Doppler tracings and B-mode images were recorded continuously and Doppler tracings were averaged over 16 heart cycles at the time of blood sampling. Vessel diameter was determined after each Doppler recording. Arterial diameter measures were assessed during the systole from arterial B-mode images with the vessel parallel to the transducer. Intra-arterial pressure was monitored with transducers (Pressure Monitoring Kit, Baxter Deerfield, IL, USA) positioned at the level of the heart. Blood samples were drawn after 2 min of exercise/infusion and blood gases, haemoglobin and lactate were measured using an ABL725 analyser (Radiometer Copenhagen, Denmark). Leg mass was calculated from whole-body dual-energy X-ray absorptiometry scanning (Prodigy, PI3K inhibitor
GE Healthcare). The stable metabolites of NO, nitrite and nitrate were measured using a fluorometric assay kit (Cayman Chemical Co., Ann Harbor, MI, USA). Freeze dried tissue samples of m. vastus lateralis were homogenized in lysis buffer and Western blot analysis was performed as previously described (Bangsbo et al. 2009). Low temperature SDS-PAGE (LT-PAGE) was performed for detection of eNOS monomers using reported procedures (Klatt et al. 1995). Briefly, total proteins were incubated in 1 �� Laemmli buffer without dithiothreitol (DTT) and then subjected to SDS-PAGE with 5% gel. Gels and buffers were equilibrated at 4��C before electrophoresis, and the buffer tank was placed in cooling conditions to maintain temperature of the gel <15��C. Subsequent <a href="http://www.selleck.cn/products/Nutlin-3.html
">Nutlin3 to LT-PAGE, the gels were transferred and the blots probed as routine Western blot. Antibodies used were: eNOS, eNOS-PThr495, nNOS (BD Transduction Laboratories, USA), and eNOS-PSer1177 (Calbiochem Millipore, http://www.selleckchem.com/
Billerica, MA, USA). To control for loading differences the blots were also probed with an antibody against GAPDH (Abcam, Cambridge, UK). A two-way ANOVA was performed to test significance between the normotensive and hypertensive subjects and a two-way repeated measures ANOVA was performed to detect training-induced changes within each group. After a significant F test, pairwise differences were identified using the Student�CNewman�CKeuls post hoc procedure. Differences in baseline characteristics were assessed with Student's t test for unpaired and paired data when comparing between groups and within each group, respectively. The significance level was set at P < 0.05 and data are means �� SEM. The 8 week training period lowered systolic (P < 0.05) and diastolic (P < 0.05) blood pressure in the hypertensive group, whereas was increased in both normotensive (P < 0.001) and hypertensive (P < 0.05; Table 1) subjects. In the hypertensive group, the improvement in tended (P= 0.084, r2= 0.57) to correlate with the reduction in blood pressure. During infusion of ACh, there was no difference in the change in LBF and LVC with ACh infusion between the normotensive and hypertensive group before or after training (Fig. 1).