Abstract: SA-PO1096

A Novel Approach for Evaluation of Cleavage of Angiotensin Peptides by Their Angiotensinase Partners

Session Information

Category: Hypertension

  • 1102 Hypertension: Basic and Experimental - Renal Causes and Consequences

Authors

  • Wysocki, Jan, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
  • Müller, Tilman, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
  • Liu, Pan, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
  • Jin, Jing, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
  • Batlle, Daniel, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
Background

Angiotensin (1-7) formation from AngII (1-8) occurs via a release of a single c-terminal phenylalanine (Phe). ACE2 and prolylendopeptidase (PEP) are angiotensinases known to cleave Phe from Ang II to form Ang1-7. To capture physiologically important enzymatic conversion of other biologically active angiotensins, such as Ang III (2-8), we used a new quantitative fluorimetric assay which detects the cleaved c-terminal Phe. Since Phe is relatively stable in tissue lysis conditions, this method should provide new insights into specific angiotensinase activities involving Phe cleavage in complex biological samples, such as the kidney, an organ rich in angiotensins and angiotensinases.

Methods

An assay detecting the release of Phe from Ang Peptides in a reaction with the Phe ammonia lyase was used in vitro with recombinant (r) angiotensinases (ACE2, PEP) and ex vivo using kidneys from C57bl/6 mice.

Results

In vitro, rACE2 and rPEP caused Phe release from Ang II to form Ang 1-7, as anticipated. Ex vivo incubation of kidney lysates with Ang II and Ang III (10-3M) resulted in a marked generation of Phe. Of note, Ex vivo Phe generation from Ang II was significantly higher than that of Ang III (2373±98 vs. 1820±153 RFU/ug prot, n=5, p<0.05, respectively). Aminopeptidase A (APA), which is abundant in the kidney, converts Ang II to Ang III (2-8) and the subsequent cleavage of Ang III to Ang (2-7) could generate Phe as well. Consistent with this, an APA inhibitor amastatin (10-5M) reduced significantly Phe cleavage from Ang II in kidney lysates (2373±98 vs. 1906±101, n=5, p<0.01, respectively). In addition, purified rACE2 and rPEP caused Phe release from Ang III showing that both enzymes convert Ang III to Ang 2-7. This shows that a substantial portion of Phe formation within kidney during Ang II exposure occurs downstream of this main peptide and that Ang III (2-8), an active metabolite, is also efficiently degraded by endogenous angiotensinases such as ACE2 and PEP.

Conclusion

The new assay detects c-terminal Phe cleavage from Ang II(1-8) but also other Ang peptides, such as Ang III, by ACE2 and PEP, angiotensinases not previously known to cleave this important peptide. This method provides a new tool in evaluating kidney enzyme activities involving cleavage of Phe from bioactive peptides that have this amino acid in c-terminal position.