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(Circulation. 2000;102:e32.)
© 2000 American Heart Association, Inc.

Correspondence

Chymase-Dependent Angiotensin II Formation in Human Vascular Tissue

Petri T. Kovanen; MD Ken A. Lindstedt, PhD; Naotaka Shiota, MD; Jorma O. Kokkonen, MD

Wihuri Research Institute, Helsinki, Finland

To the Editor:

The recent article by Takai et al¹ suggests that human chymase bound to heparin plays a functional role in angiotensin II formation in the presence of natural protease inhibitors such as 1-antitrypsin (AT). This interpretation is based on one experiment (Figure 4) in which the authors use a heparin column to mimic the physiological situation in which chymase is bound to heparin proteoglycans when secreted from a mast cell during the process of degranulation. However, the experimental setup in Figure 4 raises some doubts about the validity of the obtained result and its in vivo significance.

The authors do not mention whether the heparin column was preincubated with 1-AT or chymostatin; immediate washing with PBS (3 times the column volume) would rapidly and extensively remove the inhibitors from the column. In particular, the large molecular inhibitors, such as 1-AT (Mr=50 000), may be equilibrated slower in the column compared with chymostatin (Mr=604), and the Ki value for 1-AT to chymase is considerably lower than that for chymostatin. Therefore, 1-AT may be easily washed out of the column before interacting with the enzyme under the reported experimental condition. After the washing step, a solution containing the substrate (angiotensin I) but not the inhibitors was applied on the column.

In our opinion, the authors should have performed the test in the presence of inhibitors throughout the experiment. The reaction of chymostatin with chymase results in the irreversible inhibition of chymase through the formation of a stable 1:1 complex, which has no hydrolytic activity. However, according to Schechter et al,² when the ratios of the natural inhibitors 1-antichymotrypsin and 1-AT to chymase concentration were determined, stoichiometries of 4.5 and 5.0, respectively, were obtained instead of unity. They concluded that both 1-antichymotrypsin and 1-AT are better substrates than chymase inhibitors. Thus, to achieve a full inhibition of chymase, one would have to use an inhibitor concentration >5 times molar excess, which would preferably be present throughout the experiment.

The concentration of 1-AT (2 µmol/L) used by the authors is 50 times lower than that of chymostatin (100 µmol/L). In addition, under normal physiological conditions, the concentration of 1-AT (50 µmol/L)³ is also 25 times higher than the concentration of 1-AT (2 µmol/L) used in this experiment. Taken together, these data seem to indicate that further experimentation is required to unravel the physiological function of heparin-bound chymase in the presence of serpins.

References

1. Takai S, Jin D, Sakaguchi M, et al. Chymase-dependent angiotensin II formation in human vascular tissue. Circulation. 1999;100:654–658.[Abstract/Free Full Text]
   
2. Schechter NM, Sprows JL, Schoenberger OL, et al. Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem. 1989;264:21308–21315.[Abstract/Free Full Text]
   
3. Fritz H. Proteinase inhibitors in severe inflammatory processes (septic shock and experimental endotoxaemia): biochemical, pathophysiological and therapeutic aspects. Ciba Found Symp. 1979;75:351–379.
   

Response

Shinji Takai, PhD; Denan Jin, MD, PhD; Masato Sakaguchi, PhD; Mizuo Miyazaki, MD, PhD

Department of Pharmacology, Osaka Medical College, Osaka, Japan

We appreciate and acknowledge what Kovanen et al pointed out about our article. They discuss the difference between the results of our experiment, which hypothesized heparin-bound chymase as the biological state of chymase, and the nature of extracted chymase. We agree with the report by Schechter et al^R1 that was mentioned in the letter. Certainly, -antitrypsin is a very powerful chymase inhibitor that has a higher affinity for chymase than chymostatin. However, the inhibitory activity of macromolecular inhibitors, such as secretory leukocyte proteinase inhibitor, and not that of low molecular weight inhibitors, including chymostatin, was affected by heparin.^R2 Chymase is bound to heparin in the organism, and the purpose of our article was to study the sensitivity of certain inhibitors on the complex. Consequently, the difference between heparin-bound chymase and extracted chymase is important. Previously, our experiment using both extracts and slices of human vascular tissues demonstrated that the activity of extracted chymase was inhibited by -antitrypsin, whereas chymase activity in slices was not inhibited by -antitrypsin.^R3 We think that -antitrypsin in the reactive solution had difficulty entering into the tissue slices. Our experiment indicated that -antitrypsin was unable to work on heparin-bound chymase in the heparin column to inhibit the chymase activity. We think that this phenomenon is what actually occurs in tissues.

The concentration of -antitrypsin that was used in our experiment was 2 µmol/L. The -antitrypsin concentration in tissue is lower than that in plasma, and -antitrypsin at a concentration of 2 µmol/L completely inhibits the activity of extracted chymase. Therefore, we do not think the concentration was too low.

When macromolecular substances are used as the substrate for chymase, the decomposition activity changes extensively depending on the presence or absence of heparin. Consequently, to elucidate the role played by chymase in the organism, it is important to study inhibitors and substrate specificity using heparin-bound chymase.

References

1. Schechter NM, Sprows JL, Schoenberger OL, et al. Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem. 1989;264:21308–21315.
   
2. Walter M, Plotnick M, Schechter NM. Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin. Arch Biochem Biophys. 1996;327:81–88.
   
3. Takai S, Shiota N, Jin D, et al. Functional role of chymase in angiotensin II formation in human vascular tissue. J Cardiovasc Pharmacol. 1998;32:826–833.[Medline] [Order article via Infotrieve]
   

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