Evidence for Angiotensin Ii as a Naturally Existing Suppressor for the Natriuretic Peptide System

Figure 1.. Correlations between cGMP and ANP/BNP in Healthy Subjects Grouped by ANGII Levels. (A) Linear correlation between plasma ANP and plasma cGMP. (B) Linear correlation between plasma BNP and plasma cGMP. Each dot represents one healthy subject. Blue indicates subjects with plasma ANGII ≤ 4.5 pg/mL; Red indicates subjects with plasma ANGII > 4.5 pg/mL. In (A), subjects with plasma ANP below the minimal detection by the assay (4 pg/mL) was not included. Pearson correlation coefficient r and associated P value were shown for each constructed linear regression model. * indicates P < 0.05.

Figure 2.. ANGII Attenuates Circulating and Urinary cGMP Generation Induced by ANP In Vivo. (A) The acute protocol study design to evaluate the effect of ANGII on ANP infusion in normal rats. (B) Plasma ANP levels at 90 mins after continuous ANP infusion. (C) Plasma cGMP, * indicates P<0.05 compared to ANP group; # indicates P<0.05 compared to corresponding baseline. (D) Urinary cGMP excretion. * indicates P<0.05 compared to ANP group. (E) Change in mean arterial pressure. * indicates P<0.05 compared to ANP group; # indicates P<0.05 compared to corresponding baseline. (F) Urine output represented by urine volume collected at every 30-min interval during continuous infusion. (G) Urinary sodium excretion during the entire 90-min continuous infusion. N=4 in ANP group, N=4 in ANP+ANGII group.

Figure 3.. ANGII Attenuates GC-A Mediated cGMP Generation via AT1 Receptor. (A) Construction and nomenclature of three different HEK293 transfected cells. (B) Protein expression of human GC-A and GC-B in HEK293 parental and transfected cells by western blotting. 1, HEK293 parental cells; 2, HEK293/GC-A+; 3, HEK293/GC-B+; 4, HEK293/GC-A+/AT1+; 5, HEK293/GC-A+/AT2+. Each lane was loaded with 40 μg total protein. Antibody against GFP detected protein expression incurred by either GC-A or GC-B transfection. HEK293/GC-B+ cells served as a negative control for GC-A specific overexpression in other transfected cells. (C) mRNA expression of human AGTR1 (AT1 coding gene) in HEK293/GC-A+/AT1+ cells and human AGTR2 (AT2 coding gene) in HEK293/GC-A+/AT2+ cells, compared to HEK293/GC-A+ cells. Expression levels were normalized to human GAPDH. (D) In vitro cGMP generation in HEK293 transfected cells in response to different doses of ANP with or without ANGII (10−8 M). Values of cGMP in ANGII treated group (red) were normalized to corresponding vehicle group (blue) under each dose of ANP. Absolute values of cGMP are shown in Supplemental Figure 3. * indicates P<0.05, two-way ANOVA with Sidak multiple…

Figure 4.. SPR Binding Curves for ANGII, ANP, and BNP to Human GC-A receptor. Representative of SPR sensogram for the binding of ANGII (A), ANP (B), and BNP (C) to the extracellular domain of human GC-A receptor.

Figure 5.. Involvement of PKC Signaling in the Crosstalk between ANGII/AT1 and GC-A. (A) Protein expression of different human PKC isoforms in the membrane and cytosol fractions of different HEK293 transfected cell lines (with or without treatment of ANGII). GC-A and NaK-ATPase serve as quality control for the separation of membrane and cytosol fractions. GAPDH serves as loading control. (B) Protein expression of phosphorylated PKC substrates, phosphorylated p38 MAPK, and p38 MAPK in HEK293/GC-A+/AT1+ cells treated with different PKC modulators. GAPDH serves as loading control. PMA, phorbol 12-myristate 13-acetate. (C) In vitro cGMP generation in HEK293/GC-A+/AT1+ in response to ANP (10−8 M) with or without ANGII (10−8 M) and valsartan (1 μM) or Go6983 (5 μM). Values of cGMP were normalized to vehicle group (blue) under ANP treatment. * indicates P<0.05, one-way ANOVA with Dunnett multiple comparisons test. (D) Hypothetical mechanism underlying the crosstalk between ANGII and GC-A derived from our in vitro studies.