Effects of the reactive oxygen species hydrogen peroxide and hypochlorite on endothelial nitric oxide production.

Reactive oxygen species (ROS) hydrogen peroxide (H(2)O(2)) and hypochlorite (HOCl) participate in the pathogenesis of ischemia/reperfusion injury, inflammation, and atherosclerosis. Both NO and ROS are important modulators of vascular tone and architecture and of adhesive interactions between leukocytes, platelets, and vascular endothelium. We studied the effect of H(2)O(2) and HOCl on receptor-dependent (bradykinin [10(-6) mol/L] and ADP [10(-4) mol/L]) and receptor-independent mechanisms (calcium ionophore A23187 [10(-6) mol/L]) of NO production by porcine aortic endothelial cells (ECs). Changes in the level of EC cGMP (the second messenger of NO) were used as a surrogate of NO production. EC cGMP increased 300% in response to bradykinin and A23187 and 200% in response to ADP. Exposure of ECs to H(2)O(2) (50 micromol/L) for 30 minutes significantly impaired cGMP levels in response to ADP, bradykinin, and the receptor-independent NO agonist A23187. In contrast, preincubation with HOCl (50 micromol/L) impaired cGMP production only in response to ADP and bradykinin but not A23187. These concentrations of H(2)O(2) and HOCl did not result in increased EC lethality as assessed by lactate dehydrogenase release. Neither H(2)O(2) nor HOCl affected EC cGMP production in response to NO donor sodium nitroprusside, which suggests that guanylate cyclase is resistant to these oxidants. We also demonstrated that neither H(2)O(2) nor HOCl affects endothelial NO synthase (eNOS) catalytic activity as measured by conversion of L-arginine to L-citrulline in EC homogenates supplemented with eNOS cofactors. The present studies show that H(2)O(2) impairs NO production in response to both receptor-dependent and receptor-independent agonists and that these effects are due, at least in part, to inactivation of eNOS cofactors, whereas HOCl inhibits NO production by interfering with receptor-operated mechanisms at the level of the cell membrane. Concentrations of H(2)O(2) and HOCl used in the present studies have been shown to be generated in vivo during inflammation and ischemia/reperfusion. Therefore, we infer that these effects of H(2)O(2) and HOCl on EC NO production may contribute to disregulated vascular tone and altered leukocyte-EC interactions that occur in vascular injury as a result of those causes in which ROS generation is involved.

Cigarette smoke-induced endothelium dysfunction: role of superoxide anion.

OBJECTIVES: Cigarette smoking is strongly associated with coronary artery disease and atherosclerosis. While smoking has been shown to impair endothelium-dependent vasorelaxation, the mechanisms involved are not completely understood. We investigated the role of superoxide anion and vasoconstricting prostanoids in cigarette smoke induced endothelial dysfunction. METHODS: Endothelial function was assessed in rat aortic rings exposed to cigarette smoke-treated Krebs buffer, by measuring agonist stimulated endothelium-dependent vasorelaxation. Treatment with superoxide dismutase (SOD) as well as ifetroban, thromboxane A2/prostaglandin endoperoxide H2 (TxA2/PGH2) receptor blocker and indomethacin (cyclooxygenase inhibitor) was used to investigate the role of superoxide anion and vasoconstricting eicosanoids on cigarette smoke-induced endothelial dysfunction. The effect of cigarette smoke on endothelial nitric oxide synthase (eNOS) catalytic activity was measured by conversion of L-arginine to L-citrulline in rat aortas and rat endothelial cell homogenates supplemented with eNOS cofactors. RESULTS: Relaxations to receptor-dependent agonists, acetylcholine and adenosine diphosphate (ADP), as well as to a receptor-independent agonist, A23187 (Ca2+ ionophore) were significantly impaired by cigarette smoke. Cigarette smoke did not impair relaxations to sodium nitroprusside, indicating preserved guanylate cyclase activity. Further, cigarette smoke did not affect eNOS catalytic activity in homogenates from either endothelial cells or aortas previously exposed to cigarette-smoketreated Krebs buffer. Treatment with SOD or ifetroban and in a lesser degree by indomethacin prevented cigarette-smoke-induced endothelial dysfunction. CONCLUSIONS: Taken together, our results suggest that cigarette smoking causes an increase in vascular superoxide production which results in decreased nitric oxide (NO) bioactivity and concomitantly increases production of cyclooxygenase dependent and independent vasoconstricting eicosanoids.

Workshop: hypertension and cardiovascular risk factors: role of the angiotensin II-nitric oxide interaction.

Vascular upregulation of nitric oxide (NO) is an adaptive response to increased blood pressure that may help in the prevention of end-organ damage. Differences in cardiovascular and renal morbidity and mortality in hypertensive patients may result, at least in part, from individual variations in endothelial function in response to the hemodynamic workload of hypertension. A functional feedback balance exists between both angiotensin (Ang) II and NO under normal conditions. The NO-Ang II imbalance may not explain all the vascular pathophysiology of hypertension, but it certainly appears to be an important component. In hypertension, salt sensitivity, whether primary (ie, certain populations in the United States and Japan) or secondary (ie, aging, type II diabetes), appears to be a marker of increased cardiovascular and renal risk that is often linked to a decreased bioactivity of NO. In diabetes and atherosclerosis, NO-dependent vascular relaxation is impaired and can be restored by decreasing the synthesis and/or blocking the action of Ang II. An understanding of the relations between hypertension, cardiovascular risk factors, end-organ damage, and the NO-Ang II axis leads one to believe that the combination of therapeutic agents capable of reinstating the homeostatic balance of these vasoactive molecules within the vessel wall would be most effective in preventing or arresting end-organ disease.

Antifibrotic effects of N-acetyl-seryl-aspartyl-Lysyl-proline on the heart and kidney in aldosterone-salt hypertensive rats.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits not only hematopoietic cell proliferation but also fibroblast proliferation and collagen synthesis in vitro. Ac-SDKP also prevents collagen deposition and cell proliferation in the left ventricle (LV) in rats with renovascular hypertension (renin dependent). However, it is not clear whether Ac-SDKP has similar effects in a model of renin-independent hypertension (aldosterone-salt). Using a hypertensive rat model of cardiac and renal fibrosis created by chronic elevation of circulating aldosterone (ALDO) levels, we examined the effect of Ac-SDKP on blood pressure, cardiac and renal fibrosis and hypertrophy, and proliferating cell nuclear antigen (PCNA) expression in the LV and left kidney. Uninephrectomized rats were divided into 4 groups: (1) controls that received tap water, (2) rats that received ALDO (0.75 microgram/h SC) and 1% NaCl/0.2% KCl in drinking water (ALDO-salt), (3) rats that received ALDO-salt plus Ac-SDKP 400 microgram. kg(-1). day(-1) SC, and (4) rats that received ALDO-salt plus Ac-SDKP 800 microgram. kg(-1). d(-1) SC. After 6 weeks of treatment, the ALDO-salt group was found to have significantly increased blood pressure with decreased body weight and plasma renin concentration (P<0.05), LV and renal hypertrophy as well as renal injury, significantly increased collagen content in both ventricles and kidney as well as increased collagen volume fraction in the LV (P<0.0001), and significantly increased interstitial and perivascular PCNA-positive cells in the LV and kidney (P<0.0001). Ac-SDKP at 800 microgram. kg(-1). d(-1) markedly prevented cardiac and renal fibrosis (P<0.005) without affecting blood pressure or organ hypertrophy. It also suppressed PCNA expression in the LV and kidney in a dose-dependent manner. We concluded that Ac-SDKP prevents increased collagen deposition and cell proliferation in the heart and kidney in ALDO-salt hypertensive rats. Because ACE inhibitors increase plasma and tissue Ac-SDKP and decrease cardiac and renal fibrosis, we speculate that Ac-SDKP may participate in the antifibrotic effect of ACE inhibitors.

Postmenopausal hypertension.

Cardiovascular disease is the leading cause of death in women and claims the lives of more than half a million women every year. Hypertension is one of the most prevalent and powerful contributors to atherosclerotic cardiovascular disease. Hypertension affects more men than women until 55 years of age, but after age 55, the percentage of women is higher. Estrogen deficiency has been linked to the rapid increase in cardiovascular disease in women who have undergone natural or surgical menopause. Hormone replacement therapy (HRT) has been shown to decrease the incidence of cardiovascular disease and, in some studies, to reduce blood pressure in postmenopausal women. However, little information is available on the effects of HRT on blood pressure in hypertensive postmenopausal patients. The cardioprotective effects of estrogens are not completely understood but may involve direct effects on blood vessels through modulation of endogenous vasoconstrictors and vasodilators and through reductions in serum lipoprotein and cholesterol levels. Experimental evidence suggests that estrogen increases the biological actions of nitric oxide and decreases the actions of angiotensin. After menopause, loss of the vascular protective effects of estrogens may unmask a population of women particularly prone to hypertension who would be at higher risk for cardiovascular disease.

Nitric oxide--angiotensin II axis in renal and cardiovascular injury.

The knowledge and the information about Renin Angiotensin System and Arginine-Nitric Oxide pathway helped us in understanding more about cardiac, vascular and renal systems, their function, the physiology, and pathophysiology of different diseases that may affect them. The existence of a Nitric Oxide/Angiotensin II axis, brought modification to our ways of thinking with new and more pathophysiological approaches to the treatment of hypertension and end-organ disease. There appears to be a link between hypertension, salt sensitivity, endothelial function and NO-AnglI axis on one side and end organ damage on the other side. Anti-hypertensive agents that can restore endothelial function beside their antihypertensive effect, may be better in preventing or at least slowing end-organ damage.

Relationship between hypercholesterolaemia, endothelial dysfunction and hypertension.

OBJECTIVES: We have previously shown that in the rat a diet high in cholesterol and deficient in vitamin E and selenium results in hypercholesterolaemia and increased lipid oxidation. We utilized this model to determine whether rats given this diet develop impaired endothelium-dependent relaxation mediated by nitric oxide (NO) in mesenteric and in renal vessels. In addition, we tested whether the impairment is due to (i) decreased endothelial NO synthase activity, (ii) increased NO inactivation and/or (iii) increased production of the endothelium-derived constricting factors thromboxane A2/prostaglandin H2 and endothelin-1. We also investigated whether endothelial dysfunction induced by dyslipidaemia increases the sensitivity for the development of hypertension in response to high dietary salt. METHODS: Male Dahl salt-sensitive (DSS) rats were divided into three groups and received a standard diet (control group), a high (4%) cholesterol diet (HChol), or a high cholesterol diet deficient in the anti-oxidants vitamin E and selenium (HChol-Def). The NaCl content of these diets was 0.5%. After 18 weeks we studied endothelium-dependent relaxation in response to acetylcholine (ACh) in aortas and in isolated perfused preparations of mesenteric arteries and kidneys. In some experiments, ifetroban, a thromboxane A2/prostaglandin H2 receptor antagonist, was added to the organ bath or the perfusion buffer. Vascular responses to endothelin-1 as well as to BQ-123, an endothelin A receptor blocker, were studied in the isolated perfused kidneys. In addition, two extra groups of rats were fed a diet high in sodium chloride (2%): one of the groups received the normal cholesterol diet whereas the other group received the diet high in cholesterol and deficient in vitamin E and selenium. RESULTS: Compared to normocholesterolemic rats, responses to ACh were significantly impaired in aortas, mesenteric arteries and kidneys of HChol-Def rats (P < 0.01). Endothelial NO synthase activity (conversion of [14C]L-arginine to [14C]L-citrulline) was similar in aortas of control, HChol and HChol-Def rats; thus suggesting that impaired endothelium-dependent relaxation in the HChol-Def rats was not due to decreased cNOS catalytic activity. Ifetroban improved the impaired endothelium-dependent relaxation in mesenteric vessels, but not in aortas and kidneys. Endothelin-1 (ET-1: 10(-13)-10(-11) mol/l) elicited NO-mediated relaxations in kidneys of control rats but not in kidneys of HChol-Def; blockade of ET-1 with BQ-123, an ET(A) receptor blocker, did not improve NO-mediated relaxation of HChol-Def. Despite impaired endothelium-dependent relaxation in renal and mesenteric vessels, HChol-Def DSS rats failed to develop hypertension (systolic blood pressure 144 +/- 1 in control and 150 +/- 2 mmHg in HChol-Def) but manifested a significant increase in sensitivity to the pressor effects of a high (2% NaCl) dietary salt content during the initial 10 weeks of the study, although the final blood pressure at 18 weeks was similar in both groups. CONCLUSION: These studies support the notion that (i) products of lipid oxidation may reduce NO bioactivity without affecting endothelial NO synthase mass or catalytic activity, (ii) the mechanisms involved in the endothelial dysfunction induced by hypercholesterolaemia and oxidized lipids may differ among vascular beds, and (iii) decreased NO bioavailability does not necessarily result in systemic hypertension, but it may enhance the sensitivity to the hypertensinogenic effect of dietary salt.

Nitric oxide, salt sensitivity, and cardiorenal injury in hypertension.

A connection between salt sensitivity and hypertension seems certain, but a number of issues remain unresolved, e.g., the mechanisms involved in salt sensitivity, its role in pathogenesis and/or maintenance of hypertension, and its ability to predict cardiorenal risk. The role of nitric oxide (NO) has been studied extensively. Evidence shows that NO, along with the vasoactive substances angiotensin II and endothelin-1, is important in modulating vascular tone. In addition, imbalances among these substances may participate in the abnormal cardiovascular and renal remodeling that occurs in hypertension. What causes such imbalances remains unclear. Animal studies show that in salt-sensitive subjects, the activity of NO synthase (NOS) fails to upregulate in response to increases in blood pressure, but that such activity upregulates rapidly in similar circumstances in non-salt-sensitive subjects. Human studies of essential hypertensives have shown an association between salt sensitivity and a number of conditions, including impaired endothelium-dependent relaxation mediated by NO, insulin resistance, microalbuminuria, and ventricular hypertrophy and cardiovascular events. These findings have intriguing implications in regard to whether, in hypertension, salt sensitivity might be a marker of increased cardiovascular and renal risk that is linked to abnormalities in the bioactivity of NO.

Angiotensin II, nitric oxide, and end-organ damage in hypertension.

The adaptive changes that accompany hypertension and involve the kidney, heart, and vessels, namely, muscle hypertrophy/hyperplasia, endothelial dysfunction and extracellular matrix increase can, in fact, be maladaptive and eventually lead to end-organ disease, such as renal failure, heart failure, and coronary disease. However, these changes vary markedly between individuals with similar levels of hypertension. Nitric oxide (NO), an endogenous vasodilator and inhibitor of vascular smooth muscle and mesangial cell growth, is synthesized in the endothelium by a constitutive NO synthase (NOS). NO antagonizes the effects of angiotensin II on vascular tone and growth and also down-regulates the synthesis of angiotensin converting enzyme (ACE) and angiotensin II type 1 (AT-1) receptors. In hypertension, the physiologic response to the increased shear stress and cyclic strain is to upregulate NOS activity in endothelial cells. Upregulation of vascular NOS activity is a homeostatic adaptation to the increased hemodynamic workload that may help in preventing end-organ damage. Indeed, hypertension-prone salt-sensitive rats manifest a decrease (instead of an increase) in vascular NOS activity when hypertensive; these rats develop severe vascular hypertrophy, left ventricular hypertrophy, and renal injury. Studies in hypertensive humans suggest that, independent of the effects of salt on blood pressure, salt sensitivity may be a marker for susceptibility to the development of endothelial dysfunction as well as cardiovascular and renal injury. We hypothesize that in hypertension, recognition of markers of cardiovascular susceptibility to injury and the understanding of the pathophysiological mechanisms involved may open new opportunities for therapeutic intervention. In this context, only those antihypertensive agents that lower blood pressure and concomitantly restore the homeostatic balance of vasoactive agents such as angiotensin II and NO within the vessel wall would be effective in preventing or arresting end-organ disease.

Angiotensin II induces superoxide anion production by mesangial cells.

BACKGROUND: The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC. METHODS: MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. RESULTS: Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy. CONCLUSIONS: The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.

Efficacy and tolerability of losartan in hypertensive patients with renal impairment. Collaborative Group.

We evaluated the blood pressure-lowering activity, tolerability, and safety of losartan in 112 hypertensive (sitting diastolic blood pressure, 90 to 115 mm Hg) patients with chronic renal insufficiency including mild renal insufficiency (30 to 60 mL/min per 1.73 m2; n=51), moderate to severe renal insufficiency (10 to 29 mL/min per 1.73 m2; n=33), or hemodialysis (n=28). After a 3-week placebo period, once-daily losatan was administered for 12 weeks. The daily dose of 50 mg was increased to 100 mg after 4 weeks in patients whose sitting diastolic blood pressure remained > or = 90 mm Hg or was reduced by < 5 mm Hg. A second, non-angiotensin-converting enzyme inhibitor, antihypertensive drug was added after 8 weeks as needed. Twenty-four-hour creatinine clearance was determined and renal clearance studies of inulin and para-aminohippurate were done in a subset of 11 patients. Trough sitting blood pressures were reduced at the end of the first week in all groups. At weeks 4, 8, and 12, the reductions in systolic blood pressure/diastolic blood pressure averaged -11.9/-8.7, -10.8/-9.4, and -14.7/-12.1 mm Hg in patients with mild renal insufficiency; -7.7/-6.3, -13.1/-11.8, and -14.1/-10.6 mm Hg, in moderate to severe renal insufficiency; -17.0/-12.7, -19.1/-14.4, and -22.7/-18.0 mm Hg in hemodialysis. Creatinine clearance, glomerular filtration rate, and effective renal plasma flow were stable. Losartan was withdrawn in only 6 patients because ofa clinical or laboratory adverse experience. Hyperkalemia (> 6 mEq/L) requiring discontinuation of losartan occurred in only one (group 2) patient. We conclude that once-daily losartan, given as monotherapy at doses of 50 or 100 mg or in combination with other antihypertensive drugs, was effective in reducing blood pressure in hypertensive patients with chronic renal disease and that losartan regimens were well tolerated in all groups, including those on hemodialysis.

Nitric oxide in hypertension: relationship with renal injury and left ventricular hypertrophy.

Hypertension is accompanied by architectural changes in the kidney, heart, and vessels that are often maladaptive and can eventually contribute to end-organ disease such as renal failure, heart failure, and coronary disease. Nitric oxide, an endogenous vasodilator and antithrombotic agent synthesized in the endothelium by a constitutive nitric oxide synthase, inhibits growth-related responses to injury in vascular cells. Specifically, in the presence of hypertension, nitric oxide may work in the kidney by inhibiting both mesangial cell hypertrophy and hyperplasia as well as synthesis of extracellular matrix and in the heart and systemic vessels by modulating smooth muscle cell hypertrophy and hyperplasia. The effects of nitric oxide are antagonistic of the effects of angiotensin II. Shear stress and cyclic strain, physical forces known to operate in hypertension, are accompanied by increases in endothelial nitric oxide synthase expression, nitric oxide synthase protein, and nitric oxide synthase activity in endothelial cells. Experimental studies using genetic models of hypertension show a variation in hypertension-modulated vascular nitric oxide synthase activity in different strains of rats. These studies suggest that upregulation of vascular nitric oxide synthase activity is a homeostatic adaptation to increased hemodynamic workload in hypertension and that this may help prevent end-organ damage. If these findings apply to humans, differences in end-organ disease seen in patients with similar degrees of hypertension may be due in part to genetic differences in vascular nitric oxide synthase activity in response to hypertension.

Nitric oxide synthase activity and renal injury in genetic hypertension.

Nitric oxide (NO) is an endogenous vasodilator synthesized in the endothelium by constitutive NO synthase (cNOS). We have shown that upregulation of cNOS activity in hypertension may contribute to forestalling left ventricular and aortic hypertrophy (Hypertension. 29: 235, 1997). NO has been shown to inhibit growth-related responses affecting vascular smooth muscle, and mesangial cells, as well as reduce production of extracellular matrix in response to injury. Here, we investigated the relationship between renal cNOS activity (conversion of [14C] L-arginine to [14C] L-citrulline) and glomerular (GIS) and tubulointerstitial (TIS) injury scores and urinary protein excretion, indices of renal injury, in age and blood pressure matched spontaneously hypertensive rats (SHR, SBP 220+/-9 mm Hg) fed 0.5% NaCl diet and Dahl salt-sensitive (DS) rats fed 4% NaCl diet (DS-4%, SBP 228+/-8 mm Hg) as well as their normotensive counterparts Wistar Kyoto rats fed 0.5% NaCl diet (WKY, 137+/-3 mm Hg) and DS rats fed 0.5% NaCl diet (DS-0.5%, SBP 135+/-4 mm Hg). In SHR, renal medullary cNOS activity was 89% higher than in WKY (8.91+/-0.98 vs 4.71+/-0.37 nmol/min/g protein, P<0.05) whereas, in hypertensive DS-4% rats cNOS activity was 43% lower than in DS-0.5% rats (1.98+/-0.16 vs 3.48+/-0.29 nmol/min/g protein, P<0.05). Renal cortical cNOS was lower than in medulla but similar in all groups; inducible NOS activity was not detected. Despite hypertension of similar severity and duration, hypertensive DS-4% developed 9 fold more GIS (190+/-42 vs 21+/-11), 20 fold more TIS (4.0+/-0.7 vs 0.2+/-0.3), and 5 fold more proteinuria (54+/-11 vs 8.5+/-3.0 mg/day), all P<0.05. The current studies, in conjunction with our recent studies in heart and aorta, strongly suggest that in hypertension, increased cNOS activity may provide a protective homeostatic role in all the end-organs that are targets of hypertensive injury.

Nitric oxide production during Vibrio cholerae infection.

Vibrio cholerae induces massive intestinal fluid secretion that continues for the life of the stimulated epithelial cells. Enhanced regional blood flow and peristalsis are required to adapt to this obligatory intestinal secretory challenge. Nitric oxide (NO) is a multifunctional molecule that modulates blood flow and peristalsis and possesses both cytotoxic and antibacterial activity. We demonstrate that, compared with those in asymptomatic control subjects, levels of stable NO metabolites (NO2-/NO3-) are significantly increased in sera from acutely ill Peruvian patients with natural cholera infection as well as from symptomatic volunteers from the United States infected experimentally with V. cholerae. In a rabbit ileal loop model in vivo, cholera toxin (CT) elicited fluid secretion and dose-dependent increases in levels of NO2-/NO3- in the fluid (P < 0.01). In contrast, lipopolysaccharide (LPS) elicited no such effects when applied to the intact mucosa. NO synthase (NOS) catalytic activity also increased in toxin-exposed tissues (P < 0.05), predominantly in epithelial cells. The CT-induced NOS activity was Ca2+ dependent and was not suppressed by dexamethasone. In conclusion, symptomatic V. cholerae infection induces NO production in humans. In the related animal model, CT, but not LPS, stimulated significant production of NO in association with increases in local Ca(2+)-dependent NOS activity in the tissues.

Effect of doxazosin on endothelial dysfunction in hypercholesterolemic/antioxidant-deficient rats.

Hypertension, hypercholesterolemia, atherosclerosis, and coronary heart disease are associated with abnormal endothelium-dependent, nitric oxide-mediated vasorelaxation. In rats, hypercholesterolemia in combination with deficiencies of vitamin E and selenium results in increased endogenous lipid oxidation and endothelial dysfunction. Two hydroxymetabolites of doxazosin, an alpha 1-adrenergic blocking antihypertensive agent, inhibit human lipid oxidation in vitro in a dose-dependent fashion. The present studies were performed to determine the effect of in vivo treatment with doxazosin on endothelial dysfunction in hypercholesterolemic/ antioxidant-deficient rats. Dahl rats were fed 1) a standard diet, 2) a high cholesterol (4%) diet, or 3) a high cholesterol, vitamin E- and selenium-deficient diet. A subgroup of animals in each group were administered doxazosin (3.5 mg/100 g/day) for 16 weeks. In the aortas, vascular relaxations induced by acetylcholine were significantly decreased (P < .05) in high cholesterol/antioxidant-deficient rats compared with normal and high cholesterol animals. Doxazosin treatment prevented the impairment in endothelium-dependent vascular relaxation in the high cholesterol/antioxidant-deficient group. Vasorelaxation in response to the exogenous nitric oxide donor diethylamine nanoate, which was significantly impaired (P < .05) in aortas from high cholesterol/antioxidant-deficient animals compared with normal and high cholesterol animals, was normalized in aortas from high cholesterol/ antioxidant-deficient animals that had received doxazosin. The antioxidant effect of doxazosin may have therapeutic implications in diseases associated with endothelial dysfunction linked to products of lipid oxidation.

Endothelial dysfunction and cardiorenal injury in experimental salt-sensitive hypertension: effects of antihypertensive therapy.

BACKGROUND: Pharmacological control of hypertension has contributed to a significant decrease in cardiovascular morbidity and mortality, although the beneficial effect on cardiac and renal diseases has been far more modest than the reduction in stroke. The endothelium plays a crucial homeostatic role in the regulation of vascular tone thrombogenesis and vascular remodeling. We studied the relationship between endothelial dysfunction and cardiorenal injury in hypertensive rats and evaluated the effects of two classes of antihypertensive agents commonly used in the clinical setting, a diuretic (DIU) and an ACE inhibitor (CEI). METHODS AND RESULTS: Dahl salt-sensitive rats (DS) given high dietary salt (4% NaCl) developed hypertension (systolic blood pressure [SBP], 218+/-9 versus 147+/-3 mm Hg in DS given 0.5% NaCl; P<.001), which was associated with impaired endothelium-dependent relaxations (EDRs) in aortic rings (ED50, 5.44+/-.18 versus 7.51+/-.10; P<.05) and mesenteric vessels (area under the curve, 299+/-11 versus 217+/-11 arbitrary units; P<.05). Hypertensive DS also demonstrated depressed nitric oxide synthase activity in the aorta (0.76+/-.15 versus 2.83+/-.17 nmol x min(-1) x g protein(-1); P<.05), left ventricular hypertrophy (0.43+/-.02 versus 0.29+/-.02 g ventricular weight/100 g body weight; P<.05), glomerular injury (histological injury score: 151+/-8 versus 11+/-2; P<.05), and increased urinary protein excretion (95+/-21 versus 25+/-5 mg/24 hours; P<.05). Treatment of DS rats with the CEI perindopril (4.56 mg x kg(-1) x d(-1)) did not affect SBP (225+/-6 mm Hg) but modestly improved EDR (ED50: 6.07+/-.37; P<.05 versus hypertensive DS) as well as proteinuria and glomerular histology. Addition of the DIU indapamide (1.44 mg x kg(-1) x d(-1)) normalized SBP (151+/-2 mm Hg; P<.05), EDR (ED50, 7.33+/-.08; P<.05), left ventricular hypertrophy (0.27+/-.01 g/100 g body weight; P<.05), and proteinuria (31+/-4 mg/24 hours; P<.05) and prevented glomerular injury (injury score: 30+/-2; P<.05). Monotherapy with DIU reduced SBP (175+/-3 mm Hg; P<.05) and normalized EDR and left ventricular hypertrophy but did not provide effective renal protection. In hypertensive DS, impaired EDR and left ventricular hypertrophy were positively correlated with SBP. In addition, we found a significant correlation between cardiac hypertrophy and endothelial dysfunction. Indeed, a hierarchical regression analysis revealed that impaired aortic EDR, and therefore decreased aortic compliance, positively contributed to left ventricular hypertrophy in addition to but independently of SBP [F(2,37)=6.29; P=.004]. CONCLUSIONS: These studies suggest a dissociation of the endothelial, cardiac, and renal effects of antihypertensive therapy in hypertension and may explain the variable success of antihypertensive regimens in treating hypertension while preventing cardiac and renal damage.

Glomerular and vascular tissues do not down-regulate nitric oxide synthesis during protracted endotoxemia.

Down-regulation of cytokines is implicated as an important component of the phenomenon of tolerance to bacterial products in humans and animals. Since many effects of endotoxin and cytokines are mediated by nitric oxide, this study was designed to evaluate in vivo the L-arginine:NO pathway in endotoxin tolerance. Sublethal injections of E. coli lipopolysaccharide (LPS, 1 mg/kg body wt, i.p.) were given to rats daily for five days. Blood levels of NO2-/NO3-, stable metabolites of nitric oxide (NO), significantly increased on day 1 (baseline, 89.64 +/- 40, day 1, 260.32 +/- 36 nmol/ml; P < 0.05) but returned to baseline levels on day 5 (77.60 +/- 5 nmol/ml). However, urinary NO2-/NO3- remained significantly elevated several-fold throughout the study period (baseline, 121.25 +/- 11.4, day 1, 899.35 +/- 15.8, day 5, 250.23 +/- 21.4 nmol/hr/100 g body wt). Glomeruli and aortae obtained from these rats showed increased NO production that was maintained at similar levels even at day 5 (glomeruli: control, 0.01 +/- 0.0, day 1, 22.4 +/- 0.3, and day 5, 22.0 +/- 2.5, P < 0.05 vs. control; aortae; control, 0.01 +/- 0.0, day 1, 4.3 +/- 2.2, and day 5, 5.4 +/- 1.0 nmol/hr/mg protein, P < 0.05 vs. control, respectively); this further increased significantly in response to in vitro LPS challenge. However, peritoneal macrophages, liver and spleen showed an initial increase in NO production that decreased significantly by the fifth day of LPS and could not be further stimulated by in vitro LPS challenge. Thus, in vivo NO synthesis is down-regulated during protracted LPS. Our results show that the process is relatively specific to the liver, spleen and macrophages, and is qualitatively and quantitatively different in vascular tissues such as aortae and glomeruli.

Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis.

In glomerulonephritides, autacoids such as nitric oxide (NO), reactive oxygen species, and prostanoids are produced in increased amounts in response to cytokines such as interleukin-1 (IL-1). These autacoids influence the expression of glomerular injury by their direct as well as interactive actions. We studied the effect of hydrogen peroxide (H2O2) on NO production in rat mesangial cells. We demonstrate that transient exposure of mesangial cells to H2O2 prior to sustained exposure to IL-1 decreased extracellular accumulation of NO2/NO3 and cellular guanosine 3,'5'-cyclic monophosphate (cGMP) content. H2O2 markedly impaired inducible nitric oxide synthase (iNOS) activity induced by IL-1 directly measured by the conversion of L-[14C]arginine to L-[14C]citrulline. Such impairment in iNOS activity was accompanied by a parallel reduction in iNOS protein abundance but not by a reduced expression of iNOS mRNA. The inhibitory effect of H2O2 on NOS activity was further supported by peroxide-induced impairment in IL-1-driven, NO-dependent synthesis of prostaglandin E2. Our studies thus provide the first direct evidence of a posttranscriptional inhibitory effect of H2O2 on iNOS activity. Additionally, our studies uncover the existence of a previously unrecognized effect of H2O2 on the production of NO that may exert influence on the severity of glomerular injury during glomerular inflammation.

The link among nitric oxide synthase activity, endothelial function, and aortic and ventricular hypertrophy in hypertension.

The adaptive changes that occur in the left ventricle (LV) and vessels in response to hypertension, namely, muscle hypertrophy/hyperplasia, endothelial dysfunction, and extracellular matrix increase, do not depend solely on blood pressure elevation. These changes are in fact, maladaptive since they are forerunners of cardiac failure, stroke, and renal failure. Nitric oxide, an endogenous vasodilator and inhibitor of vascular smooth muscle cell growth, is synthesized in the endothelium by constitutive nitric oxide synthase (cNOS). We investigated the relationships among LV and aortic cNOS activity (conversion of [14C] L-arginine to [14C] L-citrulline), with LV hypertrophy (LV weight/body weight), and (2) aortic hypertrophy (aortic weight/ length) in spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DS) rats matched for blood pressure (219 +/- 12 versus 211 +/- 7 mm Hg, P = NS) and age. Compared with their normotensive counterparts, aortic cNOS activity was increased 106% in SHR but reduced by 73% in DS rats. The correlation between blood pressure and aortic cNOS activity was positive (r = .74, P < .01) in SHR and negative (r = -.82, P < .01) in DS rats, LV cNOS activity was increased 73% in SHR compared with normotensive Wistar-Kyoto rats (P < .01). On the other hand, LV cNOS activity was not increased in hypertensive DS rats compared with normotensive DS rats. In SHR, aortic hypertrophy did not increase significantly and LV hypertrophy increased only 15%, whereas in hypertensive DS rats the aorta and LV hypertrophied 36% and 88%, respectively (both P < .01). Moreover, in DS rats there was a negative correlation between cNOS activity and aortic hypertrophy (r = -.70, P < .01). In DS rats, antihypertensive therapy consisting of an angiotensin-converting enzyme inhibitor, perindopril, and a diuretic, indapamide, normalized blood pressure, aortic cNOS activity, and LV hypertrophy and reduced aortic hypertrophy. Our studies imply that upregulation of vascular cNOS activity has a protective cardiovascular homeostatic role in hypertension. Clinically, the variable end-organ disease observed in individuals with similar severity of hypertension may be explained, at least in part, by genetically conditioned differences in vascular cNOS activity in response to hypertension.

Induction of endothelial cell injury by cigarette smoke.

Cigarette smoke contains different populations of free radicals which may be responsible for endothelial cell (EC) injury of smokers. The purpose of this study was to examine the effects of gas-phase cigarette smoke on EC endothelium-derived relaxing factor (EDRF)/NO-guanylate cyclase (GC)-cGMP pathway and on EC detachment-type injury after incubation with smoke. Furthermore, we examined whether different kind of antioxidants can prevent smoke-caused EC injury. We measured cGMP pathway using direct (sodium nitroprusside, SNP) and indirect (A23187, the calcium ionophore and bradykinin, BK) activators of GC. Directly and indirectly stimulated EC cGMP production dose-dependently decreased and EC detachment increased after incubation with smoke. Externally added thiols (glutathione, GSH; D-Penicillamine, DP; N-acetylcysteine, NAC) protected EC from damage of cGMP production and cell detachment. Other antioxidants (catalase, deferoxamine and superoxide dismutase) were ineffective. These results suggest that the thiol containing GC in EC is destroyed or inactivated or thiol like species responsible for activation of GC is incomplete in EC after incubation with smoke. It is also possible that externally added thiols bind an unknown component of smoke and this way, EC is protected. EC injury may contribute to vascular diseases associated with cigarette smoking.