Martin McIntyre

Superoxide Anion Production Is Increased in a Model of Genetic Hypertension: Role of the Endothelium

The hypothesis that the decreased nitric oxide (NO) availability observed in spontaneously hypertensive stroke-prone rats (SHRSP) is due to excess superoxide (O2-) was examined. O2- generation, measured by lucigenin chemiluminescence, was studied in 12- to 16-week male and female Wistar-Kyoto rats (WKY) and SHRSP. In addition, expression of the gene encoding endothelial NO synthase, the enzyme involved in NO generation, was investigated. O2- generation was increased in male and female SHRSP (4.11+/-0.24 and 3. 84+/-0.28 nmol O2-. min-1. mg-1 respectively) compared with their WKY counterparts and was significantly higher in male than female WKY (1.22+/-0.08 in males and 0.8+/-0.08 nmol O2-. min-1. mg-1 respectively) (SHRSP versus WKY P<0.0001, 95% CI -3.39, -2.51; male versus female WKY P=0.0029, 95% CI -0.67, -0.17). Removal of the endothelium by rubbing or addition of NO synthase inhibitors attenuated O2- generation in SHRSP but not WKY. In males, removal of the endothelium reduced O2- generation from 3.86+/-0.12 to 1.35+/-0. 08 nmol. min-1. mg-1 (P<0.0001, 95% CI 2.29, 2.81), whereas addition of L-NAME caused a reduction from 4.13+/-0.17 to 1.32+/-0.16 nmol. min-1. mg-1 (P<0.0001, 95% CI 2.36, 2.83). Similar reductions were observed in females. L-arginine had no significant effect, but tetrahydrobiopterin significantly decreased O2- generation in SHRSP from 4.04+/-0.11 to 2.36+/-0.40 nmol. min-1. mg-1 (P=0.0026, 95% CI 0.89, 2.44). Endothelial NO synthase mRNA expression was significantly greater in SHRSP than in WKY and in WKY males than in WKY females. These results show that O2- generation is increased in SHRSP and that the tissue and enzymatic sources of this excess O2- appear to be the endothelium and eNOS, respectively. The increase in O2- generation could explain the decreased availability of basal NO observed in this model of genetic hypertension.

Superoxide Excess in Hypertension and Aging: A Common Cause of Endothelial Dysfunction

There is evidence in humans that hypertension and aging similarly impair endothelial function, although the mechanism remains unclear. Superoxide anion (O2−) is a major determinant of nitric oxide (NO) bioavailability and thus endothelial function. We sought to determine the relationship between endothelial function, O2−, and age in normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Aortic rings were removed from female WKY and SHRSP at 3 to 4 months (young) and 9 to 12 months (old). O2− generation by aortic rings was measured before and after removal of the endothelium or incubation with NG nitro-l-arginine methyl ester, diphenyleneiodonium, or apocynin. Levels of p22phox were studied with immunohistochemistry and used as a marker of NAD(P)H oxidase expression. NO bioavailability was significantly lower in old WKY compared with young WKY (P =0.0009) and in old SHRSP compared with young SHRSP (P =0.005). O2− generation was significantly greater in old WKY compared with young WKY (P =0.0001). Removal of the endothelium and NG nitro-l-arginine methyl ester treatment resulted in a significant reduction in O2− generation in old SHRSP (P =0.009 and 0.001, respectively). Diphenyleneiodonium significantly reduced O2− generation in 12-month WKY (P =0.008) and 12-month SHRSP (P =0.009). Apocynin attenuated O2− generation by older WKY (P =0.038) and SHRSP (P =0.028). p22phox was increased in older animals compared with young. We conclude that NO bioavailability decreases with age in female WKY and SHRSP. O2− generation increases with age in WKY and is higher in SHRSP and may contribute to the reduced NO by scavenging. NAD(P)H oxidase may contribute to the age-related increase in O2−.

Endothelial Function in Hypertension: The Role of Superoxide Anion

Much attention has been focused on the role of nitric oxide in hypertension and cardiovascular disease. More recently, the role of superoxide anion and its interaction with nitric oxide has been investigated in this context. This review will concentrate on the role of superoxide in human and experimental hypertension, paying particular attention to the potential sources of superoxide within the vasculature and discussing some of the molecular mechanisms surrounding its production and dismutation. We discuss what is known about the human superoxide dismutase enzymes. We conclude that the balance between nitric oxide and superoxide is more important than the absolute levels of either alone.

Effects of nitric oxide and superoxide on relaxation in human artery and vein

Endothelium-derived relaxing and contracting factors play an important role in atherosclerosis, re-stenosis and graft survival. Internal thoracic artery (ITA) and saphenous vein (SV) are used as conduit vessels in coronary artery bypass graft surgery (CABG). The long-term graft patency rate is higher with ITA than SV. Effects of nitric oxide and superoxide on vascular relaxation in isolated rings of ITA and SV from patients undergoing CABG were investigated. NG-nitro-L-Argenine methylester (L-NAME) was used to block nitric oxide synthesis and superoxide dismutase (SOD) and tiron to scavenge superoxide. Responses to carbachol were taken as a measure of stimulated nitric oxide release and increased responses to phenylephrine after addition of L-NAME as a measure of basal nitric oxide release. Immunocytochemical demonstration of endothelial nitric oxide synthase was performed using anti-endothelial nitric oxide synthetase (anti-eNOS) NOS antibody. Stimulated nitric oxide release was observed in ITA and SV but basal release was reduced or absent in SV. Treatment with SOD and tiron potentiated carbachol stimulated relaxation in ITA and SV. Tiron treatment resulted in a significant increase in basal nitric oxide in veins. eNOS immunoreactivity was more intense in ITA than SV, compatible with reduced nitric oxide production in veins. This may contribute to the reduced patency of venous grafts.

Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo

To assess the vascular and cardiac response to NO (nitric oxide) synthase (NOS) blockade in vivo, Wistar-Kyoto rats (WKY) were treated for 3 wk with N G-nitro-l-arginine methyl ester (l-NAME; 10 mg ⋅ kg-1 ⋅ day-1).l-NAME treatment induced hypertension that was associated with increased plasma renin activity. Flow cytometry cell cycle DNA analysis showed that aortic vascular smooth muscle cells (VSMC) froml-NAME-treated WKY had a significantly higher polyploid population compared with WKY controls. Using organ bath experiments, we have shown that aortic rings froml-NAME-treated WKY have an increased contractile response to phenylephrine and impaired relaxation to carbachol compared with control rings. NOS blockade in vivo caused a significant increase in cardiac and left ventricular hypertrophy. Northern mRNA analysis of the myocardium showed thatl-NAME treatment caused reexpression of the fetal skeletal α-actin isoform without alterations in collagen type I expression, a pattern indicating true hypertrophy of the cardiomyocytes. These studies provide further insight to confirm that NO deficiency in vivo results in the development of vascular and cardiac hypertrophy.

Nitric oxide and cardiovascular disease.

Endothelium-derived nitric oxide is an important regulatory molecule in cardiovascular function. Reduced availability of nitric oxide has been implicated in the pathogenesis of hypertension and atherosclerosis.