Aminopeptidase A Effect on Angiotensin Peptides and Their Blood Pressure Action

Abstract

Aminopeptidase A (APA) cleaves a single aspartate residue from the amino terminus of peptides within the renin angiotensin system (RAS). Since several RAS peptides contain an N-terminal aspartate, we developed an assay to evaluate the effect of recombinant APA on the cleavage of Ang I, Ang II, Ang-(1-7), Ang-(1-9), and Ang-(1-12). The latter peptide has been proposed to be a functional Ang II-forming substrate with a hypertensive action attributable to the formed Ang II acting on AT1 receptors. Here we investigated the following: (a) the hydrolytic action of APA on Ang-(1-12), Ang I (1-10), Ang-(1-9), Ang II and Ang-(1-7) and (b) whether Ang-(1-12) pressor activity is altered by recombinant APA (r-APA) or genetic APA deficiency. We found that (a) r-APA cleaves the N-terminal aspartate of not only Ang II but also [Ang-(1-12), Ang I (1-10), Ang-(1-9)] and [Ang-(1-7)]; (b) the pressor activity of Ang-(1-12) was abolished in the presence of Lisinopril or Telmisartan; (c) r-APA significantly attenuated the pressor activities of infused Ang I and Ang II but not Ang-(1-12); and (d) r-ACE2 also did not attenuate the pressor effect of infused Ang-(1-12). Thus, in addition to increasing blood pressure indirectly via the formation of Ang II, Ang-(1-12) increases blood pressure by an Ang II-independent mechanism. We conclude that APA has an antihypertensive effect attributable to rapid degradation of Ang II, and this action may have a therapeutic potential in forms of hypertension that are Ang II-dependent. In addition, APA metabolizes Ang-(1-12), a peptide that has a prohypertensive action, in part, as a source of Ang II formation but also by a yet to be determined action independent of Ang II.

Keywords: aminopeptidase A; angiotensin; angiotensin II; angiotensin-(1-12); renin angiotensin system.

Figure 1

Amino acid sequence of several Angiotensin peptides with an N-terminal aspartate residue (yellow-highlighted circle).

Figure 2

Cleavage of N-terminal aspartate from various angiotensin peptides by murine recombinant aminopeptidase A (rAPA) measured by fluorescence formation using the in vitro assay described in the Methods. Each bar represents the mean ± standard error of 4–12 experiments of each of the peptides tested; ns—not significant.

Figure 3

Systolic blood pressure (SBP) and mean SBP change from baseline after i.p. injection of different Ang peptides (0.2 μg/g body weight). Changes in systolic blood pressure (SBP) from the baseline in mice after a single i.p. injection of five Ang peptides (Ang I, Ang II, Ang-(1-12), Ang-(1-7), and Ang-(1-9) each at 0.2 μg/g body weight). (Left): Average difference (Δ) between the SBP measured during 5 min before and the SBP recorded during 5 min after an intraperitoneal (IP) administration of the angiotensin peptides (N = 3 to N = 11). (Right): Average differences (Δ) in SBP between baseline (SBP measured during 5 min before i.p. injection of the Ang peptide) and SBP measured 5 min, 10 min, and 15 min after i.p. injection of the Ang peptide; analyzed by 2-way ANOVA and Tukey post-hoc comparisons; ns—not significant differences between Ang-(1-7) and Ang-(1-9) and between Ang I, Ang II, and Ang-(1-12); **—denotes p < 0.01 or p < 0.001 regarding Ang-(1-7) and Ang-(1-9) vs. Ang I, Ang II, and Ang-(1-12) (N = 3 to N = 5).

Figure 4

Systolic blood pressure in WT mice after injection of Ang I, Ang II, and Ang-(1-12) pre-administered with recombinant (r)APA or vehicle (PBS). Systolic blood pressure (SBP) measured before and after injection of 0.2 μg/g BW Ang I, Ang II, and Ang-(1-12) (arrow) in male C57BL/6J mice which were pre-administrated with recombinant (r)APA (1 μg/g BW) or vehicle (PBS) 30 min before SBP measurement. (Left): Recombinant APA significantly enhances the rate of recovery from the SBP elevation induced by Ang I (p = 0.0266) compared to PBS (N = 3 vs. N = 3). (Middle): Recombinant APA significantly enhances the rate of recovery from the SBP elevation induced by Ang II (p = 0.0082) compared to PBS (N = 5 vs. N = 3). (Right): Recombinant APA does not significantly enhance the rate of recovery from the SBP elevation induced by Ang-(1-12) (p = 0.1403) compared to PBS (N = 9 vs. N = 8).

Figure 5

Systolic blood pressure in WT and APA Ko mice before and after injection of Ang II and Ang-(1-12). Systolic blood pressure (SBP) in WT (squares) and APAKO mice (circles) before (baseline) and after injection of 0.2 μg/g BW (arrow) of Ang II (left panel) or Ang-(1-12) (right panel). (Left): Ang II causes a significantly bigger increase in SBP in APAKO as compared to WT mice (p = 0.0196) (N = 6 vs. N = 5). (Right): Ang-(1-12) causes a significantly bigger increase in SBP in APAKO as compared to WT mice (p = 0.0015) (N = 4 vs. N = 3).

Figure 6

Effect of Telmisartan (Telm) and Lisinopril (Lis) on injection of Ang-(1-12) in WT mice. The effect of the AT1 receptor blocker, Telmisartan (1 μg/g BW, left panel), and ACE inhibitor Lisinopril (1 μg/g BW, right panel) on systolic blood pressure (SBP) responses caused by the injection of Ang-(1-12) (0.2 μg/g BW, arrow). Telmisartan and Lisinopril were injected 30 min before SBP measurements. (Left): The SBP increase caused by Ang-(1-12) injection (arrow) is completely blocked by Telmisartan as compared to the PBS pre-injected control group (p = 0.0002) (N = 15 vs. N = 5). (Right): The SBP increase caused by Ang-(1-12) injection (arrow) is completely blocked by Lisinopril as compared to the PBS pre-injected control group (p < 0.0001) (N = 15 vs. N = 4).

Figure 7

Effect of recombinant mouse ACE2 (740) on systolic blood pressure increases induced by Ang II and Ang-(1-12) injection. The effect of recombinant mouse ACE2 (1 μg/g BW) on systolic blood pressure (SBP) responses caused by the injection of Ang II (left panel), Ang-(1-12) (middle panel) (both 0.2 μg/g BW, arrow), and Ang II and Ang-(1-12) superimposed over each other. (Left): Compared to a PBS pre-injected control group, rACE2 reduced the SBP response to Ang II over ten minutes after injection by p = 0.0248 (rACE2) by 2-way ANOVA. When comparing over fifteen minutes, the injection of rACE2 reduced the SBP response by p = 0.058 (N = 4 vs. N = 4). (Middle): As compared to a PBS pre-injected control group, rACE2 did not reduce the SBP response to Ang-(1-12) either over ten minutes after injection (p = 0.5461) or over fifteen minutes after injection (p = 0.6610) (N = 5 vs. N = 3). (Right): Direct comparison of the effect of rACE2 on reducing the SBP response between the Ang II-induced (blue) and Ang-(1-12) (red)-induced SBP increase which was significantly different both over ten minutes from the injection (p = 0.0176) and over fifteen minutes from the injection of the peptides (p = 0.0230) (N = 4 vs. N = 5).

Figure 8

Metabolism of Angiotensin (1-12) by APA and other enzymes may support the downstream formation of prohypertensive and non-hypertensive peptides. This process can be renin-independent. A hypothetical Ang-(1-12) receptor is indicated to reflect the possibility of a direct signaling mechanism, independent of known metabolites such as Ang II.