Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation: Role of Perivascular Adipose Tissue

Abstract

Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin-angiotensin-aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase-derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue-derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Keywords: adipose tissue; aorta; losartan; mineralocorticoid receptor.

Figure 1.

Spironolactone, but not losartan, prevented the enhanced contraction to phenylephrine induced by β-adrenergic overstimulation. Concentration–response curves to phenylephrine obtained in aortic rings from rats treated with vehicle (CT) or isoproterenol (ISO; A) combined with losartan (LOS; B) or with spironolactone (SPI; C). Data are expressed as mean±SEM; number of animals is indicated in parenthesis. Two-way ANOVA: *P<0.001 vs CT or LOS.

Figure 2.

Effect of Nω-nitro-L-arginine methyl ester (L-NAME) (A, square symbols), superoxide dismutase (superoxide dismutase (SOD, superoxide scavenger); C, triangle symbols), and tetrahydrobiopterin (BH₄, eNOS cofactor) (E, diamond symbol) on the concentration–response curves to phenylephrine of aortic rings from control (CT) and isoproterenol (ISO) groups without or with spironolactone (SPI) treatment. B, D, and F, The difference of the area under the curve (dAUC) to phenylephrine in the presence and absence of L-NAME, SOD, or BH4, respectively. Data are expressed as mean±SEM (n=4–15 in each group). Two-way ANOVA: P<0.05 +vs respective group without incubation; *vs CT; #vs ISO.

Figure 3.

Reduced HSP90 expression and endothelial nitric oxide synthase (eNOS) uncoupling after β-AR overstimulation: prevention by mineralocorticoid receptor blockade. Diaminofluorescein (DAF-2) fluorescence (A), protein expression of total eNOS (B), dimer:monomer eNOS ratio (C), HSP90 (D), and hidroethidine (DHE) fluorescence (E) obtained in aorta from control (CT) and isoproterenol (ISO) groups without or with spironolactone (SPI) treatment. Protein expression was calculated as percentage of CT group. Representative images (20×, bar=100 µm) of DAF-2 and DHE fluorescence are shown in left side of A and E, respectively. In A, E=endothelium. The DHE fluorescence signal was evaluated under basal condition or after incubation with Nω-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) or MnTMPyP (25 µmol/L). Data represent mean±SEM; number of animals used for each group is indicated in the bars. Two-way ANOVA: P<0.05 *vs CT; +vs SPI; #vs ISO.

Figure 4.

Genomic and nongenomic pathways of vascular mineralocorticoid receptor (MR) activity after isoproterenol treatment. Nuclear translocation of MR (A), mRNA expression of osteopontin (B), and protein expression of γ-epithelial sodium channel (γENaC; C), Src (D), and ERK1/2 (E) phosphorylation in thoracic aorta of control (CT) and isoproterenol (ISO) groups without or with spironolactone (SPI) treatment. Data represent mean±SEM; number of animals used in each group is indicated into the bars. Two-way ANOVA: P<0.05 *vs CT; #vs ISO.

Figure 5.

β-AR overstimulation enhanced corticosterone content and protein expression of 11β-HSD1 in perivascular adipose tissue (PVAT) of aorta. Aldosterone (A and B) and corticosterone (C and D) content measured in plasma and aortic PVAT from control (CT) and isoproterenol (ISO) groups without or with spironolactone (SPI) treatment. Protein expression of 11β-HSD1 (E) and 11β-HSD2 (F) were evaluated in PVAT from CT, ISO, SPI, and ISO+SPI groups. Data represent mean±SEM; number of animals is indicated in the bars. Two-way ANOVA: P<0.05 *vs CT; +vs SPI; #vs ISO.

Figure 6.

Impaired anticontractile function of perivascular adipose tissue (PVAT) after isoproterenol treatment is rescued by spironolactone. Concentration–response curves to phenylephrine were obtained in aortic rings without (−) or with (+) PVAT from control (CT, A) and isoproterenol (ISO, B) groups without or with spironolactone (SPI) treatment (C and D). E, The difference of the area under the curve (dAUC) to phenylephrine in the absence and presence of PVAT. Fluorescence to NO-sensitive dye diaminofluorescein (DAF-2) was evaluated in thoracic PVAT sections from CT, ISO, SPI, and ISO+SPI groups under basal conditions or after incubation with tetrahydrobiopterin (BH4, eNOS cofactor) (100 µmol/L, F). Representative images (20×, white bar=100 µm) of DAF-2 fluorescence are shown. Data are expressed as mean±SEM (n=5–15 in each group). Two-way ANOVA: P<0.05 +vs PVAT; *vs CT; #vs ISO.