Role of ACE/AT2R complex in the control of mesenteric resistance artery contraction induced by ACE/AT1R complex activation in response to Ang I

Abstract

Objectives: In this study, we will determine the function of the interaction between AT2R and ACE, and AT1R and ACE in the control of mesenteric resistance artery (MRA) tone from normotensive (NT) and Angiotensin II (AII)-dependent hypertensive (HT) mice.

Methods-results: Hypertension was induced by infusion of Ang-II (200 ng/kg/day) for 3 weeks. Freshly MRA (100-120 microm) were isolated from HT and NT mice and mounted in an arteriograph. Dose-response of Ang-I induced a similar contraction of MRA from NT and HT mice, which was increased after endothelium removal. AT2R antagonist (PD123319, 1 microM) significantly increased Ang-I-induced contraction of MRA from NT but not from HT mice. In addition, PD123319 significantly increased in vivo blood pressure in response to Ang-I. Luminal incubation with ACE-antibody (50 ng/ml) to block only endothelial ACE function significantly enhanced Ang-I-induced contraction of MRA from NT mice. ACE inhibitor (captopril, 10 microM) completely blocked Ang-I-induced contraction of MRA from both animals and prevented the increased blood pressure. Freshly isolated MRA subjected to immunoprecipitation, Western blot analysis and RT-PCR revealed AT1R/ACE and AT2R/ACE complexes formation, and similar AT1R, AT2R, and ACE expression level in both groups.

Conclusion: The present findings show the existence of ACE/AT2R and ACE/AT1R complexes on endothelial cells and VSMC, respectively. ACE/AT2R complex plays a modulator effect on ACE/AT1R-SMC-induced contraction of MRA, which is altered in hypertension.

Figure 1

Left panel: Dose-response to Ang-I (10⁻¹⁰ to 10⁻⁷ mol/L) and diameter decrease relationship in normotensive (NT) and hypertensive (HT) mouse mesenteric resistance artery held at 50 mmHg without and with ACE inhibitor (captopril, 10 μM), n=6 per group. *P<0.05, statistically significant, NT vs. NT+captopril; HTN vs. HTN+captopril; Right panel: Dose-response to Ang-I (10⁻¹⁰ to 10⁻⁷ mol/L) and diameter decrease relationship in normotensive (NT) and hypertensive (HT) mouse mesenteric resistance artery held at 50 mmHg without and with intraluminal specific ACE antibody (50 ng/ml), n=6 per group. *P<0.05, statistically significant, NT vs. NT+ACE antibody at 10⁻⁷ of Ang-I

Figure 2

Left panel: Dose-response to Ang-I (10⁻¹⁰ to 10⁻⁷ mol/L) and diameter decrease relationship in normotensive (NT) and hypertensive (HT) mouse mesenteric resistance artery held at 50 mmHg without and with AT₂R inhibitor (PD123, 1 μM), n=6 per group. *P<0.05, statistically significant, NT vs. NT+PD123; Right panel: Dose-response to Ang-I (10⁻¹⁰ to 10⁻⁷ mol/L) and diameter decrease relationship in normotensive (NT) and hypertensive (HT) mouse mesenteric resistance artery held at 50 mmHg without and with endothelium, n=6 per group. *P<0.05, statistically significant, NT vs. NT without endothelium; HTN vs. HTN without endothelium

Figure 3

Dose-response to Ang-II (10⁻¹⁰ to 10⁻⁷ mol/L) and diameter decrease relationship in normotensive (NT) mouse mesenteric resistance artery held at 50 mmHg without and with AT₂R inhibitor (PD123, 1 μM), n=6 per group.

Figure 4

4A: Western blot analysis showing the presence of AT₂R (upper panel) and AT₁R (Lower panel) protein expression in normotensive (NT) and hypertensive (HT) mouse resistance arteries; 4B: RT-PCR showing no difference of AT₂R (upper panel) and AT₁R (Lower panel) mRNA level in resistance arteries from normotensive (NT) and hypertensive (HT) mouse; 4C: immunoprecipitation and western blot analysis showing the presence of ACE in both groups (Upper panel), the interaction between ACE and AT₂R (Middle panel) and between ACE and AT₁R (Lower panel) in resistance arteries from both groups. n=6 per group.

Figure 5

Effect of acute injection of Ang I on in vivo blood pressure in the absence or presence of AT₂R antagonist (PD123) or ACE inhibitor (captopril); n=6 per group. *P<0.05, statistically significant, Ang I vs. Ang I+PD123; Ang I vs Ang I+captopril.

Figure 6

This model shows the physiological role of ACE/AT₂R and ACE/AT₁R complexes on endothelial and smooth muscle cells respectively in the regulation of resistance artery tone. This model illustrates the access and the activation of AT₂R and AT₁R under Ang I leading to a module contraction. On the other hand, under Ang II, there is a fast access of Ang II to AT₁R and less to AT₂R leading to a high contraction.