Angiotensin converting enzyme 2 activity is not detectable in mouse plasma

Abstract

Angiotensin converting enzymes (ACE & ACE2) are critical in metabolism of Ang peptides. There is evidence for ACE and ACE2 activity in heart, kidney and brain. In addition, there is abundant ACE activity in plasma which is activated in diabetic mice and humans. Data is limited regarding the presence and activity of circulating plasma ACE2 in normal and in disease states. Further, there is a need for a reliable and specific ACE2 assay. The aim of this study was to develop, screen and quantify ACE2 activity in plasma using a new mass spectrometric (MS) method. Plasma from normal C57BL/6, streptozotocin (STZ) and db/db diabetic mice and kidney from wildtype and ACE2 knockout mice were tested. Plasma (1 μl) or tissue extracts (1–2 μg protein) were incubated with Ang II (0.1–10 μM) in the presence of protease inhibitors. Substrates and generated peptides were analyzed in the Ciphergen ProteinChips® Reader using selective enhanced lazer desorption time of flight MS (SELDI-TOF-MS). ACE2 activity was expressed as the ratio of the generated Ang (1–7) (899, m/z) to Ang II (1045, m/z). ACE2 activity was not detected in plasma obtained from normal or diabetic mice. It was present in kidney from control wild type mice, but was absent in tissue from ACE2 knockout mice. Unlike ACE, tissue ACE2 activity was not inhibited by captopril (10 μM), but was blocked with selective antagonists MLN 4760 (100 μM) and DX 600 (10 μM). We demonstrate the development of a novel assay for the measurement of ACE2 activity using endogenous peptide substrates and MS technology. Results show 1) specificity of the method since ACE2 was absent in tissues from gene deletion mice and could be blocked with an ACE 2 inhibitor and 2) absence of detectable plasma ACE2 activity in normal, STZ or genetically diabetic mice.