Brain angiotensin converting enzymes: Evaluation using mass spectrometry and Western blot.

Abstract

Angiotensin converting enzymes, ACE and ACE2, have different functional and substrate specificity. ACE produces the vasoconstrictor, angiotensin II (Ang II) from Ang I, while ACE2 mainly acts on Ang II to form the vasodilator, Ang (1–7). The aim was to evaluate brain ACE/ACE2 activity and protein expression in mice using a new mass spectrometric (MS) assay and Western blot. Brains from C57BL/6 mice and kidney from wildtype and ACE2 knockout mice were used. For enzyme activity, tissue extracts (1–2 μg protein) were incubated with Ang I or Ang-II (0.1–10 μM). Using WXC2 ProteinChips, the substrates and generated peptides were analyzed using selective enhanced laser desorption time of flight MS (SELDI-TOF-MS). ACE2 activity was expressed as the ratio of generated Ang (1–7) (899, m/z) to Ang II (1045, m/z). ACE activity was expressed as the ratio of generated Ang II to Ang I (1296, m/z). Western blot was expressed in arbituitary units normalized to βactin. Western blot demonstrated low levels of brain ACE protein expression compared to kidney. In plasma there was significant activity for ACE but undetectable levels of ACE2. ACE2 activity was absent in tissue from ACE2 knockout mice and was blocked with selective ACE2 antagonists. Results show: specificity of the MS method for measurement of ACE2 activity evidence for the presence of ACE2 in kidney and brain predominance of brain ACE2 activity over ACE Western blot analysis demonstrated low brain ACE expression, which correlated with reduced enzyme activity.