Lucigenin-enhanced Chemiluminescence Research Articles

Hypoxia-Induced Generation of Methane in Mitochondria and Eukaryotic Cells - An Alternative Approach to Methanogenesis

Background/Aims: Electrophilic methyl groups bound to positively charged nitrogen moieties may act as electron acceptors, and this mechanism could lead to the generation of methane from choline. The aims were to characterize the methanogenic potential of phosphatidylcholine metabolites, and to define the in vivo relevance of this pathway in hypoxia-induced cellular responses. Methods: The postulated reaction was investigated (1) in model chemical experiments, (2) in rat mitochondrial subfractions and (3) in bovine endothelial cell cultures under hypoxic conditions and in the presence of hydroxyl radical generation. The rate of methane formation was determined by gas chromatography with flame-ionisation detectors. The lucigenin-enhanced chemiluminescence assay was used to determine the reactive oxygen species-scavenging capacity of the choline metabolites. Results: Significant methane generation was demonstrated in all three series of experiments. Phosphatidylcholine metabolites with alcoholic moiety in the molecule (i.e. choline, N,N-dimethylethanolamine and N-methylethanolamine), inhibited oxygen radical production both in vitro and in vivo, and displayed an effectiveness proportional to the amount of methane generated and the number of methyl groups in the compounds. Conclusion: Methane generation occurs in aerobic systems. Phosphatidylcholine metabolites containing both electron donor and acceptor groups may have a function to counteract intracellular oxygen radical production.

Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus

Objective HMG-CoA reductase inhibitors have been shown to upregulate GTP cyclohydrolase I (GTPCH-I), the key enzyme for tetrahydrobiopterin de novo synthesis and to normalize tetrahydrobiopterin levels in hyperglycemic endothelial cells. We sought to determine whether in vivo treatment with the HMG-CoA reductase inhibitor atorvastatin is able to upregulate the GTPCH-I, to recouple eNOS and to normalize endothelial dysfunction in an experimental model of diabetes mellitus. Methods and results In male Wistar rats, diabetes was induced by streptozotocin (STZ, 60 mg/kg). In STZ rats, atorvastatin feeding (20 mg/kg/d, 7 weeks), normalized vascular dysfunction as analyzed by isometric tension studies, levels of circulating endothelial progenitor cells (FACS-analysis), superoxide formation (assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining), vascular levels of the phosphorylated vasodilator-stimulated phosphoprotein (P-VASP), tyrosine nitration of the prostacyclin synthase, expression of GTPCH-I, dihydrofolate reductase and eNOS, translocation of regulatory NADPH oxidase subunits rac1, p47phox and p67phox (assessed by Western blot) and vascular tetrahydrobiopterin levels as measured by HPLC. Dihydroethidine staining revealed that the reduction of vascular superoxide was at least in part due to eNOS recoupling. Conclusion HMG-CoA reductase inhibition normalizes endothelial function and reduces oxidative stress in diabetes by inhibiting activation of the vascular NADPH oxidase and by preventing eNOS uncoupling due to an upregulation of the key enzyme of tetrahydrobiopterin synthesis, GTPCH-I.

Lucigenin-enhanced chemiluminescence of animal tissues

Lucigenin-enhanced chemiluminescence (LcCL) allows one to investigate the reactions of superoxide anion radical (*O2-) generated by mitochondria and is applied to study the superoxide production in enzymatic and membrane systems by isolated mitochondria and cells, and in whole organs. The application of lucigenin-enhanced chemiluminescence to estimate the respiration of human tissues involves the use of small tissue pieces, which can be obtained, for instance, by biopsia; however, no systematic investigations have been performed on these objects. In the present paper, a comparative study of lucigenin-enhanced chemiluminescence of tissues isolated from different organs of the rat was carried out to elucidate its dependence on the extent of tissue defragmentation, storage time, and access for oxygen. It was shown that the addition of lucigenin to a piece of tissue, a suspension of fine tissue fragments, and homogenates greatly enhanced chemiluminescence, and a whole piece of tissue possessed a much lesser (by 1-1.5 order of magnitude) intensity of chemiluminescence than homogenate or gruel. In the absence of stirring of the surrounding solution, the lucigenin-enhanced chemiluminescence of tissue quickly decreased, apparently due to a decrease in the level of oxygen in the tissue, as the result of its consumption. The chemiluminescence consisted of two components: a lucigenin-dependent and lucigenin-independent one (intrinsic chemiluminescence). Thus, the tissue was a source of lucigenin-enhanced chemiluminescence, and this luminescence was observed only at a sufficient access for oxygen. The lucigenin-independent component did not practically depend on oxygen and was determined by the components coming out of the tissue into the surrounding solution. Nitric oxide (NO) inhibited chemiluminescence as its concentration increased and did not affect considerably the rate of oxygen consumption by the tissue. The results obtained allow one to conclude that lucigenin can be used as a rather effective chemiluminescent probe for the production of superoxide radicals by tissue pieces.

Angiotensin-(1-7) Counterregulates Angiotensin II Signaling in Human Endothelial Cells

Angiotensin (Ang)-(1-7), acting through the Mas receptor, opposes the actions of Ang II. Molecular mechanisms for this are unclear. Here we sought to determine whether Ang-(1-7) influences Ang II signaling in human endothelial cells, focusing specifically on Src homology 2-containing inositol phosphatase 2 (SHP-2) and its interaction with c-Src. Ang II-induced phosphorylation of c-Src, extracellular signal regulated kinase (ERK)1/2, and SHP-2 and activation of NAD(P)H oxidase were assessed in the absence and presence of Ang-(1-7) (10(-6) mol/L, 15 minutes) by immunoblotting and lucigenin-enhanced chemiluminescence, respectively. (D-Ala(7))-Ang I/II (1-7) (Ang fragment 1-7 receptor antagonist) was used to block Ang-(1-7) effects. Association between SHP-2 and c-Src was assessed by immunoprecipitation/immunoblotting studies. Ang II significantly increased activation of c-Src, ERK1/2, and NAD(P)H oxidase and reduced phosphorylation of SHP-2 (P<0.05) in human endothelial cells. These effects were abrogated in cells pre-exposed to Ang-(1-7). Ang fragment 1-7 receptor antagonist pretreatment blocked the negative modulatory actions of Ang-(1-7) on Ang II-induced signaling. Ang-(1-7) alone did not significantly alter phosphorylation of c-Src, ERK1/2, and SHP-2 and had no effect on basal activity of NAD(P)H oxidase. SHP-2 and c-Src were physically associated in the basal state. This association was increased by Ang-(1-7) and blocked by Ang fragment 1-7 receptor antagonist. Our findings demonstrate that, in human endothelial cells, Ang-(1-7) negatively modulates Ang II/Ang II type 1 receptor-activated c-Src and its downstream targets ERK1/2 and NAD(P)H oxidase. We also show that SHP-2-c-Src interaction is enhanced by Ang-(1-7). These phenomena may represent a protective mechanism in the endothelium whereby potentially deleterious effects of Ang II are counterregulated by Ang-(1-7).

Growth-related oncogene-α induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.

Aspirin Prevents and Partially Reverses Adrenocorticotropic Hormone-Induced Hypertension in the Rat

Glucocorticoid-induced hypertension is associated with increased oxidative stress. The aim of the present study was to investigate the effects of aspirin, a potent antioxidant, on adrenocorticotropic hormone (ACTH) and dexamethasone (Dex)-induced hypertension. Male Sprague-Dawley (SD) rats were treated with saline, ACTH (0.2 mg/kg/d subcutaneously) or Dex (10 mug/rat/d subcutaneously). Aspirin (100 mg/kg/d in drinking water) was given 4 days before and during glucocorticoid-treatment (prevention studies). In reversal studies, saline, ACTH, or Dex was administered for 13 days and at day 8 (T8), rats were co-administered aspirin for 5 days. Systolic blood pressure (BP) was measured by the tail-cuff method. Thymus wet weight was measured as a marker of glucocorticoid activity and lucigenin-enhanced chemiluminescence as a marker of aortic superoxide production. Saline or aspirin alone did not change systolic BP. Systolic BP was increased by ACTH (mean +/- SEM; from 99 +/- 2 to 133 +/- 4 mm Hg, n = 10, P < .001) and Dex (from 102 +/- 3 to 125 +/- 5 mm Hg, n = 10, P < .001). Aspirin prevented the development of hypertension caused by ACTH (P' < .01) and tended to prevent Dex-induced hypertension (P' = .07). ACTH- but not Dex-induced hypertension was partially reversed by aspirin. Both ACTH and Dex decreased thymus weight. Aspirin had no effect on thymus weight. ACTH tended to increase lucigenin-enhanced chemiluminescence (P' = .07). Aspirin had no effect on this marker of tissue superoxide production. Aspirin prevented and partially reversed ACTH-induced hypertension in the SD rats.

TYPE 1 DIABETES AND HYPERCHOLESTEROLAEMIA REVEAL THE CONTRIBUTION OF ENDOTHELIUM‐DERIVED HYPERPOLARIZING FACTOR TO ENDOTHELIUM‐DEPENDENT RELAXATION OF THE RAT AORTA

1. The present study evaluated the effect of diabetes, hypercholesterolaemia and their combination on the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to relaxation of rat isolated aortic rings and the potential contribution of oxidant stress to the disturbance of endothelial function. 2. Thoracic aortic rings from control, diabetic, hypercholesterolaemic and diabetic plus hypercholesterolaemic rats were suspended in organ baths for tension recording. Generation of superoxide by the aorta was measured using lucigenin-enhanced chemiluminescence. 3. The maximal response to acetylcholine (ACh) was significantly reduced in diabetic or hypercholesterolaemic rats compared with control rats. In rats with diabetes plus hypercholesterolaemia, both the sensitivity and maximal response to ACh was impaired. In control rats, the response to ACh was abolished by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) or inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In contrast, in rats with diabetes, hypercholesterolaemia or both, relaxation to ACh was resistant to inhibition by L-NNA or ODQ, but abolished by additional inhibition of K(Ca) channels with charybdotoxin plus apamin. 4. The generation of superoxide was not significantly enhanced in aortic rings from either diabetic or hypercholesterolaemic rats, but was significantly increased in aortic rings from rats with diabetes plus hypercholesterolaemia. 5. These results suggest that when diabetes and hypercholesterolaemia impair endothelium-dependent relaxation, due to a diminished contribution from NO, a compensatory contribution of EDHF to endothelium-dependent relaxation of the aorta is revealed. The attenuation of NO-mediated relaxation, at least in the presence of both diabetes and hypercholesterolaemia, is associated with enhanced superoxide generation.

Abstract 1205: Role for Nox1 NADPH Oxidase in Atherosclerosis

NADPH oxidase-derived reactive oxygen species (ROS) contribute to the pathobiology of vascular disease. However, studies investigating NADPH oxidases in atherosclerosis have been limited in their ability to distinguish between the role of vascular cell and inflammatory cell -derived ROS. In this study, we examined the contribution of Nox1-derived ROS in a mouse model of atherosclerosis. Nox1 is a primary catalytic subunit of vascular cell, but not inflammatory cell, NADPH oxidase. At weaning, male apolipoprotein E deficient mice (AS, n=12) and male mice deficient in both apolipoprotein E and Nox1 (AS/Nox, n=16) received an atherogenic diet for 18 weeks. Mean blood pressures (116±3 vs. 110 ±3 mmHg; AS vs. AS/Nox, n=6), weights, and serum cholesterol levels (1578±150 vs. 1631±80 mg/dl; AS vs. AS/Nox) were similar between the AS and AS/Nox mice. As measured by lucigenin-enhanced chemiluminescence, superoxide levels were increased in segments of thoracic aorta from AS mice as compared to aorta from control mice (22±2 vs. 14±2 RLU/sec/mm 2 ; AS vs. C57BL/6; p&lt;0.05, n=6). In contrast, superoxide levels in segments of thoracic aorta from AS/Nox mice were significantly lower (9±1 RLU/sec/mm 2 , n=6) than both AS and C57BL/6 mice. Dihydroethidium staining confirmed decreased superoxide levels in aorta of AS/Nox mice. Atherosclerotic lesion area was measured by staining the aorta en face with Oil Red O. Although atherosclerotic lesion area was reduced over the entire length of aorta in AS/Nox mice as compared to AS mice (10±1% vs. 14±1%, p&lt;0.01), the reduction in lesion was primarily limited to the aortic arch (28±3% vs. 43±2%, p&lt;0.001). In summary, Nox1 contributes to generation of ROS and lesion formation in atherosclerosis. These data confirm a role for vascular cell NADPH oxidases, and in particular Nox1, in vascular disease.

Abstract 477: Selective Heart Rate Reduction Via Inhibition Of The I F -current Prevents Atherosclerosis And Improves Endothelial And Erectile Function In Apoe −/− Mice

Introduction : Elevated resting heart rate (HR) is associated with increased risk for cardiovascular morbidity and mortality. Erectile dysfunction is associated with endothelial dysfunction and atherosclerosis due to similar risk factors. We hypothesized that HR reduction may influence endothelial- and erectile function and tested the effects of the I f -current inhibitor ivabradine in ApoE−/− mice. Methods and Results: Male ApoE−/− mice fed a high-cholesterol diet were treated with ivabradine (iva) (10 mg/kg/d p.o.) or vehicle (n=10 per group) for 6 weeks. Treatment with ivabradine reduced HR by 13.4% (iva 472±9 bpm vs vehicle 545±11 bpm, p&lt;0.01). High cholesterol feeding led to severe atherosclerosis in the aortic sinus and the aorta. Ivabradine reduced atherosclerotic plaque size in the aortic root from 32±4% to 18±2% and in the ascending aorta from 26±4% to 7±1% (p&lt;0,05). Endothelium dependent relaxation of aortic rings and corpora cavernosa to carbachol was significantly improved in ivabradine fed ApoE−/− mice compared to controls (p&lt;0.01). Aortic eNOS mRNA expression, quantified by real time PCR with the TaqMan system was upregulated to 156±11% (p&lt;0.05) in aortic rings after treatment with ivabradine. HR reduction led to attenuation of NADPH oxidase activity to 48±6% as measured by a lucigenin-enhanced chemiluminescence assay and decreased L-012 chemiluminescence to 24±9% (both p&lt;0.05). Lipidperoxidation was reduced to 65±8% in the vasculature of the iva group compared to vehicle treatment (p&lt;0.05). DHE fluorescence microscopy in aortic sections detected reduction of ROS release to 62±4% in ivabradine treated mice (p&lt;0.01). Conclusions : Selective HR reduction improves endothelial as well as erectile function and reduces atherosclerotic plaque formation in ApoE−/− mice. Those functional effects are mediated by decreased oxidative stress and upregulation of endothelial NOS (eNOS). These results identify HR as a risk factor for vascular disease and support the importance of heart rate reduction in vascular prevention.

Modulation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase Expression and Function by 3′,4′-Dihydroxyflavonol in Phagocytic and Vascular Cells

Previously we have demonstrated that 3',4'-dihydroxyflavonol (DiOHF), a novel synthetic flavonol, protects against ischemia reperfusion injury in both heart and brain. In this study, we characterized the pharmacological effects of DiOHF on phagocytic and vascular NADPH oxidase. Superoxide release (lucigenin-enhanced chemiluminescence or cytochrome c reduction), NADPH oxidase activation (membrane translocation of p47phox), and subunit expression (real-time polymerase chain reaction and Western blot) were examined in differentiated HL-60 cells, human neutrophils, vascular endothelial and smooth muscle cells, and mouse aorta. DiOHF concentration dependently suppressed superoxide accumulation (EC(50) = 8.4 +/- 1.7 microM) in vascular smooth muscle cells, which appears to be attributable to its superoxide scavenging activity (EC(50) = 6.1 +/- 1.1 microM measured in a cell-free system). DiOHF had similar effects in HL-60 cells and isolated aortic rings. In HL-60 cells, but not endothelial or smooth muscle cells, DiOHF and quercetin (10 and 30 microM) significantly reduced the protein expression of p47phox, whereas p67phox was not altered. DiOHF did not affect phorbol ester-induced membrane translocation of either p47phox or protein kinase C in leukocytes. Our results suggest that suppression of NADPH oxidase-dependent superoxide accumulation may contribute to the cytoprotective actions of DiOHF during ischemia-reperfusion injury.

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 × 10 6 cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017–167 ng ml −1) for 1.25 × 10 6 neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.

α‐Lipoic acid modulates gut inflammation induced by trinitrobenzene sulfonic acid in rats

Alpha-lipoic acid (ALA) has been shown to combat oxidative stress by quenching a variety of reactive oxygen species. It is involved in the regeneration of exogenous and endogenous antioxidants, chelation of metal ions, and repair of oxidized proteins. This study aimed to evaluate the potential beneficial effect of ALA on trinitrobenzenesulfonic acid (TNBS)-induced gut ileitis and colitis in rats. After 48 h of fasting, Sprague-Dawley rats underwent a laparotomy under ether anesthesia. TNBS solution 30 mg/mL in 40% ethanol (1 mL) was injected into the lumen, 10 cm proximal to the ileocolonic junction to induce ileitis or intrarectally 8 cm proximal to the anal sphincter to induce colitis. ALA (25 mg/kg intraperitoneally, twice a day) was given after induction of inflammation and continued for 3 days. All animals were decapitated 3 days after induction of the inflammation. The mucosal lesions of the ileum and colon were scored macroscopically and microscopically. Samples were taken for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, tissue-associated myeloperoxidase (MPO) activity and luminol- or lucigenin-enhanced chemiluminescence (CL). Macroscopic scores, morphological changes and increased tissue lipid peroxidation with a concomitant reduction in GSH of the ileitis or colitis groups were all reversed by treatment with ALA. ALA treatment was also effective in improving tissue MPO activity and CL values, which were elevated in untreated ileitis or colitis groups. ALA is beneficial in TNBS-induced gut inflammation in rats via suppression of neutrophil accumulation, preservation of endogenous glutathione and inhibition of reactive oxidant generation.

Contribution of polyol pathway to arteriolar dysfunction in hyperglycemia. Role of oxidative stress, reduced NO, and enhanced PGH2/TXA2 mediation

Hyperglycemia increases glucose metabolism via the polyol pathway, which results in elevations of intracellular sorbitol concentration. Thus we hypothesized that elevated level of sorbitol contributes to the development of hyperglycemia-induced dysfunction of microvessels. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles (approximately 150 microm), high glucose treatment (25 mM) induced reduction in flow-dependent dilation (from maximum of 39 +/- 2% to 15 +/- 1%), which was significantly mitigated by an aldose reductase inhibitor, zopolrestat (maximum 27 +/- 2%). Increasing doses of sorbitol (10(-10)-10(-4) M) elicited dose-dependent constrictions (maximum 22 +/- 3%), which were abolished by endothelium removal, a prostaglandin H(2)/thromboxane A(2) (PGH(2)/TXA(2)) receptor (TP) antagonist SQ-29548, or superoxide dismutase (SOD) plus catalase (CAT). Incubation of arterioles with sorbitol (10(-7) M) reduced flow-dependent dilations (from maximum of 39 +/- 2% to 20 +/- 1.5%), which was not further affected by inhibition of nitric oxide synthase by N(omega)-nitro-l-arginine methyl ester but was prevented by SOD plus CAT and mitigated by SQ-29548. Nitric oxide donor sodium nitroprusside-induced (10(-9)-10(-6) M) dilations were also decreased in a SQ-29548 and SOD plus CAT-reversible manner, whereas adenosine dilations were not affected by sorbitol exposure. Sorbitol significantly increased arterial superoxide production detected by lucigenin-enhanced chemiluminescence, which was inhibited by SOD plus CAT. Sorbitol treatment also increased arterial formation of 3-nitrotyrosine. We suggest that hyperglycemia by elevating intracellular sorbitol induces oxidative stress, which interferes with nitric oxide bioavailability and promotes PGH(2)/TXA(2) release, both of which affect regulation of vasomotor responses of arterioles. Thus increased activity of the polyol pathway may contribute to the development of microvascular dysfunction in diabetes mellitus.

Simvastatin Reverses Target Organ Damage and Oxidative Stress in Angiotensin II Hypertension: Comparison With Apocynin, Tempol, and Hydralazine

The ability of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor simvastatin to reverse established cardiovascular and renal alterations and oxidative stress was assessed in angiotensin II (AngII) hypertension. Sprague-Dawley rats infused with AngII (200 ng/kg per minute for 17 days) were concomitantly treated or not for the last 7 days with simvastatin, apocynin, tempol, and hydralazine (60, 60, 30, and 15 mg/kg per day, respectively). Only hydralazine lowered AngII hypertension. Simvastatin and apocynin lowered cardiac hypertrophy by 52% and 54% and reversed the marked rise in albuminuria by 25% and 70%. Neither tempol nor hydralazine affected cardiac mass or albuminuria. None of the treatments modified the AngII-induced increase in carotid media thickness. The rise in cardiac superoxide anion production (lucigenin-enhanced chemiluminescence method) induced by AngII was reversed by all treatments. Enhanced plasma concentration of advanced oxidation protein products (spectrophotometry using chloramine T) was unaffected by simvastatin and tempol, but it was reversed by apocynin and hydralazine. Our results indicate that simvastatin reverse established cardiac and renal alterations in AngII hypertension independently of arterial pressure. It is suggested that oxidative stress participates in the maintenance of target organ damage and that antioxidant properties are involved in the beneficial influence of the statin.

Endothelial Superoxide Production Is Altered in Sheep Programmed by Early Gestation Dexamethasone Exposure

Background: Animal models have demonstrated that maternal undernutrition or early gestation glucocorticoid exposure induces endothelial dysfunction in the offspring. Objectives: We sought to determine whether early gestation dexamethasone (DEX) exposure is further associated with increased vascular superoxide anion production. Methods: DEX (0.28 mg/kg/day i.v. for 48 h) was administered to pregnant ewes at 27–28 days’ gestation (term 145 days). Tissues were harvested from DEX-exposed and control lambs at 125 days’ gestation (n = 6 for each group) and 4 months following delivery (n = 9 and 12, respectively). Results: By lucigenin-enhanced chemiluminescence, coronary and mesenteric arteries from DEX-exposed fetuses exhibited diminished basal superoxide production (both p < 0.01). Similarly, DEX-exposed carotid arteries from 4-month-old lambs had decreased superoxide production (p < 0.01) that localized to the endothelium by endothelial cell culture and dihydroethidium fluorescence. In contrast, DEX-exposed coronary arteries from the 4-month-old sheep had increased superoxide production (p < 0.05). Although early gestation DEX exposure did not alter lipid peroxidation, DEX exposure was associated with significantly increased renal and cerebral cortex aconitase activity (consistent with decreased protein oxidation). These changes occurred in the absence of alterations in renal cortex superoxide dismutase activity. Conclusions: We conclude that in a DEX-exposure model of fetal programming, endothelial superoxide production and protein oxidation are decreased in the mesenteric and carotid circulation. This contrasts with the postnatal coronary artery-specific increase in superoxide production.

The role of free radicals and nitric oxide in the ischemia–reperfusion injury mediated by acute bladder outlet obstruction

Free-radical generation and nitric oxide (NO) generation were detected in the rat bladder following acute bladder outlet obstruction (BOO), and the results were compared with those for vascular ischemia and reperfusion (I-R). Forty male Sprague Dawley rats were used. In the acute BOO plus I-R group (group 1), rats were catheterized with a 3-Fr catheter and an inflated balloon was positioned at the bladder neck. The bladder was overdistended after administration of Ringer solution and furosemide (12 mg/kg, each) for 60 min, and was then drained to allow reperfusion for 30 min. In the acute BOO plus nerve stimulation group (group 2), the pelvic nerve was stimulated in the distended bladder for 60 min (5 s every 5 min, 10 V/0.1 ms, 20 Hz). Pelvic nerve stimulation was performed in nonobstructed animals in group 3. In the I-R group (group 4), the distal aorta was occluded for 60 min followed by reperfusion for 30 min. Sham-operated animals served as the control group (group 5). At the end of the protocols, the levels of hydroxyl and superoxide radicals and NO levels were measured in the bladder tissues with luminol- and lucigenin-enhanced chemiluminescence methods. The results were compared by a one-way analysis of variance test. The levels of hydroxyl radicals were not significantly different between the study groups. In contrast, superoxide radicals and NO levels were significantly increased in both group 1 and group 4 compared with those in control animals (P<0.05 for all comparisons). Superoxide radical generation in group 2 was comparable to the levels in group 1 (P>0.05), whereas NO levels were substantially lower than in group 1 (P=0.06). In summary, vascular I-R causes significant oxidative damage to the bladder. Acute BOO with overdistension of the bladder mimics the effects of true vascular I-R injury. The NO pathway has possibly a major role in I-R-induced bladder damage. Prolonged BOO may therefore significantly enhance the oxidative damage to the bladder and further accentuate the effects of generalized atherosclerotic processes in the elderly adult.

Hypoxic preconditioning attenuates lipopolysaccharide-induced oxidative stress in rat kidneys.

Chronic hypoxic (CH) preconditioning reduces superoxide-induced renal dysfunction via the upregulation of superoxide dismutase (SOD) activity and contents. Endotoxaemia reduces renal antioxidant status. We hypothesize that CH preconditioning might protect the kidney from subsequent endotoxaemia-induced oxidative injury. Endotoxaemia was induced by intraperitoneal injection of lipopolysaccharide (LPS; 4 mg kg(-1)) in rats kept at sea level (SL) and rats with CH in an altitude chamber (5500 m for 15 h day(-1)) for 4 weeks. LPS enhanced xanthine oxidase (XO) and gp91phox (catalytic subunit of NADPH oxidase) expression associated with burst amount of superoxide production from the SL kidney surface and renal venous blood detected by lucigenin-enhanced chemiluminescence. LPS induced a morphologic-independent renal dysfunction in baseline and acute saline loading stages and increased renal IL-1beta protein and urinary protein concentration in the SL rats. After 4 weeks of induction, CH significantly increased Cu/ZnSOD, MnSOD and catalase expression (16 +/- 17, 128 +/- 35 and 48 +/- 21, respectively) in renal cortex, and depressed renal cortex XO (44 +/- 16%) and renal cortex (20 +/- 9%) and medulla (28 +/- 11%) gp91phox when compared with SL rats. The combined effect of enhanced antioxidant proteins and depressed oxidative proteins significantly reduced LPS-enhanced superoxide production, renal XO and gp91phox expression, renal IL-1beta production, and urinary protein level. CH also ameliorated LPS-induced renal dysfunction in the baseline and acute saline loading periods. We conclude that CH treatment enhances the intrarenal antioxidant/oxidative protein ratio to overcome endotoxaemia-induced reactive oxygen species formation and inflammatory cytokine release.

Effect of Aging, MnSOD Deficiency, and Genetic Background on Endothelial Function

The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD+/+ and MnSOD-deficient (MnSOD+/-) mice. Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD+/+ (n=9) and young MnSOD+/- (n=6) mice. This response was impaired in old MnSOD+/+ (n=8) mice and old MnSOD+/- mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 micromol/L ACh produced 77+/-3% [mean+/-SE], 77+/-3%, 70+/-4%, and 57+/-4% relaxation in young MnSOD+/+, young MnSOD+/-, old MnSOD+/+, and old MnSOD+/- mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD+/+ and MnSOD+/- mice compared with both groups of young mice (P<0.05). Superoxide levels, as measured using lucigenin-enhanced chemiluminescence, were increased more than 2-fold in old MnSOD+/- mice compared with old MnSOD+/+ and young mice (P<0.05). These data provide the first direct evidence that MnSOD haploinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.

Effect of Gender on NADPH-Oxidase Activity, Expression, and Function in the Cerebral Circulation

This study tested whether NADPH-oxidase activity, expression, and functional effects on vascular tone are influenced by gender in the rat cerebral circulation and whether such differences are estrogen-dependent. NADPH-stimulated superoxide production by cerebral (basilar [BA]; middle cerebral) arteries from male and female Sprague-Dawley rats was measured using lucigenin-enhanced chemiluminescence and dihydroethidium. Protein expression of Nox1, Nox2, Nox4, superoxide dismutase 1 (SOD1), SOD2, and SOD3 was measured using Western blotting. Vascular responses of BA to NADPH were assessed in a myograph. Some female rats were ovariectomized and treated with either vehicle (dimethyl sulfoxide) or 17beta-estradiol. NADPH-stimulated superoxide production by BA and middle cerebral arteries from males was approximately 2-fold greater than vessels from females. Superoxide production was virtually abolished by the NADPH-oxidase inhibitor, diphenyleneiodonium. Protein expression of Nox1 and Nox4 in BA was also higher in males than in females (2.4- and 2.8-fold, respectively), whereas Nox2, SOD1, SOD2, and SOD3 expression did not differ between genders. NADPH induced greater vasorelaxant effects in BA from males versus females (P<0.05). The hydrogen peroxide scavenger, catalase, abolished these NADPH-induced relaxations. NADPH-stimulated superoxide production by BA from ovariectomized rats treated with vehicle was 3-fold greater than levels in intact females. Treatment of ovariectomized rats with 17beta-estradiol decreased superoxide production (P<0.05). NADPH-induced relaxations of BA were smaller in 17beta-estradiol-treated than in vehicle-treated ovariectomized rats (P<0.05). NADPH-oxidase activity and function are lower in cerebral arteries of female rats. These gender differences are estrogen-dependent and are associated with lower Nox1 and Nox4 expression.

Chronic Granulomatous Disease (CGD) and Complete Myeloperoxidase Deficiency Both Yield Strongly Reduced Dihydrorhodamine 123 Test Signals but Can Be Easily Discerned in Routine Testing for CGD

The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD). We measured NADPH-oxidase and MPO activity in neutrophils from MPO-deficient patients, CGD patients, NADPH-oxidase-transfected K562 cells and cells with inhibited and substituted MPO. Eosinophils from MPO-deficient individuals retain eosinophilic peroxidase and therefore generate a normal DHR signal. The addition of recombinant human MPO enhances the DHR signal when simply added to a suspension of MPO-deficient cells but not when added to NADPH-oxidase-deficient (CGD) cells. Lucigenin-enhanced chemiluminescence (LCL) is increased in neutrophils from MPO-deficient patients, whereas neutrophils from patients with CGD show a decreased response. A false-positive result caused by MPO deficiency can be easily ascertained because, unlike cells from a CGD patient, cells from MPO-deficient patients (a) contain functionally normal eosinophils, (b) show a significant enhancement of the DHR signal following addition of rhMPO, and (c) generate a strong LCL signal.