Role of prolylcarboxypeptidase in angiotensin II type 2 receptor-mediated bradykinin release in mouse coronary artery endothelial cells

Abstract

Activation of angiotensin II type 2 receptors (AT(2)R) causes the release of kinins, which have beneficial effects on the cardiovascular system. However, it is not clear how AT(2)R interact with the kallikrein-kinin system to generate kinins. Prolylcarboxypeptidase is an endothelial membrane-bound plasma prekallikrein activator that converts plasma prekallikrein to kallikrein, leading to generation of bradykinin from high-molecular-weight kininogen. We hypothesized that AT(2)R-induced bradykinin release is at least in part mediated by activation of prolylcarboxypeptidase. Cultures of mouse coronary artery endothelial cells were transfected with an adenoviral vector containing the AT(2)R gene (Ad-AT(2)R) or green fluorescent protein only (Ad-GFP) as control. We found that overexpression of AT(2)R increased prolylcarboxypeptidase mRNA by 1.7-fold and protein 2.5-fold compared with Ad-GFP controls. AT(2)R overexpression had no effect on angiotensin II type 1 receptor mRNA. Bradykinin release was increased 2.2-fold in AT(2)R-transfected cells. Activation of AT(2)R by CGP42112A, a specific AT(2)R agonist, increased bradykinin further in AT(2)R-transfected cells. These effects were diminished or abolished by AT(2)R blockade or a plasma kallikrein inhibitor. Furthermore, blocking prolylcarboxypeptidase with a small interfering RNA partially but significantly reduced bradykinin release by transfected AT(2)R cells either at the basal condition or when stimulated by the AT(2)R agonist CGP42112A. These findings suggest that overexpression of AT(2)R in mouse coronary artery endothelial cells increases expression of prolylcarboxypeptidase, which may contribute to kinin release.

Fig. 1

Characterization of mouse coronary artery endothelial cells (ECs). A: Uptake of Dil-labeled acetylated low-density lipoprotein (Ac-LDL; red fluorescence); magnification 200x. B: Immunostaining with an antibody to von Willebrand factor (vWF; green fluorescence). 4′,6-diamidino-2-phenylindole (DAPI) was used to indicate the nuclei (blue fluorescence); magnification 400x.

Fig. 2

Adenovirus-induced angiotensin II type 2 receptor (Ad-AT2R) transfection in ECs. A: Representative images taken 24 hrs after transfection of Ad-AT2R (green fluorescence) at a multiplicity of infection (MOI) of 40, 80 or 160 (magnification 100x). B: Quantitative evaluation of AT2R mRNA expression in Ad-AT2R-transfected cells by real-time PCR. GAPDH was used as an internal control. Data are shown as fold change relative to MOI 40, n = 3.

Fig. 3

Effect of Ad-AT2R transfection on AT1R mRNA expression. Cells were transfected with Ad-GFP or Ad-AT2R at MOI 40, 80 or 160 for 24 hrs. mRNA levels were evaluated by real-time PCR and normalized to GAPDH. Data are shown as fold change relative to Ad-GFP-transfected cells at MOI 40; n = 3.

Fig. 4

Effect of AT2R overexpression on PRCP protein and mRNA expression. Cells were transfected with Ad-GFP or Ad-AT2R at MOI 80 for 24 hrs. A: Representative Western blots showing the increase in PRCP protein expression induced by Ad-AT2R transfection. B. Quantitative evaluation of PRCP protein expression normalized to GAPDH. C. PRCP mRNA expression evaluated by real-time PCR and normalized to GAPDH. Data are shown as fold change relative to Ad-GFP-transfected cells, n = 3–4.

Fig. 5

Effect of AT2R overexpression on bradykinin release. Ad-AT2R- transfected cells (MOI 80) were either left untreated or treated with the AT2R antagonist PD123319 (PD, 100 μM) or the AT1R antagonist valsartan (Val, 1 μM). Ad-GFP cells (MOI 80) served as controls. Bradykinin concentration was normalized to total protein and expressed as pg/μg protein. n = 5–18. NS: no statistical significance.

Fig. 6

Effect of the AT2R agonist CGP42112A (CGP) on bradykinin release. Ad-AT2R-transfected cells (MOI 80) were treated with CGP (0.1 μM) with or without the AT2R antagonist PD123319 (PD, 100 μM). Ad-GFP cells (MOI 80) served as controls. n = 9–18.

Fig. 7

Effect of soybean trypsin inhibitor (SBTI) on bradykinin release. Ad-AT2R- transfected cells (MOI 80) were treated with SBTI (1 μM) with or without the AT2R agonist CGP42112A (CGP, 0.1 μM). Ad-GFP cells (MOI 80) served as controls. n = 4–18.

Fig. 8

Effect of prolylcarboxypeptidase (PRCP) blockade by siRNA on bradykinin release in Ad-AT2R-transfected ECs. A: Top panel: Representative Western blot showing PRCP protein expression. Lower panel: Quantitative data. B: fold change in bradykinin release relative to Ad-GFP-transfected cells. n = 4.