Cherax destructor were analyzed. For comparison, myofibrillar protein isoform compositions of slow (long-sarcomere) fibers from claw and abdomen were also determined; our results indicate that the slow fibers in the claw closer were the slow-twitch (S1) type and those in the abdominal superficial flexor were primarily slow-tonic (S2) type. F1 fibers had shorter resting sarcomere lengths (2.93 microm in unstretched fibers and 3.06 microm in stretched fibers) and smaller fiber diameter (256 microm) than F2 fibers (sarcomere lengths 3.48 microm in unstretched and 3.46 microm in stretched; 747 microm diameter). Moreover, F1 fibers showed a narrower range in sarcomere lengths than F2 fibers (2.81 to 3.28 microm vs. 2.47 to 4.05 micro m in unstretched fibers). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the fast fibers from claw and abdomen differed in troponin-I composition; F1 fibers expressed two isoforms of troponin-I (TnI1 and TnI2) in approximately equal amounts, whereas F2 fibers expressed primarily TnI3 and lower levels of TnI1. F1 fibers were more sensitive to Ca2+, as shown by higher pCa values at threshold activation (pCa(10)=6.50+/-0.07) and at 50% maximum force (pCa(50)=6.43+/-0.07) than F2 fibers (pCa(10)=6.12+/-0.04 and pCa(50)=5.88+/-0.03, respectively). F1 fibers also had a greater degree of co-operativity in Ca2+ activation, as shown by a higher maximum slope of the force-pCa curve (n(Ca)=12.98+/-2.27 vs. 4.34+/-0.64). These data indicate that there is a greater fast fiber-type diversity in crustacean muscles than was previously supposed. Moreover, the differences in activation properties suggest that the TnI isoform composition influences the Ca2+ sensitivity of the contractile mechanism.     

Thick Filaments in Unstretched Mammalian Smooth Muscle

THE controversy concerning the organization of myosin in mammalian smooth muscle was reviewed (Nature New Biology, 231, 225; 1971) at a time when the studies of Rice's laboratory and our own demonstrated a regular, quasi-rectangular array of thick filaments in guinea-pig taenia coli (TC) and rabbit portal-anterior mesenteric vein (MV), and, further, that, by excessive stretch and by the use of hypertonic incubation solutions, the thick filaments in this lattice could be aggregated into ribbon-like structures^1,2. These observations were made on muscles stretched to approximately 1.5 times their excised length. Both the TC³ and the rabbit MV^2,4 are spontaneously active smooth muscles, which shorten to less than their in vivo length when excised from the body: stretching by approximately 1.5 times brings these muscles close to their in vivo length. Nevertheless, recent reports^5,6, indicating that thick filaments were more readily visualized (but see Figs. 2 and 3 in ref. 5) in stretched smooth muscles, prompted the editorial writer of Nature (231, 423; 1971) to consider it a debatable question whether thick filaments are present in unstretched muscle. Thick filaments have been observed in relaxed muscles^1,5,6 and we now show that an array of thick filaments can also be observed in completely unstretched guinea-pig and rabbit MV smooth muscle (excised and dropped into the fixative) and that such arrays are present after two different modes of fixation.     

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.     

Effect of pyruvate, octanoate and glucose on leucine degradation in skeletal muscle from fed and fasted chicks

1. Pyruvate at 5 mM decreased the rate of leucine oxidative decarboxylation and increased the rate of 2-oxoisocaproate production in extensor digitorum communis (EDC) muscles from both fed and 24-hr fasted chicks. Pyruvate at 5 mM increased the net rate of leucine transamination in EDC muscle from fed chicks and had no effect in EDC muscles from 24-hr fasted chicks. 2. Octanoate at 0.2 and 1 mM markedly increased the rates of net leucine transamination, leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6 in EDC muscles from fed chicks, but had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks. 3. Glucose at 5 and 12 mM decreased the rates of leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6, and increased the net rate of 2-oxoisocaproate production as compared to control (no glucose) group in muscles from fed chicks. Glucose had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks.     

Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles

A state of hypokinesia and hypodynamia has been induced in the hindlimb muscles of the rat (100 g) using a suspension model. The ensuing muscle atrophy was assessed by reference to muscles in fully mobile control animals, which were either fed ad libitum or fed the same lower food intake of the suspended animals. Over a total of 7 days of suspension the slow-twitch postural soleus muscle underwent a much greater atrophy than the fast-twitch phasic extensor digitorum longus. Changes with respect to the position of the suspended foot, and hence muscle length, necessitate caution in comparing the extent of the atrophy between different muscle types. After 3 days of inactivity the atrophy of the soleus muscle was explained by a 21% decrease in the fractional rate of synthesis (measured in vivo) and a 100% increase in the rate of protein breakdown. The reduction in the synthetic rate was associated with a net loss (23%) of RNA and hence muscle ribosomes. In contrast when this inactive soleus muscle was permanently stretched the RNA content (44%) and protein synthetic rate increased (59%) markedly above control values. Although protein breakdown remained elevated in this stretched muscle, the extent of the atrophy in response to hypokinesia and hypodynamia was greatly reduced.     

Cevadine-induced changes of membrane potential and sodium transport of muscle membrane in a chloride-free solution

Cevadine-induced changes in membrane potential, sodium transport, intracellular Na, K, and water content were investigated in sartorius muscles incubated in chloride-free (glutamate) Ringer. Cevadine sensitivity of muscles incubated in glutamate Ringer was about five times greater than that of muscles incubated in normal Ringer. Therefore, even 0.005 mmol/l cevadine could induce depolarization and membrane potential oscillations. The membrane potential oscillations were recorded much longer from muscles incubated in chloride-free Ringer (even in the 15th hour of treatment) than in normal Ringer. Depolarization and membrane potential oscillations reversed more slowly in cevadine-free glutamate Ringer than in alkaloid-free normal Ringer. The rhythmic activity could be recorded even in the 10th-15th hour of incubation in cevadine-free glutamate Ringer. Cevadine increased the 24Na uptake of muscles incubated in glutamate Ringer by an average of 230%. In comparison, the cevadine-induced increase of 24Na uptake of muscles incubated in normal Ringer was approximately 350%. In the presence of cevadine the 24Na loss of muscles incubated either in glutamate or in normal Ringer increased to the same degree, i.e. three times. The increase of 24Na loss developed faster in glutamate Ringer than in the presence of chloride. The water content of muscles incubated in cevadine containing, chloride-free (glutamate) Ringer did not increase significantly. Muscles incubated in normal Ringer with cevadine showed a 42.7% increase of water content in 2 hours. Intracellular Na content and Na concentration increased by about 60% during a 2-hour-treatment with cevadine in a chloride-free environment. At the same time, cevadine treatment increased the intracellular Na content and Na concentration of muscles incubated in normal Ringer by about 160% and 80%, respectively. The cevadine-induced decrease of intracellular K content and concentration of muscles incubated in glutamate Ringer was 5% and 10%, respectively, in 2 hours. On the other hand, the decrease of intracellular K concentration in muscles incubated in cevadine-containing normal Ringer occasionally reached 30% due to the increase of water content of the muscles. The cevadine-induced increase of the wet weight of muscles incubated in normal Ringer was practically irreversible. It was not possible to eliminate the increase of wet weight even by washout lasting for 10-15 hours.     

Effect of stretch frequency and sex on the rate of gain and rate of loss in muscle flexibility during a hamstring-stretching program: a randomized single-blind longitudinal study

This study evaluated the effects of 4 different weekly stretching protocols on the rate of gain and decline in hamstring flexibility over an 8-week period, across sex. Using a randomized single-blind design, 53 healthy subjects aged 18-46 years were assigned to 1 of 4 stretching protocols or a control group. The stretching protocols consisted of either daily or 3 times per week stretching and performed once or twice each day. These protocols differed in terms of frequency and total weekly stretching time. All the subjects stretched their hamstring muscles for 4 weeks and were measured weekly for their hip range of motion (ROM). Stretching ceased the final 4 weeks as the weekly measurements continued. The results revealed no significant differences in the rate of gain or the rate of loss between the different stretching protocols (2-way analysis of variance, F = 2.60, p > 0.05). All the stretching groups gained in hip ROM from pre to week 4 (F = 269.24, p < 0.001). After cessation, the rate of loss was similar for all the 4 stretching groups (F = 102.86, p < 0.001); all the groups retained significant gains at the end of the study (p < 0.001). The control group did not change over time. Those who stretched at least 6 times per week gained more than those who stretched 3 times per week (24 and 16.8%, respectively, F = 5.20, p < 0.05). Subject sex did not influence ROM changes (p > 0.05). Stretching appears to be equally effective, whether performed daily or 3 times per week, provided individuals stretch at least 2 times each day. Moreover, although women are more flexible than men are, there was no sex difference in terms of stretching response.     

Hindlimb unloading increases muscle content of cytosolic but not nuclear Id2 and p53 proteins in young adult and aged rats.

This study tested the hypothesis that inhibitor of differentiation-2 (Id2), p53, and heat shock proteins (HSP) are responsive to suspension-induced muscle atrophy. Fourteen days of hindlimb suspension were used to unload the hindlimbs and induce atrophy in gastrocnemius muscles of young adult and aged rats. Following suspension, medial gastrocnemius muscle wet weight was reduced by approximately 30%, and the muscle wet weight normalized to the animal body weight decreased by 11 and 15% in young adult and aged animals, respectively. mRNA abundances of Id2, p53, HSP70-2, and HSP27 did not change with suspension, whereas HSP70-1 mRNA content was lower in the suspended muscle compared with the control muscle in both young adult and aged animals. Our immunoblot analyses indicated that protein expressions of HSP70 and HSP60 were not different between suspended and control muscles in both ages, whereas HSP27 protein content was increased in suspended muscle relative to control muscle only in young adult animals. Id2 and p53 protein contents were elevated in the cytosolic fraction of suspended muscle compared with the control muscle in both young and aged animals, but these changes were not found in the nuclear protein fraction. Furthermore, compared with young adult, aged muscles had a lower HSP70-1 mRNA content but higher HSP70-2 mRNA content and protein contents of Id2, p53, HSP70, and HSP27. These findings are consistent with the hypothesis that Id2 and p53 are responsive to unloading-induced muscle atrophy. Moreover, our data indicate that aging is accompanied with altered abundances of HSP70-1 and HSP70-2 mRNA, in addition to Id2, p53, HSP70, and HSP27 protein in rat gastrocnemius muscle.     

Muscle acetylcholinesterase adapts to compensatory overload by a general increase in its molecular forms

We have investigated the impact of compensatory overload on the content of acetylcholinesterase (AChe) molecular forms in the rat fast-twitch medial gastrocnemius (MG). Overload was induced by way of a bilateral tenotomy of the MG's functional synergists coupled to a daily walking training program (15 m/min, 30% incline, up to 60 min per session, 12-18 wks). This latter condition ensured that the MG were used on a regular basis. In comparison to control values, overloaded MG showed 25 and 19% increases (P less than 0.05) in muscle wet weight and protein concentration, respectively. The content in AChe (activity per muscle) was also increased in these MG (28%, P less than 0.05). Sedimentation analyses revealed a general elevation in the content of AChe molecular forms, with A8, G2, and G1 displaying significant changes (35-42%, P less than 0.05). In a second group of rats, daily running training (27 m/min, 30% incline, using the same timetable) was supplemented to the compensatory overload. In this group, the additional running training led to a greater hypertrophic response as attested to by increases (P less than 0.05) in the MG wet weight (41%) and protein concentration (35%) in comparison to controls. However, total AChe content of these muscles was increased to an extent similar to that observed in the MG subjected only to compensatory overload (24%, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses to cafeteria feeding in mice after the removal of interscapular brown adipose tissue

Feeding acafeteria diet to mice resulted in an increased energy intake of approximately 30% and this led to increases in the wet weight, total protein content , and total cytochrome oxidase activity of interscapular and dorso-cervical brown adipose tissue. Surgical removal of interscapular brown adipose tissue, followed by cafeteria feeding, gave rise to an elevation in dorso-cervical brown adipose tissue wet weight, total protein content, and total cytochrome oxidase activity, compared to intact cafeteria-fed mice. Cafeteria feeding with or without the removal of interscapular brown adipose tissue did not lead to significant increases in body weight compared to stock-fed control mice, but both cafeteria-fed groups of mice showed significant elevations in body fat content indicating that the induced hyperphagia led to a relative obesity in the cafeteria-fed groups. The results presented are consistent with an increased thermogenic activity in the brown adipose tissue of cafeteria-fed mice, and the effect of the removal of interscapular brown adipose tissue further indicates the quantitative importance of the tissue in the control of body weight.     

Comparison of cold storage and perfusion of dog livers on function of tissue slices

We compared how two methods of hypothermic preservation affect physiological functions of tissue slices of dog liver. Livers were preserved by either (i) cold storage (CS) in Collins' solution or (ii) continuous perfusion (P) with a perfusate, containing hydroxyethyl starch, sodium gluconate, adenosine, and potassium phosphate, recently developed in our laboratory. Livers were cold stored for 6 to 8, 24, or 48 hr, and perfused for 24 or 72 hr. Tissue slices of preserved livers were incubated at 30 degrees C and analyzed for volume control, electrolyte-pump activity (K and Na), and adenine nucleotide concentration. Also, mitochondria were isolated after preservation to quantify respiratory activity. Slice functions of livers preserved for short periods (6 to 8 hr by CS and 24 hr by P) were similar to those for control livers. After normothermic incubation, the mean (+/- SD) water content of tissue (expressed per unit dry mass of tissue) was 2.3 +/- 0.3 kg/kg for control, 2.6 +/- 0.4 kg/kg for 6- to 8-hr CS, and 2.5 +/- 0.5 kg/kg for 24-hr P. Longer periods of preservation resulted in cell swelling, and water content was 3.3 +/- 0.4 kg/kg for 24- to 48-hr CS and 2.8 +/- 0.3 kg/kg for 72-hr P. The mean (+/- SD) K/Na ratio was nearly normal for livers preserved for short periods: 3.7 +/- 0.5 for control, 4.1 +/- 0.2 for 6- to 8-hr CS, and 3.3 +/- 0.4 for 24-hr P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical stretch induces activation of skeletal muscle satellite cells in vitro.

Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. In response to stretch, satellite cells entered the cell cycle earlier than if they were under control conditions. Only a brief period of stretch, as short as 2 h, was necessary to stimulate activation. Additionally, conditioned medium from stretched cells could activate unstretched satellite cells. The presence of HGF on c-met-positive myogenic cells was detected by immunofluorescence at 12 h in culture, and immunoblots demonstrated that HGF was released by stretched satellite cells into medium. Also, stretch activation could be abolished by the addition of anti-HGF antibodies to stretched cultures, and activity in conditioned medium from stretched cells could be neutralized by anti-HGF antibodies. In addition, stretch appeared to cause release of preexisting HGF from the extracellular matrix. These experiments suggest that HGF may be involved in linking mechanical perturbation of muscle to satellite cell activation.