Effects of acute and chronic endurance exercise on intracellular nitric oxide and superoxide in circulating CD34⁺ and CD34⁻ cells

Abstract

We investigated the influence of acute and chronic endurance exercise on levels of intracellular nitric oxide (NO), superoxide (O₂·⁻), and expression of genes regulating the balance between these free radicals in CD34⁺ and CD34⁻ peripheral blood mononuclear cells (PBMCs; isolated by immunomagnetic cell separation). Blood samples were obtained from age- and body mass index (BMI)-matched endurance-trained (n = 10) and sedentary (n = 10) men before and after 30 min of exercise at 75% maximal oxygen uptake (·VO(₂max)). Baseline levels of intracellular NO (measured by DAF-FM diacetate) and O₂·⁻ (measured by dihydroethidium) were 26% (P < 0.05) and 10% (P < 0.05) higher, respectively, in CD34⁺ PBMCs from the sedentary group compared with the endurance-trained group. CD34⁺ PBMCs from the sedentary group at baseline had twofold greater inducible nitric oxide synthase (iNOS) mRNA and 50% lower endothelial NOS (eNOS) mRNA levels compared with the trained group (P < 0.05). The baseline group difference in O₂·⁻ was eliminated by acute exercise. Experiments with apocynin indicated that the training-related difference in O₂·⁻ levels was explained by increased NADPH oxidase activity in the sedentary state. mRNA levels of additional angiogenic and antioxidant genes were consistent with a more angiogenic profile in CD34⁺ cells of trained subjects. CD34⁻ PBMCs, examined for exploratory purposes, also displayed a more angiogenic mRNA profile in trained subjects, with vascular endothelial growth factor (VEGF) and eNOS being more highly expressed in trained subjects. Overall, our data suggest an association between the sedentary state and increased nitro-oxidative stress in CD34⁺ cells.

Fig. 1.

Nitric oxide (NO; A) and superoxide (B) fluorescence in CD34⁺ cells of endurance-trained and sedentary men before and after acute exercise. *Statistically significant difference between groups within the given experimental condition (P < 0.05). PBMCs, peripheral blood mononuclear cells. †Statistically significant within-group change relative to baseline control sample (P < 0.05).

Fig. 2.

NO (A) and superoxide (B) fluorescence in CD34⁻ cells of endurance-trained and sedentary men before and after acute exercise. *Statistically significant difference between groups (P < 0.05) within the given experimental condition. †Statistically significant within-group change relative to baseline control sample (P < 0.05).

Fig. 3.

mRNA levels of endothelial nitric oxide synthase (eNOS) (A), inducible NOS (iNOS) (B), VEGF (C), superoxide dismutase 1 (SOD1) (D), SOD2 (E), GPX-1 (F), p47^phox (G), and gp91^phox (H) in CD34⁺ cells of endurance-trained and sedentary men before and after acute exercise. I: RT-PCR agarose gel electrophoresis products shown are representative of data presented in A–H; separate images from the same gel are shown for each target gene. ET, endurance-trained group; S, sedentary group. *Statistically significant difference between groups within the given experimental condition (P < 0.05). †Statistically significant within-group change relative to baseline control sample (P < 0.05).

Fig. 4.

mRNA levels of eNOS (A), iNOS (B), VEGF (C), SOD1 (D), SOD2 (E), GPX-1 (F), p47^phox (G), and gp91^phox (H) in CD34⁻ cells of endurance-trained and sedentary men before and after acute exercise. I: RT-PCR agarose gel electrophoresis products shown are representative of data presented in A–H; separate images from the same gel are shown for each target gene. *Statistically significant difference between groups within the given experimental condition (P < 0.05). †Statistically significant within-group change relative to baseline control sample (P < 0.05).