5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.

The present study examined in vitro vasomotor function and expression of enzymes controlling nitric oxide (NO) bioavailability in thoracic aorta of adult male normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) that either remained sedentary (Sed) or performed 6 wk of moderate aerobic exercise training (Ex). Training efficacy was confirmed by elevated maximal activities of both citrate synthase (P = 0.0024) and beta-hydroxyacyl-CoA dehydrogenase (P = 0.0073) in the white gastrocnemius skeletal muscle of Ex vs. Sed rats. Systolic blood pressure was elevated in SHR vs. WKY (P < 0.0001) but was not affected by Ex. Despite enhanced endothelium-dependent relaxation to 10(-8) M ACh in SHR vs. WKY (P = 0.0061), maximal endothelium-dependent relaxation to 10(-4) M ACh was blunted in Sed SHR (48 +/- 12%) vs. Sed WKY (84 +/- 6%, P = 0.0067). Maximal endothelium-dependent relaxation to 10(-4) M ACh was completely restored in Ex SHR (93 +/- 9%) vs. Sed SHR (P = 0.0011). N(omega)-nitro-l-arginine abolished endothelium-dependent relaxation in all groups (P

Cardiovascular disease is the single leading cause of death and morbidity for Canadians. A universal feature of cardiovascular disease is dysfunction of the vascular endothelium, thus disrupting control of vasodilation, tissue perfusion, hemostasis, and thrombosis. Nitric oxide bioavailability, crucial for maintaining vascular endothelial health and function, depends on the processes controlling synthesis and destruction of nitric oxide as well as on the sensitivity of target tissue to nitric oxide. Evidence supports a major contribution by oxidative stress-induced destruction of nitric oxide to the endothelial dysfunction that accompanies a number of cardiovascular disease states including hypertension, diabetes, chronic heart failure, and atherosclerosis. Regular physical activity (exercise training) reduces cardiovascular disease risk. Numerous studies support the hypothesis that exercise training improves vascular endothelial function, especially when it has been impaired by preexisting risk factors. Evidence is emerging to support a role for improved nitric oxide bioavailability with training as a result of enhanced synthesis and reduced oxidative stress-mediated destruction. Molecular targets sensitive to the exercise training effect include the endothelial nitric oxide synthase and the antioxidant enzyme superoxide dismutase. However, many fundamental details of the cellular and molecular mechanisms linking exercise to altered molecular and functional endothelial phenotypes have yet to be discovered. The working hypothesis is that some of the cellular mechanisms contributing to endothelial dysfunction in cardiovascular disease can be targeted and reversed by signals associated with regular increases in physical activity. The capacity for exercise training to regulate vascular endothelial function, nitric oxide bioavailability, and oxidative stress is an example of how lifestyle can complement medicine and pharmacology in the prevention and management of cardiovascular disease.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.

Cardiovascular diseases such as hypertension are associated with a generalized skeletal myopathy including a proapoptotic phenotype. Current evidence suggests that exercise may alter apoptosis-related signaling in skeletal muscle; however, the effect of exercise on skeletal muscle DNA fragmentation and apoptotic signaling is unclear in hypertensive animals. Male normotensive Wistar Kyoto (WKY; n = 24) and spontaneously hypertensive rats (SHR; n = 24) were assigned to a sedentary (SED) condition or exercise (EX) consisting of progressive treadmill running 5 days/wk for 6 wks. Consistent with our previous work we found that soleus muscle of hypertensive animals had significantly higher DNA fragmentation (a hallmark of apoptosis), elevated proapoptotic factors (Bax, caspase-3 activity), and lower antiapoptotic proteins (apoptosis repressor with caspase recruitment domain, Bcl-2, X-linked inhibitor of apoptosis protein) compared with normotensive rats. In addition, soleus muscle of hypertensive animals displayed myosin accumulation and fragmentation, had elevated cytosolic cytochrome c, second mitochondrial-derived activator of caspase (Smac), apoptosis inducing factor (AIF), and endonuclease G protein levels, higher nuclear AIF content, and greater muscle reactive oxygen species generation compared with normotensive animals. Interestingly, exercise training significantly lowered DNA fragmentation and myosin accumulation/fragmentation in soleus muscle of hypertensive rats. Furthermore, exercise training significantly reduced cytosolic levels of cytochrome c as well as cytosolic and nuclear AIF in soleus muscle of hypertensive animals. This beneficial response is likely due to exercise-mediated elevations in Bcl-2, heat shock protein 70, and manganese superoxide dismutase protein content, as well as reductions in Bax protein levels and the Bax-to-Bcl-2 ratio. These results suggest that regular exercise training provides protection against skeletal muscle apoptosis by altering a number of apoptosis regulatory proteins and by influencing mitochondrial-mediated apoptotic signaling mechanisms.