Rat Quadriceps Muscle Research Articles

Effect of Hypoxia on Glucose Tolerance and GLUT4 Gene Expression on Spinal Cord Injury Rats

2262 PURPOSE: To investigate the effect of intermittent systematical hypoxia on wholebody glucose tolerance and GLUT4 gene expression in atrophied muscle of spinal cord injury (SCI) rats. METHODS: Sixteen female SD rats (279 ± 3.5 g) were weightmatched into four groups, including SCI-hypoxia (TH), SCI-normaxia (TN), shamehypoxia (CH), and shame-normaxia (CN) for 2 weeks of treatment; and normobaric intermittent hypoxia was used for hypoxic exposure (90 min/day; O2%=12%). OGTT was used for evaluating glucose tolerance, and Western and Northern Blot were used for analyzing GLUT4 protein and mRNA, respectively. RESULTS: In the present study we found that the SCI rats exhibited higher glucose levels during OGTT. After two weeks hypoxia intervention, glucose tolerance was improved in both SCI and shame-operated rats. Hypoxia also enhanced GLUT4 protein level in soleus and red quadriceps muscle of SCI rats (P<0.05). CONCLUSION: We observed that the shortterm intermittent hypoxia reversed the reduction of glucose tolerance following SCI. Moreover, GLUT4 expression induced by hypoxia may be a possible mechanism of whole-body glucose tolerance improvement after SCI.

Effect of Hypoxia on Glucose Tolerance and GLUT4 Gene Expression on Spinal Cord Injury Rats

2262 PURPOSE: To investigate the effect of intermittent systematical hypoxia on wholebody glucose tolerance and GLUT4 gene expression in atrophied muscle of spinal cord injury (SCI) rats. METHODS: Sixteen female SD rats (279 ± 3.5 g) were weightmatched into four groups, including SCI-hypoxia (TH), SCI-normaxia (TN), shamehypoxia (CH), and shame-normaxia (CN) for 2 weeks of treatment; and normobaric intermittent hypoxia was used for hypoxic exposure (90 min/day; O2%=12%). OGTT was used for evaluating glucose tolerance, and Western and Northern Blot were used for analyzing GLUT4 protein and mRNA, respectively. RESULTS: In the present study we found that the SCI rats exhibited higher glucose levels during OGTT. After two weeks hypoxia intervention, glucose tolerance was improved in both SCI and shame-operated rats. Hypoxia also enhanced GLUT4 protein level in soleus and red quadriceps muscle of SCI rats (P<0.05). CONCLUSION: We observed that the shortterm intermittent hypoxia reversed the reduction of glucose tolerance following SCI. Moreover, GLUT4 expression induced by hypoxia may be a possible mechanism of whole-body glucose tolerance improvement after SCI.

Involuntary leg movements affect interstitial nutrient gradients and blood flow in rat skeletal muscle.

To evaluate the effect of passive muscle shortening and lengthening (PSL) on the transcapillary exchange of glucose, lactate, and insulin in the insulin-stimulated state, microdialysis was performed in rat quadriceps muscle. Electrical pulsatile stimulation (0.1 ms, 0.3-0.6 V, 1 Hz) was performed on the sciatic nerve in one leg to induce passive tension on the quadriceps during a hyperinsulinemic-euglycemic clamp (10 mU x kg(-1) x min(-1)). In the non-insulin-stimulated (basal) state, the muscle arterial-interstitial (A-I) concentration difference of glucose was 1.6 +/- 0.3 mM (P < 0.01). During insulin infusion, it remained unaltered in resting muscle (1.3 +/- 0.3 mM) but diminished during PSL. In the basal state there was no A-I concentration difference of lactate, whereas in the insulin infusion state it increased significantly and was significantly greater in moving (2.8 +/- 0.5 mM, P < 0.01) than in resting muscle (0.7 +/- 0.4 mM). The A-I concentration difference of insulin was equal in resting and moving muscle: 86 +/- 7 and 100 +/- 8 microU/ml, respectively. Muscle blood flow estimated by use of radiolabeled microspheres increased during PSL from 17 +/- 4 to 34 +/- 6 ml x 100 g(-1) x min(-1) (P < 0.05). These results confirm that diffusion over the capillary wall is partly rate limiting for the exchange of insulin and glucose and lactate in resting muscle. PSL, in addition to insulin stimulation, increases blood flow and capillary permeability and, as a result, diminishes the A-I concentration gradient of glucose but not that of insulin or lactate.

What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent K m ADP of oxidative phosphorylation increased significantly, by 70–100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent K m ADP value (234±24 μM ADP) compared with other preparations (14–28 μM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.

Differential effects of endurance training and creatine depletion on regional mitochondrial adaptations in rat skeletal muscle

To examine the combined effects of 2-week endurance training and 3-week feeding with β-guanidinopropionic acid (GPA) on regional adaptability of skeletal muscle mitochondria, intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated from quadriceps muscles of sedentary control, trained control, sedentary GPA-fed and trained GPA-fed rats. Mitochondrial oxidative phosphorylation was assessed polarographically by using pyruvate plus malate, succinate (plus rotenone), and ascorbate plus N,N,N´,N´-tetramethyl-p-phenylenediamine (TMPD) (plus antimycin) as respiratory substrates. Assays of cytochrome c oxidase and F1-ATPase activities were also performed. In sedentary control rats, IFM exhibited a higher oxidative capacity than SSM, whereas F1-ATPase activities were similar. Training increased the oxidative phosphorylation capacity of mitochondria with both pyruvate plus malate and ascorbate plus TMPD as substrates, with no differences between IFM and SSM. In contrast, the GPA diet mainly improved the overall SSM oxidative phosphorylation capacity, irrespective of the substrate used. Finally, the superimposition of training to feeding with GPA strongly increased both oxidase and enzymic activities in SSM, whereas no cumulative effects were found in IFM mitochondria. It therefore seems that endurance training and feeding with GPA, which are both known to alter the energetic status of the muscle cell, might mediate distinct biochemical adaptations in regional skeletal muscle mitochondria.

Differential effects of endurance training and creatine depletion on regional mitochondrial adaptations in rat skeletal muscle

To examine the combined effects of 2-week endurance training and 3-week feeding with beta-guanidinopropionic acid (GPA) on regional adaptability of skeletal muscle mitochondria, intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated from quadriceps muscles of sedentary control, trained control, sedentary GPA-fed and trained GPA-fed rats. Mitochondrial oxidative phosphorylation was assessed polarographically by using pyruvate plus malate, succinate (plus rotenone), and ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (plus antimycin) as respiratory substrates. Assays of cytochrome c oxidase and F(1)-ATPase activities were also performed. In sedentary control rats, IFM exhibited a higher oxidative capacity than SSM, whereas F(1)-ATPase activities were similar. Training increased the oxidative phosphorylation capacity of mitochondria with both pyruvate plus malate and ascorbate plus TMPD as substrates, with no differences between IFM and SSM. In contrast, the GPA diet mainly improved the overall SSM oxidative phosphorylation capacity, irrespective of the substrate used. Finally, the superimposition of training to feeding with GPA strongly increased both oxidase and enzymic activities in SSM, whereas no cumulative effects were found in IFM mitochondria. It therefore seems that endurance training and feeding with GPA, which are both known to alter the energetic status of the muscle cell, might mediate distinct biochemical adaptations in regional skeletal muscle mitochondria.

Mechanical loading regulates tenascin-C expression in the osteotendinous junction.

Elastic extracellular matrix protein tenascin-C (TN) has very restricted expression in normal tissues, but is expressed in large quantities during embryogenesis and hyperplastic processes. To examine the importance of mechanical stress on the regulation of TN expression in vivo, the effects of various mechanical loading states (immobilization and three forms of subsequent remobilization) on the expression of TN were studied immunohistochemically at the bone-tendon attachment of the rat quadriceps muscle. This osteotendinous junction (OTJ) was selected as study site, since it receives its mechanical stimuli only from muscle contracting activity, which is easy to block by cast immobilization. TN was expressed abundantly in the normal OTJ. Following the removal of the mechanical stress from the junction by cast-immobilization of three weeks, the immunoreactivity of TN was almost completely absent. Normal mechanical stress in the form of free remobilization of eight weeks (free cage activity) resulted in a slight increase in TN expression, but could not restore the expression of TN to the level of the healthy contralateral leg. After the application of the increased mechanical stress (intensified remobilization of the eight weeks by low- or high-intensity treadmill running), the distribution and immunoreactivity of TN reached the level of the healthy contralateral limb in the low-intensity running group or even exceeded that in the high-intensity running group. High TN expression was seen around the chondrocytes and fibroblasts of the OTJ as well as around the collagen fibers of the tendon belly. We conclusively show that mechanical strain regulates the expression of TN in vivo, and propose that mechanical stress is a major regulator of TN expression in fibroblasts and chondrocytes. This may be an important aspect of the regulation of TN expression during embryogenesis, tendon degeneration, wound healing, bone formation, and in the other normal or regenerative morphogenetic processes TN is postulated to take part in.

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Role of patellar tendon on the population of muscle fiber types and the contractile properties of single glycerinated muscle fibers in quadriceps muscles of rats

The role of the patellar tendon (PT) in the contractile properties of quadriceps muscle was investigated. After PT resection (partial or complete), PT reconstruction, or a sham operation, rats were forced to run on a rodent treadmill. The histochemical and mechanical characteristics of the vastus medialis (VM) and vastus lateralis (VL) muscles were examined 2, 4, and 8 weeks after treatment. There were no significant changes in either fiber type population or the contractile properties of glycerinated single muscle fibers from VM and VL 2 and 4 weeks after partial PT resection. Complete PT resection caused a decrease in the population of type I fibers in VM, while the running training after PT reconstruction increased the population of type I fibers in VM. For both the PT resection and PT reconstruction groups, the Hill coefficient, an indicator of the Ca2+ sensitivity of myofilaments, was calculated from the pCa-tension relationship of glycerinated single muscle fibers. In the PT reconstruction group, the Hill values for fibers isolated from VM were significantly decreased by running training, but the Hill values for VL were not. The effect of running training on the Ca2+ sensitivity of myofilaments in muscle fibers with PT reconstruction was different from that in muscle fibers without PT treatment. Both resection and partial resection of the PT in this animal model had heterogeneous effects on the contractile properties of quadriceps muscles; we presume that, clinically, we must give careful consideration to PT treatment.

Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting.

The newly described uncoupling protein 3 (UCP3) may make an important contribution to thermogenesis in humans because of its high level of expression in skeletal muscle. Contrary to expectations, fasting, a condition that reduces resting energy expenditure, has been reported to increase UCP3 expression in muscle. We have confirmed that a 10-fold increase in UCP3 mRNA levels occurs in rat quadriceps muscle between 12 and 24 h of food removal. A less consistent twofold increase in muscle UCP2 mRNA levels was observed in animals fasted for up to 72 h. Administration of recombinant leptin to prevent a fall in circulating leptin levels did not eliminate the fasting-induced increase in quadriceps UCP3 expression. Administration of a high dose of glucocorticoid to fed animals to mimic the increase in corticosterone induced by fasting did not reproduce the increase in UCP3 expression observed in fasted animals. In contrast, elevation of circulating free fatty acid levels in fed animals by Intralipid plus heparin infusion caused significant increases in the UCP3/actin mRNA ratio compared with saline-infused fed controls in both extensor digitorum longus (2.01 +/- 0.34 vs. 0.68 +/- 0.11, P = 0.002) and soleus muscles (0.31 +/- 0.07 vs. 0.09 +/- 0.02, P = 0.014). We conclude that free fatty acids are a potential mediator of the increase in muscle UCP3 expression that occurs during fasting. This seemingly paradoxical induction of UCP3 may be linked to the use of free fatty acid as a fuel rather than an increased need of the organism to dissipate energy.

Endurance training attenuates the decrease in skeletal muscle malonyl-CoA with exercise.

Muscle malonyl-CoA has been postulated to regulate fatty acid metabolism by inhibiting carnitine palmitoyltransferase 1. In nontrained rats, malonyl-CoA decreases in working muscle during exercise. Endurance training is known to increase a muscle's reliance on fatty acids as a substrate. This study was designed to investigate whether the decline in malonyl-CoA with exercise would be greater in trained than in nontrained muscle, thereby allowing increased fatty acid oxidation. After 6-10 wk of endurance training (2 h/day) or treadmill habituation (5-10 min/day), rats were killed at rest or after running up a 15% grade at 21 m/min for 5, 20, or 60 min. Training attenuated the exercise-induced drop in malonyl-CoA and prevented the exercise-induced increase in the constant for citrate activation of acetyl-CoA carboxylase in the red quadriceps muscle of rats run for 20 and 60 min. Hence, contrary to expectations, the decrease in malonyl-CoA was less in trained than in nontrained muscle during a single bout of prolonged submaximal exercise.

Use of Bone Morphogenetic Protein 2 on Ectopic Porous Coated Implants in the Rat

The ability of recombinant human bone morphogenetic protein 2 to remain osteoinductive and stimulate appositional bone formation on a porous coated implant was tested in a rat quadriceps muscle pouch. Implants with or without hydroxyapatite were used to compare the effects on bone formation of two different does (23 micrograms or 46 micrograms) of recombinant human bone morphogenetic protein 2 against controls as evidenced by contact radiography, histologic examination, and backscatter scanning electron microscopic analysis. Cylindrical plasma sprayed porous titanium implants were placed bilaterally within a muscle pouch surgically created in 48 Lewis rats. Implants treated with recombinant human bone morphogenetic protein 2 formed significantly more bone than did control implants independent of the dose or presence of hydroxyapatite. In all implants with bone formation, osteoinduction via endochondral ossification began within 7 days. By 21 days, cartilage largely was replaced by bone and marrow. The results of this ectopic, nonweightbearing in vivo assay suggest that recombinant human bone morphogenetic protein 2 remains biologically active after application to a titanium implant and may be used to enhance appositional bone formation by direct application to the implant surface.

A high-fat diet influences insulin-stimulated posttransport muscle glucose metabolism in rats

Because of a failure to detect significant quantities of intracellular glucose, it has been generally accepted that transport rather than phosphorylation is the rate-limiting process of muscle glucose metabolism under most (but not all) physiological conditions. Here, we have measured tissue free levels of the glucose analog 2-deoxy- d-glucose (2DG) in red quadriceps muscle of rats fed a high-fat diet (59% of energy from fat) for 3 weeks, to identify the barrier to insulin-stimulated glucose uptake previously seen in such animals. Measurements were performed on pentobarbital-anesthetized rats following exogenous infusion of radiolabeled 2DG. A glucose clamp was used to maintain plasma insulin at high physiological levels (≈ 120 mU/L). Three other treatment groups representing normal insulin action (chow-fed), extreme glucose uptake (maximal insulin stimulation + hyperglycemia), and insulin resistance with elevated free intracellular glucose (epinephrine infusion) were also studied for comparison. In chow-fed animals, no muscle free 2DG was detected, confirming transport as the rate-limiting process. In fat-fed animals, a significant elevation in muscle free 2DG was observed ( P < .01 v) chow-fed controls). The elevation was similar in magnitude to that in epinephrine-infused rats, and implied a limitation of insulin action at a posttransport step. This result was confirmed with a more complex modeling analysis. We conclude that posttransport steps influence insulin-stimulated in vivo muscle glucose metabolism in long-term high-fat—fed rats.

Cytochrome c protein synthesis rate in rat skeletal muscle.

Endurance training is associated with increases in mitochondrial density, of which cytochrome c protein is an index. Increases in the synthesis rates of cytochrome c protein in skeletal muscle during endurance training have been inferred (Biochem. Biophys. Res. Commun. 66: 173, 1975; J. Biol. Chem. 252: 416, 1977). One purpose of the present study was to test these indirect approximations with direct measurements of the synthesis rates of cytochrome c protein in skeletal muscles postexercise. No change in the fractional synthesis rate of cytochrome c was detected in the red quadriceps muscle of rats either 2-7 h after a 104-min run on a motor-driven treadmill or 17-22 h after the final bout of 4 days of running 100 min/day. If the 16% increase in cytochrome c protein concentration in the red quadriceps muscle on the 5th day of training is used to calculate the nanomoles of cytochrome c synthesized per gram of wet muscle weight, the normalized rate of cytochrome c protein synthesis is increased 29% on the 5th day of training. The observation of no significant alteration in cytochrome c mRNA in the red quadriceps muscle of rats during the 1st wk of training implies that the initial increase in the synthesis rate of cytochrome c protein normalized per unit of muscle mass during treadmill training is likely to occur at a translational or posttranslational step. These results suggest that the control of increased cytochrome c expression in skeletal muscle during exercise training involves a complex mechanism.

In vivo location of the rate-limiting step of hexose uptake in muscle and brain tissue of rats

The uptake of glucose proceeds via facilitated transport from the plasma followed by phosphorylation of intracellular glucose. We have quantified the relative contribution of transport and phosphorylation to the overall rate of hexose utilization into the quadriceps muscle (red and white) and cerebellum of rats anesthetized with pentobarbital sodium. The method employed simultaneous infusions of radiolabeled 3-O-methyl-D-glucose and 2-deoxy-D-glucose. Results were expressed in terms of a parameter ft*, which has theoretical limits of 0 and 1 corresponding to phosphorylation and transport limitation, respectively. In cerebellum, basal rates of transport and phosphorylation were comparable (ft* = 0.32 +/- 0.02). Under conditions of hyperglycemia plus maximum insulin stimulation, phosphorylation limited glucose utilization to a greater extent (ft* = 0.12 +/- 0.02). No effect of hyperinsulinemia alone was observed. In red muscle, transport determined overall glucose utilization in the basal (ft* = 0.96 +/- 0.05) and euglycemic insulin-stimulated states (ft* = 0.90 +/- 0.02). A shift of the rate-limiting step from transport toward phosphorylation was observed in insulin-stimulated red muscle when blood glucose (ft* = 0.64 +/- 0.05) or epinephrine levels (ft* = 0.66 +/- 0.07) were elevated. Neither effect was seen in white muscle. We conclude that the transport step dominates but is not the only determinant of muscle hexose utilization under all conditions.

Proximal skeletal muscle alterations in streptozotocin‐diabetic rats: A histochemical and morphometric analysis

The response of rat quadriceps muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from diabetic and weight-matched control rats were assayed for myofibrilar adenosine triphosphatase (ATPase) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). A quantitative analysis was carried out by an automatic interactive analysis system focused on the fiber type size and distribution. STZ-induced diabetes caused important effects in this muscle, with changes in the distribution of oxidative enzyme reactions, type I fiber hypertrophy, and type II fiber atrophy, which was greater in type IIB than in type IIA. It is concluded that hypoinsulinism produces morphological alterations in proximal skeletal muscle fibers that are similar to those of neurogenic myopathy. Thus the pathological changes in these mammalian muscle fibers could explain the clinical syndrome seen in diabetic patients called "diabetic symmetrical proximal motor neuropathy," perhaps the least understood of the major neuropathic complications of diabetes.

Glutamine metabolism in skeletal muscle of septic rats

The metabolism of skeletal muscle glutamine was studied in rats made septic by cecal ligation and puncture technique. Blood glucose was not significantly different in septic rats, but lactate, pyruvate, glutamine, and alanine were markedly increased. Conversely, blood ketone body concentrations were markedly decreased in septic rats. Both plasma insulin and glucagon were markedly elevated in septic rats. Sepsis increased the rates of glutamine production in muscle, but without marked effects on skin and adipose tissue preparations, with muscle production accounting for over 87% of total glutamine produced by the hindlimb. Sepsis produced decreases in the concentrations of skeletal muscle glutamine, glutamate, 2-oxoglutarate, and adenosine monophosphate (AMP). The concentrations of ammonia, pyruvate, and inosine monophosphate (IMP) were increased. Hindlimb blood flow showed no marked change in response to sepsis, but was accompanied by an enhanced net release of glutamine and alanine. The maximal activity of glutamine synthetase was increased only in quadriceps muscles of septic rats, whereas that of glutaminase was decreased in all muscles studied. Tyrosine release from incubated muscle preparation was markedly increased in septic rats; however, its rate of incorporation was markedly decreased. It is concluded that there is an enhanced rate of production of glutamine from skeletal muscle of septic rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine; these suggestions are discussed.

A Comparative Study of the Effect of Arterial and Venous Occlusion after Various Periods of Ischemia

The author attempted to quantify any additional tissue damage resulting from one hour of venous occlusion after controlled periods of ischemia in skeletal muscle, compared to equal time periods of ischemia. The rat quadriceps muscle was used. pH and temperature were monitored during the experiment and the damage was evaluated on histochemical sections. Of the other two parameters monitored in this study, the pH recovery rate during reperfusion best reflected tissue perfusion and served as a predictor for the extent of tissue damage, when global ischemia or the combination of ischemia and postischemic venous stasis did not exceed five hours. Apart from the nonstained lethally damaged areas, interstitial tissue edema and cellular infiltration were constant findings in all muscles that sustained ischemia or ischemia and subsequent venous occlusion. However, both findings were more pronounced in the muscles that underwent venous obstruction. Muscle fibers were more edematous after venous occlusion, compared to ischemic muscles. Thrombosed veins and collapsed arteries, surrounded by excessive edema and inflammatory reaction, were common in muscles after ischemia and venous occlusion. Comparing the difference in percent of lethal damage from one hour of postischemic venous stasis versus the damage from additional ischemia of equal duration, findings demonstrated comparable damage when reperfusion was established in less than five hours.

In vivo nmr diffusion spectroscopy: 31p application to phosphorus metabolites in muscle

Apparent diffusion coefficients (Da) of individual metabolites can be studied in vivo by diffusion NMR spectroscopy using an echo sequence sensitized to molecular motion. The methods are based on the echo attenuation due to phase dispersion resulting from incoherent displacement during the diffusion time. As the displacement of metabolites by diffusion in vivo can be affected by compartment size, temperature, adsorption processes, etc., the presented methods are potentially useful in studying such phenomena in vivo. Here, the methods are applied to phosphocreatine in the rat quadriceps muscle. It is demonstrated that the displacement of phosphocreatine resembles free diffusion for short diffusion times but becomes limited as a result of boundaries due to compartmentation for longer diffusion times. The limit of the displacement indicates an apparent average size of 44 microns of the compartment in the direction of the diffusion gradient. As the gradient was applied approximately parallel (angle less than 25 degrees) to the muscle fiber, this result indicates that phosphocreatine moves freely in the cytosol but is limited by the boundaries of the muscle cells. Error analyses are performed with regard to motion artifacts and gradient performance. The methods were tested extensively for distilled water and free metabolites.

Anaerobic chemical changes and mechanical output during isometric tetani of rat skeletal muscle in situ

The time course was examined of the energy-rich phosphate usage and exerted isometric tetanic force in electrically stimulated rat quadriceps muscle. The maximal rate of energy-rich phosphate usage was calculated from the changes in the intramuscular concentrations of phosphocreatine, lactate, ATP and inosine monophosphate (IMP) and was somewhat higher than those calculated on the basis of exercise in vivo. The IMP concentration increased directly from the onset of the contraction until after about 11 s it remained constant. The increase in the IMP concentration coincided with a decrease in the ATP concentration. The relationship between mechanical output and energy usage was examined in two ways (i) by calculating the ratio time integral of the force (FTI) and the total energy-rich usage (Ptot) and (ii) by calculating the ratio Force (Ft) to the energy flux (dPtot/dt) at a certain time t. Whereas the ratio FTI/Ptot showed a hyperbolic relationship, the ratio Ft/(dPtot/dt) showed a parabolic relationship. From the latter finding and from the results described in the literature it is concluded that the ratio mechanical output/energy-rich phosphate usage depends on the conditions under which exercise is carried out. Recovery under aerobic conditions from a maximal tetanic isometric contraction sustained for 15 s was slow compared to results of experiments in vivo.