MANP Activation Of The cGMP Inhibits Aldosterone Via PDE2 And CYP11B2 In H295R Cells And In Mice

Abstract

Background: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear.

Methods: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice.

Results: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased.

Conclusions: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.

Keywords: aldosterone; hypertension; knockout; natriuretic peptide; sacubitril.

Figure 1.

cGMP production and aldosterone inhibition in H295R cells. (A) intracellular cGMP generation by MANP treatment (10⁻⁸, 10⁻⁷, or 10⁻⁶ M) for 10 min. (B) aldosterone production stimulated by ANGII (10⁻⁸, 10⁻⁷, or 10⁻⁶ M). (C) aldosterone inhibition by MANP (10⁻⁸, 10⁻⁷, or 10⁻⁶ M) in the presence of 10⁻⁸ M ANGII. (D) aldosterone inhibition by MANP (10⁻⁷ or 10⁻⁶ M) in the presence of 10⁻⁶ M forskolin (FSK, a cAMP activator). Aldosterone levels were described as fold changes from Control (Ctrl) group. n=3 each group. * p<0.05 vs. Control, # p<0.05 vs. ANGII or FSK. Data are expressed as means ± SE. Unpaired t-test or non-parametric Mann-Whitney test was performed.

Figure 2.

Aldosterone suppression by MANP is not affected by NPRC or PKG actions in H295R cells. (A) aldosterone production by 10⁻⁷ M MANP or 10⁻⁶ M NPRC agonist, cANF(4-23) in the presence of 10⁻⁸ M ANGII. (B) aldosterone production by 10⁻⁷ M MANP in the presence of 10⁻⁸ M ANGII with or without 10⁻⁶ M NPRC antagonist, AP-811 (AP). (C) aldosterone production by various concentrations of a membrane-permeable, non-specific cGMP analog, cGMP-AM (cGAM) in the presence of 10⁻⁸ M ANGII. (D) aldosterone production by a range of membrane-permeable, PKG-specific cGMP analog, 8-pCTP-cGMP (10⁻¹², 10⁻¹¹, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, or 10⁻⁵ M) in the presence of 10⁻⁸ M ANGII. (E) aldosterone production by 10⁻⁷ M MANP in the presence of 10⁻⁸ M ANGII and 10⁻⁶ M PKG-specific blocker, Rp-8-pCPT-cGMPS. Aldosterone levels were described as fold changes from Control (Ctrl) group. n=3 each group. * p<0.05 vs. Control, # p<0.05 vs. ANGII. Data are expressed as means ± SE. Unpaired t-test or non-parametric Mann-Whitney test was performed.

Figure 3.

Aldosterone suppression by MANP is mediated by phosphodiesterase 2 (PDE2) in H295R cells. (A) aldosterone production by 10⁻⁷ M MANP in the presence of 10⁻⁸ M ANGII with 5X10⁻⁴ M of the PDE non-specific inhibitor, IBMX. (B) aldosterone production by 10⁻⁷ M MANP in the presence of 10⁻⁸ M ANGII and PDE2 specific inhibitor, Bay 60-7550 (10⁻⁸, 10⁻⁷, or 10⁻⁶ M) or in cells transfected with 10⁻⁷ M PDE2 specific siRNA. Aldosterone levels were described as fold changes from Control (Ctrl) group. n=3 each group. * p<0.05 vs. Control, # p<0.05 vs. ANGII, $ p<0.05 vs. MANP. Data are expressed as means ± SE. Unpaired t-test or non-parametric Mann-Whitney test was performed.

Figure 4.

PDE2 activity measured by fluorescence resonance energy transfer (FRET) based biosensor Epac2-camps expressed in H295R cells using adenovirus. The use of FRET provides us with a tool to measure cAMP activity in which FRET ratio is inversely related to PDE2 activity. (A) Examples of live cell imaging traces. Changes in FRET signal (expressed as CFP/YFP ratio reflecting intracellular cAMP levels) by 10⁻⁷ M MANP, or by 10⁻⁶ M Bay 60-7550 plus MANP, in the presence of 10⁻⁵ M forskolin (a cAMP activator) were measured. Control group was 10⁻⁵ M forskolin stimulation only. For MANP group, cells were pretreated with 10⁻⁷ M MANP for 10 min followed by forskolin stimulation. For Bay group, cells were pretreated with 10⁻⁶ M Bay 60-7550, MANP followed by forskolin stimulation. (B) Quantification of the experiments shown in (A) as a % FRET changes. PDE2 activity is inversely related to FRET signals. Control group, n=6. MANP group, n=11, Bay group, n=7. (C) Examples of live cell imaging traces in siRNA study. Control siRNA group used negative control siRNA. In PDE2 siRNA group, cells were transfected with PDE2 siRNA. 48 hours post-transfection, both groups received 10⁻⁷ M MANP pretreatment for 10 min and 10⁻⁵ M forskolin treatment. (D) Quantification of the experiments shown in (C) as a % FRET changes. Control siRNA group, n=10. PDE2 siRNA group, n=10. * p<0.05 vs. Control. Data are expressed as means ± SE. Unpaired t-test was performed.

Figure 5.

Intracellular Ca²⁺ levels and aldosterone production regulated by Ca²⁺ levels in H295R cells. (A) Relative Ca²⁺ levels were calculated based upon recorded fluorescence signals in Fura-2 loaded H295R cells. Fold difference F/F0 was used as relative Ca²⁺ concentration values. The groups are as follows: ANGII group, 10⁻⁸ M ANGII perfusion; MANP group, 10⁻⁷ M MANP 5 min pretreatment plus ANGII perfusion; Bay 60-7550 group, 10⁻⁶ M Bay 60-7550 15 min pretreatment followed by MANP 5 min treatment plus ANGII perfusion; efonidipine (Efo) group, 10⁻⁶ M efonidipine 15 min pretreatment and then Bay 60-7550 15 min treatment followed by MANP 5 min treatment plus ANGII perfusion. n=7-10 for each group. (B) aldosterone production by a serial of concentrations of Ca²⁺ blocker, efonidipine (10⁻⁸, 10⁻⁷, or 10⁻⁶ M) in the presence of 10⁻⁸ M ANGII, 10⁻⁷ M MANP, and 10⁻⁶ M Bay 60-7550. (C) aldosterone production by 10⁻⁶ M Efo in cells transfected with 10⁻⁷ M PDE2 siRNA. Aldosterone levels were described as fold changes from Control group (in siRNA experiments, control group transfected with negative control siRNA) that received only treatment buffer (panel B and C). (D) Aldosterone synthase CYP11B2 gene expression by real-time PCR in H295R cells. CYP11B2 expression by 10⁻⁸ M ANGII, 10⁻⁷ M MANP in the presence of ANGII, or 10⁻⁶ M Bay 60-7550 plus MANP in the presence of ANGII. CYP11B2 gene expression was normalized to GAPDH. n=3 each group. * p<0.05 vs. Control, # p<0.05 vs. ANGII group, $ p<0.05 vs. Bay 60-7550 group or PDE2 siRNA group. Data are expressed as means ± SE. 2-way ANOVA was used for panel A and unpaired t-test or non-parametric Mann-Whitney test was performed was performed for panels B-D.

Figure 6.

Cyp11b2 gene expression in Pde2 genetic mice. (A) Adult adrenal relative Cyp11b2 expression in wildtype (WT) and heterozygotes (HET) mice. (B) Embryonic adrenal relative Cyp11b2 expression in WT and knockout (KO) mice embryos. n=3-6 for different groups. * p<0.05 vs. wild type. Data are expressed as means ± SE. Unpaired t-test or non-parametric Mann-Whitney test was performed.