0 500 1000 1500 2000 2500 kcat/Km [mM-1 sec-1 ]
Figure 2. Comparison of the efficiency constants for the formation of Ang-(1-7) from Ang II, Ang-(1-9) and Ang I. Peptidase abbreviations: ACE, angiotensin-converting enzyme; NEP, neprilysin; PCP, prolyl carboxypeptidase; PEP, prolyl (oligo) endopeptidase Source: Kinetic data from Rice et al 2004 & Welches et al 1993
Figure 3. The ACE2 inhibitor MLN-4760 (ACE2-I) increases the half life (t1/2) of Ang II in the serum of male and female sheep. Inset: comparison of ACE and ACE2 activities in female serum. ACE and ACE2 were determined by the conversion of Ang I to Ang II and Ang II to Ang-(1-7), respectively, by HPLC analysis in the absence or presence of the ACE2 MLN-4760 and the ACE inhibitor lisinopril. Data are n = 4-5, mean ± SEM;* P<0.05 vs. control at equimolar concentrations under identical incubation conditions. Interestingly, as shown in Fig. 3, male sheep exhibited higher ACE2 activity than females that likely contributes to the lower half-life (t^2) of serum Ang II in males (Westwood et al 2006). Addition of the specific ACE2 inhibitor abolished the conversion of Ang II to Ang-(1-7) as measured by a HPLC-125I-detector and markedly increased the Ang II-[t1/2] in both male (6 fold) and female (3 fold) sheep (Fig. 3). These c50 I
Figure 3. The ACE2 inhibitor MLN-4760 (ACE2-I) increases the half life (t1/2) of Ang II in the serum of male and female sheep. Inset: comparison of ACE and ACE2 activities in female serum. ACE and ACE2 were determined by the conversion of Ang I to Ang II and Ang II to Ang-(1-7), respectively, by HPLC analysis in the absence or presence of the ACE2 MLN-4760 and the ACE inhibitor lisinopril. Data are n = 4-5, mean ± SEM;* P<0.05 vs. control
Female c50 I
Female ex vivo data in sheep serum demonstrate that circulating ACE2 constitutes a major pathway in the metabolism of Ang II and support the increase in circulating Ang II levels in the ACE2 null mouse (Crackower et al 2002). Furthermore, we did not find that soluble ACE2 in the serum contributed to the direct conversion of Ang I to Ang-(1-9) even in the presence of complete ACE inhibition (Shaltout et al 2007).
Within the kidney, ACE2 is primarily localized to the apical aspect of the proximal tubule epithelium. Indeed, expression of ACE2 in the renal MDCK cell line revealed exclusive trafficking of the enzyme to the apical side, while the distribution of expressed ACE was different, trafficking to the basolateral and luminal aspects of the cell (Guy et al 2005). Consistent with the apical expression of ACE2 in the renal epithelium, we found significant urinary ACE2 activity that converted Ang II to Ang-(1-7), but did not process Ang I to Ang-(1-9) (Shaltout et al 2007). The glycosylated form of ACE2 is approximately 120,000 Daltons and the filtration of the enzyme into the tubular fluid is highly unlikely (Shaltout et al 2007). In this regard, Lambert and colleagues report that the metallopeptidase ADAM 17 may function as a secretase to release ACE2 from extracellular side of the cell membrane (Lambert et al 2005). Interestingly, ADAM 17 does not release ACE suggesting that the regulation for the secretion for ACE and ACE2 is distinct. The localization of ACE2 in the proximal tubule epithelium along with other elements of the RAS (ACE, angiotensinogen, Ang receptors) supports a role for the enzyme in the processing of angiotensin peptides. In the rat kidney, Burns and colleagues found no evidence that ACE2 or other pepti-dases metabolize Ang II in proximal tubule preparations or in perfused proximal tubule segments isolated from male Sprague Dawley rats (Li et al 2004). However, ACE2 activity was clearly evident in the rat tubules as the conversion of exogenous Ang I to Ang-(1-9) was sensitive to the ACE2 peptide inhibitor DX-600 (Li et al 2004). Ang-(1-9) was subsequently converted to Ang-(1-7) by ACE, a pathway similar to that reported for Ang I metabolism in isolated cardiomyocytes (Donoghue et al 2000). In contrast to the rat, we found that ACE2 was the predominant activity to convert Ang II to Ang-(1-7) in sheep proximal tubules (Shaltout et al 2007). The addition of the non-peptide ACE2 inhibitor MLN-4760 significantly attenuated the metabolism of Ang II at early time points. However, as shown in Fig. 4, the significant ACE and neprilysin activities required prior inhibition to protect Ang-(1-7) from rapid degradation in the proximal tubules. We could not demonstrate that ACE2 participated in the direct metabolism of Ang I, particularly under conditions where other enzymatic pathways were blocked (Shaltout et al 2007). Indeed, Ang I was directly converted to Ang II and Ang-(1-7) via ACE and neprilysin, respectively. The preferred conversion of Ang II to Ang-(1-7) by ACE2 in the sheep kidney is entirely consistent with kinetic studies on various peptide substrates by the human enzyme (Rice et al 2004; Vickers et al 2002), as well studies in membrane fractions of mouse kidney and rat renal cortex that demonstrated ACE2-dependent conversion of Ang II to Ang-(1-7) (Elased et al 2006; Ferrario etal 2005b). An explanation for the discrepancy in the metabolism studies for angiotensin metabolism is not readily apparent; however, if the rat exhibits different kinetic properties for Ang I and Ang II than sheep or human, then the role of ACE2 is likely to be quite different among species. Additionally, these studies have important
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