Received December 6, 2007
Revised January 4, 2008
Accepted after revision February 7, 2008

¹ Wright State University
² State University of Campinas

^* To whom correspondence should be addressed. E-mail: mariana.morris{at}wright.edu.

	Abstract

Angiotensin (Ang) converting enzyme-2 (ACE2) metabolizes Ang II to the vasodilatory peptide, Ang (1-7), while neprilysin (NEP) generates Ang (1-7) from Ang I. Experiments used novel SELDI-TOF mass spectroscopic (MS) assays to study Ang processing. MS was used to measure proteolytic conversion of Ang peptide substrates to their specific peptide products. We compared ACE/ACE2 activity in plasma, brain and kidney from C57BL/6 and NEP -/- mice. Plasma or tissue extracts were incubated with Ang I or Ang II (1296 or 1045 m/z, respectively) and generated peptides were monitored with MS. ACE2 activity was detected in kidney and brain, but not plasma. Brain ACE2 activity was highest in hypothalamus. ACE2 activity was inhibited by the specific ACE2 inhibitor, DX600 (10 µM, 99% inhibition), but not by the ACE inhibitor, captopril (10 µM). Both MS and colorimetric assays showed high ACE activity in plasma and kidney with low levels in brain. To extend these findings, ACE measurements were made in ACE over-expressing mice. ACE 4-copy mice showed higher ACE activity in kidney and plasma with low levels in hypothalamus. In hypothalamus from NEP-/-, generation of Ang (1-7) from Ang I was decreased, suggesting a role for NEP in Ang metabolism. With Ang II as substrate, there was no difference between NEP (-/-) and wild type controls, indicating that other enzymes may contribute to generation of Ang (1-7). Data suggest a predominant role of hypothalamic ACE2 in the processing of Ang II in contrast to ACE which is most active in plasma

Key Words: Angiotensin, Enzyme, Hypothalamus

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