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 Ϫ scavenging, as well as removal of H2O2, the byproduct of O 2 Ϫ 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 2O2 production as evidenced by enhanced diaminofluorescein and dichlorodihydrofluorescein fluorescence, respectively, whereas aging reduced flow-induced NO and H 2O2 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 M), the H 2O2 scavenger catalase (100 U/ml), or Tempol plus catalase. Catalase reduced flowinduced dilation in both groups, whereas Tempol and Tempol plus catalase diminished vasodilation in young but not old rats. Tempol plus deferoxamine (100 M), 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 2O2-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.reactive oxygen species; superoxide dismutase; hydroxyl radical; deferoxamine; nitric oxide; hydrogen peroxide INCREASING EVIDENCE INDICATES that aging contributes significantly to the development of ischemic heart disease. Vascular resistance increases, leading to impairments in coronary blood flow and flow reserve (15). Endothelial dysfunction similarly progresses with aging (4) and may be mediated by impaired nitric oxide (NO) activity (7, 21) or elevated oxidant stress (6, 10). Previous studies have demonstrated age-associated declines in flow-mediated dilation in coronary arterioles due to impaired phosphatidylinositol 3-kinase signaling (21) or elevated superoxide (O 2 Ϫ ) production (7). Because flow-induced vasodilation is inextricably linked to NO-mediated signaling, it is possible that other factors directly involved in the NO pathway may be altered with advancing age.In (6,7,12). Thus ROS production and scavenging are likely carefully modulated to produce appropriate flow-induced dilation. Therefore, the purpose of our study was 1) to investigate age-related changes in NO and H 2 O 2 signaling during flow-induced vasodilation and 2) to examine the effects of ROS scavenging on flow-mediated dilation in coronary arterioles. METHODSAnimals. All procedures in this study were approved by the Institutional Animal Care and U...

October 7, 2009; doi:10.1152/ajpregu.00178.2009.-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 flowinduced 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 ϩ estrogen replaced (OVE) female Fischer-344 rats to assess flowinduced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 Ϯ 4 vs. old: 34 Ϯ 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 ϳ160%. 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 flowinduced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flowinduced, 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.endothelial nitric oxide synthase; Akt; nitric oxide; ovariectomy THE RISK FOR CARDIOVASCULAR disease (CVD) and heart failure increase with advancing age; however, sexual dimorphism exists in the chronological development of these risks (22,47). Although the chronological rate of aging is independent of sex, mechanisms that regulate the cardiovascular system across the lifespan may differ dramatically between men and women. The risk for CVD in men begins to increase at approximately the same age that flow-mediated vasodilation begins to decline (5). Women also exhibit this age-related impairment of flow-mediated vasodilation; however, significant reduction of flow-mediated dilation becomes apparent at the age of menopause, more than a decade later than in men (5). The cardioprotection that women experience until menopause is presumably due to the presence of ovarian estrogen and results in a sex-related delay of the expression of CVD (49). Chronic estrogen treatment has been shown to enhance endothelial function in a number of vascular beds (27, 31, 39), in part, through a pathway involving activation of Akt/PKB and subsequent phosphorylation of endothelial nitric oxide synthase (eNOS) (3,10,15,43,44). Endothelium-dependent vasodilation to a...

Sindler AL, Reyes R, Chen B, Ghosh P, Gurovich AN, Kang LS, Cardounel AJ, Delp MD, Muller-Delp JM. Age and exercise training alter signaling through reactive oxygen species in the endothelium of skeletal muscle arterioles. J Appl Physiol 114: [681][682][683][684][685][686][687][688][689][690][691][692][693] 2013. First published January 3, 2013; doi:10.1152/japplphysiol.00341.2012.-Exercise training ameliorates age-related impairments in endotheliumdependent 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 traininginduced enhancement of endothelial function occurs, in part, through an increase in ROS signaling. hydrogen peroxide; superoxide; peroxynitrite; nitric oxide; acetylcholine ENDOTHELIAL FUNCTION IN THE skeletal muscle resistance vasculature declines with age primarily due to decreased nitric oxide (NO) bioavailability (10,38,51,55). In feed arteries from soleus muscle, reductions in NO-dependent vasodilation are accompanied by reduced expression of endothelial NO synthase (eNOS) (65). In contrast, our laboratory has previously reported that NO-mediated vasodilation of soleus muscle arterioles declines with advancing age, despite an increase in eNOS protein levels (51). Thus the age-related decline in bioavailability of NO may be dependent on numerous other factors that regulate both NO production and degradation. eNOS activity, and subsequent NO production, is regulated by availability of substrate and cofactors, by protein-...

Kang LS, Kim SJ, Dominguez JM 2nd, Sindler AL, Dick GM, Muller-Delp JM. Aging and muscle fiber type alter K ϩ channel contributions to the myogenic response in skeletal muscle arterioles. J Appl Physiol 107: 389 -398, 2009. First published April 30, 2009 doi:10.1152/japplphysiol.91245.2008.-Aging diminishes myogenic tone in arterioles from skeletal muscle. Recent evidence indicates that both large-conductance Ca 2ϩ -activated (BKCa) and voltage-dependent (K V) K ϩ channels mediate negative feedback control of the myogenic response. Thus we tested the hypothesis that aging increases the contributions of K V and BKCa channels to myogenic regulation of vascular tone. Because myogenic responsiveness differs between oxidative and glycolytic muscles, we predicted that K V and BK Ca channel contributions to myogenic responsiveness vary with fiber type. Myogenic responses of first-order arterioles from the gastrocnemius and soleus muscles of 4-and 24-mo-old Fischer 344 rats were evaluated in the presence and absence of 4-aminopyridine (5 mM) or iberiotoxin (30 nM), inhibitors of K V and BKCa, respectively. 4-Aminopyridine enhanced myogenic tone with aging and normalized age-related differences in both muscle types. By contrast, iberiotoxin eliminated age-related differences in soleus arterioles and had no effect in gastrocnemius vessels. KV1.5 is an integral component of KV channels in vascular smooth muscle; therefore, we determined the relative protein expression of KV1.5, as well as BKCa, in soleus and gastrocnemius arterioles. Immunoblot analysis revealed no differences in KV1.5 protein with aging or between variant fiber types, whereas BKCa protein levels declined with age in arterioles from both muscle groups. Collectively, these results suggest that the contribution of BKCa to myogenic regulation of vascular tone changes with age in soleus muscle arterioles, whereas increased KV channel expression and negative feedback regulation of myogenic tone increases with advancing age in arterioles from both oxidative and glycolytic muscles. delayed rectifier potassium channel; calcium-activated potassium channel; iberiotoxin; 4-aminopyridine; KV1.5 THE MYOGENIC RESPONSE IS ESSENTIAL in regulating peripheral vascular resistance as well as tissue-specific blood flow. This autoregulatory mechanism, in which vessels contract or dilate in response to changes in transmural pressure, may be impaired with advancing age. The magnitude of myogenic tone development declines with aging in both mesenteric (19) and skeletal muscle arterioles (32), and age-induced modifications of vascular smooth muscle contractile mechanisms may contribute to this reduced myogenic reactivity (32). Myogenic tone development depends on an increase in intracellular Ca2ϩ and subsequent initiation of contractile activity, which is mediated by voltage-gated Ca 2ϩ channels (VGCC). In the presence of steady Ca 2ϩ influx from VGCC, an opposing, hyperpolarizing force is likely required to prevent regenerative Ca 2ϩ entry and tonic contraction (13,21,35). Severa...

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P

Kang LS, Chen B, Reyes RA, LeBlanc AJ, Teng B, Mustafa SJ, Muller-Delp JM. Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles. Am J Physiol Heart Circ Physiol 300: H2105-H2115, 2011. First published March 25, 2011 doi:10.1152/ajpheart.00349.2010.-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 Ϫ and H2O2 in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX ϩ estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flowinduced NO production, whereas flow-induced H 2O2 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 M) or the H2O2 scavenger catalase (100 U/ml). Removal of H 2O2 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 Ϫ and H2O2 are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O 2 Ϫ buffering by SOD contributes to impaired flowinduced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O 2 Ϫ homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age. superoxide dismutase; hydrogen peroxide; ovariectomy; estrogen replacement

Nitric oxide (NO) mediates a major portion of arteriolar endothelium-dependent dilation in adults, but indirect evidence has suggested that NO contributes minimally to these responses in the young. Isolated segments of arterioles were studied in vitro to verify this age-related increase in NO release and investigate the mechanism by which it occurs. Directly measured NO release induced by ACh or the Ca(2+) ionophore A-23187 was five- to sixfold higher in gracilis muscle arterioles from 42- to 46-day-old (juvenile) rats than in those from 25- to 28-day-old (weanling) rats. There were no differences between groups in arteriolar endothelial NO synthase (eNOS) expression or tetrahydrobiopterin levels, and arteriolar l-arginine levels were lower in juvenile vessels than in weanling vessels (104 ± 6 vs.126 ± 3 pmol/mg). In contrast, agonist-induced eNOS Thr(495) dephosphorylation and eNOS Ser(1177) phosphorylation (events required for maximal activity) were up to 30% and 65% greater, respectively, in juvenile vessels. Juvenile vessels did not show increased expression of enzymes that mediate these events [protein phosphatases 1 and 2A and PKA and PKB (Akt)] or heat shock protein 90, which facilitates Ser(1177) phosphorylation. However, agonist-induced colocalization of heat shock protein 90 with eNOS was 34-66% greater in juvenile vessels than in weanling vessels, and abolition of this difference with geldanamycin also abolished the difference in Ser(1177) phosphorylation between groups. These findings suggest that growth-related increases in arteriolar NO bioavailability may be due at least partially to changes in the regulation of eNOS phosphorylation and increased signaling activity, with no change in the abundance of eNOS signaling proteins.

The aging kidney exhibits slowly developing injury and females are usually protected compared to males, in association with maintained renal nitric oxide (NO). Here, we compared renal injury in Fischer 344 (F344) rats in intact, ovariectomized, and ovariectomized + estrogen replaced young (6 month) and old (24 month) female rats with young and old intact male rats and measured renal protein abundance of NO synthase isoforms and oxidative stress. There was no difference in age-dependent glomerular damage between young or old intact male and female F344, and neither ovariectomy nor estrogen replacement affected renal injury; however, tubulointerstitial injury was greater in old males than old females. These data suggest that ovarian hormones do not influence these aspects of kidney aging in F344 rats and that the greater tubulointerstitial injury is caused by male sex. Old males had greater kidney cortex NOS3 abundance than females, and NOS1 abundance (alpha and beta isoforms) was increased in old males compared to both young males and old females. NOS abundance was preserved with age in intact females, ovariectomy did not reduce NOS1 or 3 protein abundance, and estrogen replacement did not uniformly elevate NOS proteins, suggesting that estrogens are not primary regulators of renal NOS abundance in this strain. NADPH oxidase-dependent superoxide production and nitrotyrosine immunoreactivity were increased in the aging male rat kidney compared to the female which could compromise renal NO production and/or bioavailability. In conclusion, the kidney damage expressed in the aging F344 is fairly mild and is not related to loss of renal cortex NOS3 or NOS1 alpha. This is in contrast to the aging male Sprague Dawley rat [1] where kidney damage is exacerbated and NOS3 and NOS1 alpha are lost compared to the old female.