Ten microliters of maxadilan were added to each well, and the plates were incubated at 37uC for 1 h

ed as means 6 SD of 5 animals in each group. p,0.05 versus lean untrained, p,0.001 versus lean untrained, p,0.05 versus lean trained, p,0.001 versus lean trained, p,0.05 versus obese untrained. doi:10.1371/journal.pone.0046114.t001 Cardiac angiotensin-converting enzyme 2 activity ACE2 activity was determined in LV tissue by the same method described above. However, this method uses a fluorescent peptide Abz-APK-OH in 0.2 M Tris-HCl buffer, 200 mM NaCl, 2 mg BSA, pH 7.5, which is hydrolyzed by ACE2. ACE2 activity was expressed in UF.min21.mg21of protein. Measurement of Angiotensin II Plasma and LV Ang II levels were determined by ELISA, according to the manufacturer’s instructions. To determine plasma Ang II concentration the first 3 ml of trunk blood was rapidly collected in chilled glass tubes containing a mixture of protease inhibitors to prevent the in vitro production and degradation of angiotensin peptides. The blood was centrifuged, and plasma was separated and stored at 220uC. LV was homogenized in lyses buffer containing a mixture of protease inhibitors and centrifuged at 10 0006g, 4uC, for 10 min., to determine the measurement of cardiac Ang II. The supernatant and plasma were collected and passed through phenyl silica cartridges, and the absorbed angiotensin was eluted with methanol. Eluate was dried in a vacuum centrifuge and the pellet was re-suspended in EIA buffer, vortexed and centrifuged at 3000 g for 10 minutes at 4uC. The results were expressed pg/ml. The tissue protein PG-490 biological activity content was determined by the Bradford methods by using bovine serum albumin as the standard. Western blot analysis The frozen ventricles were thawed and minced into small pieces and homogenized in cell lysis buffer containing 100 mM Tris, 50 mM NaCl, 10 mM EDTA, 1% TritonX-100 and a mixture of protease inhibitors. Insoluble heart tissues were removed by centrifugation at 3,0006g, 4uC, for 10 min. Data are reported as means 6 SD of 5 animals in each group. p,0.001 versus lean untrained, p,0.0001 versus lean untrained, p,0.001 versus lean trained, p,0.0001 versus lean trained p,0.05 versus obese untrained, p,0.001 versus obese untrained. The blot membrane was then incubated in a blocking buffer for 2 h at room temperature and then probed with a polyclonal antibody directed against AT1 or AT2 at room temperature. Primary antibody binding was detected with the use of peroxidase-conjugated secondary antibodies, and enhanced chemiluminescence reagents were used to visualize the autoradiogram, which was later exposed to photographic film. The film was developed and the bands were analyzed using Scion Image software. GAPDH expression levels were used to normalize the results. the control gene were used to standardize the results in order to compensate for differences in RNA content among the samples. The comparative threshold cycle method was used for data analyses. CT indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold, and DCT is the difference in threshold cycle for target and control. The levels of gene expression were given by 22DDCT; where DDCT is the DCT value subtracted from DCT of lean untrained rats. Finally the 2 fold DDCT was calculated. Statistical Analysis All the data were subjected to statistical analyses using the SAS program. A mixed model was used, with body composition and exercise training as fixed factors for all variables, except for body weight, for which a time factor was inclu