Activation of the mineralocorticoid receptor increases striatin levels

Abstract

Background: Aldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells.

Methods: We studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue.

Results: We observed that dietary sodium restriction was associated with increased striatin levels in mouse heart and aorta and that striatin and MR are present in the human endothelial cell line, (EA.hy926), and in mouse aortic endothelial cells (MAEC). Further, we show that MR co-precipitates with striatin in vascular tissue. Incubation of EA.hy926 cells with ALDO (10(-8) mol/l for 5-24 h) increases striatin protein and mRNA expression, an effect that was inhibited by canrenoic acid, an MR antagonist. Consistent with these observations, incubation of MAEC with ALDO increased striatin levels that were likewise blocked by canrenoic acid. To test the in vivo relevance of these findings, we studied two previously described mouse models of increased ALDO levels. Intraperitoneal ALDO administration augmented the abundance of striatin protein in mouse heart. We also observed that in a murine model of chronic ALDO-mediated cardiovascular damage following treatment with N(G)-nitro-L-arginine methyl ester plus angiotensin II an increased abundance of striatin protein in heart and kidney tissue.

Conclusion: Our results provide evidence that increased striatin levels is a component of MR activation in the vasculature and suggest that regulation of striatin by ALDO may modulate estrogen's nongenomic effects.

Conflict of interest statement

Disclosure: The authors declared no conflict of interest.

Figures

Figure 1

Striatin and the mineralocorticoid receptor are expressed and co-immunoprecipitate in early cultures of mouse aortic endothelial cells, EA.hy926 cells, and mouse heart tissue. (a) EA.hy926 cells were lysed and protein levels quantified as described in the Methods. Cell lysates (6.5–26.0 μg) were analyzed by western blotting for striatin levels. (b) Prior to cell lysis and western blotting for striatin, EA.hy926 cells were either untransfected (blank, lane 1) or transfected with scrambled siRNA (lane 2) or anti-striatin duplex (lanes 3–4), as described under Methods. Early cultures of mouse aortic endothelial cells (MAEC), EA.hy926 cells and mouse heart tissue were processed for western blotting for striatin (c) or (d) mineralocorticoid receptor (MR). (e) Immunoprecipitation with striatin flowed by western blot for MR. (f) Immunoprecipitation with MR followed by western blot for striatin as described under Methods. EA.hy926, human endothelial cell line.

Figure 2

Dietary sodium restriction increases striatin levels in mouse hearts. (a) Mouse heart tissues were obtained from mice maintained on a high-sodium (HS) (n = 7) or low-sodium (LS) diet (n = 6) for 11 days. (b) Mouse aorta were obtained from mice maintained on a high- (n = 3) or low-sodium diet (n = 4) for 11 days. Tissue homogenates were analyzed for striatin and β-actin protein levels as described in Methods.

Figure 3

The aldosterone-induced increases in striatin expression are mediated via activation of the mineralocorticoid receptor (MR) in EA.hy926 cells. (a) EA.hy926 cells were incubated with 10 nmol/l ALDO for 1, 5, 12, 24 h. Cell lysates were analyzed for striatin protein levels as described in Methods. Data were normalized to β-actin. (b) EA.hy926 cells were incubated with vehicle (white bars) or 10 nmol/l ALDO (gray bars) for 5 h and 24 h in the absence or presence (black bars) of the MR antagonist, canrenoic acid (MRA) and analyzed for striatin protein normalized to β-actin (b) and mRNA normalized to (c) 18S rRNA. *P < 0.05 vs. vehicle-treated cells. **P < 0.05 vs. ALDO-treated cells. ALDO, aldosterone; EA.hy926, human endothelial cell line.

Figure 4

The in vivo and in vitro effects of increased aldosterone on striatin levels in mice. (a) Aldosterone (ALDO) increases striatin expression via activation of the mineralocorticoid receptor (MR) in early cultures of mouse aortic endothelial cells. Mouse aortic endothelial cells (MAEC) were incubated with 10 nmol/l ALDO for 5 h, in the presence or absence of the MR antagonist canrenoic acid. Cell lysates were analyzed for striatin and β-actin protein levels as described in Methods. (b) Acute in vivo administration of aldosterone increases striatin levels in mouse heart. Heart tissues were obtained from mice at 0, 1, 2, or 3 h after acute intraperitoneal administration of ALDO (10 μg/kg, gray squares) or vehicle (Sham, white circles). Tissue homogenates were analyzed for striatin and β-tubulin protein levels as described in Methods. *P < 0.05 vs. Sham. (c,d) Treatment of mice with Ang II/L-NAME increases striatin levels in mouse (c) heart and (d) kidney. Heart and kidney tissues were obtained from mice treated with placebo (control) or L-NAME/AngII for 11 days. Tissue homogenates were analyzed for striatin and β-actin protein levels as described in Methods. EA.hy926, human endothelial cell line. AngII, angiotensin II; L-NAME, NG-nitro-L-arginine methyl ester.