Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release

The present study was performed to determine the importance of nitric oxide in eliciting epicardial coronary artery dilation during sustained increases in shear stress in the absence of pulsatile flow. Isolated first-order porcine epicardial coronary conduit arteries (approximately 500 microm) were preconstricted (U-46619) and subjected to steady-state changes in flow in vitro. Nonpulsatile flow (shear stress range from 0 to approximately 100 dyn/cm2) produced a graded dilation of epicardial arteries. Inhibiting nitric oxide synthase with 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) blocked bradykinin-induced vasodilation but did not affect the flow-diameter relation or the maximum change in diameter from static conditions (67 +/- 10 microm in control vs. 71 +/- 8 microm after L-NAME, P = not significant). The addition of indomethacin (10(-5) M) had no effect on flow-mediated vasodilation. Depolarizing vascular smooth muscle with KCl (60 mM) or removing the endothelium blocked bradykinin vasodilation and completely abolished flow-mediated responses. The K+ channel blocker tetraethylammonium chloride (TEA; 10(-4)M) attenuated flow-mediated vasodilation (maximum diameter change was 110 +/- 18 microm under control conditions vs. 58 +/- 10 microm after TEA, P < 0.001). These data indicate that epicardial coronary arteries dilate to steady-state changes in nonpulsatile flow via a mechanism that is independent of nitric oxide production. The ability to completely block this with KCl and attenuate it with TEA supports the hypothesis that epicardial coronary arteries dilate to steady levels of shear stress through hyperpolarization of vascular smooth muscle. This may be secondary to the release of an endothelium-dependent hyperpolarizing factor.     

Aging enhances pressure-induced arterial superoxide formation

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.     

Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats

This study determined alterations to hypoxic dilation of isolated skeletal muscle resistance arteries (gracilis arteries; viewed via television microscopy) from obese Zucker rats (OZR) compared with lean Zucker rats (LZR). Hypoxic dilation was reduced in OZR compared with LZR. Endothelium removal and cyclooxygenase inhibition (indomethacin) severely reduced this response in both groups, although nitric oxide synthase inhibition (N(omega)-nitro-L-arginine methyl ester) reduced dilation in LZR only. Treatment of vessels with a PGH(2)-thromboxane A(2) receptor antagonist had no effect on hypoxic dilation in either group. Arterial dilation to arachidonic acid, iloprost, acetylcholine, and sodium nitroprusside was reduced in OZR versus LZR, although dilation to forskolin and aprikalim was unaltered. Treatment of arteries from OZR with oxidative radical scavengers increased dilation to hypoxia and agonists, with no effect on responses in LZR. The restored hypoxic dilation in OZR was abolished by indomethacin. These results suggest that hypoxic dilation of skeletal muscle microvessels from LZR represents the summated effects of prostanoid and nitric oxide release, whereas the impaired response of vessels in OZR may reflect scavenging of PGI(2) by superoxide anion.     

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

Age-related changes in adenosine-mediated relaxation of coronary and aortic smooth muscle

We tested whether adenosine mediates nitric oxide (NO)-dependent and NO-independent dilation in coronary and aortic smooth muscle and whether age selectively impairs NO-dependent adenosine relaxation. Responses to adenosine and the relatively nonselective analog 5'-N-ethylcarboxamidoadenosine (NECA) were studied in coronary vessels and aortas from immature (1-2 mo), mature (3-4 mo), and moderately aged (12-18 mo) Wistar and Sprague-Dawley rats. Adenosine and NECA induced biphasic concentration-dependent coronary vasodilation, with data supporting high-sensitivity (pEC(50) = 5.2-5.8) and low-sensitivity (pEC(50) = 2.3-2.4) adenosine sites. Although sensitivity to adenosine and NECA was unaltered by age, response magnitude declined significantly. Treatment with 50 microM N(G)-nitro-L-arginine methyl ester (L-NAME) markedly inhibited the high-sensitivity site, although response magnitude still declined with age. Aortic sensitivity to adenosine declined with age (pEC(50) = 4.7 +/- 0.2, 3.5 +/- 0.2, and 2.9 +/- 0.1 in immature, mature, and moderately aged aortas, respectively), and the adenosine receptor transduction maximum also decreased (16.1 +/- 0.8, 12.9 +/- 0.7, and 9.6 +/- 0.7 mN/mm(2) in immature, mature, and moderately aged aortas, respectively). L-NAME decreased aortic sensitivity to adenosine in immature and mature tissues but was ineffective in the moderately aged aorta. Data collectively indicate that 1) adenosine mediates NO-dependent and NO-independent coronary and aortic relaxation, 2) maturation and aging reduce NO-independent and NO-dependent adenosine responses, and 3) the age-related decline in aortic response also involves a reduction in the adenosine receptor transduction maximum.     

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.     

Chronic nitric oxide synthase inhibition blunts endothelium-dependent function of conduit coronary arteries, not arterioles

Current literature suggests that chronic nitric oxide synthase (NOS) inhibition has differential effects on endothelium-dependent dilation (EDD) of conduit arteries vs. arterioles. Therefore, we hypothesized that chronic inhibition of NOS would impair EDD of porcine left anterior descending (LAD) coronary arteries but not coronary arterioles. Thirty-nine female Yucatan miniature swine were included in the study. Animals drank either tap water or water with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/l), resulting in control and chronic NOS inhibition (CNI) groups, respectively. Treatment was continued for 1-3 mo (8.3 +/- 0.6 mg x kg(-1) x day(-1)). In vitro EDD of coronary LADs and arterioles was assessed via responses to ADP (LADs only) and bradykinin (BK), and endothelium-independent function was assessed via responses to sodium nitroprusside (SNP). Chronic NOS inhibition diminished coronary artery EDD to ADP and BK. Incubating LAD rings with L-NAME decreased relaxation responses of LADs from control pigs but not from CNI pigs such that between-group differences were abolished. Neither indomethacin (Indo) nor sulfaphenazole incubation significantly affected relaxation responses of LAD rings to ADP or BK. Coronary arteries from CNI pigs showed enhanced relaxation responses to SNP. In contrast to coronary arteries, coronary arterioles from CNI pigs demonstrated preserved EDD to BK and no increase in dilation responses to SNP. L-NAME, Indo, and L-NAME + Indo incubation did not result in significant between-group differences in arteriole dilation responses to BK. These results suggest that although chronic NOS inhibition diminishes EDD of LAD rings, most likely via a NOS-dependent mechanism, it does not affect EDD of coronary arterioles.     

A novel vascular EET synthase: role of CYP2C7

We demonstrated previously that cytochrome P-450 (CYP) 2C29 is the epoxyeicosatrienoic acid (EET) synthase responsible for the EET-mediated flow/shear stress-induced dilation of vessels of female nitric oxide (NO)-deficient mice (Sun D, Yang YM, Jiang H, Wu H, Ojami C, Kaley G, Huang A. Am J Physiol Regul Integr Comp Physiol 298: R862-R869, 2010). In the present study, we aimed to identify which specific CYP isoform(s) is the source of the synthesis and release of EETs in response to stimulation by shear stress in vessels of rats. Cannulated mesenteric arteries isolated from both sexes of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats were perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress stimulated release of EETs in the perfusate of female (but not male) NO-deficient vessels, associated with an EET-mediated vasodilation, in which 11,12- and 14,15-EET contributed predominantly to the responses. Rat CYP cDNA array screened a total of 32 CYP genes of mesenteric arteries, indicating a significant upregulation of CYP2C7 in female L-NAME-treated rats. Endothelial RNA and protein were extracted from intact single vessels. Expression of CYP2C7 mRNA and protein in pooled extractions of endothelial lysate was identified by PCR and Western blot analyses. Transfection of the vessels with CYP2C7 short interfering RNA eliminated the release of EETs, consequently abolishing the EET-mediated flow-induced dilation; these responses, however, were maintained in vessels transfected with nonsilencing short interfering RNA. Knockdown of endothelial CYP2C7 was confirmed by PCR and Western blot analyses. In conclusion, CYP2C7 is an endothelial EET synthase in the female rat vasculature, by which, in NO deficiency, shear stress stimulates the release of EETs to initiate vasodilation.     

Contribution of epoxyeicosatrienoic acids to flow-induced dilation in arteries of male ERalpha knockout mice: role of aromatase

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ERalpha-knockout (ERalpha-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ERalpha-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ERalpha-KO arteries, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited FID by approximately 26%, whereas indomethacin inhibited dilations by approximately 50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/cm(2) shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ERalpha-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ERalpha-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ERbeta in ERalpha-KO arteries. Treatment of ERalpha-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ERalpha-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone.     

Exercise training enhances flow-mediated dilation in spontaneously hypertensive rats

This study investigated the effect of exercise training on the flow-mediated dilation (FMD) in gastrocnemius muscle arteries from spontaneously hypertensive rats (SHR). SHR and WKY rats were divided into sedentary and exercised groups. After swimming exercise for eight weeks, the isolated arteries were mounted on pressurized myograph and FMD responses examined. The role of nitric oxide (NO), prostaglandins (PGs) and endothelium derived hyperpolarizing factor (EDHF) on FMD were assessed by obtaining dilation responses in the presence and absence of pharmacological antagonists. N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin (INDO) and tetraethylamonium (TEA) were used to inhibit nitric oxide synthase, cyclooxygenase and EDHF-mediated responses, respectively. The FMD response was significantly blunted in arteries of SHR compared with WKY rats, and, improved by exercise training in SHR (SHR-ET) group. In SHR arteries, L NAME and TEA did not affect dilation responses to flow, while INDO led to a significant enhancement in this response. Although dilation response was not altered by L-NAME in arteries obtained from trained SHR, TEA caused a significant attenuation and INDO led to significant increases. These results demonstrate that exercise training improves FMD in SHR, and, this enhancement induced by exercise training occurs through EDHF-mediated mechanism(s).     

Adenosine and hypoxic dilation of rat coronary small arteries: roles of the ATP-sensitive potassium channel, endothelium, and nitric oxide

The aims of the study were to examine the roles of the ATP-sensitive potassium (K(ATP)) channel, the endothelium, and nitric oxide (NO) in the responses of rat coronary small arteries to adenosine and hypoxia. Segments of rat coronary vessel were investigated in vitro using pressure myography; all vessels studied developed stable spontaneous myogenic tone during equilibration. Glibenclamide (a K(ATP) channel inhibitor) reversed pinacidil but not 2-deoxyglucose-induced dilation. Both adenosine and hypoxia dilated the vessels, and glibenclamide did not reverse these responses. Endothelial removal or N(G)-nitro-L-arginine methyl ester (L-NAME) inhibited the dilation to adenosine by approximately 50%; subsequent addition of glibenclamide was without effect. Hypoxic dilation was completely inhibited by endothelium removal or L-NAME. We conclude that adenosine- and hypoxia-induced dilation of rat coronary arteries does not appear to involve the K(ATP) channel. Adenosine-induced dilation is partially and hypoxic dilation is completely dependent on endothelium-derived NO.     

Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(2) (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.     

Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-alpha (TNF-alpha) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-alpha (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of N(G)-nitro-L-arginine methyl ester, TNF-alpha produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-alpha. Pretreatment of the arteries with desipramine (10 microM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-alpha on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-alpha activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-alpha inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.     

Impaired endothelium-mediated relaxation in isolated cerebral arteries from insulin-resistant rats

Insulin resistance (IR) impairs vascular responses in peripheral arteries. However, the effects of IR on cerebrovascular control mechanisms are completely unexplored. We examined the vascular function of isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. Endothelium-dependent vasodilation elicited by bradykinin (BK) was reduced in IR compared with control MCAs. Maximal dilation to BK (10(-6) M) was 38 +/- 3% (n = 13) in control and 19 +/- 3% (n = 10) in IR arteries (P < 0.01). N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) decreased responses to BK in control arteries by approximately 65% and inhibited the already reduced responses completely in IR MCAs. Indomethacin (10 microM) reduced relaxation to BK in control MCAs by approximately 40% but was largely ineffective in IR arteries. Combined L-NAME and indomethacin treatments eliminated the BK-induced dilation in both groups. Similarly to BK, endothelium-mediated and mainly cyclooxygenase (COX)-dependent dilation to calcium ionophore A23187 was reduced in IR arteries compared with controls. In contrast, vascular relaxation to sodium nitroprusside was similar between the IR and control groups. These findings demonstrate that endothelium-dependent dilation in cerebral arteries is impaired in IR primarily because of a defect of the COX-mediated pathways. In contrast, nitric oxide-mediated dilation remains intact in IR arteries.     

Exercise training enhances flow-induced vasodilation in skeletal muscle resistance arteries of aged rats: role of PGI2 and nitric oxide

Flow-induced vasodilation is attenuated with old age in rat skeletal muscle arterioles. The purpose of this study was to determine whether diminished cyclooxygenase (COX) signaling contributes to the age-induced attenuation of flow-induced vasodilation in gastrocnemius muscle arterioles and to determine whether, and through which mechanism(s), exercise training restores this deficit in old rats. Fischer 344 rats (3 and 22 mo old) were assigned to a sedentary or exercise-trained group. First-order arterioles were isolated from the gastrocnemius muscles, cannulated, and pressurized to 70 cm H(2)O. Diameter changes were determined in response to graded increases in intraluminal flow in the presence and absence of nitric oxide synthase (NOS) inhibition [10(-5) M N(G)-nitro-L-arginine methyl ester (L-NAME)], COX inhibition (10(-5) M indomethacin), or combination NOS (10(-5) M L-NAME) plus COX (10(-5) M indomethacin) inhibition. Aging reduced flow-induced vasodilation in gastrocnemius muscle arterioles. Exercise training restored responsiveness to flow in arterioles of aged rats and enhanced flow-induced vasodilation in arterioles from young rats. L-NAME inhibition of flow-induced vasodilation was greater in arterioles from old rats compared with those from young rats and was increased after exercise training in arterioles from both young and old rats. Although the indomethacin-sensitive portion of flow-induced dilation was not altered by age or training, both COX-1 mRNA expression and PGI(2) production increased with training in arterioles from old rats. These data demonstrate that exercise training restores flow-induced vasodilation in gastrocnemius muscle arterioles from old rats and enhances flow-induced vasodilation in gastrocnemius muscle arterioles from young rats. In arterioles from both old and young rats, the exercise training-induced enhancement of flow-induced dilation occurs primarily through a NOS mechanism.     

Shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries.

We tested the hypothesis that increased intraluminal shear stress induces endothelial nitric oxide (NO) synthase (eNOS) mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries (SFA) by increasing NO production. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two resistance-matched glass micropipettes. SFA were exposed to no flow (NF), low flow (LF), intermediate flow (IF), or high flow (HF) for 4 h. Mean intraluminal shear stress ranged from 0 to 82 dyn/cm(2). At the end of the 4-h treatment period, eNOS mRNA expression was assessed in each SFA. eNOS mRNA expression was significantly lower in old NF SFA than in young NF SFA. In old SFA, eNOS mRNA expression was induced by IF (+154%) and HF (+136%), resulting in a level of expression that was not different from that of young SFA. In a separate series of experiments, SFA were pretreated with NF or HF for 4 h, and endothelial function was assessed by examining vasodilator responses to ACh. ACh-induced dilation was less in old NF SFA than young NF SFA. Pretreatment with HF improved ACh-induced dilation in old SFA such that the response was similar to that of young SFA. In the presence of N(omega)-nitro-L-arginine to inhibit NOS, ACh-induced dilation was inhibited in old HF SFA such that the response was no longer greater than that of old NF SFA. These results indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production.     

Effects of aging on adipose resistance artery vasoconstriction: possible implications for orthostatic blood pressure regulation.

The purpose of this investigation was to determine mean arterial pressure (MAP) and regional vascular conductance responses in young and aged Fisher-344 rats during orthostatic stress, i.e., 70 degrees head-up tilt (HUT). Both groups demonstrated directionally different changes in MAP during HUT (young, 7% increase; aged, 7% decrease). Vascular conductance during HUT in young rats decreased in most tissues but largely remained unchanged in the aged animals. Based on the higher vascular conductance of white adipose tissue from aged rats during HUT, resistance arteries from white visceral fat were isolated and studied in vitro. There was diminished maximal vasoconstriction to phenylephrine and norepinephrine (NE: young, 42 +/- 5%; old, 18 +/- 6%) in adipose resistance arteries from aged rats. These results demonstrate that aging reduces the ability to maintain MAP during orthostatic stress, and this is associated with a diminished vasoconstriction of adipose resistance arteries.     

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.