Ang II (Angiotensin II) Conversion to Angiotensin-(1-7) in the Circulation Is POP (Prolyloligopeptidase)-Dependent and ACE2 (Angiotensin-Converting Enzyme 2)-Independent

Abstract

The Ang II (Angiotensin II)-Angiotensin-(1-7) axis of the Renin Angiotensin System encompasses 3 enzymes that form Angiotensin-(1-7) [Ang-(1-7)] directly from Ang II: ACE2 (angiotensin-converting enzyme 2), PRCP (prolylcarboxypeptidase), and POP (prolyloligopeptidase). We investigated their relative contribution to Ang-(1-7) formation in vivo and also ex vivo in serum, lungs, and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In wild-type (WT) mice, infusion of Ang II resulted in a rapid increase of plasma Ang-(1-7). In ACE2^-/-/PRCP^-/- mice, Ang II infusion resulted in a similar increase in Ang-(1-7) as in WT (563±48 versus 537±70 fmol/mL, respectively), showing that the bulk of Ang-(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a POP inhibitor, Z-Pro-Prolinal reduced the rise in plasma Ang-(1-7) after infusing Ang II to control WT mice. In POP^-/- mice, the increase in Ang-(1-7) was also blunted as compared with WT mice (309±46 and 472±28 fmol/mL, respectively P=0.01), and moreover, the rate of recovery from acute Ang II-induced hypertension was delayed (P=0.016). In ex vivo studies, POP inhibition with ZZP reduced Ang-(1-7) formation from Ang II markedly in serum and in lung lysates. By contrast, in kidney lysates, the absence of ACE2, but not POP, obliterated Ang-(1-7) formation from added Ang II. We conclude that POP is the main enzyme responsible for Ang II conversion to Ang-(1-7) in the circulation and in the lungs, whereas Ang-(1-7) formation in the kidney is mainly ACE2-dependent.

Keywords: angiotensins; hypertension; renin-angiotensin system.

Figure 1:. Angiotensin peptide levels in serum measured by LC/MS-MS in Ang II-infused mice.

Plasma showed high levels of Ang II (upper), Ang-(1-7) (middle), and Ang-(1-5) (lower panel) measured by Liquid Chromatography Tandem Mass Spectrometry (LC/MS-MS). Blood was obtained by cardiac puncture from mice (n=6) at 5 minutes after Ang II i.p. infusion.

Figure 2:

Ang II and Ang-(1-7) levels in plasma measured 5 minutes post infusion of Ang II. (A) Plasma Ang II levels in WT and ACE2^−/−/PRCP^−/− mice were not significantly different from each other in the absence or with administration of ZPP. (B) Ang-(1-7) levels in WT mice with ZPP administration (WT+ZPP) were markedly reduced as compared to WT without ZPP (WT). In the ACE2^−/−/PRCP^−/− mice, ZPP administration also led to much lower Ang 1-7 levels as compared to ACE2^−/−/PRCP^−/− mice without ZPP (*p<0.05; **p<0.01). In these studies, blood was obtained from tail vein at 5 minutes and also at 30 minutes (see supplement) after Ang II.

Figure 3:. Studies in POPKO mice

(A) Endogenous Ang II levels in plasma of POP^−/− mice were higher but not significantly different from WT controls. (B) Endogenous plasma Ang-(1-7) levels in POP^−/− mice were not detectable whereas in WT mice the levels were low, but detectable (p=0.032) (C) Ang II levels in plasma measured 5 minutes after i.p. injection of Ang II were significantly higher in POP^−/− mice (n=8) than in WT mice (*p<0.05). (D) Ang-(1-7) levels in plasma also measured 5 minutes after i.p. injection of Ang II were lower in POP^−/− mice than in WT mice(**p<0.01). Baseline levels were measured in blood obtained by cardiac puncture while post Ang II infusion blodd was obtained by tail snip. (E) Systolic blood pressure (SBP) after single i.p. bolus injection of Ang II (time point “0”) under light ketamine anaesthesia WT and POP^−/− mice (n=10 in each group). The SBP was recorded every 30 sec. for 15 minutes after Ang II injection. The slope of SBP decline was significantly slower in POP^−/− than in WT (3.06±0.48 vs. 4.81±0.45 mmHg/min respectively, *p<0.05.)

Figure 4:. Ex vivo Ang-(1-7) formation from Ang II in kidney and lung lysates as assessed by the peak levels of Ang-(1-7) at 15 minutes (see methods).

Experiments with each lysate were performed on two different occasions in duplicate. A) In WT kidney lysates, the POP inhibitor, ZPP, did not affect Ang-(1-7) formation from Ang II significantly as compared to WT lysates without ZPP. In ACE2^−/−/PRCP^−/− kidney lysates Ang-(1-7) was markedly decreased. In ACE2^−/−/PRCP^−/− kidney lysates ZPP addition decreased Ang-(1-7) to essentially non-detectable levels. B) In WT lung lysates, ZPP completely prevented Ang-(1-7) formation from Ang II (compare WT and WT+ZPP). In ACE2^−/−/PRCP^−/− lung lysates Ang-(1-7) levels were not reduced (actually, they were higher) and ZPP resulted in undetectable Ang-(1-7) levels. C) In POP^−/− kidney lysates, Ang-(1-7) formation from Ang II was not different from that of WT lysates. In contrast, in POP^−/− kidney lysates an addition of MLN-4760, a specific inhibitor of ACE2, was associated with no detectable Ang-(1-7) formation from Ang II. D) In POP^−/− lung lysates Ang-(1-7) formation was completely prevented as compared to WT lung lysates and addition of MLN-4760 had no further effect.

Figure 5:. Ex vivo formation of Ang-(1-7) from Ang II in serum.

Ex vivo Ang-(1-7) formation after incubation with Ang II in sera from A) WT mice, without ZPP and with ZPP and B) serum from from ACE2^−/−/PRCP^−/− mice and POP^−/− mice. A) In WT sera (n=6) in the presence of the dual POP/PRCP inhibitor, ZPP (10⁻⁵M), Ang-(1-7) formation from Ang II is markedly lower than in the absence of ZPP (p<0.01 by GLM statistics over time). B) In sera from POP^−/− mice (n=4), Ang-(1-7) formation from Ang II is markedly lower than in the sera from ACE2^−/−/PRCP^−/− mice (n=3) (p<0.01 by GLM statistics over time).

Figure 6:. ACE2 and POP proteins in WT sera, lung and kidneys and the effect of rACE2 and rPOP on Ang-(1-7) and Phenylalanine formation.

A) ACE2 and POP proteins in WT sera, lung and kidneys by Western blot. ACE2 protein is abundant in kidneys, but not in serum or lungs. POP protein is present in serum, lungs and kidneys. B) in vitro Ang II to Ang-(1-7) conversion assessed by equivalent amounts of recombinant (r)ACE2 and rPOP is higher with rACE2 than with rPOP (***p<0.001). C) generation of free Phenylalanine (Phe) from Ang II as a substrate by equivalent amounts of recombinant (r)ACE2 and rPOP is higher with rACE2 than with rPOP (*** p<0.001). For Western blot, different amounts of mouse recombinant protein standards were loaded to estimate ACE2 and POP protein expression levels in sera (1ul), lung and kidney lysates (50 ug total protein) from two WT mice.