Voluntary wheel running prevents salt-induced endothelial dysfunction: role of oxidative stress

Abstract

Diets high in salt can lead to endothelial dysfunction, a nontraditional risk factor for cardiovascular disease (CVD). Exercise is known to reduce CVD risk; however, it remains unknown whether chronic physical activity can attenuate salt-induced endothelial dysfunction independent of blood pressure (BP) and whether these changes are due to an upregulation in endogenous antioxidants. Eight-week-old Sprague-Dawley rats were fed either a normal (NS; 0.49%)- or a high (HS; 4.0%)-salt diet and further divided into voluntary wheel running (NS-VWR, HS-VWR) and sedentary (NS, HS) groups for 6 wk. BP was measured weekly and remained unchanged within groups ( P = 0.373). Endothelium-dependent relaxation (EDR) was impaired in the femoral artery of HS compared with NS (38.6 ± 4.0% vs. 65.0 ± 3.6%; P = 0.013) animals, whereas it was not different between NS and HS-VWR (73.4 ± 6.4%; P = 0.273) animals. Incubation with the antioxidants TEMPOL ( P = 0.024) and apocynin ( P = 0.013) improved EDR in HS animals, indicating a role for reactive oxygen species (ROS). Wheel running upregulated the antioxidant superoxide dismutase-2 (SOD-2) ( P = 0.011) under HS conditions and lowered NOX4 and Gp91-phox, two subunits of NADPH oxidase. Wheel running elevated phosphorylated endothelial nitric oxide synthase (eNOS) ( P = 0.014) in HS-fed rats, demonstrating a role for physical activity and eNOS activity under HS conditions. Finally, there was a reduction in EDR ( P = 0.038) when femoral arteries from NS-VWR animals were incubated with TEMPOL or apocynin, suggesting there may be a critical level of ROS needed to maintain endothelial function. In summary, physical activity protected HS-fed rats from reductions in endothelial function, likely through increased SOD-2 levels and reduced oxidative stress. NEW & NOTEWORTHY Our data suggest that voluntary wheel running can prevent impairments in endothelium-dependent relaxation in the femoral artery of rats fed a high-salt diet. This appears to be independent of blood pressure and mediated through a decrease in expression of NADPH oxidases as a result of physical activity. These data suggest that increased chronic physical activity can protect the vasculature from a diet high in salt, likely through a reduction in oxidative stress.

Keywords: endothelium; exercise; sodium.

Fig. 1.

Endothelium-dependent relaxation (EDR) in sedentary and voluntary wheel running (VWR) rats fed either a normal-salt (NS) or a high-salt (HS) diet: dose responses of femoral rings to acetylcholine (ACh) under control (solid lines) and N^G-nitro-l-arginine methyl ester (l-NAME)-treated (dashed lines) conditions (A), maximal relaxation (E_max) to ACh (B), and area under the curve (AUC) to ACh (C). Values are means ± SE; n: NS = 12, NS-VWR = 13, HS = 14, HS-VWR = 12. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. *vs. HS, α vs. NS, †vs. NS-VWR, ‡vs. HS-VWR, §vs. respective control group (P < 0.05).

Fig. 2.

Endothelium-independent relaxation (EIR) in sedentary and voluntary wheel running (VWR) rats fed either a normal-salt (NS) or a high-salt (HS) diet. Values are means ± SE; n: NS = 12, NS-VWR = 13, HS = 14, HS-VWR = 12. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. SNP, sodium nitroprusside.

Fig. 3.

A–C: total endothelial nitric oxide synthase (eNOS) protein expression in the femoral artery normalized to β-actin (A), phosphorylated eNOS (eNOS-P, ser1177) relative to total eNOS (B), and representative Western blots (C). D–F: total eNOS protein expression in the aorta normalized to β-actin (D), eNOS-P (ser1177) relative to total eNOS (E), and representative Western blots (F). Values are means ± SE; n = 9 or 10 per group. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. HS, high salt; NS, normal salt; Sed, sedentary; VWR, voluntary wheel running. *vs. HS, †vs. NS-VWR (P < 0.05).

Fig. 4.

Femoral nitrotyrosine expression normalized to β-actin (A), total aortic NOX4 protein expression normalized to β-actin (B), aortic Gp91phox normalized to β-actin (C), and representative Western blots (D). Values are means ± SE; n = 8 or 9 per group. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. HS, high salt; NS, normal salt; VWR, voluntary wheel running. α vs. NS, †vs. NS-VWR, ‡ vs. HS-VWR (P < 0.05).

Fig. 5.

Superoxide dismutase-1 (SOD-1) protein expression in the femoral artery relative to β-actin (A), total SOD-2 protein expression in the aorta normalized to β-actin (B), representative Western blots of SOD-1 and SOD-2 (C), catalase protein expression in the aorta normalized to β-actin (D), GPx-1/2 expression in the aorta normalized to β-actin (E), and representative Western blots of catalase and GPx-1/2 (F). Values are means ± SE; n = 7 or 8 per group. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. HS, high salt; NS, normal salt; VWR, voluntary wheel running. *vs. HS, α vs. NS (P < 0.05).

Fig. 6.

A: endothelium-dependent relaxation (EDR) in the femoral artery in normal-salt (NS) rats in response to acetylcholine (ACh) with and without addition of the superoxide dismutase mimetic TEMPOL. B: EDR in the femoral artery in high-salt (HS) rats in response to ACh with and without the addition of TEMPOL. C: EDR in the femoral artery in NS rats in response to ACh with and without addition of the NADPH oxidase inhibitor apocynin (APO). D: EDR in femoral artery in HS rats in response to ACh with and without the addition of the NADPH oxidase inhibitor APO. E: EDR in the femoral artery in NS rats in response to ACh with and without addition of TEMPOL + APO (T/A). F: EDR in femoral artery in HS rats in response to ACh with and without the addition of T/A. G: maximal relaxation with and without the addition of TEMPOL, APO, or T/A. H: area under the curve (AUC) with and without the addition of TEMPOL, APO, or T/A. Values are means ± SE; n: NS = 12, NS-voluntary wheel running (VWR) = 13, HS = 14, HS-VWR = 12. Data analysis was performed with a (4 × 3) 2-way repeated-measures ANOVA.*vs. HS, §vs. respective control group (P < 0.05).