Chymase-dependent angiotensin II formation in human vascular tissue

Abstract

Background - Some reports have suggested that, in vitro, human heart chymase in homogenates contributes little to angiotensin (Ang) II formation in the presence of natural protease inhibitors such as α-antitrypsin. We studied whether chymase bound to heparin, resembling an in vivo form, could contribute to Ang II formation in the presence of natural protease inhibitors. Methods and Results - The Ang II formation was increased time- dependently after incubation in an extract (1 mg of protein/mL) of human vascular tissues containing Ang I. The concentration of Ang II in the extract after incubation for 30 minutes was 1.67 ± 0.06 nmol/mL, and we regarded this quantity of Ang II as 100%. The Ang II formation was inhibited 10%, 95%, and 96% by 1 μmol/L lisinopril, 100 μmol/L chymostatin, and 0.1 g/L α- antitrypsin, respectively. The extract was applied to a heparin affinity column. After the column was washed with PBS, the eluted PBS contained a weak Ang II-forming activity, which was completely inhibited by lisinopril. The eluted PBS, to which > 0.8 mol/L NaCl had been added, showed a strong Ang II- forming activity which was inhibited by chymostatin and α-antitrypsin. After the application of the extract, the column was washed with PBS and then an Ang I solution in PBS was applied to the column. The Ang II formation in the PBS eluted from the incubated column was increased time-dependently. The concentration of Ang II in the PBS (1 mL) eluted from the column after incubation for 30 minutes was 2.56 ± 0.28 nmol/mL, and we regarded this quantity of Ang II as 100%. To study the effects of inhibitors, the extract (1 mg of protein/mL) was applied to a heparin affinity column (1 mL) which was preequilibrated with PBS (3 mL); 100 μmol/L chymostatin or 0.1 g/L α- antitrypsin in PBS (1 mL) was then applied to the column. After the column was washed with PBS (3 mL), Ang I solution (1 mg/mL) in PBS was applied to the column, and the column was incubated for 30 minutes. The Ang II formation in the PBS eluted from the column was suppressed up to 5% by application of chymostatin, although this was not affected by application of α-antitrypsin. Conclusions - These findings suggest that human chymase bound to heparin plays a functional role in Ang II formation in the presence of natural protease inhibitors such as α-antitrypsin.