Effects of exercise training on vasodilatory protein expression and activity in rats.

Increased endothelium-dependent vasodilatation is associated with endurance exercise training. The purpose of this study was to test the hypothesis that increased endothelial nitric oxide synthase (eNOS) protein function, but not increased vascular smooth muscle sensitivity to NO, underlies augmented endothelium-dependent dilatation with training. To test these hypotheses, rats ran on a treadmill at 30 m/min (10% grade) for 60 min/day, 5 days/week, over 8-12 weeks (Trn). Training efficacy was demonstrated by greater (P < 0.05) hindlimb muscle citrate synthase activity and left ventricular mass-body mass ratio in Trn compared with sedentary control rats (Sed). Expression of eNOS protein in the aorta was increased with training (Sed, 1.00 ± 0.18 normalized units; Trn, 1.55 ± 0.23; P < 0.05). Aortic NOS activity was, however, unchanged by training (Sed, 1,505 ± 288 fmol/h/mg protein; Trn, 1,650 ± 247; n.s.). Expression of heat shock protein 90 and protein kinase B/Akt was not different between groups, nor was their association with eNOS. In follow-up series of rats, phosphorylated eNOS content (Serine 1177) was similar for Sed and Trn in both the aorta and gastrocnemius feed artery. Aortic NOS activity with eNOS phosphorylation status preserved was also similar between groups. Finally, cGMP concentration with a NO donor did not differ between groups (Sed, 73.0 ± 20.2 pmol/mg protein; Trn, 62.5 ± 12.9; n.s.). These findings indicate that training-induced increases in eNOS protein expression are not coupled to augmented function, illustrating the complexity of eNOS regulation. Further, they show that vascular sensitivity to NO is not altered by exercise training.

Effects of high-density lipoprotein on endothelium-dependent vasorelaxation.

Studies using cultured endothelial cells have shown that high-density lipoprotein (HDL) positively modulates endothelial nitric oxide synthase (eNOS). The purpose of this study was to test the hypotheses that positive modulation of eNOS by HDL occurs in whole vessels and that it augments endothelium-dependent vasorelaxation. To test these hypotheses, brachial arteries were obtained from swine. Endothelium-dependent and endothelium-independent vasorelaxation were determined in vitro to assess the effects of acute administration of HDL (50 microg.mL-1; n = 8) and chronic exposure to relatively high HDL concentration on vascular function (low HDL, 0.89 +/- 0.02 mmol.L-1, n = 4; high HDL, 1.16 +/- 0.05 mmol.L-1, n = 4; p < 0.005). Acute administration of HDL did not augment maximal endothelium-dependent vasorelaxation to bradykinin (BK) (no HDL, 82.6% +/- 2.2%; HDL, 76.7% +/- 3.5%; not significant (ns)). Similarly, maximal relaxation to BK was not enhanced by chronic exposure to high HDL concentrations. NO synthase (NOS) activity was also similar between groups (low HDL, 129.0 +/- 19.2 counts.h-1.microg-1 protein; high HDL, 113.9 +/- 47.1 counts.h-1.microg-1; ns). Consistent with NOS activity, the extent of eNOS phosphorylation at several sites was similar between low HDL and high HDL. Both apolipoprotein A-I (ApoA-I) and scavenger receptor class B type I (SR-BI) were associated with eNOS. Similar to cultured cell studies, this study demonstrates that both ApoA-I and SR-BI associate with eNOS in the vascular wall. Binding of ApoA-I and SR-BI to eNOS does not, however, result in modulation of either NO formation or endothelial function.

Chronic inhibition of nitric oxide synthase augments the ACTH response to exercise.

Exercise can activate the hypothalamo-pituitary-adrenocortical (HPA) axis, and regular exercise training can impact how the HPA axis responds to stress. The mechanism by which acute exercise induces HPA activity is unclear. Therefore, the purpose of this study was to test the hypothesis that nitric oxide modulates the neuroendocrine component of the HPA axis during exercise. Female Yucatan miniature swine were treated with N-nitro-l-arginine methyl ester (l-NAME) to test the effect of chronic nitric oxide synthase (NOS) inhibition on the ACTH response to exercise. In addition, we tested the effect of NOS inhibition on blood flow to tissues of the HPA axis and report the effects of handling and treadmill exercise on the plasma concentrations of ACTH and cortisol. Chronic NOS inhibition decreased plasma NO(x) levels by 44%, increased mean arterial blood pressure by 46%, and increased expression of neuronal NOS in carotid arteries. Vascular conductance was decreased in the frontal cortex, the hypothalamus, and the adrenal gland. Chronic NOS inhibition exaggerated the ACTH response to exercise. In contrast, chronic NOS inhibition decreased the ACTH response to restraint, suggesting that the role of NO in modulating HPA activity is stressor dependent. These results demonstrate that NOS activity modulates the response of the neuroendocrine component of the HPA axis during exercise stress.

Vascular nitric oxide: effects of exercise training in animals.

Exercise training is known to induce several adaptations in the cardiovascular system, one of which is increased skeletal muscle blood flow at maximal exercise. Improved muscle blood flow, in turn, could in part be accounted for by augmented endothelium-dependent, nitric oxide (NO)-mediated vasodilation. Studies have indeed demonstrated that endothelium-dependent, NO-mediated dilation of conductance-type vessels is augmented after endurance exercise training; recently, this adaptation has been extended into resistance-type vessels within rodent skeletal muscle. With the latter, however, it appears that only resistance vessels supplying muscle active during training sessions exhibit this adaptation. These findings in rats are in contrast to those from human studies, in which increased endothelium-dependent dilation has been observed in vasculatures not associated with elevated blood flow during exercise. Increased expression of endothelial NO synthase (eNOS) appears to underlie enhanced endothelium-dependent, NO-mediated dilation of both conductance and resistance vessels. Greater eNOS expression may also underlie the preventive and (or) rehabilitative effect(s) of exercise training on atherosclerosis, given that NO inhibits several steps of the atherosclerotic disease process. Thus, exercise training may induce adaptations that benefit both vasodilation and vascular health.

Effects of chronic nitric oxide synthase inhibition on responses to acute exercise in swine.

Nitric oxide (NO) is potentially involved in several responses to acute exercise. We tested the hypotheses that inhibition of NO formation reduces maximal O(2) delivery to muscle, but does not affect O(2) utilization by muscle, therefore lowering maximal O(2) consumption. To test these hypotheses, swine (approximately 30 kg) drank either tap water (Con, n = 25) or water with N(G)-nitro-l-arginine methyl ester (8.0 +/- 0.4 mg x kg(-1) x day(-1) for >or=4 wk; LN, n = 24). Treatment efficacy was reflected by higher mean arterial pressure and lower plasma NO metabolite concentration in LN than Con (both P < 0.05). Swine completed two graded treadmill running tests to maximum. In the first test, O(2) consumption was determined at rest through maximal exercise intensity. O(2) consumption did not differ between groups at rest or at most exercise intensities, including maximum (Con, 40.8 +/- 1.8 ml x min(-1) x kg(-1); LN, 40.4 +/- 2.9; not significant). In the second test, tissue-specific blood flows were determined using the radiolabeled-microsphere technique. At rest, blood flows were lower (P < 0.05) in LN compared with Con for a number of tissues, including kidney, adrenal, lung, and several skeletal muscles. During both submaximal and maximal exercise, however, blood flows were similar between Con and LN for all 16 muscles examined; only blood flows to kidney (Con, 99 +/- 16 ml x min(-1) x 100 g; LN, 55 +/- 15; P < 0.05) and pancreas (Con, 25 +/- 7; LN, 6 +/- 2; P < 0.05) were lower in LN at maximum. Endothelium-dependent, but not -independent, relaxation of renal arterial segments was reduced (P < 0.05) in vitro. These data indicate that exercise-induced increases in muscle blood flows are maintained with chronic inhibition of NO formation and that maximal O(2) consumption is therefore preserved. Redundant vasodilatory pathways and/or upregulation of these pathways may underlie these findings.

Chronic nitric oxide synthase inhibition blunts endothelium-dependent function of conduit coronary arteries, not arterioles.

Current literature suggests that chronic nitric oxide synthase (NOS) inhibition has differential effects on endothelium-dependent dilation (EDD) of conduit arteries vs. arterioles. Therefore, we hypothesized that chronic inhibition of NOS would impair EDD of porcine left anterior descending (LAD) coronary arteries but not coronary arterioles. Thirty-nine female Yucatan miniature swine were included in the study. Animals drank either tap water or water with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/l), resulting in control and chronic NOS inhibition (CNI) groups, respectively. Treatment was continued for 1-3 mo (8.3 +/- 0.6 mg x kg(-1) x day(-1)). In vitro EDD of coronary LADs and arterioles was assessed via responses to ADP (LADs only) and bradykinin (BK), and endothelium-independent function was assessed via responses to sodium nitroprusside (SNP). Chronic NOS inhibition diminished coronary artery EDD to ADP and BK. Incubating LAD rings with L-NAME decreased relaxation responses of LADs from control pigs but not from CNI pigs such that between-group differences were abolished. Neither indomethacin (Indo) nor sulfaphenazole incubation significantly affected relaxation responses of LAD rings to ADP or BK. Coronary arteries from CNI pigs showed enhanced relaxation responses to SNP. In contrast to coronary arteries, coronary arterioles from CNI pigs demonstrated preserved EDD to BK and no increase in dilation responses to SNP. L-NAME, Indo, and L-NAME + Indo incubation did not result in significant between-group differences in arteriole dilation responses to BK. These results suggest that although chronic NOS inhibition diminishes EDD of LAD rings, most likely via a NOS-dependent mechanism, it does not affect EDD of coronary arterioles.

Vascular nitric oxide: effects of physical activity, importance for health.

NO (nitric oxide), formed in the vascular endothelium and derived from a biochemical reaction catalysed by eNOS (endothelial NO synthase), appears to play a role in exercise-induced dilation of blood vessels supplying cardiac and skeletal muscle. Endothelium-dependent, NO-mediated vasodilation is augmented by exercise training. Increases in eNOS gene transcription, eNOS mRNA stability and eNOS protein translation appear to contribute to increased NO formation and, consequently, enhanced NO-mediated vasodilation after training. Enhanced endothelial NO formation may also have a role(s) in the prevention and management of atherosclerosis because several steps in the atherosclerotic disease process are inhibited by NO. A growing body of work suggests that exercise training, perhaps via increased capacity for NO formation, retards atherosclerosis. This has significant implications for human health, given that atherosclerosis is the leading killer in Western society.

Effects of cyclosporine-A on rat soleus muscle fiber size and phenotype.

PURPOSE: Organ transplant patients treated with cyclosporine-A (CsA) often exhibit weight loss and muscle weakness. The cellular target of CsA, calcineurin, has been implicated in maintenance of muscle fiber size and in expression of the type I skeletal muscle phenotype. We hypothesized that CsA treatment would cause fiber atrophy, as well as increase type IIa myosin heavy chain (MHC) content and oxidative enzyme activities in the soleus muscle. METHODS: Rats were treated with CsA for 21 d (20 mg.kg(-1).d(-1); N = 16) and compared with control rats given olive oil vehicle (Veh; N = 16). Soleus muscles were excised bilaterally. MHC content was determined by gel electrophoresis, oxidative enzyme activities by spectrophotometric methods, and fiber type and size by histochemistry. RESULTS: Lymphocyte count was depressed in CsA rats (P < 0.05), indicating treatment efficacy. Type IIa MHC content was increased in the soleus muscle with CsA (Veh, 10.4 +/- 1.7%; CsA, 15.1 +/- 2.0; P < 0.05) at the expense of type I MHC. Soleus muscle oxidative enzyme activities were also increased with CsA treatment (P < 0.05). Soleus muscle atrophy occurred, reflected by a 22% decrease in fiber cross-sectional area (Veh, 3255 +/- 105 microm(2); CsA, 2533 +/- 125; P < 0.05). CONCLUSION: These findings indicate that CsA treatment is associated with changes in skeletal muscle fiber size and phenotype. The former may underlie clinical symptoms of transplant patients treated with CsA.

Measurement of muscle microvascular oxygen pressures: compartmentalization of phosphorescent probe.

OBJECTIVE: To determine whether the phosphorescent probe Oxyphor R2 (a palladium porphyrin dendrimer) becomes extravasated within normotensive skeletal muscle, R2 perfusion and washout studies were performed using a perfused rat hindlimb preparation. METHODS: Phosphorescence signals were monitored in tibialis anterior muscles after 35 min of R2 blood perfusion and across a subsequent washout period that included vasodilation (sodium nitroprusside, SNP, approximately 3 x 10(-2) M). RESULTS: Two responses were evident: Group 1 (n = 4)--Inflowing blood pressure and vascular conductance remained stable close to initial values and subsequently a marked vasodilation was evident with SNP (vascular conductance; R2 blood perfusion, 0.096 +/- 0.005; washout, pre-SNP, 0.085 +/- 0.005, post-SNP, 0.110 +/- 0.005 mL/min/mmHg, p <.05, for pre- vs. post-SNP). Baseline phosphorescence signals could be monitored up to 99 +/- 36 s post-SNP when the phosphorescence signal disappeared. For these muscles, palladium content was undetectable. Group 2 (n = 3)--Inflowing blood pressure increased 112% and vascular conductance fell approximately 50%. These hindlimbs were unresponsive to SNP, phosphorescence signal was undiminished by washout and SNP, and muscles became edematous. CONCLUSIONS: These results suggest that in normotensive muscle (i.e., Group 1 above), extravasation of phosphorescent probe R2 over 35 min of perfusion is insufficient to yield a detectable phosphorescence signal in skeletal muscle.

Training-induced changes in skeletal muscle Na+-K+ pump number and isoform expression in rats with chronic heart failure.

The mechanisms responsible for the decrements in exercise performance in chronic heart failure (CHF) remain poorly understood, but it has been suggested that sarcolemmal alterations could contribute to the early onset of muscular fatigue. Previously, our laboratory demonstrated that the maximal number of ouabain binding sites (B(max)) is reduced in the skeletal muscle of rats with CHF (Musch TI, Wolfram S, Hageman KS, and Pickar JG. J Appl Physiol 92: 2326-2334, 2002). These reductions may coincide with changes in the Na(+)-K(+)-ATPase isoform (alpha and beta) expression. In the present study, we tested the hypothesis that reductions in B(max) would coincide with alterations in the alpha- and beta-subunit expression of the sarcolemmal Na(+)-K(+)-ATPase of rats with CHF. Moreover, we tested the hypothesis that exercise training would increase B(max) along with producing significant changes in alpha- and beta-subunit expression. Rats underwent a sham operation (sham; n = 10) or a surgically induced myocardial infarction followed by random assignment to either a control (MI; n = 16) or exercise training group (MI-T; n = 16). The MI-T rats performed exercise training (ET) for 6-8 wk. Hemodynamic indexes demonstrated that MI and MI-T rats suffered from severe left ventricular dysfunction and congestive CHF. Maximal oxygen uptake (Vo(2 max)) and endurance capacity (run time to fatigue) were reduced in MI rats compared with sham. B(max) in the soleus and plantaris muscles and the expression of the alpha(2)-isoform of the Na(+)-K(+)-ATPase in the red portion of the gastrocnemius (gastrocnemius(red)) muscle were reduced in MI rats. After ET, Vo(2 max) and run time to fatigue were increased in the MI-T group of rats. This coincided with increases in soleus and plantaris B(max) and the expression of the alpha(2)-isoform in the gastrocnemius(red) muscle. In addition, the expression of the beta(2)-isoform of the gastrocnemius(red) muscle was increased in the MI-T rats compared with their sedentary counterparts. This study demonstrates that CHF-induced alterations in skeletal muscle Na(+)-K(+)-ATPase, including B(max) and isoform expression, can be partially reversed by ET.

Endothelium-dependent vasodilation in different rat hindlimb skeletal muscles.

Few studies have examined potential for endothelium-dependent vasodilation in skeletal muscles of different fiber-type composition. We hypothesized that muscles composed of slow oxidative (SO)- and/or fast oxidative glycolytic (FOG)-type fibers have greater potential for endothelium-dependent vasodilation than muscles composed of fast glycolytic (FG)-type fibers. To test this hypothesis, the isolated perfused rat hindlimb preparation was used with a constant-flow, variable-pressure approach. Perfusion pressure was monitored continuously, and muscle-specific flows were determined by using radiolabeled microspheres at four time points: control, at peak effect of acetylcholine (ACh I; 1-2 x 10(-4) M), at peak effect of ACh after infusion of an endothelial inhibitor (ACh II), and at peak effect of sodium nitroprusside (SNP; 4-5 x 10(-4) M). Conductance was calculated by using pressure and flow data. In the SO-type soleus muscle, conductance increased with ACh and SNP, but the increase in conductance with ACh was partially abolished by the endothelial inhibitor N(G)-nitro-l-arginine methyl ester (control, 0.87 +/- 0.19; ACh I, 2.07 +/- 0.29; ACh II, 1.32 +/- 0.15; SNP, 1.76 +/- 0.19 ml. min(-1). 100 g(-1). mmHg(-1); P < 0.05, ACh I and SNP vs. control). In the FOG-type red gastrocnemius muscle, similar findings were obtained (control, 0.64 +/- 0.11; ACh I, 1.36 +/- 0.21; ACh II, 0.73 +/- 0.16; SNP, 1.30 +/- 0.21 ml. min(-1). 100 g(-1). mmHg; P < 0.05, ACh I and SNP vs. control). In the FG-type white gastrocnemius muscle, neither ACh nor SNP increased conductance. Similar findings were obtained when muscles were combined into high- and low-oxidative muscle groups. Indomethacin had no effect on responses to ACh. These data indicate that endothelium-dependent vasodilation is exhibited by high-oxidative, but not low-oxidative, rat skeletal muscle. Furthermore, endothelium-dependent vasodilation in high-oxidative muscle appears to be primarily mediated by nitric oxide.

Influence of peak VO2 and muscle fiber type on the efficiency of moderate exercise.

PURPOSE: The purpose of this study was to determine if %Type I fibers and/or aerobic fitness (as peak .VO(2)) would predict Delta efficiency (DeltaEff) and Delta.VO(2)/Deltawork rate (WR) for moderate (below lactate threshold 0.05). CONCLUSION: These results suggest that aerobic fitness affects the energetic response to changes in power output during moderate exercise, such that the more aerobically fit a subject, the greater the increase in oxygen cost (.VO(2)) (reduced efficiency) as work rate increases. Further, Delta.VO(2)/DeltaWR reflects the inverse of DeltaEff for moderate-intensity exercise in healthy fed subjects.

Changes in skeletal muscle myosin heavy chain isoform content during congestive heart failure.

Recent investigations have suggested that changes in contractile protein expression contribute to reductions in skeletal muscle function during congestive heart failure (CHF). Myosin heavy chain (MHC), a major contractile protein, has been shown to undergo alterations in protein isoform expression during CHF. The purpose of this investigation was twofold: (1) to determine whether muscles of the same functional group undergo similar changes in MHC expression, and (2) determine whether the magnitude of alterations in MHC is related to the severity of CHF. Using the rat coronary ligation model, mild and severe forms of CHF were produced and muscles of the plantar flexor group were analyzed. Whole-muscle MHC isoform proportions were not altered in the soleus and white gastrocnemius muscle, however significant increases in the percentage of fast MHC isoforms (7-9% increases in MHC IIx and IIb expression) were found in the red gastrocnemius muscle. In addition, there were significant proportional increases (8%) in MHC type IIb at the expense of MHC type IIx in the plantaris muscle. Many of the changes in the proportions of MHC isoforms were significantly correlated with indices of CHF severity. This indicates that changes in skeletal muscle MHC isoform expression are related to the severity of CHF and suggests that some peripheral skeletal muscles are more susceptible to shifts in MHC expression due to CHF. These changes in MHC isoform expression may contribute to alterations in the physiological performance of skeletal muscle and exercise capacity during CHF.