Rafael A. Reyes

Effects of ageing and exercise training on eNOS uncoupling in skeletal muscle resistance arterioles

Reduced availability of tetrahydrobiopterin (BH4) contributes to the age‐related decline of nitric oxide (NO)‐mediated vasodilatation of soleus muscle arterioles. Depending on availability of substrate and/or necessary co‐factors, endothelial nitric oxide synthase (eNOS) can generate NO and/or superoxide (O2−). We evaluated the effects of age and chronic exercise on flow‐induced vasodilatation and levels of NO and O2− in soleus muscle arterioles. Young (3 months) and old (22 months) male rats were exercise trained or remained sedentary (SED) for 10 weeks. Flow‐stimulated NO and O2−, as well as BH4 and l‐arginine content, were determined in soleus muscle arterioles. Flow‐induced vasodilatation was assessed under control conditions and during the blockade of O2− and/or hydrogen peroxide. Exercise training enhanced flow‐induced vasodilatation in arterioles from young and old rats. Old age reduced, and exercise training restored, BH4 content and flow‐stimulated NO availability. Flow‐stimulated, eNOS‐derived O2− levels were higher in arterioles from old SED compared to those from young SED rats. Exercise training increased flow‐stimulated eNOS‐derived O2− levels in arterioles from young but not old rats. O2− scavenging with Tempol reduced flow‐induced vasodilatation from all groups except young SED rats. Addition of catalase to Tempol‐treated arterioles eliminated flow‐induced vasodilatation in arterioles from all groups. Catalase reduced flow‐induced vasodilatation from all groups. In Tempol‐treated arterioles, flow‐induced vasodilatation was restored by deferoxamine, an iron chelator. These data indicate that uncoupling of eNOS contributes to the age‐related decline in flow‐induced vasodilatation; however, reactive oxygen species are required for flow‐induced vasodilatation in soleus muscle arterioles from young and old rats.

Aging impairs flow-induced dilation in coronary arterioles: role of NO and H2O2

Aging contributes significantly to the development of cardiovascular disease and is associated with elevated production of reactive oxygen species (ROS). The beneficial effects of nitric oxide (NO)-mediated vasodilation are quickly abolished in the presence of ROS, and this effect may be augmented with aging. We previously demonstrated an age-induced impairment of flow-induced dilation in rat coronary arterioles. Therefore, the purpose of this study was to determine the effects of O(2)(-) scavenging, as well as removal of H(2)O(2), the byproduct of O(2)(-) scavenging, on flow-mediated dilation in coronary resistance arterioles of young (4 mo) and old (24 mo) male Fischer 344 rats. Flow increased NO and H(2)O(2) production as evidenced by enhanced diaminofluorescein and dichlorodihydrofluorescein fluorescence, respectively, whereas aging reduced flow-induced NO and H(2)O(2) production. Endothelium-dependent vasodilation was evaluated by increasing intraluminal flow (5-60 nl/s) before and after treatment with the superoxide dismutase mimetic Tempol (100 muM), the H(2)O(2) scavenger catalase (100 U/ml), or Tempol plus catalase. Catalase reduced flow-induced dilation in both groups, whereas Tempol and Tempol plus catalase diminished vasodilation in young but not old rats. Tempol plus deferoxamine (100 muM), an inhibitor of hydroxyl radical formation, reversed Tempol-mediated impairment of flow-induced vasodilation in young rats and improved flow-induced vasodilation in old rats compared with control. Immunoblot analysis revealed increases in endogenous superoxide dismutase, catalase, and nitrotyrosine protein levels with aging. Collectively, these data indicate that NO- and H(2)O(2)-mediated flow-induced signaling decline with age in coronary arterioles and that elevated hydroxyl radical formation contributes to the age-related impairment of flow-induced vasodilation.

Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats

The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 +/- 4 vs. old: 34 +/- 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by approximately 160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME), declined with age. l-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, l-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age.

Age and exercise training alter signaling through reactive oxygen species in the endothelium of skeletal muscle arterioles

Exercise training ameliorates age-related impairments in endothelium-dependent vasodilation in skeletal muscle arterioles. Additionally, exercise training is associated with increased superoxide production. The purpose of this study was to determine the role of superoxide and superoxide-derived reactive oxygen species (ROS) signaling in mediating endothelium-dependent vasodilation of soleus muscle resistance arterioles from young and old, sedentary and exercise-trained rats. Young (3 mo) and old (22 mo) male rats were either exercise trained or remained sedentary for 10 wk. To determine the impact of ROS signaling on endothelium-dependent vasodilation, responses to acetylcholine were studied under control conditions and during the scavenging of superoxide and/or hydrogen peroxide. To determine the impact of NADPH oxidase-derived ROS, endothelium-dependent vasodilation was determined following NADPH oxidase inhibition. Reactivity to superoxide and hydrogen peroxide was also determined. Tempol, a scavenger of superoxide, and inhibitors of NADPH oxidase reduced endothelium-dependent vasodilation in all groups. Similarly, treatment with catalase and simultaneous treatment with tempol and catalase reduced endothelium-dependent vasodilation in all groups. Decomposition of peroxynitrite also reduced endothelium-dependent vasodilation. Aging had no effect on arteriolar protein content of SOD-1, catalase, or glutathione peroxidase-1; however, exercise training increased protein content of SOD-1 in young and old rats, catalase in young rats, and glutathione peroxidase-1 in old rats. These data indicate that ROS signaling is necessary for endothelium-dependent vasodilation in soleus muscle arterioles, and that exercise training-induced enhancement of endothelial function occurs, in part, through an increase in ROS signaling.

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O(2)(-) and H(2)O(2) in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H(2)O(2) production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.

Divergent Effects of Aging and Sex on Vasoconstriction to Endothelin in Coronary Arterioles

The risk for cardiovascular disease increases with advancing age; however, the chronological development of heart disease differs in males and females. The purpose of this study was to determine whether age‐induced alterations in responses of coronary arterioles to the endogenous vasoconstrictor, endothelin, are sex‐specific.