Angiotensin‐converting enzymes, ACE 1 and ACE 2 activity in mouse brain

ACE2 was first discovered in heart, kidney and testis, and thought to be associated with cardiovascular disease. Our studies extend these results to show a predominance of ACE2 activity in normal brain. For this, we developed new mass spectrometric (MS) assays which provide quantitative data on ACE1 and ACE2 activity. The method is based on the proteolysis of the endogenous peptide substrates, Ang I (ACE1) and Ang II (ACE2), with measurement of Ang II and Ang 1-7. Kidney, hypothalamus, hippocampus, brainstem, pituitary and cerebral cortex from C57BL/6 mice were homogenized in Tris HCl (50mM). Tissue extracts (1–2 μg protein) or plasma were added to MES buffer, containing protease inhibitors, and spiked with Ang I or Ang II (10 μM). Generated peptides were analyzed using weak cation exchange Protein Chips and MS. Peak heights were measured and ACE1 and ACE2 activity were quantified as the ratio between substrate and processed peptide. ACE2 activity was detected in kidney and brain, but not in plasma. Brain ACE2 activity was dependent on the region, highest in hypothalamus (1.35± 0.22) followed by cortex (0.69±0.05), hippocampus (0.53±0.18), pituitary (0.27±0.16) and brainstem (0.06±0.02). There was a linear relationship between protein concentration and product formation (R2=0.92). ACE2 activity was inhibited by the specific ACE2 inhibitor, DX600 (10 μM, 99%). ACE1 activity was detected in plasma (0.78±0.12), but was almost undetectable in hypothalamus (0.026±0.004). This study shows 1) development of a novel, sensitive and specific assay for ACE2 activity using natural peptide substrates 2) first evidence for brain ACE2 activity 3) evidence for a predominance of ACE2, rather than ACE1 activity in brain. TSC supported by CAPES/PDEE. Research supported by NIH grant HL 69319.