Chronic exercise impairs nitric oxide pathway in rabbit carotid and femoral arteries

Abstract

Key points: Some of the beneficial effects of exercise in preventing vascular related diseases are mediated by the enhancement of endothelial function where the role of nitric oxide (NO) is well documented, although the relevance of calcium activated potassium channels is not fully understood. The impact of oxidative stress induced by training on endothelial function remains to be clarified. By evaluating different endothelial vasodilator pathways on two vascular beds in a rabbit model of chronic exercise, we found a decreased NO bioavailability and endothelial nitric oxide synthase expression in both carotid and femoral arteries. Physical training induced carotid endothelial dysfunction as a result of an increase in oxidative stress and a reduction in superoxide dismutase expression. In the femoral artery, the lower production of NO was counteracted by an increased participation of large conductance calcium activated potassium channels, preventing endothelial dysfunction.

Abstract: The present study aimed to evaluate the effects of chronic exercise on vasodilator response in two different arteries. Rings of carotid and femoral arteries from control and trained rabbits were suspended in organ baths for isometric recording of tension. Endothelial nitric oxide synthase (eNOS), Cu/Zn and Mn-superoxide dismutase (SOD), and large conductance calcium activated potassium (BKCa) channel protein expression were measured by western blotting. In the carotid artery, training reduced the relaxation to ACh (10^-9 to 3 × 10^-6 m) that was reversed by N-acetylcysteine (10^-3 m). l-NAME (10^-4 m) reduced the relaxation to ACh in both groups, although the effect was lower in the trained group (in mean ± SEM, 39 ± 2% vs. 28 ± 3%). Physical training did not modify the relaxation to ACh in femoral arteries, although the response to l-NAME was lower in the trained group (in mean ± SEM, 41 ± 5% vs. 17 ± 2%). Charybdotoxin (10^-7 m) plus apamin (10^-6 m) further reduced the maximal relaxation to ACh only in the trained group. The remaining relaxation in both carotid and femoral arteries was abolished by KCl (2 × 10^-2 m) and BaCl₂ (3 × 10^-6 m) plus ouabain (10^-4 m) in both groups. Physical training decreased eNOS expression in both carotid and femoral arteries and Cu/Zn and Mn-SOD expression only in the carotid artery. BKCa channels were overexpressed in the trained group in the femoral artery. In conclusion, chronic exercise induces endothelial dysfunction in the carotid artery as a result of oxidative stress. In the femoral artery, it modifies the vasodilator pathways, enhancing the participation of BKCa channels, thus compensating for the impairment of NO-mediated vasodilatation.

Keywords: Nitric oxide; calcium activated potassium channels; chronic exercise; endothelial dysfunction; oxidative stress.

Figure 1. Curves to ACh of carotid rings

Relaxation–response curves to ACh of carotid rings from control and trained rabbits and in the presence of NAC (10^–3 m) in the trained group. Data are the mean ± SEM.

Figure 2. Acetylcholine curves for carotid rings in the presence of indomethacin, indomethacin plus L‐NAME and indomethacin plus L‐NAME combined with charybdotoxin plus apamin, or KCl or BaCl₂ plus ouabain

Relaxation–response curves to ACh of carotid rings from control and trained rabbits in the absence of inhibitors and in the presence of indomethacin (10^–5 m), indomethacin plus l‐NAME (10^–4 m) and indomethacin plus l‐NAME combined with charybdotoxin (10^–7 m) plus apamin (10^–6 m), or KCl (2 × 10^–2 m) or BaCl₂ (3 × 10^–6 m) plus ouabain (10^–4 m). Data are the mean ± SEM.

Figure 3. Curves to ACh of femoral rings

Relaxation–response curves to ACh of femoral rings from control and trained rabbits. Data are the mean ± SEM.

Figure 4. Acetylcholine curves for femoral rings in the presence of indomethacin, indomethacin plus L‐NAME and indomethacin plus L‐NAME combined with charybdotoxin plus apamin, or KCl or BaCl₂ plus ouabain

Relaxation–response curves to ACh of femoral rings from control and trained rabbits in the absence of inhibitors and in the presence of indomethacin (10^–5 m), indomethacin plus l‐NAME (10^–4 m) and indomethacin plus l‐NAME combined with charybdotoxin (10^–7 m) plus apamin (10^–6 m), or KCl (2 × 10^–2 m) or BaCl₂ (3 × 10^–6 m) plus ouabain (10^–4 m). Data are the mean ± SEM.

Figure 5. Blockade of remaining relaxation to Ach and protein expression levels of BKCa channels in femoral artery

A, control represents the remaining relaxation to ACh after blockade with indomethacin (10⁻⁵ m) plus l‐NAME (10⁻⁴ m) in femoral artery from the trained group. Effects of apamin (10⁻⁶ m) (APA), TRAM‐34 (10⁻⁶ m) (TRAM), charybdotoxin (10⁻⁷ m) (CHTX), iberiotoxin (10⁻⁷ m) (IBTX), and the combination of charybdotoxin (10⁻⁷ m) + apamin (10⁻⁶ m) (CHTX + APA) on the remaining relaxation to ACh after blockade with indomethacin (10⁻⁵ m) + l‐NAME (10⁻⁴ m). Data are the mean ± SEM. B, BKCa channels protein expression by WB analysis in femoral artery from control (C) and trained (T) group. A representative immunoblot is shown and α‐tubulin was used as the control amount of protein. Data are the mean ± SD of four independent experiments. ^* P < 0.05 vs. control.

Figure 6. Curves to sodium nitroprusside of carotid and femoral rings

Relaxation–response curves to sodium nitroprusside of carotid and femoral rings from control and trained rabbits. Data are the mean ± SEM.

Figure 7. Protein expression levels of eNOS, Mn‐SOD and Cu/Zn‐SOD

Protein expression levels of eNOS, Mn‐SOD and Cu/Zn‐SOD in carotid and femoral rings from control (C) and trained (T) rabbits. A representative immunoblot of each protein is shown and α‐tubulin was used as the control amount of protein. Data are the mean ± SD of four independent experiments. ^* P < 0.05 vs. control.

Figure 8. Representative morphological analysis

Representative morphological analysis showing haematoxylin and eosin staining in carotid and femoral artery sections from the control and trained groups. L, lumen; TI, tunica intima; TM, tunica media; TA, tunica adventitia. Scale bars = 400 or 100 μm.