Protease Inhibitors Extracted from Caesalpinia echinata Lam. Affect Kinin Release during Lung Inflammation

Figure 3

ACE activity in lungs. Rats were pretreated intravenously with buffer (negative or positive controls), CeKI (7.8 or 2.6 mg), or rCeEI (2.6 or 0.84 mg). After 20 min, they received 75 μg of LPS/animal (positive control and CeKI and rCeEI groups) or buffer (negative control) injected via the trachea directly into their lungs. Six hours later, lungs were extracted and homogenized. Samples of lung (5 µL) were maintained in 50 mM Tris buffer at pH 7.4 containing 50 mM NaCl for 5 min at 37°C before the addition of the substrate Abz-F-R-K(Dnp)-P-OH (10 µM) in a final volume of 200 µL. Fluorescence changes were monitored continuously for 30 min at  nm and  nm. The slope of the generated fluorescence signal was converted into micromoles of substrate hydrolyzed per minute based on a calibration curve obtained from the complete hydrolysis of peptide and adjusted for total protein quantity. difference compared to negative control . difference compared to CeKI-treated groups .