Short-term exercise training improves vascular function in hypercholesterolemic rabbit femoral artery

Chronic exercise in healthy or hypercholesteremic animals for at least two months improves their vascular functions. This study is to examine whether short-term exercise training protocols can correct early-stage vascular dysfunction induced by high-cholesterol diet feeding. Male New Zealand White rabbits were fed for 2, 4 or 6 weeks with rabbit chow with or without the addition of 2% (w/w) cholesterol. They were further divided into control and exercise groups. Animals in exercise groups ran on a leveled treadmill for the same time periods as diet intervention. At the end of experiments, femoral arteries were dissected, loaded with fura 2-AM, and mounted in a tissue flow chamber. Phenylephrine-precontracted vessel specimens were exposed to acetylcholine. The endothelial intracellular calcium elevation and vasorelaxation were determined simultaneously under an epifluorescence microscope with ratio imaging capability. En face oil red O staining was used to evaluate fatty streak formation. Our results showed that 1) high-cholesterol diet feeding for > or = 4 weeks caused lipid deposition, reduced the acetylcholine-evoked endothelial calcium signaling, and impaired both endothelium-dependent and endothelium-independent vascular responses in a time-dependent manner; 2) vasorelaxation at given levels of endothelial intracellular calcium elevation decreased in hypercholesterolemia; 3) concomitant exercise program had reverse effects. We conclude that high-cholesterol diet intervention for as short as 4 weeks induces vascular structural changes, impairs endothelial intracellular calcium signaling and vasodilatation in rabbit femoral arteries. Short-term exercise training in parallel completely eliminates these adverse effects so long as the diet intervention is no more than 6 weeks.     

Invited review: effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo.

To study the effects of flow on in situ endothelial intracellular calcium concentration ([Ca(2+)](i)) signaling, rat aortic rings were loaded with fura 2, mounted on a tissue flow chamber, and divided into control and flow-pretreated groups. The latter was perfused with buffer at a shear stress of 50 dyns/cm(2) for 1 h. Endothelial [Ca(2+)](i) responses to ACh or shear stresses were determined by ratio image analysis. Moreover, ACh-induced [Ca(2+)](i) elevation responses were measured in a calcium-free buffer, or in the presence of SKF-96365, to elucidate the role of calcium influx in the flow effects. Our results showed that 1) ACh increased endothelial [Ca(2+)](i) in a dose-dependent manner, and these responses were incremented by flow-pretreatment; 2) the differences in ACh-induced [Ca(2+)](i) elevation between control and flow-pretreated groups were abolished by SKF-96365 or by Ca(2+)-free buffer; and 3) in the presence of 10(-5) M ATP, shear stress induced dose-dependent [Ca(2+)](i) elevation responses that were not altered by flow-pretreatment. In conclusion, flow-pretreatment augments the ACh-induced endothelial calcium influx in rat aortas ex vivo.     

Effects of chronic exercise on calcium signaling in rat vascular endothelium

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca(2+) signaling, we examined intracellular Ca(2+) concentration ([Ca(2+)](i)) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca(2+)](i) responses; 2) ACh induced heterogeneous [Ca(2+)](i) elevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca(2+)](i) elevation was inhibited by the Ca(2+) influx blocker SKF-96365, by a Ca(2+)-free buffer, or by high concentrations of extracellular K(+). We conclude that chronic exercise increases ACh-induced [Ca(2+)](i) elevation in rat aortic endothelium in situ, possibly by facilitating Ca(2+) influx.     

Effect of tetrahydrobiopterin and exercise training on endothelium-dependent vasorelaxation in SHR

We examined whether the improvement of impaired NO-dependent vasorelaxation by exercise training could be mediated through a BH₄-dependent mechanism. Male spontaneously hypertensive rats (SHR, n?=?20) and Wistar-Kyoto rats (WKY, n?=?20) were trained (Tr) for 9 weeks on a treadmill and compared to age-matched sedentary animals (Sed). Endothelium-dependent vasorelaxation (EDV) was assessed with acetylcholine by measuring isometric tension in rings of femoral artery precontracted with 10^?5?M phenylephrine. EDV was impaired in SHR-Sed as compared to WKY-Sed (p?=?0.02). Training alone improved EDV in both WKY (p?=?0.01) and SHR (p?=?0.0001). Moreover, EDV was not different in trained SHR than in trained WKY (p?=?0.934). Pretreatment of rings with L-NAME (50 μM) cancelled the difference in ACh-induced relaxation between all groups, suggesting that NO pathway is involved in these differences. The presence of 10^?5?M BH₄ in the organ bath significantly improved EDV for sedentary SHR (p?=?0.030) but not WKY group (p?=?0.815). Exercise training turned the beneficial effect of BH₄ on SHR to impairment of ACh-induced vasorelaxation in both SHR-Tr (p?=?0.01) and WKY-Tr groups (p?=?0.04). These results suggest that beneficial effect of exercise training on endothelial function is due partly to a BH₄-dependent mechanism in established hypertension.     

Effect of chronic lithium administration on endothelium-dependent relaxation of rat mesenteric bed: role of nitric oxide

The mechanism of action of lithium, an effective treatment for bipolar disease, is still unknown. In this study, the mesenteric vascular beds of control rats and rats that were chronically treated with lithium were prepared by the McGregor method, and the mesenteric vascular bed vasorelaxation responses were examined. NADPH-diaphorase histochemistry was used to determine the activity of NOS (nitric oxide synthase) in mesenteric vascular beds. We demonstrated that ACh-induced vasorelaxation increased in the mesenteric vascular bed of rats treated with lithium. Acute No-nitro-L-arginine methyl ester (L-NAME) administration in the medium blocked ACh-induced vasorelaxation in the control group more effectively than in lithium-treated rats, while the vasorelaxant response to sodium nitroprusside, a NO donor, was not different between lithium-treated and control groups. Acute aminoguanidine administration blocked ACh-induced vasorelaxation of lithium-treated rats, but had no effect in the control rats. Furthermore, NOS activity, determined by NADPH-diaphorase staining, was significantly greater in the mesenteric vascular beds from chronic lithium-treated rats than in those from control rats. These data suggest that the enhanced ACh-induced endothelium-derived vasorelaxation in rat mesenteric bed from chronic lithium-treated rats might be associated with increased NOS activity, likely via iNOS. Simultaneous acute L-NAME and indomethacin administration suggests the possible upregulation of EDHF (endothelium-derived hyperpolarizing factor) in lithium-treated rats.     

Enhancement of vasorelaxation in hypertension following high-intensity exercise

Exercise can ameliorate vascular dysfunction in hypertension, but its underlying mechanism has not been explored thoroughly. We aimed to investigate whether the high-intensity exercise could enhance vasorelaxation mediated by insulin and insulin-like growth factor-1 (IGF-1) in hypertension. Sixteen-week-old spontaneously hypertensive rats were randomly divided into non-exercise sedentary (SHR) and high-intensity exercise (SHR+Ex) groups conducted by treadmill running at a speed of 30 m/ min until exhaustion. Age-matched Wistar-Kyoto rats (WKY) were used as the normotensive control group. Immediately after exercise, the agonist-induced vasorelaxation of aortas was evaluated in organ baths with or without endothelial denudation. Selective inhibitors were used to examine the roles of nitric oxide synthase (NOS) and phosphatidylinositol-3 kinase (PI3K) in the vasorelaxation. By adding superoxide dismutase (SOD), a superoxide scavenger, the role of superoxide production in the vasorelaxation was also clarified. We found that, the high-intensity exercise significantly (P < 0.05) induced higher vasorelaxant responses to insulin and IGF-1 in the SHR+Ex group than that in the SHR group; after endothelial denudation and pre-treatment of the PI3K inhibitor, NOS inhibitor, or SOD, vasorelaxant responses to insulin and IGF-1 became similar among three groups; the protein expression of insulin receptor, IGF-1 receptor, and endothelial NOS (eNOS) was significantly (P < 0.05) increased in the SHR+Ex group compared with the SHR group;] the relaxation to sodium nitroprusside, a NO donor, was not different among three groups. Our findings suggested that the high-intensity exercise ameliorated the insulin- and IGF-1-mediated vasorelaxation through the endothelium-dependent pathway, which was associated with the reduced level of superoxide production.     

Cellular Responses to Mechanical Stress: Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

To study the effects of flow on in situendothelial intracellular calcium concentration([Ca²⁺]_i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm² for 1 h. Endothelial[Ca²⁺]_i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca²⁺]_i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca²⁺]_i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca²⁺]_i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca²⁺-free buffer; and 3) in the presenceof 10⁵ M ATP, shear stress induced dose-dependent[Ca²⁺]_i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo.

     

Effects of high-cholesterol diet and parallel exercise training on the vascular function of rabbit aortas: a time course study.

It is plausible to assume that exercise training, when applied early enough, can completely correct atherosclerotic defects. Using rabbit aortic specimens, we examined the effects of chronic exercise and high-cholesterol diet feeding on vascular function for different time periods. Male New Zealand White rabbits were divided into four groups: the normal diet groups with or without exercise training and the high-cholesterol diet groups with or without exercise training. Animals in high-cholesterol diet groups were fed 2% cholesterol rabbit chow for 2, 4, or 6 wk. Those in exercise training groups ran on a treadmill at 0.88 km/h for up to 40 min/day, 5 days/wk for the same period of time as the diet feeding. Thoracic aortas were isolated for functional and immunohistochemical analyses. We found that 1). although high-cholesterol diet feeding (>or=2 wk) elevated serum cholesterol levels and impaired acetylcholine-evoked vasorelaxation, only the latter effect was reversed by exercise training; 2). the effects of diet and exercise on acetylcholine-evoked vasorelaxation were mainly due to altered release of nitric oxide and endothelium-derived hyperpolarizing factor; and 3). diet feeding for 4 or 6 wk caused significant lipid deposition and expression of P-selectin, VCAM-1, monocyte chemoattractant protein-1, and inducible nitric oxide synthase, which were largely reduced by exercise training. In conclusion, parallel exercise training almost completely reverses the early-stage endothelial dysfunction caused by high-cholesterol diet feeding.     

Lysophosphatidylcholine alters vascular tone in rat aorta by suppressing endothelial [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> signaling

The detailed mechanism of how lysophosphatidylcholine (LPC) suppresses endothelium-dependent vasodilatation is unclear at present. We investigated the effects of LPC on endothelial intracellular calcium (EC [Ca(2+)](i)) signaling and vascular tone simultaneously using a new technique we developed. Fura-2-labeled rat aortic specimens were mounted in a tissue flow chamber and precontracted with phenylephrine (5 x 10(-8) M). Under either basal or agonist-stimulated conditions, the EC [Ca(2+)](i) level was calculated from fura 2 fluorescence ratio images, and the vascular tone was estimated by measuring the relative displacement of the fluorescence images. Although both acetylcholine (ACh)-induced EC [Ca(2+)](i) elevation and the concomitant vasorelaxation were partially suppressed in specimens pretreated with LPC (20 microM), the quantitative relationship between EC [Ca(2+)](i) elevation and the corresponding vasorelaxation was unaffected. A high concentration of LPC (40 microM) completely eliminated ACh-evoked [Ca(2+)](i) elevation and vasodilatation. It has been reported that exposing vascular tissue to a calcium-free buffer causes a reduction in the EC [Ca(2+)](i) level and the accompanying vasoconstriction. Pretreatment with 20 microM LPC reduced the basal EC [Ca(2+)](i) level and abolished the calcium-free solution-induced EC [Ca(2+)](i) reduction and vasoconstriction. We conclude that LPC impairs endothelium-dependent vasorelaxation mainly by reducing the basal EC [Ca(2+)](i) level and suppressing agonist-evoked EC [Ca(2+)](i) signaling.     

Exercise training preserves endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hyperlipidemia on endothelial function by enhancing NO-mediated vasorelaxation in porcine brachial (Br) arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after initiation of the diet, pigs underwent Ex or remained sedentary (Sed) for 16 wk. Relaxation to ACh was impaired by HF (P = 0.03). The combination of HF and Sed impaired ACh-induced relaxation more than HF or Sed alone (P = 0.0002). Relaxation to high doses of bradykinin (BK) was impaired by HF (P = 0.0002). Ex significantly improved ACh-induced relaxation (P = 0.01) and tended to improve relaxation to BK (P = 0.38). To determine the mechanism(s) by which HF and Ex affected relaxation to ACh and BK, relaxation was assessed in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. In the presence of l-NAME, Indo, or l-NAME + Indo, ACh-induced relaxation was no longer different between HF and NF arteries; however, relaxation remained greater in Ex than in Sed arteries. In the presence of l-NAME or Indo, BK-induced relaxation was no longer altered by HF but was enhanced by Ex. In the presence of l-NAME + Indo, BK-induced relaxation was enhanced by HF and Ex. These data indicate that hyperlipidemia impairs ACh- and BK-induced relaxation by impairing NO- and PGI(2)-mediated relaxation. Ex attenuates the effects of HF by enhancing a vasodilator mechanism independent of NO and PGI(2).     

Endothelium-derived microparticles impair endothelial function in vitro

Endothelial cell dysfunction (ECD) is emerging as the common denominator for diverse and highly prevalent cardiovascular diseases. Recently, an increased number of procoagulant circulating endothelial microparticles (EMPs) has been identified in patients with acute myocardial ischemia, preeclampsia, and diabetes, which suggests that these particles represent a surrogate marker of ECD. Our previous studies showed procoagulant potential of endothelial microparticles and mobilization of microparticles by PAI-1. The aim of this study was to test the effects of isolated EMPs on the vascular endothelium. EMPs impaired ACh-induced vasorelaxation and nitric oxide production by aortic rings obtained from Sprague-Dawley rats in a concentration-dependent manner. This effect was accompanied by increased superoxide production by aortic rings and cultured endothelial cells that were coincubated with EMPs and was inhibited by a SOD mimetic and blunted by an endothelial nitric oxide synthase inhibitor. Superoxide was also produced by isolated EMP. In addition, p22(phox) subunit of NADPH-oxidase was detected in EMP. Our data strongly suggest that circulating EMPs directly affect the endothelium and thus not only act as a marker for ECD but also aggravate preexisting ECD.     

Enhanced endothelium-dependent vasodilatation in human skin vasculature induced by physical conditioning

Functional alterations to the endothelial cells of the vascular system may contribute to the improved circulatory performance induced by physical conditioning. We evaluated microvascular reactivity to iontophoretic application of acetylcholine (ACh) and sodium nitroprusside (SNP) through the skin and blood perfusion measurements in the same area using laser Doppler flowmetry. Whereas ACh acts on smooth muscle cells of the vascular system via the production of vasodilator substances from the endothelium, SNP is an endothelium-independent vasodilator acting on vascular smooth muscle cells directly. The study was performed using two groups of subjects with different levels of aerobic endurance, long distance runners competing at national level (n = 9) and controls (n = 9). The subjects were tested for 40 min on a treadmill before and after an exercise test at 80% of their maximal oxygen uptake. During stimulation by ACh cutaneous perfusion increased to a higher level in the athletes than in the controls (overall P<0.05), whereas an acute period of exercise abolished this difference (overall P>0.6). There was no significant difference between the athletes and the controls with respect to the SNP-induced increase in cutaneous perfusion either before (P>0.9) or after (P>0.9) exercise. The higher cutaneous perfusion responses to stimulation with ACh in the athletes than in the controls may support the hypothesis that regular exercise modifies the responsiveness of the cutaneous endothelium. The difference in ACh-induced perfusion and in unstimulated forearm perfusion between the two groups was present only at rest. This finding indicated that mechanisms were introduced during exercise, which compensated for the lower endothelial sensitivity to stimulation in the controls at rest.     

Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction

Congestive heart failure (CHF) after myocardial infarction is associated with diminished endothelial nitric oxide (NO)-mediated vasorelaxation. The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors have been shown to modulate vascular tone independent of the effects on lipid lowering. We hypothesized that simvastatin restores NO-dependent vasorelaxation with CHF. We found that incubation of the normal rat aorta with 0.1 mM simvastatin for 24 h enhanced ACh-mediated vasorelaxation (P < 0.05). Moreover, simvastatin increased (P < 0.05) endothelial NO synthase (eNOS) protein content by >200% (82.0 +/- 14.0 vs. 21.6 +/- 7.9% II/microg). In cultured endothelial cells, simvastatin (10 and 20 microM) increased eNOS levels by 114.7 +/- 39.9 and 212.0 +/- 75.0% II/microg protein, respectively (both P < 0.05; n = 8). In the rat coronary artery ligation model, oral gavage with 20 mg. kg(-1). day(-1) simvastatin for 3 wk decreased (P < 0.05) mean arterial pressure (121 +/- 20 vs. 96.5 +/- 10.8 mmHg) and left ventricular change in pressure with time (4,500 +/- 700 vs. 4,091 +/- 1,064 mmHg/s, n = 6). Simvastatin reduced (P < 0.05) basal vasoconstriction and improved ACh-mediated vasorelaxation in CHF arterial rings. Inhibition of NO generation by N(G)-nitro-L-arginine methyl ester (100 microM) abolished the ACh-induced vasorelaxation in all rats. In conclusion, chronic treatment of CHF with simvastatin restores endothelial NO-dependent dysfunction and upregulates eNOS protein content in arterial tissue.     

Effect of nitric oxide on exercise-induced proteinuria in rats.

Temporary proteinuria occurring after exercise is a common finding, and it is explained predominantly by alterations in renal hemodynamics. In this study, we investigated whether nitric oxide (NO), which is known to have an effect on renal hemodynamics and to increase during exercise, has a role in postexercise proteinuria. In the first step of this study, the effect of acute NO synthase blockage on exercise proteinuria was evaluated. The urinary protein levels in animals that performed acute exhaustive treadmill running exercise were considerably elevated compared with the control animals. Significantly elevated urinary protein levels were also detected in animals that received Nomega-nitro-L-arginine methyl ester before exhaustion, compared with both control and exhausted groups, and mixed-type proteinuria was detected in electrophoresis, as in all exhausted animals. In the second step of the study, a NO donor (isosorbide mononitrate) was given to rats 1 h before exhaustive exercise. Mixed-type proteinuria and the elevation in urinary protein levels that occur as a consequence of exhaustive exercise were prevented by NO donor treatment. Finally, in the third step of our study, a calcium channel blocker (diltiazem), another vasodilator, was applied to the rats 1 h before exhaustive exercise. Urinary protein levels were not different in exhausted rats with or without calcium channel blocker treatment. On the other hand, in both groups, urinary protein levels were higher than in the control group. The tail-cuff blood pressure alterations caused by vasodilator drug applications before exercise were not different for NO donor and calcium channel blocker groups. These results suggest that endogenous NO might prevent the postexercise proteinuria from becoming more severe by affecting hemodynamic changes that occur during exercise.     

Chronic exercise training does not alter pulmonary vasorelaxation in normal pigs.

Exercise training increases acetylcholine-induced pulmonary vasorelaxation in pigs with coronary occlusion. The present study tested the hypothesis that chronic exercise training enhances endothelium-mediated vasorelaxation in pulmonary arteries from normal pigs. Yucatan miniswine exercised for 16 wk on a treadmill (Ex); control pigs (Sed) remained in pens. Pulmonary artery rings (2- to 3-mm OD) were studied using standard isometric techniques. Contractile responses to 80 mM KCl and norepinephrine (NE) were determined. Vessels were constricted with levels of NE that resulted in half-maximal contraction to examine endothelium-dependent relaxation to ACh and endothelium-independent relaxation to sodium nitroprusside in the presence and absence of nitric oxide synthase inhibition, cyclooxygenase inhibition, and endothelial denudation. Arteries from Ex pigs developed increased contraction to 80 mM KCl, but the response to NE did not differ between groups. Endothelium-dependent and endothelium-independent responses did not differ between Sed and Ex in the presence or absence of pharmacological inhibitors or denudation. We conclude that chronic exercise training does not alter endothelium-dependent or endothelium-independent vasorelaxation responses of pulmonary arteries from normal pigs.     

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.     

Exercise training improves insulin-induced and insulin-like growth factor-1-induced vasorelaxation in rat aortas

Improved vasorelaxant response is one of the beneficial effects of exercise training on vascular function. The mechanism for this response is, however, poorly understood. The aim of this study was to investigate the effects of exercise training on insulin-induced and insulin-like growth factor-1 (IGF-1)-induced vasorelaxation. Fourteen 6-week-old male Wistar rats were randomly divided into sedentary control and exercise groups. For 12 weeks, the exercise group ran on a treadmill 60 min/day, 5 days/week. After exercise training, insulin-induced and IGF-1-induced vasorelaxant responses were evaluated by measuring the isometric tension of aortic rings. The vasorelaxant role of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) was examined by applying inhibitors, such as wortmannin (an inhibitor of PI3K) and N(omega)-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor). In addition, we examined the vascular response to the NO donor, sodium nitroprusside (SNP). We found that: (1) exercise training significantly enhanced both insulin-induced and IGF-1-induced vasorelaxation in rat aortas; (2) this vasorelaxant effect disappeared after the use of wortmannin or L-NAME; (3) there was no significant difference in SNP-induced vasorelaxation between control and exercise groups. Our findings indicate that exercise training enhances insulin-induced and IGF-1-induced vasorelaxant responses which are mediated through the PI3K-NOS-dependent pathway.     

Oxidative stress contributes to vascular endothelial dysfunction in heart failure

Congestive heart failure (HF) is characterized by inadequate nitric oxide (NO) production in the vasculature. Because NO is degraded by oxygen radicals, we hypothesized that NO is degraded faster in HF from inadequate peripheral arterial antioxidant reserves. HF was induced in male Sprague-Dawley rats by left coronary artery ligation. Vascular endothelial function was evaluated by measuring the NO-mediated vasorelaxation response to acetylcholine (ACh; 10(-9)-10(-4) M) in excised aortas. This was repeated with the free radical generator pyrogallol (20 microM) and again with pyrogallol and superoxide dismutase (SOD; 60 U/ml). Aortic and myocardial SOD activity was also determined. ACh-induced vasorelaxation was reduced in HF (n = 9) compared with normal control rats (n = 11; P < 0.001). Pyrogallol further reduced vasorelaxation in HF: 74 +/- 11% at 10(-4) M ACh versus 58 +/- 10% in normal control rats (P < 0.004). There was a trend (P = 0.06) toward reduced SOD activity in HF aortas. In conclusion, altered NO-dependent vasorelaxation in HF is in part due to excessive degradation of NO and is likely related to reduced vascular SOD activity.     

Interleukin-10 counteracts impaired endothelium-dependent relaxation induced by ANG II in murine aortic rings

ANG II stimulates the production of reactive oxygen species and activates proinflammatory cytokines leading to endothelial dysfunction. We hypothesized that the anti-inflammatory cytokine IL-10 counteracts the impairment in endothelium-dependent ACh relaxation caused by ANG II. Aortic rings of C57BL/6 mice were incubated in DMEM in the presence of vehicle (deionized H(2)O), ANG II (100 nmol/l), recombinant mouse IL-10 (300 ng/ml), or both ANG II and IL-10 for 22 h at 37 degrees C. After incubation, rings were mounted in a wire myograph to assess endothelium-dependent vasorelaxation to cumulative concentrations of ACh. Overnight exposure of aortic rings to ANG II resulted in blunted ACh-induced vasorelaxation compared with that shown in untreated rings (maximal response = 44 +/- 3% vs. 64 +/- 3%, respectively; P<0.05). IL-10 treatment significantly restored this impairment in relaxation (63 +/- 2%). In addition, the NADPH oxidase inhibitor apocynin restored the impairment in relaxation (maximal response = 76 +/- 3%). Western blotting showed increased gp91(phox) expression (a subunit of NADPH oxidase) in response to ANG II. Vessels treated with a combination of ANG II and IL-10 showed decreased expression of gp91(phox). Immunohistochemical analysis showed increased gp91(phox) expression in ANG II-treated vessels compared with those treated with combined ANG II and IL-10. We found that the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation in response to long-term incubation with ANG II via decreasing NADPH oxidase expression.     

Acute exercise enhances receptor-mediated endothelium-dependent vasodilation by receptor upregulation

The effects of acute exercise on receptor-mediated endothelium-dependent vasodilation and its possible mechanisms were investigated in the presence of indomethacin. Male Wistar rats (16–20 weeks old) were divided into control and exercise groups. The exercise group ran on a drum exerciser until exhaustion, followed by immediate decapitation. Acetylcholine (ACh)- or clonidine (CLO)-induced vasodilating responses in thoracic aortae of the control and exercise groups were compared. Receptor-binding assays were performed to determine whether there were any upregulations of endothelial receptors after acute exercise. Our results indicated that acute exercise induced the following effects: (1) the dose-response curves of ACh and CLO shifted to the left; (2) the high-affinity M₃ binding sites increased in number but not in affinity; (3) the ₂ binding sites decreased in number but increased in affinity. We conclude that acute exercise enhances receptor-mediated vasodilation responses, at least in part, by regulating either endothelial receptor number or receptor affinity.