GPR30 expression is required for the mineralocorticoid receptor-independent rapid vascular effects of aldosterone

Abstract

It has been increasingly appreciated that steroids elicit acute vascular effects through rapid, so-called nongenomic signaling pathways. Though aldosterone, for example, has been demonstrated to mediate rapid vascular effects via both mineralocorticoid receptor-dependent and -independent pathways, the mechanism(s) of this mineralocorticoid receptor-independent effect of aldosterone is yet to be determined. For estrogen, its rapid effects have been reported to be, at least in part, mediated via the 7-transmembrane-spanning, G protein-coupled receptor GPR30. Previous studies have demonstrated common response outcomes in response to both aldosterone and estrogen on GPR30 expression, ie, activation of phosphatidylinositol 3-kinase-dependent contraction and extracellular signal-regulated kinase activation in vascular smooth muscle cells. The present studies were undertaken to test the hypothesis that the rapid response to aldosterone in smooth muscle is dependent on the availability of a GPR30-dependent signaling pathway. These findings not only reconcile differences in the literature for aldosterone response in freshly isolated versus cultured aortic smooth muscle cells but also suggest alternative therapeutic strategies for modulating aldosterone actions on the vasculature in vivo.

Figure 1.

Aldosterone-mediated effects on ERK phosphorylation in freshly isolated endothelium-denuded aortic ring segments. A, Aldosterone mediates ERK1/2 activation. Freshly isolated aortic ring segments were incubated with increasing concentrations of aldosterone (Aldo; 1 to 1000 pM) for 15 minutes. The effect of aldosterone on phospho-ERK1/2 was assessed by Western blotting. B, Aldosterone-mediated ERK phosphorylation: effects of antagonists. Aortic ring segments were preincubated with the specific MR antagonist eplerenone (EPL; 1 μmol/L) or the GPR30-selective antagonist G15 (1 μmol/L) for 15 minutes, followed by the incubation with aldosterone. The effect of these agents on aldosterone-mediated ERK1/2 phosphorylation was assessed by Western blotting. Data represent the mean±SEM from 3 independent experiments performed under identical conditions. *P<0.05 vs control cells by 1-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 2.

Effect of transduction of MR or GPR30 on aldosterone-mediated regulation of ERK phosphorylation. VSMCs infected with adenoviral GFP (A), MR (B), or GPR30 (C) and serum starved for 24 hours were treated with an increasing concentration of aldosterone (Aldo; 1 to 1000 pM for 15 minutes) in the absence or presence of eplerenone (EpL; 1 μmol/L). Data represent the mean±SEM from 3 to 4 independent experiments performed under identical conditions. *P<0.05 vs control VSMCs by 1-way ANOVA followed by Dunnett’s multiple comparison tests; #P<0.05 vs aldosterone-alone-treated VSMCs. Control levels were defined as the extent of ERK phosphorylation in the absence of aldosterone.

Figure 3.

Effects of antagonists on aldosterone- and Gl-mediated ERK activation in GPR30-expressing cells. A, Effect of G15 on aldosterone-mediated responses in GPR30-expressing cells. VSMCs infected with adenoviral GFP or GPR30 were incubated with aldosterone in the presence or absence of the GPR30 antagonist G15 (1 μmol/L). Data represent the mean±SEM from 4 independent experiments. *P<0.05 vs control. B, Effect of eplerenone (EPL) or spironolactone (SPIRO) on G1-mediated responses in GPR30-expressing cells with minimal MR expression. Cells were incubated with G1 in the presence or absence of EPL (1 μmol/L) or SPIRO (1 μmol/L). Data represent the mean±SEM from 6 independent experiments. *P<0.05 vs control.

Figure 4.

The rapid effects of aldosterone and E2 on ERK phosphorylation in rat vascular endothelial cells. A, RT-PCR analysis of mRNA expression of GPR30 and MR as well as ERα, ERβ, and the androgen receptor (AR) in cultured rat aortic vascular endothelial cells. These studies demonstrate endogenous expression of GPR30 but not MR. One microgram of total RNA from freshly isolated VSMCs or cultured VSMCs was utilized for RT-PCR analysis using specific primers (cf Methods). Samples from endothelial cells without reverse transcriptase were used as a negative control. The resultant DNA was separated by electrophoresis on a 2.5% agarose gel and visualized with ethidium bromide staining. B, Aldosterone-mediated increase in ERK phosphorylation. Endothelial cells were incubated with increasing concentrations of aldosterone (Aldo; 1 to 1000 pM) for 15 minutes. The effect of aldosterone on phospho-ERK1/2 was assessed by Western blotting. Data represent the mean±SEM from 3 independent experiments. *P<0.05 vs control. C, E2-mediated decrease in ERK phosphorylation. Endothelial cells were incubated with increasing concentrations of E2 (0.1 to 100 nmol/L) for 15 minutes. The effect of aldosterone on phospho-ERK1/2 was assessed by Western blotting. Data represent the mean±SEM from 3 independent experiments. *P<0.05 vs control.

Figure 5.

The rapid effects of aldosterone and E2 on ERK phosphorylation in endothelial cells: impact of shRNA-mediated inhibition of endogenous GPR30 expression. A, Effects of G15 (1 μmol/L) and eplerenone (EPL; μmol/L) on aldosterone (Aldo; 10 pM)-stimulated ERK phosphorylation in vascular endothelial cells. Data represent the mean±SEM from 3 independent experiments. *P<0.05 vs control; #P<0.05 vs aldosterone alone. B, Effects of G15 (1 μmol/L) and the ER-specific antagonist ICI-182780 (ICI; 1 μmol/L) on E2 (10 nmol/L)-mediated inhibition of ERK phosphorylation was assessed by Western blotting. Data represent the mean±SEM from 3 independent experiments. *P<0.05 vs control. C, Dose-dependent effects of shGPR30 on GPR30 expression as assessed by RT-PCR. In contrast, GAPDH expression was not altered. Furthermore, the shGFP vector had no effects on the expression of either GAPDH or GPR30. Results are from 3 identical experiments. D, shGPR30 inhibits the aldosterone (Aldo; 10 pM)- and G1 (1 μmol/L)-mediated stimulation of ERK phosphorylation, but not the angiotensin II (ANG II; 100 nmol/L)-mediated stimulation of ERK phosphorylation nor E2 (E2, 10 nmol/L)-mediated inhibition of ERK phosphorylation. The effect of shGPR30 expression on hormone-stimulated phospho-ERK1/2 activities was assessed by Western blotting. Data represent the mean±SEM from 3 independent experiments. *P<0.05 vs control; #P<0.05 vs E2 for shGPR30.

Figure 6.

The effect of aldosterone on serum starvation-provoked apoptosis is ERK and PI3 kinase dependent. A, Concentration-dependent effects of aldosterone. Serum-starved VSMCs were incubated with increasing concentrations of aldosterone (Aldo; to 1000 pM), and the effect of aldosterone on apoptosis was assessed by annexin labeling. B, The aldosterone-mediated enhancement in VSMC apoptosis was inhibited by the MEK inhibitor U0126 (10 μmol/L), but not the inactive analog U0124 (10 μmol/L). Aldosterone-mediated apoptosis was not affected by the p38-MAPK inhibitor SB203580 (10 μmol/L) or the inactive analog SB202474 (10 μmol/L). C, Aldosterone-mediated apoptosis is inhibited by the Pl3 kinase inhibitor LY29004 (1 μmol/L). Data represent the mean±SEM from 3 to 7 independent experiments performed under identical conditions. *P<0.05 vs untreated VSMCs. For each assay, data are normalized relative to the control levels of annexin-positive staining (APCs) specific for each condition.

Figure 7.

Effect of MR and GPR30 expression on aldosterone-mediated apoptosis. VSMCs infected with adenoviral GFP (A), GPR30 (B), or MR (C) and serum starved for 24 hours were treated with aldosterone (Aldo) in the absence or presence of eplerenone (EPL; 1 μmol/L). Eplerenone treatment inhibited aldosterone-mediated apoptosis in both GFP- and MR-infected vSMCs but only resulted in a partial inhibition in GPR30-infected VSMCs. Data represent the mean±SEM from 4 independent experiments performed under identical conditions. *P<0.05 vs untreated VSMCs; #P<0.05 vs aldosterone-alone-treated VSMCs. D, Effect of the GPR30 antagonist G15 on aldosteronemediated apoptosis. VSMCs infected with adenoviral GFP or GPR30 were incubated with aldosterone in the presence or absence of the GPR30 antagonist G15 (1 μmol/L). Data represent the mean±SEM from 4 independent experiments. *P<0.05 vs control.

Figure 8.

Aldosterone stimulates MLC phosphorylation in freshly isolated aortic ring segments via a GPR30-dependent mechanism. Freshly isolated ring segments denuded of endothelium were treated with aldosterone (Aldo; 10 nmol/L), G1 (1 μmol/L), or phenylephrine (PE; 10 μmol/L) with or without pretreatment with G15 (1 μmol/L). Stimulation of MLC phosphorylation by aldosterone and by G1 was inhibited by G15, whereas the effects of PE were unaltered. Data represent the mean±SEM from 4 independent experiments. *P<0.05 vs control.

Figure 9.

Effect of MR or GPR30 gene transfer on aldosterone-mediated MLC phosphorylation. A, Concentration-dependent effects of aldosterone on MLC phosphorylation. VSMCs infected with adenoviral GFP, MR, or GPR30 were treated with an increasing concentration of aldosterone (Aldo; 0.1 to 100 nmol/L). Aldosterone mediated a dose-dependent increase in MLC phosphorylation, which was enhanced following either MR or GPR30 gene transfer. B, Effect of eplerenone on aldosterone-mediated MLC phosphorylation. VSMCs infected with adenoviral GFP, MR, or GPR30 were treated with aldosterone (100 nmol/L) in the absence or presence of eplerenone (EPL; 1 μmol/L). Data represent the mean±SEM from 3 to 4 independent experiments performed under identical conditions. *P<0.05 vs control VSMCs by 1-way ANOVA followed by Dunnett’s multiple comparison tests; #P<0.05 vs aldosterone-alone-treated VSMCs. C, Effect of the GPR30 antagonist G15 on aldosterone-mediated MLC phosphorylation. VSMCs infected with adenoviral GFP or GPR30 were incubated with aldosterone (100 nmol/L) in the presence or absence of the GPR30 antagonist G15 (1 μmol/L). Data represent the mean±SEM from 4 independent experiments. *P<0.05 vs control.