Cytoskeletal proteins and heat shock proteins 27 under eccentric exercise of rats: effects of calcium L-type channel blockade

It is known that during eccentric exercise the calcium accumulation and degradation of cytoskeletal proteins take place. This study was aimed to investigation into the role of baseline calcium accumulation in cytoskeletal degradation and the way of calcium inflow into skeletal muscle un- der conditions of eccentric exercise. Wister rats were divided into control (C), eccentric exercise (EE) (one set of downhill treadmill running (-16 degrees) at a speed of 20 m. min(-1) for 40 min) and eccentric exercise plus nifedipine administration (EEN) groups (with a daily supplementation of 6.25 mg/kg nifedipine in drinking water during two days). After a 24-hour post-exercise dystrophin, layer integrity and desmin level in m. soleus were declined in EE and didn't change in EEN gr. vs. C (p < 0.05). HSP27 were decreased in EEN in comparison with EE gr. (p < 0.05). Titin and nebullin were not changed after exercise. It seems that calcium L-type channel blocker attenuates or reduces the contraction-induced damage to cytoskeletal proteins.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

L-arginine Supplementation Protects Exercise Performance and Structural Integrity of Muscle Fibers after a Single Bout of Eccentric Exercise in Rats

Eccentric exercise is known to disrupt sarcolemmal integrity and induce damage of skeletal muscle fibers. We hypothesized that L-arginine (L-Arg; nitric oxide synthase (NOS) substrate) supplementation prior to a single bout of eccentric exercise would diminish exercise-induced damage. In addition, we used N-nitro-L-arginine methyl ester hydrochloride (L-NAME; NOS inhibitor) to clarify the role of native NOS activity in the development of exercise-induced muscle damage. Rats were divided into four groups: non-treated control (C), downhill running with (RA) or without (R) L-Arg supplementation and downhill running with L-NAME supplementation (RN). Twenty four hours following eccentric exercise seven rats in each group were sacrificed and soleus muscles were dissected and frozen for further analysis. The remaining seven rats in each group were subjected to the exercise performance test. Our experiments showed that L-Arg supplementation prior to a single bout of eccentric exercise improved subsequent exercise performance capacity tests in RA rats when compared with R, RN and C rats by 37%, 27% and 13%, respectively. This outcome is mediated by L-Arg protection against post-exercise damage of sarcolemma (2.26- and 0.87-fold less than R and RN groups, respectively), reduced numbers of damaged muscle fibers indicated by the reduced loss of desmin content in the muscle (15% and 25% less than R and RN groups, respectively), and diminished µ-calpain mRNA up-regulation (42% and 30% less than R and RN groups, respectively). In conclusion, our study indicates that L-Arg supplementation prior to a single bout of eccentric exercise alleviates muscle fiber damage and preserves exercise performance capacity.     

Eccentric contractions leading to DOMS do not cause loss of desmin nor fibre necrosis in human muscle

High force eccentric muscle contractions can result in delayed onset muscle soreness (DOMS), prolonged loss of muscle strength, decreased range of motion, muscle swelling and an increase of muscle proteins in the blood. At the ultrastructural level Z-line streaming and myofibrillar disruptions have been taken as evidence for muscle damage. In animal models of eccentric exercise-induced injury, disruption of the cytoskeleton and the sarcolemma of muscle fibres occurs within the first hour after the exercise, since a rapid loss of staining of desmin, a cytoskeletal protein, and the presence of fibronectin, a plasma and extracellular protein, are observed within the muscle fibres. In the present study, biopsies from subjects who had performed different eccentric exercises and had developed DOMS were examined. Our aim was to determine whether eccentric exercise leading to DOMS causes sarcolemmal disruption and loss of desmin in humans. Our study shows that even though the subjects had DOMS, muscle fibres had neither lost staining for desmin nor contained plasma fibronectin. This study therefore does not support previous conclusions that there is muscle fibre degeneration and necrosis in human skeletal muscle after eccentric exercise leading to DOMS. Our data are in agreement with the recent findings that there is no inflammatory response in skeletal muscle following eccentric exercise in humans. In combination, these findings should stimulate the search for other mechanisms explaining the functional and structural alterations in human skeletal muscle after eccentric exercise.     

Desmin and actin alterations in human muscles affected by delayed onset muscle soreness: a high resolution immunocytochemical study

Lack of staining for desmin in muscles in animal models of eccentric exercise has been suggested to reflect disruption of the desmin intermediate filament network and proposed to cause disruption of the myofibrillar apparatus and deterioration of muscle fibers. In a recent study, we examined muscle biopsies from persons who had performed different eccentric exercise protocols, which induced delayed onset muscle soreness (DOMS). We were unable to verify that loss of staining for desmin was a feature of sore muscles. Nevertheless, we observed changes in the desmin cytoskeleton, but the meaning of the observations was not conclusive. In the present study, a high resolution immunocytochemical method was used to investigate the changes of desmin and actin in human muscles following a bout of eccentric exercise that lead to DOMS 2-3 days post-exercise. Biopsies were taken before exercise and 1 h and 2-3 and 7-8 days after exercise. Phalloidin, a ligand that labels filamentous actin, and anti-desmin antibodies were used to stain semithin (approximately 0.5 micro m) cryosections. At 1 h post-exercise, the staining of actin and desmin did not differ from the controls, whereas in biopsies taken 2-3 and 7-8 days after exercise, 12.5% (SD 5.8%) and 6.1% (SD 2.3%) fibers showed areas of increased staining for actin. Corresponding values for fibers with increased staining for both actin and desmin were 8.7% (SD 3.9%) and 11.4% (SD 4.6%), respectively. We suggest that the increased staining of actin and desmin reflects an increased synthesis of these proteins as part of an adaptation process following the unaccustomed eccentric exercise.     

Exercise-induced muscle damage and inflammation: re-evaluation by proteomics

Using proteomics combined with immunohistochemistry (IHC), we re-evaluated our previous hypothesis that voluntary eccentric exercise does not result in inflammation or necrosis while it does lead to muscular adaptation/remodeling through Z-band related proteins. Muscle biopsies from m. vastus lateralis were taken from five control and five exercised subjects 48 h after 45 min of downhill running. General muscle morphology was examined using histology and histochemistry. Proteomics was used to reveal protein profiles and novel proteins. IHC with specific antibody against three Z-band related proteins identified by proteomics was also performed. General morphology showed no muscle degeneration or inflammation in any exercised biopsy. Proteomics revealed that out of 612 individual protein spots, the exercised biopsy presented three proteins with significant (p < 0.05) higher expression ratio and four proteins of lower ratio compared to controls. Four of the proteins desmin, actin, Rab-35 and LDB3 are Z-band related; the former two have long been the focus of interest and were found to be up-regulated in the study; the latter two are Z-band assembly/stabilization protein and were for the first time observed to be down-regulated in exercised muscles. The other three proteins are related with either cellular metabolism or calcium homeostasis and none is related with muscle necrosis or inflammation. IHC observations that both desmin and actin were increased whereas LDB3 was completely absent in some focal areas are consistent with proteomic results and with our previous observations. The results of the study confirmed our previous findings and therefore strengthened the hypothesis that voluntary eccentric exercise does not cause human muscle necrosis or inflammation; instead, muscular remodeling occurs specifically through Z-band related proteins.     

Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells

The goal of this research was to evaluate the roles of calpains and their interactions with the proteasome and the lysosome in degradation of individual sarcomeric and cytoskeletal proteins in cultured muscle cells. Rat L8-CID muscle cells, in which we expressed a transgene calpain inhibitor (CID), were used in the study. L8-CID cells were grown as myotubes after which the relative roles of calpain, proteasome and lysosome in total protein degradation were assessed during a period of serum withdrawal. Following this, the roles of proteases in degrading cytoskeletal proteins (desmin, dystrophin and filamin) and of sarcomeric proteins (alpha-actinin and tropomyosin) were assessed. Total protein degradation was assessed by release of radioactive tyrosine from pre-labeled myotubes in the presence and absence of protease inhibitors. Effects of protease inhibitors on concentrations of individual sarcomeric and cytoskeletal proteins were assessed by Western blotting. Inhibition of calpains, proteasome and lysosome caused 20, 62 and 40% reductions in total protein degradation (P<0.05), respectively. Therefore, these three systems account for the bulk of degradation in cultured muscle cells. Two cytoskeletal proteins were highly-sensitive to inhibition of their degradation. Specifically, desmin and dystrophin concentrations increased markedly when calpain, proteasome and lysosome activities were inhibited. Conversely, sarcomeric proteins (alpha-actinin and tropomyosin) and filamin were relatively insensitive to the addition of protease inhibitors to culture media. These data demonstrate that proteolytic systems work in tandem to degrade cytoskeletal and sarcomeric protein complexes and that the cytoskeleton is more sensitive to inhibition of degradation than the sarcomere. Mechanisms, which bring about changes in the activities of the proteases, which mediate muscle protein degradation are not known and represent the next frontier of understanding needed in muscle wasting diseases and in muscle growth biology.     

Temporal response of desmin and dystrophin proteins to progressive resistance exercise in human skeletal muscle.

We have investigated the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 wk of progressive resistance training or after a single bout of unaccustomed resistance exercise. Muscle biopsies were taken from the vastus lateralis of 12 untrained men. For the single-bout group (n=6) biopsies were taken 1 wk before the single bout of exercise (week 0) and 1, 2, 4, and 8 wk after this single bout of exercise. For the training group (n=6), biopsies were taken 1 wk before the beginning of the program (week 0) and at weeks 1, 2, 4, and 8 of the progressive resistance training program. Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS-PAGE at each time point for each group. In the training group, desmin was significantly increased compared with week 0 beginning at week 4 (182% of week 0; P<0.0001) and remained elevated through week 8 (172% of week 0; P<0.0001). Desmin did not change at any time point for the single-bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single-bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and it further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5,719+/-382 and 6,582+/-640 microm2, weeks 0 and 4, respectively; P<0.05). Finally, a significant negative correlation was observed between the desmin-to-actin ratio and the percentage of MHC IIx (R=-0.31; P<0.05, all time points from both groups). These data demonstrate a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but before changes in MHC isoforms, whereas dystrophin remains unchanged.     

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

The mode of myofibril remodelling in human skeletal muscle affected by DOMS induced by eccentric contractions

Myofibrillar Z-disc streaming and loss of the desmin cytoskeleton are considered the morphological hallmarks of eccentric contraction-induced injury. The latter is contradicted by recent studies where a focal increase of desmin was observed in biopsies taken from human muscles with DOMS. In order to determine the effects of eccentric contraction-induced alterations of the myofibrillar Z-disc, we examined the distribution of alpha-actinin, the Z-disc portion of titin and the nebulin NB2 region in relation to actin and desmin in DOMS biopsies. In biopsies taken 2-3 days and 7-8 days after exercise, we observed a significantly higher number of fibres showing focal areas lacking staining for alpha-actinin, titin and nebulin than in biopsies taken from control or 1 h after exercise. None of these proteins were part of Z-disc streamings but instead they were found in distinct patterns in areas characterised by altered staining for desmin and actin. These were preferentially seen in regions with increased numbers of sarcomeres in parallel myofibrils. We propose that these staining patterns represent different stages of sarcomere formation. These findings therefore support our previous suggestion that muscle fibres subjected to eccentric contractions adapt to unaccustomed activity by the addition of new sarcomeres.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.     

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated mice.     

Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.     

Muscular nitric oxide synthase (muNOS) and utrophin.

Duchenne muscular dystrophy (DMD), the severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. Three types of treatment are envisaged: pharmacological (glucocorticoid), myoblast transplantation, and gene therapy. An alternative to the pharmacological approach is to compensate for dystrophin loss by the upregulation of another cytoskeletal protein, utrophin. Utrophin and dystrophin are part of a complex of proteins and glycoproteins, which links the basal lamina to the cytoskeleton, thus ensuring the stability of the muscle membrane. One protein of the complex, syntrophin, is associated with a muscular isoform of the neuronal nitric oxide synthase (nNOS). We have demonstrated an overexpression of utrophin, visualised by immunofluorescence and quantified by Western blotting, in normal myotubes and in mdx (the animal model of DMD) myotubes, as in normal (C57) and mdx mice, both treated with nitric oxide (NO) donor or L-arginine, the NOS substrate. There is evidence that utrophin may be capable of performing the same cellular functions as dystrophin and may functionally compensate for its lack. Thus, we propose to use NO donors, as palliative treatment of Duchenne and Becker muscular dystrophies, pending, or in combination with, gene and/or cellular therapy. Discussion has focussed on the various isoforms of NOS that could be implicated in the regeneration process. Dystrophic and healthy muscles respond to treatment, suggesting that although NOS is delocalised in the cytoplasm in the case of DMD, it conserves substantial activity. eNOS present in mitochondria and iNOS present in cytoplasm and the neuromuscular junction could also be activated. Lastly, production of NO by endothelial NOS of the capillaries would also be beneficial through increased supply of metabolites and oxygen to the muscles.     

Just say NO to muscle degeneration?

The devastating muscle degeneration characteristic of Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. The dystrophin complex has two functions: a structural role in maintaining sarcolemmal integrity during contraction and a scaffolding function that recruits signaling proteins such as neuronal nitric oxide synthase to the membrane. New studies indicate that transgenic restoration of nitric oxide (NO) production in the mdx dystrophic mouse improves muscle pathology. Although NO-mediated killing of inflammatory cells might be involved, other mechanisms are also possible. These results point to the therapeutic potential of manipulating the signaling activity of the dystophin complex as a way to ameliorate the progression of muscle degeneration.     

Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans

In this study the stress protein response to unaccustomed maximal eccentric exercise in humans was investigated. Eleven healthy males performed 300 maximal eccentric actions with the quadriceps muscle. Biopsies from vastus lateralis were collected at 30 min and 4, 8, 24, 96, and 168 h after exercise. Cellular regulation and localization of heat shock protein (HSP) 27, alpha B-crystallin, and HSP70 were analyzed by immunohistochemistry, ELISA technique, and Western blotting. Additionally, mRNA levels of HSP27, alpha B-crystallin, and HSP70 were quantified by Northern blotting. After exercise (30 min), 81 +/- 8% of the myofibers showed strong HSP27 staining (P < 0.01) that gradually decreased during the following week. alpha B-Crystallin mimicked the changes observed in HSP27. After exercise (30 min), the ELISA analysis showed a 49 +/- 13% reduction of the HSP27 level in the cytosolic fraction (P < 0.01), whereas Western blotting revealed a 15-fold increase of the HSP27 level in the myofibrillar fraction (P < 0.01). The cytosolic HSP70 level increased to 203 +/- 37% of the control level 24 h after exercise (P < 0.05). After 4 days, myofibrillar-bound HSP70 had increased approximately 10-fold (P < 0.01) and was accompanied by strong staining on cross sections. mRNA levels of HSP27, alpha B-crystallin, and HSP70 were all elevated the first day after exercise (P < 0.01); HSP70 mRNA showed the largest increase (20-fold at 8 h). HSP27 and alpha B-crystallin seemed to respond immediately to maximal eccentric exercise by binding to cytoskeletal/myofibrillar proteins, probably to function as stabilizers of disrupted myofibrillar structures. Later, mRNA and total HSP protein levels, especially HSP70, increased, indicating that HSPs play a role in skeletal muscle recovery and remodeling/adaptation processes to high-force exercise.     

Protective effect of L-arginine administration on proteins of unloaded <Emphasis Type="Italic">m. soleus</Emphasis>

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.     

Aging alters the contribution of nitric oxide to regional muscle hemodynamic control at rest and during exercise in rats

Advanced age is associated with altered skeletal muscle hemodynamic control during the transition from rest to exercise. This study investigated the effects of aging on the functional role of nitric oxide (NO) in regulating total, inter-, and intramuscular hindlimb hemodynamic control at rest and during submaximal whole body exercise. We tested the hypothesis that NO synthase inhibition (N(G)-nitro-l-arginine methyl ester, l-NAME; 10 mg/kg) would result in attenuated reductions in vascular conductance (VC) primarily in oxidative muscles in old compared with young rats. Total and regional hindlimb muscle VCs were determined via radiolabeled microspheres at rest and during treadmill running (20 m/min, 5% grade) in nine young (6-8 mo) and seven old (27-29 mo) male Fisher 344 × Brown Norway rats. At rest, l-NAME increased mean arterial pressure (MAP) significantly by ～17% and 21% in young and old rats, respectively. During exercise, l-NAME increased MAP significantly by ～13% and 19% in young and old rats, respectively. Compared with young rats, l-NAME administration in old rats evoked attenuated reductions in 1) total hindlimb VC during exercise (i.e., down by ～23% in old vs. 43% in young rats; P < 0.05), and 2) VC in predominantly oxidative muscles both at rest and during exercise (P < 0.05). Our results indicate that the dependency of highly oxidative muscles on NO-mediated vasodilation is markedly diminished, and therefore mechanisms other than NO-mediated vasodilation control the bulk of the increase in skeletal muscle VC during the transition from rest to exercise in old rats. Reduced NO contribution to vasomotor control with advanced age is associated with blood flow redistribution from highly oxidative to glycolytic muscles during exercise.     

Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats

We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. l-Nitroarginine methyl ester (l-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.