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.