Role of SCTR/AT1aR heteromer in mediating ANGII-induced aldosterone secretion

Figures

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  Fig 1. Functional SCTR and AT1R are present in mouse ZG cells. (A) The presence of SCTR in adrenal ZG cells. Representative images of immunohistochemical staining of sections obtained from at least 5 independent animals for each genotype and they were captured from at least 3 stained sections of every independent animal. Sections from SCTR-/- were employed as a negative control for SCTR staining. Scale bar represents 50 μm in outer panel and 5 μm in the zoomed fields. (B) cAMP assays showing the presence of functional SCTR in mouse adrenal gland. ***, P < 0.001 vs control group in respective genotype. Data are presented as the mean + SEM of at least three independent experiments performed in duplicates. (C) Transcripts of AT1aR, AT1bR and AT2R in ZG tissue determined by Taq-man probes normalized with internal control gene gapdh. *, P < 0.05 vs corresponding control in C57 mice. N = 4–5. ***, P < 0.001; *, P < 0.05 vs DMSO group. Data are presented as the mean + SEM of four independent experiments performed at least in duplicates.

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  Fig 2. SCTR potentiates ANGII-induced increase in [Ca2+]i. (A) [Ca2+]i change in primary ZG cells obtained from C57 and SCTR-/- mice, in response to ANGII (ranging from 0.1 nM to 1 μM). ANGII dose-dependently increased [Ca2+]i in C57 ZG cells. The tested ANGII dosages lead to a minor increase in [Ca2+]i in SCTR-/-, similar to the 0.1 nM ANGII-treated ZG cells of C57 mice. The maximal ANGII-induced [Ca2+]i increase was recorded at around 9 min, 11 min and 12 min post-ANGII loading, depending on the dosage of ANGII. Loading buffer was loaded as negative control. *, P <0.05 vs 0.1 nM ANGII-treatment in corresponding genotype. #, P < 0.05, vs counterparts in C57 mice. Data are present as mean + SEM from at least five independent experiments. (B) Extracellular calcium is a prerequisite for ANGII-induced Ca2+ mobilization and is the major source of [Ca2+]i influx. Pre-exposure to EGTA (5 mM) prior to ANGII (10 nM) abolished extracellular Ca2+ mobilization in C57 ZG cells. In contrary, increased [Ca2+]i upon ANGII was observed, until EGTA was loaded. The [Ca2+]i influx induced by ANGII was gradually and eventually chelated by EGTA nearly back to the resting level. Data are present as mean ±…

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  Fig 3. SCTR/AT1R hetero-complex mediates ANGII-induced [Ca2+]i in C57 mice. (A) STM-II/-IV, but not their mutants, inhibited ANGII-induced [Ca2+]i. Non-dimer-involved peptide STM-I had no effect on the ANGII-induced [Ca2+]i. (B) and (C) The real-time records of [Ca2+]i and the maximal [Ca2+]i, respectively, in ZG cells from C57 and SCTR-/- in response to ANGII (10 nM) with the presence or absence of candesartan (1 μM) or STM peptides (20 μg/ml). The primary ZG cells were pre-incubated with/-out candesartan or STM-II/-IV for 10 min before ANGII loading. In C57 mice, compared to ANGII-only treatment, the presence of STM-II/-IV abolished the [Ca2+]i increase like candesartan. This inhibitory effect of STM-II/-IV was absent in SCTR-/-. Candesartan prevented ANGII-induced [Ca2+]i elevation in both C57 and SCTR-/- mice. STM-IIm/-IVm showed much less pronounced inhibitory effects than STM-II/-IV in C57 mice. +++, P < 0.001; ++, P < 0.01 vs ANGII treatment. ***, P < 0.001; *, P < 0.05 vs candesartan-pretreated group in corresponding genotype. ###, P < 0.001; ##, P < 0.01; #, P < 0.05 vs C57 mice in respective treatment. Data are present as mean + SEM from at least five independent experiments.

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  Fig 4. SCTR potentiates ANGII-induced aldosterone release. The dispersed primary ZG cells obtained from C57 and SCTR-/- were exposed to ANGII (10 nM). A significant increase in aldosterone release was generated by the presence of ANGII within 20 min in C57 mice, but not until 60 min in SCTR-/- mice in tested time points. This stimulatory effects lasted for at least four hours as observed. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs start point in C57. ###, P < 0.001; ##, P < 0.05 vs 0 min in SCTR-/-. N = 4–5. Data are presented as the mean ± SEM.

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  Fig 5. SCTR/AT1R hetero-complex is responsible for ANGII-induced aldosterone release in C57 mice. Primary ZG cells prepared from C57 or SCTR-/- mice were pre-incubated with candesartan (1 μM), STM-II (20 μg/ml) or STM-IIm (20 μg/ml) for 30 min, respectively, before co-incubation with ANGII (10 nM) for another 1 h. (A) STM-II, but not STM-IIm, prevented ANGII-induced aldosterone secretion in primary C57 ZG cells, similar to the effects of candesartan. (B) The inhibitory effects of STM-II on the ANGII-induced aldosterone release was absent in SCTR-/-. With the presence or absence of STM-II, SCTR-/- secreted similar aldosterone levels upon ANGII, significantly higher than the control group. Besides, the 1 h aldosterone production in control SCTR-/- ZG cells showed the attenuated intrinsic secretion. ***, P < 0.0001; **, P < 0.01; *, P < 0.05 vs respective treatment in basal group (A) or control group in corresponding genotype (B). ##, P < 0.01 vs C57 mice in respective treatment. N = 8–12. Data are present as mean + SEM.