Measurement of angiotensin peptides.

Ang I, Ang II, and Ang 1-7 peptides in cardiac tissue were measured by ELISA (Mybiosource). On the basis of instructions provided by the manufacturer, pulverized whole hearts were homogenized in a sample buffer and centrifuged at 1,000 g for 10 min, and the supernatant fraction was collected. In a 96-well plate, 50 μl of samples or peptide standard solution was added to each well, followed by the addition of 100 μl of horseradish peroxidase-conjugate reagent. The plate was gently mixed, covered with a plastic membrane, and incubated at 37°C for 60 min. Following incubation, the plate was washed with wash buffer four times. The plate was subsequently blotted dry, and 50 μl of each Chromogen Solution A and Chromogen Solution B were added to each well. The plate was gently mixed and incubated for an additional 15 min at 37°C, and 50 μl of stop solution was then immediately added. The absorbance was read at 450 nm using a SpectraMax 96-well plate spectrophotometer (Molecular Devices). The amount of angiotensin peptides was calculated by comparing absorbance readings of samples to those of the standard curve. Values were normalized to milligrams of tissue protein.