Inhibitory mechanism of ANP on aldosterone production in human adrenocortical cells

Figure 1. Expression of NPR1 in human adrenocortical cells. Protein expression of NPR1 by immunoprecipitation and immunoblotting in human adrenocortical cells.

Figure 2. Aldosterone production from H295R cells. (A) Aldosterone production from H295R cells in the presence of ANP (0.01, 0.1, 1 μM). (B) Aldosterone production from H295R cells in the presence of various concentrations of LBQ657 (1, 10, 30 μM). (C) Aldosterone production from H295R cells in the presence of forskolin (5 μM) or (D) 8-Br-cAMP (1 mM). (E) Aldosterone production from H295R cells pretreated with Ang-II and then incubated in the presence of forskolin (1 μM) or (F) 8-Br-cAMP (1 mM), and (G) KH7 (10 μM) and H89 (20 μM). Cells were pretreated with Ang-II (10−8M) before the addition of indicated compounds including ANP, except for (C) or (D). Results are mean of s.d. Significant differences: *P < 0.05, **P < 0.01 from control values.

Figure 3. Gene expressions related to aldosterone synthesis in H295R cells. Aldosterone synthesis gene expressions in H295R cells were measured under two conditions. Cells were treated either with Ang-II or in serum-free medium after pretreatment with Ang-II for 3 h. Cells were collected and used for gene expression analysis. Gene expression in Ang-II-treated and Ang-II-pretreated cells in the presence or absence of ANP. (A) NR4A2, (B) HSD3B2, (C) StAR, (D and E) CYP11B2, and (F and G) CYP11A1. Results are mean of s.d. Significant differences: *P < 0.05, **P < 0.01, versus control, n = 3.

Figure 4. Effects of NPR1 and NPR3 on aldosterone production from H295R cells. Aldosterone production from H295R cells transfected with siNPR1 or siNPR3. (A) NPR1 or (B) NPR3 mRNA expression. The RNA used for RT-qPCR was extracted from the cell cultured four to five days after siRNA transfection (n = 3). NPR1 or NPR3 mRNA was normalized by GAPDH mRNA (C and D). Aldosterone production after the transfection of siRNA. Cells were pretreated with Ang-Ⅱ (10−8M) before the addition of ANP. Results are mean of s.d.; significant differences: *P < 0.05, **P < 0.01 from values in siRNA-negative control, n = 3. The results are represented as fold increase ± s.d.

Figure 5. Role of inhibitory G signaling in aldosterone production in H295R cells. (A) Aldosterone production from H295R cells in the presence of a cGMP agonist, 8-bromo-cGMP, (B) in the presence of a PKG inhibitor, KT5823. Cells were pretreated with Ang-II before the addition of cGMP or ANP. (C) Aldosterone production from H295R cells with or without PTX (1 μg/mL). Results are mean of s.d.; significant differences: *P < 0.05, **P < 0.01, versus control, n = 3.

Figure 6. Proposed mechanism of ANP suppression of aldosterone production stimulated by Ang-II. A schematic diagram illustrating the inhibition of aldosterone production by ANP in adrenocortical cells. Angiotensin II induces aldosterone production through an increase in cytoplasmic Ca2+ concentration and further activation of Ca2+-sensitive adenylyl cyclase (AC). On the other hand, ANP binds to NPR1 and NPR3, increasing cGMP and activating Gi signaling, thereby decreasing cAMP and inhibiting aldosterone synthesis.