Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase

Abstract

Habitual aerobic exercise is associated with enhanced endothelium-dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6-8 months) and old (29-32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age-related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l-NAME minus that with ACh alone was -28% vs. -55%, P < 0.01) were restored in old VR (EDD: 96%, NO: -46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR (P < 0.05). Aortic superoxide dismutase (SOD) activity was greater (P < 0.01), whereas NADPH oxidase protein expression (P < 0.01) and activity (P = 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression (P < 0.05) and activation (Ser1177 phosphorylation) (P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age-associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise-mediated preservation of NO bioavailability and EDD with ageing.

Figure 1. Daily running distance (n= 14–16 per group) of young and old mice

Values are means ±s.e.m.

Figure 2. Systemic (in vivo) EDD to ACh (n= 6 per group) (A) and EID to sodium nitroprusside (SNP) (B), carotid artery EDD to ACh in the absence or presence of the NOS inhibitor, l-NAME (n= 6–10 per group) (C), and carotid artery EID to SNP in young (Y) and old (O) cage control (CC) and voluntary wheel running mice (VR) (D)

Values are means ±s.e.m.*P < 0.05 vs. YCC.

Figure 3. Nitrotyrosine abundance in aortas from young (Y) and old (O) cage control (CC) and voluntary wheel running (VR) mice (n= 7–9 per group)

Nitrotyrosine abundance is expressed relative to GAPDH to account for differences in protein loading and shown normalized to the YCC mean. Values are means ±s.e.m. Representative blots shown below the summary graph. *P < 0.05 vs. YCC; †P < 0.05 vs. OCC.

Figure 4. Aortic SOD (A) protein expression (n= 7–13 per group) and (B) enzyme activity (n= 7–10 per group) in young (Y) and old (O) cage control (CC) and voluntary wheel running (VR) mice

Protein expression is expressed relative to GAPDH to account for differences in protein loading and shown normalized to YCC mean. Representative blots shown below the summary graph. Values are means ±s.e.m.*P < 0.05 vs. YCC; †P < 0.05 vs. OCC; ecSOD expression (A) P= 0.07 OVR vs. OCC; MnSOD expression (A) P= 0.06 OVR vs. OCC.

Figure 5. Aortic NADPH oxidase (A) p67 subunit protein expression (n= 4–8 per group) and (B) enzyme activity (n= 7–10 per group) in young (Y) and old (O) cage control (CC) and voluntary wheel running (VR) mice

Protein expression is expressed relative to GAPDH to account for differences in protein loading and shown normalized to YCC mean. Representative blots shown below the summary graph. Values are means ±s.e.m.*P < 0.05 vs. YCC; †P < 0.05 vs. OCC.

Figure 7. Nitric oxide (NO) component of EDD (NO bioavailability) in carotid arteries from (A) young (Y) cage control (CC), (B) old (O) CC, (C) Y voluntary wheel running (VR) and (D) OVR mice in response to ACh alone (CNT) or after pre-treatment with TEMPOL or apocynin (APO) (n= 6–10 per group)

NO bioavailability (%) = Maximum Dilatation_ACh+L−NAME– Maximum Dilatation_ACh. *P < 0.05 vs. matched CNT.

Figure 6. Dilatation of carotid arteries to ACh alone (CNT) or to ACh after pre-treatment with TEMPOL or apocynin (APO) in the absence or presence of the NOS inhibitor, l-NAME, in (A) young (Y) cage control (CC), (B) old (O) CC, (C) Y voluntary wheel running (VR) and (D) OVR mice (n= 6–10 per group)

Values are means ±s.e.m.*P < 0.05 vs. matched CNT maximal dilatation.

Figure 8. Total and Ser1177-phosphorylated eNOS (peNOS) in aortas from young (Y) and old (O) cage control (CC) and voluntary wheel running (VR) mice (n= 5–7 per group)

Protein expression is expressed relative to GAPDH to account for differences in protein loading and shown normalized to the YCC mean. Values are means ±s.e.m. Representative blots shown below the summary graph. *P < 0.05 vs. YCC; †P < 0.05 vs. OCC.