James W. E. Rush

Chronic Resveratrol Enhances Endothelium-Dependent Relaxation but Does Not Alter eNOS Levels in Aorta of Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHRs) were administered the red wine polyphenol resveratrol in drinking water at 0, 0.448, or 4.48 mg/l (control, low, or high, respectively) for 28 days. The low dosage was chosen to mimic moderate red wine consumption. After the treatment period, thoracic aorta rings were excised for in vitro assessment of vasomotor function. Chronic resveratrol significantly improved endothelium-dependent relaxation to acetylcholine (Ach), increasing maximal values to 80.8% ± 5.2% and 80.8% ± 5.0% in low and high groups, respectively, compared with 60.7% ± 1.4% in controls (P < 0.01). This treatment effect was eliminated in the presence of the endothelial nitric oxide synthase (eNOS) blocker Nω-nitro-l-arginine methyl ester. Resveratrol did not affect relaxation to sodium nitroprusside or systolic blood pressure in SHRs. In contrast to the SHR results, chronic resveratrol in Sprague Dawley rats did not affect vasomotor function in aorta rings in response to Ach. Hydrogen peroxide was reduced in the SHR thoracic aorta by a high dosage of resveratrol (P < 0.05), but it was not significantly altered in other tissues tested. Thoracic aorta immunoblots revealed no significant treatment effects in SHRs on eNOS, superoxide dismutases 1 and 2, gp91phox, or Hsp90. Thus, these data provide novel evidence of improved endothelium-dependent vasorelaxation in hypertensive, but not normotensive, animals as a result of chronic resveratrol consumption mimicking dosages resulting from moderate red wine consumption. This response was not dependent on increases in eNOS expression but was dependent on improved NO bioavailability.

Flow regulation of ecNOS and Cu/Zn SOD mRNA expression in porcine coronary arterioles

The purpose of this study was to test the hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and Cu/Zn superoxide dismutase (SOD) mRNA expression. Single porcine coronary arterioles (ID 100-160 μm; pressurized) were cannulated, perfused, and exposed to intraluminal flow sufficient to produce maximal flow-induced dilation of coronary arterioles (high flow; 7.52 ± 0.22 μl/min), low flow (0.84 ± 0.05 μl/min), or no flow for 2 or 4 h. Mean shear stress was calculated to be 5.7 ± 1.0 dyn/cm2 for high-flow arterioles and 1.6 ± 1.0 dyn/cm2 for low-flow arterioles. At the end of the treatment period, mRNA was isolated from each vessel, and ecNOS and SOD mRNA expression was assessed using a semiquantitative RT-PCR. All data were standardized by coamplifying ecNOS or SOD with glyceraldehyde-3-phosphate dehydrogenase. The results indicate that ecNOS mRNA expression is increased in arterioles exposed to 2 or 4 h of high flow. In contrast, SOD mRNA expression was increased only after 4 h of high flow. Neither gene is induced by exposure to low flow. On the basis of these data, we concluded that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, we concluded that a threshold level of flow and shear stress must be sustained to elicit the upregulation of ecNOS and SOD mRNA expression.The purpose of this study was to test the hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and Cu/Zn superoxide dismutase (SOD) mRNA expression. Single porcine coronary arterioles (ID 100-160 micrometers; pressurized) were cannulated, perfused, and exposed to intraluminal flow sufficient to produce maximal flow-induced dilation of coronary arterioles (high flow; 7.52 +/- 0.22 microliter/min), low flow (0.84 +/- 0.05 microliter/min), or no flow for 2 or 4 h. Mean shear stress was calculated to be 5.7 +/- 1.0 dyn/cm2 for high-flow arterioles and 1. 6 +/- 1.0 dyn/cm2 for low-flow arterioles. At the end of the treatment period, mRNA was isolated from each vessel, and ecNOS and SOD mRNA expression was assessed using a semiquantitative RT-PCR. All data were standardized by coamplifying ecNOS or SOD with glyceraldehyde-3-phosphate dehydrogenase. The results indicate that ecNOS mRNA expression is increased in arterioles exposed to 2 or 4 h of high flow. In contrast, SOD mRNA expression was increased only after 4 h of high flow. Neither gene is induced by exposure to low flow. On the basis of these data, we concluded that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, we concluded that a threshold level of flow and shear stress must be sustained to elicit the upregulation of ecNOS and SOD mRNA expression.

Plasma glutathione peroxidase in healthy young adults: influence of gender and physical activity

OBJECTIVES Oxidative stress is implicated in the pathophysiology of many cardiovascular diseases. Plasma glutathione peroxidase (pl x GPx, GPx3) is an antioxidant enzyme found in the extracellular fluid. This study aimed to determine reference values for serum GPx3 concentration and GPx activity in young healthy males and females of similar aerobic fitness and to determine the impact of acute physical activity on serum GPx. DESIGN AND METHODS 52 young, healthy but not physically trained subjects (24 male, 28 female; age = 20.4 +/- 0.7 yr, cycling VO(2 max) = 39.2 +/- 1.6 mL/kg/min; mean +/- SE) participated in this study. An independent group of 18 subjects participated in an acute, 90 min bout of 50% VO(2 max) cycling exercise. Serum GPx activity and GPx3 protein levels, as well as estradiol and 8-iso- prostaglandin F(2alpha) (8-iso-PGF(2alpha;) an index of lipid peroxidation) were determined. RESULTS Females had significantly higher serum GPx3 concentration (29.1 +/- 1.6 vs. 24.2 +/- 1.3 mg/L, p < 0.01) and serum GPx activity (256.4 +/- 10.4 vs. 222.8 +/- 15.6 U/L, p < 0.05) than males; specific activity (U/mg) was not different between genders. There was no significant gender difference in 8-iso-PGF(2alpha). No significant correlation was found between either GPx activity or GPx3 concentration and serum estradiol or VO(2)(max). The acute, prolonged, mild intensity exercise did not affect serum GPx activity or 8-iso-PGF(2a) levels in males or females. CONCLUSIONS The results of this study suggest that in a young, healthy but not physically well-trained population females are endowed with slightly higher serum GPx3 concentrations and GPx activities than males, but the functional significance of this has not been established. Furthermore, the results indicate that serum GPx levels are not associated with aerobic fitness level, or serum estradiol concentration and that acute, prolonged, mild exercise does not affect the activity of serum GPx in this population.

Encapsulation-stucturing of edible oil attenuates acute elevation of blood lipids and insulin in humans

Blood triglyceride, free fatty acid and insulin levels are lower after acute intake of an oil-water-monoglyceride gel versus an oil-water mixture, demonstrating that food matrix nanostructure and microstructure can be engineered to modulate the physiological response. Oil emulsification by the monoglyceride Lα liquid-crystalline lamellar phase, followed by droplet wall crystallization, encapsulates oil and creates a material with the functionality and properties of a fat. This novel phase is devoid of trans fatty acids and can be manufactured with as little as 4% added saturated monoglyceride.

Vascular biology of angiotensin and the impact of physical activity

The renin-angiotensin system (RAS) is important for regulating blood pressure and extracellular fluid. The concept of the RAS has recently evolved from a classical systemic endocrine system to an appreciation of local RASs functioning in a paracrine manner, including in the vascular wall. Angiotensin II (AII), the main effector of the RAS, is a potent vasoconstrictor formed by the action of angiotensin-converting enzyme (ACE). ACE is multifunctional and also destroys the endogenous vasodilator bradykinin. A recently discovered novel ACE2 enzyme is responsible for forming a vasodilatory compound, angiotensin 1-7, from AII. Thus, the actions of ACE and ACE2 are antagonistic. Tissue actions of AII are mediated by specific receptors, AT1 and AT2, with AT1 mediating the classical actions. AT1-stimulated vasoconstricton occurs via phospholipase-D-mediated second messenger generation directly, and indirectly via the coupling of AT1 to the prooxidant enzyme NADPH oxidase. Since the vascular NADPH oxidase is a major source of vascular reactive oxygen species generation and is responsible for the breakdown of the vasodilator nitric oxide (NO), there is another potential link between RAS and regulation of vasodilatory pathways. AT2 signaling is antagonistic to AT1 signaling, and results in bradykinin and NO formation. Chronic AII signaling induces vascular dysfunction, whereas pharmacological management of the RAS can not only control blood pressure, but also correct endothelial dysfunction in hypertensives. Exercise training can also improve endothelial function in hypertensives, raising the question of whether there is a potential role for RAS in mediating the vascular effects of exercise training. Recent studies have demonstrated reductions in the expression of NADPH oxidase components in the vascular wall in response to exercise training, thus tempering one of the main cellular effectors of AII, and this is associated with reduced vascular ROS production and enhanced NO bioavailability. Importantly, it has now been demonstrated in human arteries that exercise training also tempers vascular AT1 receptor expression and AII-induced vasoconstriction, while enhancing endothelium-dependent dilation. The signals responsible for these chronic adaptations are not clearly understood, and may include changes in RAS components prompted by acute exercise. ACE genotype may have an effect on physical activity levels and on the cardiovascular responses to exercise training, and the II genotype (compared with ID and DD) is associated with the largest endothelium-dependent dilations in athletes compared with those in sedentary individuals. Thus, the tissue location of the RAS, the complement of ACE/ACE2, the receptor expression of AT1/AT2, and the ACE genotype are all variables that could impact the vascular responses to exercise training, but the responses of most of these variables to regular exercise training and the mechanisms responsible have not been systematically studied.

Myoadenylate deaminase deficiency does not affect muscle anaplerosis during exhaustive exercise in humans.

1 Myoadenylate deaminase (AMPD) deficiency is present in 1‐2 % of the population. In theory, this deficiency may alter exercise energy metabolism by impairing the purine nucleotide cycle (PNC) and reducing tricarboxylic acid (TCA) cycle anaplerosis. The role of the PNC in TCA cycle anaplerosis is still a debated issue in physiology. Using patients with the AMPD1 mutation will allow a human ‘knockout’ approach to answering this question. 2 Muscle AMPD activity and genotype (whole blood AMPD1 analysis) was used to classify participants into three groups: n= 3 with absence of AMPD activity and ‐/‐ AMPD1 genotype (homozygous); n= 4 with less than 50 % normal AMPD activity and +/‐ genotype (heterozygous) and n= 12 with normal AMPD activity and +/+ genotype (control). Biopsies were taken from the vastus lateralis muscle before and after incremental cycle ergometry exercise to exhaustion. The muscle biopsies were analysed for AMPD activity, purine nucleotides/nucleosides and bases, creatine, phosphocreatine, amino acids, and the TCA cycle intermediates malate, citrate and fumarate. 3 Time to exhaustion on the cycle ergometer was not different between groups. Muscle adenosine monophosphate increased significantly with exercise for homozygous subjects as compared with the other groups (P < 0.05). Inosine monophosphate increased significantly after exercise for control (P < 0.05) but not for the homozygous subjects. There were no other between‐group differences for any other measured variables. 4 In summary, complete and partial muscle AMPD deficiency did not affect TCA cycle anaplerosis, phosphocreatine hydrolysis, energy charge or exercise performance.

Nitric oxide and coronary vascular endothelium adaptations in hypertension

This review highlights a number of nitric oxide (NO)-related mechanisms that contribute to coronary vascular function and that are likely affected by hypertension and thus become important clinically as potential considerations in prevention, diagnosis, and treatment of coronary complications of hypertension. Coronary vascular resistance is elevated in hypertension in part due to impaired endothelium-dependent function of coronary arteries. Several lines of evidence suggest that other NO synthase isoforms and dilators other than NO may compensate for impairments in endothelial NO synthase (eNOS) to protect coronary artery function, and that NO-dependent function of coronary blood vessels depends on the position of the vessel in the vascular tree. Adaptations in NOS isoforms in the coronary circulation to hypertension are not well described so the compensatory relationship between these and eNOS in hypertensive vessels is not clear. It is important to understand potential functional consequences of these adaptations as they will impact the efficacy of treatments designed to control hypertension and coronary vascular disease. Polymorphisms of the eNOS gene result in significant associations with incidence of hypertension, although mechanistic details linking the polymorphisms with alterations in coronary vasomotor responses and adaptations to hypertension are not established. This understanding should be developed in order to better predict those individuals at the highest risk for coronary vascular complications of hypertension. Greater endothelium-dependent dilation observed in female coronary arteries is likely related to endothelial Ca2+ control and eNOS expression and activity. In hypertension models, the coronary vasculature has not been studied extensively to establish mechanisms for sex differences in NO-dependent function. Genomic and nongenomic effects of estrogen on eNOS and direct and indirect antioxidant activities of estrogen are discussed as potential mechanisms of interest in coronary circulation that could have implications for sex- and estrogen status-dependent therapy for hypertension and coronary dysfunction. The current review identifies some important basic knowledge gaps and speculates on the potential clinical relevance of hypertension adaptations in factors regulating coronary NO function.

Endothelium-dependent vasorelaxation to the AMPK activator AICAR is enhanced in aorta from hypertensive rats and is NO and EDCF dependent

Activation of AMP-activated protein kinase (AMPK) induces vasorelaxation in arteries from healthy animals, but the mechanisms coordinating this effect are unclear and the integrity of this response has not been investigated in dysfunctional arteries of hypertensive animals. Here we investigate the mechanisms of relaxation to the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) in isolated thoracic aorta rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Although AICAR generated dose-dependent (10(-6)-10(-2) M) relaxation in precontracted WKY and SHR aortic rings with (E(+)) or without (E(-)) endothelium, relaxation was enhanced in E(+) rings. Relaxation in SHR E(+) rings was also enhanced at low [AICAR] (10(-6) M) compared with that of WKY (57 ± 8% vs. 3 ± 2% relaxation in SHR vs. WKY E(+)), but was similar and near 100% in both groups at high [AICAR]. Pharmacological dissection showed that the mechanisms responsible for the endothelium-dependent component of relaxation across the dose range of AICAR are exclusively nitric oxide (NO) mediated in WKY rings, but partly NO dependent and partly cyclooxygenase (COX) dependent in SHR vessels. Further investigation revealed that ACh-stimulated COX-endothelium-derived contracting factors (EDCF)-mediated contractions were suppressed by AICAR, and this effect was reversed in the presence of the AMPK inhibitor Compound C in quiescent E(+) SHR aortic rings. Western blots demonstrated that P(Thr(172))-AMPK and P(Ser(79))-acetyl-CoA carboxylase (indexes of AMPK activation) were elevated in SHR versus WKY E(+) rings at low AICAR (∼2-fold). Together these findings suggest that AMPK-mediated inhibition of EDCF-dependent contraction and elevated AMPK activation may contribute to the enhanced sensitivity of SHR E(+) rings to AICAR. These results demonstrate AMPK-mediated vasorelaxation is present and enhanced in arteries of SHR and suggest that activation of AMPK may be a potential strategy to improve vasomotor dysfunction by suppressing enhanced endoperoxide-mediated contraction and enhancing NO-mediated relaxation.

Fasting triacylglycerol status, but not polyunsaturated/saturated fatty acid ratio, influences the postprandial response to a series of oral fat tolerance tests

Elevated postprandial lipemia is emerging as a risk factor for obesity-related chronic diseases, such as type 2 diabetes and cardiovascular disease, and is associated with alterations in several metabolic biomarkers of disease. Our goal was to examine the effects of specific polyunsaturated/saturated fatty acid (P/S) ratios on postprandial triacylglycerol (TAG) concentrations and metabolic biomarkers in men with different fasting TAG concentrations through a series of oral fat tolerance tests (OFTT) consisting solely of emulsified lipid. Otherwise healthy men with high (>1.69 mmol/L) fasting TAG (HTAG, n=8) and low fasting TAG (LTAG, n=8) underwent three OFTTs with specific P/S ratios of 0.2, 1.0 and 2.0, respectively, and a total lipid load of 1 g/kg subject body mass. All subjects received each treatment separated by at least 1 week. Postprandial plasma TAG fatty acid composition reflected fatty acids present in the OFTT. All other metabolic responses were independent of the P/S ratio ingested. An accelerated increase in postprandial TAGs was observed in HTAG compared to LTAG. Interleukin (IL)-6 and soluble intercellular adhesion molecule (sICAM)-1 were significantly elevated in HTAG at baseline (P<.05). IL-6 increased significantly following each OFTT (P<.05) in both groups. Postprandial glucose and CRP were significantly exaggerated (P<.05) in HTAG. Overall, HTAG subjects had an accelerated postprandial TAG response and increased concentrations of several inflammatory markers following an OFTT, in the absence of an insulin response. However, P/S ratio had no influence on postprandial lipid and inflammatory parameters.

Cyclooxygenase and thromboxane/prostaglandin receptor contribute to aortic endothelium-dependent dysfunction in aging female spontaneously hypertensive rats

Cyclooxygenase (COX)-derived vasoconstrictory prostanoids contribute to impaired endothelium-dependent vasorelaxation in aging male (m) spontaneously hypertensive rats (SHR); however, vasomotor responses in aging female (f) SHR and sex differences in aging SHR are unknown. Examining mechanisms governing dysfunction in aging fSHR will contribute to understanding sex-dependent vascular complications in advanced hypertension. Aortic endothelium-dependent relaxation dose responses (ACh) of 16- and 30-wk-old mSHR and fSHR and normotensive Wistar-Kyoto rats were examined in the absence (no drug control) and presence of COX inhibition [indomethacin (Indo)] and thromboxane/prostaglandin receptor inhibition (SQ-29548). No drug control-treated 16-wk mSHR exhibited considerable blunting of the peak relaxation response to ACh (e.g., 77 +/- 4% relaxation to 10(-5) mol/l) vs. Wistar-Kyoto controls (89 +/- 6%), and greater dysfunction occurred in 30-wk mSHR (63 +/- 2%). Interestingly, ACh relaxations of fSHR were unimpaired at 16 wk (101 +/- 2% to 10(-5) mol/l), but blunted in 30 wk (76 +/- 4%). Indo and SQ-29548 restored robust ACh vasorelaxation in all groups (e.g., 113 +/- 3 and 112 +/- 3%, respectively, in Indo- and SQ-29548-treated 30-wk fSHR). Aortic COX-1 protein expression was elevated by 75% in 30-wk vs. 16-wk fSHR, whereas group-averaged ACh-stimulated aortic PGI(2) release (assessed as 6- keto-PGF(1alpha)) was 30% greater in 30-wk vs. 16-wk fSHR (9,926 +/- 890 vs. 7,621 +/- 690 pg.ml(-1).mg dry wt(-1)), although this did not reach significance (P = 0.0758). Dramatic deterioration of endothelium-dependent vasomotor function in fSHR across this age range involves COX and thromboxane/prostaglandin receptor, supporting a mechanism of impairment similar to that which occurs in aging mSHR.