A normalized value to evaluate the mRNA expression was calculated as the difference in the threshold cycle: the CT values of receptor minus CT of internal standard (β-actin), resulting in ΔCT. Since it is uncommon to use ΔCT parameter as a relative expression parameter due to this logarithmic characteristic, the 2−ΔCT parameter was used to express the relative gene expression
data . Final data are expressed as the ratio of the selleckchem fold-change in the target gene in the transgenic rat over the fold-change in the target gene of control rat. Thoracic aorta isolated from rat were quickly harvested, rinsed, blotted, frozed and homogenized in Tris–HCl buffer, pH 7.0, containing 50 mM NaCl and Tween 20, 0.2%. Subsequently, the
samples were centrifuged at 1000 g for 10 min and the supernatant was frozen at −20 °C. The protein contents of the samples were measured by the method of Bradford using bovine serum albumin as standard. The ACE activity was determined using Abz-FRK(Dnp)P-OH (Abz = ortho-amino benzoic acid; Dnp = ethylenediamine) as substrates following the methodology previously described . The increase in the selleck fluorescence was continuously measured in a Hitachi F-7000 fluorimeter set at λem = 420 nm and λex = 320 nm and the assays carried out in 96-well plates (final volume in each well = 0.2 mL). The evaluation of thoracic aorta ACE activity OSBPL9 was performed at 37 °C in 0.1 M Tris–HCl buffer, pH 7.0, containing 0.05 M NaCl, 10 μM ZnCl2, and inhibitors of the hydrolytic activities that we want to suppress (10 μM E64, 1 μM pepstatin, 1 mM PMSF, 100 μM TLCK, and 100 μM TPCK). Before starting the reaction by the addition of 10 μM of Abz-FRK(Dnp)P-OH, the tissues homogenates were pre incubated
for 5 min in the assay buffer, at 37 °C. To define the specificity for ACE, the assays were also performed in the presence of the cocktail of inhibitors plus 1 μM of the lisinopril. The slope was converted into nM of substrate hydrolyzed/min. The measurements were performed in triplicate and the results are expressed as means ± SD. BK, AngI, AngII, lisinopril, L-NAME, indomethacin and HOE-140 were purchased from Sigma Chemical Co. (Dorset, U.K). R-715 was a gift from D. Regoli, Université de Sherbrooke, Quebec, Canada. Concentrated solutions of peptides and other agents were prepared in water and kept at 20 °C until they were used. The stock solutions were serially diluted with Krebs–Ringer solution. Oligonucleotide primer and fluorogenic probe sets for Taqman™ Real-Time PCR were designed for kinin receptors and beta-actin using Assays-by-Design Service (Applied Biosystems). Values are expressed as means ± SD and (n). The Student t-test was used to determine the statistical differences, with the level of significance set as P < 0.05.