Portal pressure responses and angiotensin peptide production in rat liver are determined by relative activity of ACE and ACE2

Abstract

Angiotensin converting enzyme (ACE) 2 activity and angiotensin-(1-7) [Ang-(1-7)] levels are increased in experimental cirrhosis; however, the pathways of hepatic Ang-(1-7) production have not been studied. This study investigated the role of ACE2, ACE, and neutral endopeptidase (NEP) in the hepatic formation of Ang-(1-7) from angiotensin I (Ang I) and Ang II and their effects on portal resistance. Ang I or Ang II were administered to rat bile duct ligated (BDL) and control livers alone and in combination with the ACE inhibitor lisinopril, the ACE and NEP inhibitor omapatrilat, or the ACE2 inhibitor MLN4760 (n = 5 per group). BDL markedly upregulated ACE, ACE2, and NEP. Ang-(1-7) was produced from Ang II in healthy and in BDL livers and was increased following ACE inhibition and decreased by ACE2 inhibition. In contrast, Ang-(1-7) production from Ang I was minimal and not affected by ACE or NEP inhibition. Surprisingly, ACE2 inhibition in BDLs dramatically increased Ang-(1-7) production from Ang I, an effect abolished by ACE2/NEP inhibition. Ang II and Ang I induced greater portal pressure increases in BDL livers than controls. The effects of Ang I were closely correlated with Ang II production and were strongly attenuated by both ACE and ACE/NEP inhibition. These findings show that the major substrate for hepatic production of Ang-(1-7) is Ang II and this is catalyzed by ACE2. Ang I is largely converted to Ang II by ACE, and net conversion of Ang I to Ang-(1-7) is small. NEP has the ability to generate large amounts of Ang-(1-7) in the BDL liver from Ang I only when ACE2 activity is greatly decreased or inhibited.

Fig. 1.

Schematic representation of the pathways responsible for the generation of antifibrotic peptide angiotensin-(1-7) [Ang-(1-7)] in rat liver. The thickness of the arrows represents the relative contribution of each pathway for ex vivo formation of Ang-(1-7) from the substrates angiotensin I (Ang I) and angiotensin II (Ang II). The dashed line indicates an efficient pathway to generate Ang-(1-7) directly from Ang I by the action of neutral endopeptidase (NEP), but it appears that this pathway is masked by ACE2. Ang-(1-9), angiotensin-(1-9); Ang-(1-5), angiotensin-(1-5); ACE, angiotensin converting enzyme; ACE2, angiotensin converting enzyme 2.

Fig. 2.

Angiotensin-(1-7) production in rat liver. Ang I (99 pmol) or Ang II (60 pmol) bolus was injected into the portal vein of in situ perfused healthy (○ and open bars) and fibrotic (• and solid bars) rat liver preparations with or without the ACE inhibitor lisinopril (10⁻⁶ mol/l), ACE and NEP inhibitor omapatrilat (10⁻⁶ mol/l), ACE2 inhibitor MLN4760 (10⁻⁶ mol/l), or a combination of MLN4760 and omapatrilat. Angiotensin-(1-7) production from Ang II (A) and the baseline-corrected total area under the respective curves (B), and angiotensin-(1-7) production from Ang I (C) and the baseline-corrected total area under the respective curves (D), and Ang II-induced portal pressure responses (E) and the area under pressure response curve (F) are shown. Each symbol represents the mean ± SE profile from 4–5 rats per treatment group. Note that y-axis scale is different between A and C and between B and D. Arrow indicates the time of bolus injection. ****P < 0.0005, **P < 0.01, *P < 0.05, vs. respective healthy livers. ††††P < 0.0005, between the 2 groups indicated by brackets. In E and F, *P < 0.05 vs. other groups except bile duct ligation (BDL)/ACE inhibition group (P = 0.059).

Fig. 3.

Ang II production and portal pressure changes in response to Ang I in rat liver. Ang I bolus (99 pmol) was injected into the portal vein of in situ perfused healthy (○ and open bars) and fibrotic (• and solid bars) rat liver preparations with or without the ACE inhibitor lisinopril (10⁻⁶ mol/l), ACE and NEP inhibitor omapatrilat (10⁻⁶ mol/l), ACE2 inhibitor MLN4760 (10⁻⁶ mol/l), or a combination of MLN4760 and omapatrilat. Ang II production (A) and the area under the Ang II curve (B) and Ang I-induced portal pressure responses (C) and the area under pressure-response curve (D) are shown. Each symbol represents the mean ± SE profile from 4–5 rats per treatment group. Arrows indicate the time of bolus injection. ****P < 0.0005, **P < 0.01, *P < 0.05, vs. respective healthy livers. ††††P < 0.0005, †P < 0.05, between the 2 groups indicated by brackets.

Fig. 4.

Gene expression profiles of the components of the classic and alternative arms of the renin angiotensin system in the perfused livers from healthy (open bars) and fibrotic (solid bars) rats. Quantitative real-time polymerase reaction (QPCR) was performed by Taqman multiplexing assay with dual-labeled probes and primers. Gene expression values were normalized to ribosomal 18S, and the healthy livers were given a value of 1. Each bar represents the mean ± SE expression from 4–5 rats per treatment group. ****P < 0.0005 vs. healthy livers.

Fig. 5.

ACE2 activity of the perfused rat liver. In situ perfused healthy (open bars) and fibrotic (solid bars) rat livers, incubated with or without the ACE inhibitor lisinopril (10⁻⁶ mol/l), ACE and NEP inhibitor omapatrilat (10⁻⁶ mol/l), ACE2 inhibitor MLN4760 (10⁻⁶ mol/l), or a combination of MLN4760 and omapatrilat were used for cell membrane preparations for measurement of ACE2 activity. ACE2 activity was determined by measuring nanomoles of ACE2 substrate [Mca-APK-(Dnp)-OH] cleaved by solubilized membrane fractions. Each bar represents the mean ± SE activity from 4–5 rats per treatment group. ACE2 activity was not different between different groups of healthy livers so that the mean value from all groups is shown. ****P < 0.0005, ***P < 0.005, **P < 0.01, *P < 0.05 vs. group indicated by brackets.