[Oxidative stress and cardiovascular diseases]. / Oxidativer Stress und kardiovaskuläre Erkrankungen.

A number of diseases like hypercholesterolemia and atherosclerosis, hypertension, congestive heart failure, diabetes, ischemia-reperfusion, neurodegenerative diseases as well as acute and chronic inflammatory diseases are characterized by an increased steady-state concentration of reactive oxygen species (ROS). On a biomolecular level an enhanced oxidative stress causes damage of proteins, lipids and nucleic acids. Both the experimental and therapeutic efficiency of different antioxidative compounds (like various antioxidative enzymes) , drugs, metabolites and vitamins for the maintenance of an appropriate intracellular redox potential underline the importance of an excessive ROS-formation for these diseases. Control of excessive ROS-formation can be obtained by angiotensin converting enzyme (ACE-) inhibitors, by AT (1)-receptor blockers, by statins and other lipid lowering compounds, by improved expression of antioxidative enzymes (superoxide dismutase, catalase etc.), by compounds such as probucol, certain vitamins, pyruvate, by lipid apheresis and by physical exercise training, which displays surprising efficacy.

The effect of high-dose pentaerythritol tetranitrate on the development of nitrate tolerance in rabbits.

Experimental studies with therapeutic doses of pentaerythritol tetranitrate (PETN) have shown unexpected actions such as a lack of nitrate tolerance and vasoprotective effects in atherosclerosis. We investigated the effect of a 3-week treatment with low- (6 mg kg(-1) day(-1), n=10) and high-dose (100 mg kg(-1) day(-1), n=10) oral PETN given twice daily on the development of nitrate tolerance in rabbits. We measured aortic relaxation in response to acetylcholine, S-nitroso-N-acetyl-D,L-penicillamine and PETN, constriction in response to phenylephrine and production of reactive oxygen species (ROS). Mean aortic pressure (AOPmean) and heart rate were measured after a single oral dose of PETN (50 mg kg(-1), n=6) and after increasing doses of pentaerythritol dinitrate (PEDN, n=5) and pentaerythritol mononitrate (PEMN, n=5) in anaesthetized rabbits. Oral PETN, even at high dosage, was not associated with nitrate tolerance. None of the aortic ring studies showed a difference in the responses to the vasodilators, while the vasoconstriction to phenylephrine was slightly reduced in both PETN groups. The production of vascular ROS was also not different. Oral PETN reduced AOPmean transiently (-19.3+/-4.4%, P<0.01 vs. controls) and i.v. administration of both PEMN and PEDN reduced AOPmean dose dependently (P<0.05, ANOVA). These results suggest that oral PETN elicits minor nitrate tolerance. This unique feature might be due to the slow onset of vasodilator activity of the predominantly active metabolites PEDN and PEMN and might contribute to the vasoprotective activity of PETN in atherosclerosis.

Impact of postprandial hypertriglyceridemia on vascular responses in patients with coronary artery disease: effects of ACE inhibitors and fibrates.

We analyzed vascular responses (endothelial function, oxidant stress) to postprandial hypertriglyceridemia (PHTG) in patients with coronary artery disease (CAD) to reveal potential therapeutical effects of angiotensin converting enzyme inhibition (ACE-I) and of lipid lowering (fibrate). The study population (n=39, mean age: 60 years) consisted of four groups, all of which had angiographically documented CAD. A high fat group (n=9) consumed a high fat meal, a low fat group (n=9) a low fat meal, and ACE-I (n=10) or fibrate (n=11) groups consumed a high fat meal plus lisinopril or fenofibrate. Serum triglycerides (TG) increased significantly 2 h after eating a test meal in all groups with the exception of the low fat group. In the high and low fat groups changes of serum TG were positively correlated (r=0.664, P<0.005) with changes of phorbol ester-activated leukocyte superoxide anion radical (O(2-.)) formation and were negatively correlated (r=-0.488, P<0.05) with flow-mediated brachial artery dilation (FMD). There was a negative correlation (r=-0.419, P=0.094) between FMD and changes of O(2-.) formation in the high and low fat groups. In the ACE-I and fibrate groups, O(2-.) formation decreased 2 h after eating a test meal (from 5.34+/-1.01 to 3.81+/-1.15 nmol/10(6)cells per min, P<0.01, and from 4.66+/-0.91 to 4.26+/-0.97 nmol/10(6)cells per min, P=0.374, respectively). However, endothelial function did not show any significant changes 2 h after eating a test meal in all groups. PHTG increases oxidant stress and further deteriorates endothelial function, even in patients with CAD. Both ACE-I and fibrates have an antioxidant effect but no acute beneficial effects in terms of endothelial function under conditions of PHTG in CAD patients.

Potentiation of sildenafil-induced hypotension is minimal with nitrates generating a radical intermediate.

Recently the new specific phosphodiesterase-5 inhibitor sildenafil was introduced into therapy for erectile dysfunction. Because of the phosphodiesterase-5 inhibitor-induced increases of cyclic GMP in the vasculature, vasodilation in various vascular beds is induced, which in combination with various nitrovasodilators (e.g., when used simultaneously for the treatment of coronary artery disease), may lead to excessive hypotension. Thus nitrovasodilators are contraindicated when sildenafil may be used and reports of a number of accidents have recently been published. We therefore studied the acute interactions of glyceryl trinitrate (GTN), pentaerythritol tetranitrate (PETN), and isosorbide dinitrate (ISDN) with sildenafil in six chronically instrumented conscious dogs for each nitrate to assess the magnitude of blood pressure drops (and compensatory increases in heart rate) during a 24-h nitrate administration (infusion into the pulmonary artery). Sildenafil (3 mg/kg) was given orally (after a 24-h fast) 30 min after start of nitrate infusion. GTN, PETN, or ISDN (which follow different steps of metabolic conversion to nitric oxide) were applied at submaximal dosages leading to 90% of maximal coronary artery dilation at 1.5 microg/kg per min, 0.75 microg/kg per min, or 6 microg/kg per min, respectively. During GTN infusion sildenafil caused a maximum drop in mean blood pressure of 21 +/- 3 mm Hg (rise in heart rate from 117.0 +/- 7.2 to 126.0 +/- 6 .0/min) and during ISDN infusion of 18 +/- 3 mm Hg (rise in heart rate from 115.0 +/- 7.0 to 125 +/- 6/min), which was significantly less (p < 0.01) during PETN (only 6 +/- 1 mm Hg with a rise in heart rate from 107.0 +/- 5.0 to 122.0 +/- 7.0/min). When sildenafil is used during exposure to nitrates (e.g., in coronary artery disease), the PETN-induced drop in blood pressure at equi-effective dosages (with regard to coronary dilation) is substantially smaller compared with that of GTN or ISDN, which is probably because of lesser potentiation of phosphodiesterase-5 inhibitor-induced effects in the arteriolar bed, thus minimizing critical drops in blood pressure.

Postprandial hypertriglyceridemia impairs endothelial function by enhanced oxidant stress.

AIMS: it appears that hypertriglyceridemia (HTG) is a risk factor of atherosclerosis as demonstrated by recent studies. In this study, we analyzed the effects of acute HTG on endothelial function and oxidative stress, which are important mechanisms in the pathogenesis of atherosclerosis. METHODS AND RESULTS: in a high fat meal group (n = 11), serum triglycerides and PMA-activated leukocyte O(2)(-)* production were significantly (P < 0.005) increased from 146 +/- 69 mg/dl and 4.09 +/- 0.93 nmol/10(6) cells/min preprandially to 198 +/- 88 mg/dl and 5.49 +/- 1.19 nmol/10(6) cells/min, respectively, 2 h after eating a high-fat meal. The flow-mediated endothelium-dependent brachial artery dilation (FMD; high-resolution ultrasound) was decreased from 13.7 +/- 3.3% preprandially to 8.2 +/- 3.7%, 2 h after eating a high-fat meal (P < 0.005). However, following a low-fat meal (n = 9), there were no significant changes in triglycerides, leukocyte O(2)(-)* production and FMD. Changes of serum triglycerides were correlated negatively (r = -0.650, P < 0.005) with changes of FMD, but were correlated positively (r = 0.798, P < 0.001) with changes of leukocyte O(2)(-)* production, which - in turn - were correlated negatively (r = -0.784, P < 0.001) with changes of FMD in all study subjects (mean age: 56 years, n = 20). CONCLUSIONS: this study suggests that acute HTG causes endothelial dysfunction via enhanced oxidant stress and this may pave the way for the development of atherosclerosis under chronic conditions.

Angiotensin receptor blocker losartan suppresses platelet activity by interfering with thromboxane signaling.

UNLABELLED: Enhanced platelet activity and platelet endothelial interaction are hallmarks of different vascular and metabolic diseases with subsequent thrombus formation. In atherosclerosis, coronary artery disease, congestive heart failure, nitrate tolerance, chronic inflammation, or diabetic states, platelet activation may in part be due to a stimulation of the renin-angiotensin-aldosteron system, which also contributes to enhanced oxidant stress in these conditions. AIMS: We examined the putative role of the angiotensin receptor (AT1) and of phospholipase A2 (PLA2) in mediating platelet activation under defined in vitro conditions using the AT1 receptor antagonists losartan, EXP 3174, candesartan, and the PLA2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3), respectively. RESULTS: In washed human or canine platelet suspensions, losartan (10(-4)-10(-6) mol/L) dose-dependently suppressed thrombin-induced calcium transients as well as thromboxane (TxA2) release. In both species, aggregation of washed platelets in response to thrombin or ADP was substantially diminished by different doses of losartan. This inhibition of platelet aggregation was even maintained in ADP-stimulated platelet-rich plasma. While the PLA2 inhibitor AACOCF3 effectively inhibited thrombin-induced TxA2 release from washed human or canine platelets (similar to the effects observed with losartan), the AT1 agonist angiotensin II elicited platelet TxA2 release only at high supra-physiological doses (e.g., at 10(-4) mol/L). The AT1 specific antagonist candesartan did not diminish stimulated platelet aggregation, TxA2 formation, or calcium transients. By contrast, the active losartan metabolite EXP 3174 dose-dependently inhibited stimulated platelet calcium transients as well as TxA2 release at 1-100 micromol/L. CONCLUSIONS: Losartan significantly counteracts ex vivo platelet activation, probably via the blockade of TxA2 receptor-dependent signaling (e.g. implying activation of phospholipase A2) rather than acting at the AT1 receptor itself. This implies that the TxA2 signaling pathway plays a significant role during platelet activation, which may be successfully antagonized in vivo under different pathological states with enhanced thrombus formation or platelet-endothelium interactions.

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state.

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.

Blockade of angiotensin signaling improves myocardial function in hypercholesterolemia independent of changes in eicosanoid release.

In hypercholesterolemia in the presence or absence of atherosclerosis, cardiovascular dysfunction and altered signaling of angiotensin, nitric oxide, or prostanoids are closely related to enhanced oxidant stress. We analyzed the potentially beneficial effects of the specific angiotensin-converting enzyme inhibitor enalapril and the specific angiotensin receptor blocker losartan on cardiac performance, eicosanoid metabolism, and parameters of oxidant stress in hypercholesterolemic animals. Guinea pigs were fed a 1% cholesterol diet for 8 weeks (Chol) with or without equieffective doses of either enalapril (1.5 mg/kg/d; Ena) or losartan (3 mg/kg/d; Los). Hemodynamics were analyzed in Langendorff hearts. Detection of eicosanoids was by enzyme immunoassay. Estimation of plasma xanthine oxidase (XO) activity was determined by spectrophotometry. In hypercholesterolemic guinea pigs, enhanced oxidant stress (e.g., increased plasma XO activities) was associated with profound myocardial and coronary (e.g., endothelial) dysfunction. Both enalapril and losartan lowered plasma cholesterol levels slightly, but only the angiotensin receptor antagonist effectively suppressed the increased plasma XO activities (from 11.4 +/- 0.7 to 7.6 +/- 2.2 U/L), and at the same time decreased the augmented coronary flow (from 26.0 +/- 1.0 to 23.0 +/- 1.0 mL/min/g tissue) observed in hypercholesterolemic animals. Assessment of left ventricular pressure and contractility (e.g., dp/dtmax) as well as the diastolic relaxation parameter (tau) revealed substantial myocardial dysfunction (systolic and diastolic) in Chol that was more substantially (and comparably) improved during administration of losartan (Los) than during enalapril (Ena). Surprisingly, angiotensin signaling blockade by either antagonist further suppressed the diminished coronary dilator responses to bradykinin (BK; not significant for enalapril) or adenosine (Ado) was demonstrated in Chol Langendorff hearts [delta CPPBK/Ado: from 5.0 +/- 0.5/0.9 +/- 0.1 to 4.4 +/- 1.5/0.4 +/- 0.1 (Ena) or to 1.9 +/- 0.5/0.4 +/- 0.1 (Los) cm2 (area under the curve), respectively]. Finally, as expected from control studies using heart preparations from normocholesterolemic guinea pigs, enhanced cardiac release of eicosanoids, prostacyclin, and thromboxane in Chol (0.48 +/- 0.03 and 0.6 +/- 0.1 ng/min/g) was augmented even further by treatment with enalapril (Ena: 1.6 +/- 0.4 and 1.0 +/- 0.1 ng/min/g), but was significantly reduced to or below control levels in losartan-treated animals (Los: 0.4 +/- 0.1 and 0.2 +/- 0.1 ng/min/g). Blockade of angiotensin signaling via angiotensin-converting enzyme inhibition or receptor antagonism--although differentially acting on enhanced cardiac prostanoid metabolism and oxidant stress--efficiently restored proper systolic and diastolic myocardial performance (losartan was more beneficial than enalapril), probably by counterbalancing altered angiotensin II-->angiotensin receptor signaling in the cardiovascular system of hypercholesterolemic animals. Impaired coronary vasodilator capacity seems to be irreversible after 8 weeks of a high-cholesterol diet, as shown by the unexpected lack of a dilator effect with both enalapril and losartan.

How urine analysis reflects oxidative stress--nitrotyrosine as a potential marker.

Enhanced oxidant stress involved in the pathogenesis of cardiovascular (heart failure, atherosclerosis, ischemia-reperfusion injury), neurodegenerative (M. Alzheimer), metabolic (hypercholesterolemia, diabetes) and inflammatory disorders is mimicked by non-intermittent therapy with nitrovasodilators. We used this latter therapy model to study urinary 3-nitrotyrosine (n-tyr) excretion as a potential biomarker that may reflect the enhanced generation of reactive oxygen species. Namely, free or protein-bound n-tyr is formed in the organism by nitration of tyrosine (residues) via peroxynitrite (reaction product of NO&z.ccirf; and O(2)(-)&z. ccirf;). Free n-tyr content was analyzed by gas chromatography in urine obtained from healthy human subjects under a nitrite-limited diet during a two-day non-intermittent transdermal administration of glyceroltrinitrate (GTN; 0.4 mg/h) with or without vitamin C (Vit-C; 55 microg/kg/min) as antioxidant. Concomitant with the development of complete vascular tolerance (loss of dilator action), a progressive increase in urinary n-tyr excretion (up to 186+/-9 microg/day) was demonstrated in volunteers given GTN only. In contrast, when Vit-C was added, the GTN-induced increases in urinary n-tyr content were significantly suppressed (up to 130.20+/-6.91 microg/day), whereas Vit-C alone even decreased urinary n-tyr content (down to 34.00+/-5.66 microg/day), which was below control values (56.0+/-3.4 microg/day). Thus, urinary n-tyr may serve as a biomarker to detect changes in oxidant stress and thereby to evaluate the efficacy of therapeutic interventions aimed at reducing oxidant stress under various pathophysiological conditions.

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo.

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.

Cardiovascular dysfunction in hypercholesterolemia associated with enhanced formation of AT1-receptor and of eicosanoids.

BACKGROUND: In hypercholesterolemia with or without atherosclerosis cardiovascular dysfunction and altered signalling of angiotensin (Ang II), nitric oxide (NO), or prostanoids are intimately related to enhanced oxidant stress and concomitant changes in gene expression. We analyzed cardiac angiotensin receptor (AT1) expression and metabolism of Ang II, eicosanoids, and NO in hypercholesterolemic animals. METHODS: Guinea pigs were fed a 1% cholesterol diet for 8 weeks (Chol). Hemodynamics were analyzed in Langendorff hearts. Spectrophotometric determination of plasma lipids and radioimmunological detection of eicosanoids/cyclic guanosine monophosphate (cGMP). Activities of NO synthase III (NOS-III) or angiotensin converting enzyme (ACE) were determined by enzymatic assays. AT1 receptor density was assessed by radioligand binding assay. NOS-III mRNAs were quantitated by reverse transcription polymerase chain reaction. RESULTS: Hypercholesterolemia was associated with fatty degeneration of the liver and profound myocardial and coronary (e.g., endothelial) dysfunction. In Chol Langendorff hearts we observed significant increases in coronary flow (26.0 +/- 1.0 vs. 17.5 +/- 0.5 mL/min/g tissue) but diminished coronary responses to bradykinin (Bk, 250 ng bolus) or adenosine (Ado, 250 micrograms bolus) (delta CPPBk/Ado: 5 +/- 0.5 vs. 7.2 +/- 1/0.9 +/- 0.1 vs. 1.9 +/- 0.3 cm2 (area under the curve)). AT1 receptor expression was significantly increased in Chol hearts (72 +/- 6.8 vs. 45 +/- 5.6 fmol/mg protein), whereas marked suppression of cardiac activities of ACE (1.96 +/- 0.34 vs. 4.90 +/- 0.20 nmol/min/mg tissue) and of the entire cardiac nitric oxide-cGMP axis (e.g., NOS-III activity: 1.9 +/- 0.4 vs. 3.1 +/- 0.1 pmol/min/mg tissue; NOS-III mRNA: 0.82 +/- 0.16 vs. 1.20 +/- 0.12 arbitrary units; cGMP release: 0.41 +/- 0.02 vs. 0.54 +/- 0.04 pmol/min/g tissue) were shown in Chol. Finally, cardiac release of eicosanoids prostacyclin (PGI2) and thromboxane (TxA2) were significantly enhanced (0.48 +/- 0.05 vs. 0.38 +/- 0.05 and 0.60 +/- 0.10 vs. 0.24 +/- 0.10 ng/min/g tissue, respectively). Enhanced cardiac PGI2 release and suppression of cGMP synthesis in Chol were even more pronounced on stimulation with Bk (38.2 +/- 3.0 vs. 28.2 +/- 2.0 ng/min/g tissue and 1.9 +/- 0.3 vs. 3.0 +/- 0.3 pmol/min/g tissue, respectively). CONCLUSIONS: Altered angiotensin-mediated signal transduction probably related to augmented eicosanoid formation does not compensate for the limited endogenous NO production and for cardiovascular dysfunction in hypercholesterolemic guinea pigs. In this context, changes in redox-sensitive regulation of gene expression (AT1 receptor, NOS-III--caused by enhanced oxidant stress--could play a pivotal role.

Tolerance to nitrates with enhanced radical formation suppressed by carvedilol.

Enhanced oxidant stress occurs under many pathophysiologic conditions (e.g., inflammation) and can be induced and mimicked by continuous nitrate therapy, eliciting increases in platelet activity, enhanced formation of reactive oxygen species (ROS), and impaired nitrate-induced vasorelaxation. Analysis was performed of effects of coinfusion of glycerol trinitrate (GTN) either with a carvedilol metabolite with antioxidant properties or with antioxidant vitamin C (Vit-C) on various hemodynamic parameters during enhanced oxidant stress associated with nitrate tolerance. Carvedilol metabolite (BM910228: 4.5 microg/kg/min) or Vit-C (55 microg/kg/min) was coadministered with GTN (1.5 microg/kg/min) for 5 days in chronically instrumented dogs. Changes in coronary diameters (CD) and other hemodynamic parameters were continuously monitored, as well as changes in platelet function. At the beginning of GTN treatment, CD increased by 9.8 +/- 0.4% and progressively declined to basal control values within 3 days. However, with additional antioxidant protection either with BM910228 or with Vit-C, the GTN-induced increase in CD was maintained (8.6 +/- 0.4% or 10.5 +/- 0.6%) and remained elevated for the entire infusion period. The thrombin-stimulated intracellular Ca2+ concentrations of platelets remained nearly unchanged during Vit-C or BM910228 in contrast to the increase with GTN. The basal cyclic guanosine monophosphate (cGMP) contents of platelets after GTN coadministered with BM910228 or with Vit-C increased on day 1 to 233 or to 250% versus control and remained at that level. Additional in vitro tests with xanthine oxidase-induced oxidant stress resulted in a more or less pronounced scavenging of O2- radicals by BM920228, Vit-C, or superoxide dismutase (SOD). Coadministration of carvedilol metabolite BM910228 or of Vit-C along with GTN suppressed noxious effects of GTN-induced oxidant stress such as increased platelet activity and impaired nitrate-induced vasorelaxation.

Assembly and characterization of canine heart endothelial nitric oxide synthase cDNA and 5'-flanking sequence by homology (RT-)PCR cloning.

A broad spectrum of cardiovascular diseases is studied in canine animal models, in which dysfunction or dysregulation of the endothelial nitric oxide synthase (ecNOS) is of pivotal pathogenetic importance. To provide the tools for subsequent molecular analyses of ecNOS structure or function and to identify putative regulatory factors we isolated and characterized the canine heart ecNOS cDNA and putative regulatory (promoter) sequences. The complete coding sequence, 5'- plus part of 3'-untranslated regions (UTR) of ecNOS cDNA, and part of the 5'-flanking sequence (putative promoter region) were identified by homology (RT-)PCR cloning using canine heart total RNA or genomic DNA. Primer sequences were derived from bovine/human ecNOS cDNAs or genes. An ecNOS sequence contig of 5138 nucleotides length was established containing an open reading frame of 3618 nucleotides (1206 amino acids predicting a 133-kDa protein) and 253 bp 3'-UTR (distal to TGA codon)/1267 bp proximal to ATG codon (containing 5'-UTR and 5'-flanking sequences = putative promoter region). Comparison to human, bovine, murine, or porcine ecNOS sequences at the nucleotide or amino acid level yielded between 86 and 91% or 83 and 84% homologies, respectively. The canine ecNOS 5'-flanking sequence (putative promoter region) revealed stretches of homology up to 86% as compared to the human sequence containing a cluster of binding sites for several regulatory elements. The homology (RT-)PCR cloning strategy is presented as an alternative to common library cloning approaches. The obtained canine ecNOS sequence might serve to further analyze the structure, regulated function (promoter region consensus sites), and expression of ecNOS in different pathophysiological conditions and in other species (GenBank Accession No. BankIt264069 AF143503).

Comparison of glyceryl trinitrate-induced with pentaerythrityl tetranitrate-induced in vivo formation of superoxide radicals: effect of vitamin C.

Glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are among the most known organic nitrates that are used in cardiovascular therapy as vasodilators. However, anti-ischemic therapy with organic nitrates is complicated by the induction of nitrate tolerance. When nitrates are metabolized to release nitric oxide (NO), there is considerable coproduction of superoxide radicals in vessels leading to inactivation of NO. However, nitrate-induced increase of superoxide radical formation in vivo has not been reported. In this work, the authors studied the in vivo formation of superoxide radicals induced by treatment with PETN or GTN and determined the antioxidant effect of vitamin C. The formation of superoxide radicals was determined by the oxidation of 1-hydroxy-3-carboxy-pyrrolidine (CP-H) to paramagnetic 3-carboxy-proxyl (CP) using electron spin resonance spectroscopy. CP-H (9 mg/kg intravenous bolus and 0.225 mg/kg per minute continuous intravenous GTN or PETN 130 microg/kg) were infused into anesthetized rabbits. Every 5 min, blood samples were obtained from Arteria carotis to measure the CP formation. Both PETN and GTN showed similar vasodilator effects. Formation of CP in blood after infusions of GTN and PETN were 2.0+/-0.4 microM and 0.98+/-0.23 microM, respectively. Pretreatment with 30 mg/kg vitamin C led to a significant decrease in CP formation: 0.27+/-0.14 microM (vitamin C plus GTN) and 0.34+/-0.15 microM (vitamin C plus PETN). Pretreatment of animals with superoxide dismutase (15,000 units/kg) significantly inhibited nitrate-induced nitroxide formation. Therefore, in vivo infusion of GTN or PETN in rabbits increased the formation of superoxide radicals in the vasculature. PETN provoked a minimal stimulation of superoxide radical formation without simultaneous development of nitrate tolerance. The data suggest that the formation of superoxide radicals induced by organic nitrate correlates with the development of nitrate tolerance. The effect of vitamin C on CP formation leads to the conclusion that vitamin C can be used as an effective antioxidant for protection against nitrate-induced superoxide radical formation in vivo.

Venous Function in Patients with Venous Disease and Healthy Controls Before and After a Bathing Procedure and Subsequent Cold Stimulus

The object of this study was to determine whether bathing in thermal water acutely affects venous function in patients with venous insufficiency. Measurements were taken of venous function in patients with leg varicosities and healthy subjects before and after a thermal bath and exercise on two days, with subsequent application of a cold stimulus on 1 day. A total of 28 patients with varicose veins in one or both legs (n = 45 legs) and 30 healthy controls (n = 60 legs). On 2 consecutive days all subjects underwent a standardized bathing and exercise procedure in water at 34 degreesC for 20 minutes. On one day, determined randomly for each subject, a cold stimulus was applied to both lower legs after bathing. Measurements were done before subjects entered the bath and after the completion of bathing (including cold stimulus on one day). Without the cold stimulus, venous function of patients and controls did not change. After application of the cold stimulus the venous capacity of both patients and controls was significantly reduced compared with the basal levels. There was no change in maximum venous outflow in the controls, but that of the patient group decreased slightly compared with basal levels. Contrary to widespread belief, bathing in water at temperatures above 28 degreesC does not adversely affect venous function in patients with varicose veins. When applied in moderation (34 degreesC for 20 minutes) this physical therapy should not be regarded as contraindicated in this and related conditions, especially given its known hemodynamic benefits. Further studies are required to determine the long-term effects of repeated immersion.

Formation of reactive oxygen species by pentaerithrityltetranitrate and glyceryl trinitrate in vitro and development of nitrate tolerance.

Anti-ischemic therapy with organic nitrates is complicated by tolerance. Induction of tolerance is incompletely understood and likely multifactorial. Recently, increased production of reactive oxygen species (ROS) has been investigated, but it has not been clear if this is a direct consequence of the organic nitrate on the vessel or an in vivo adaptation to the drugs. To examine the possibility that nitrates could directly stimulate vascular ROS production, we compared the development of nitrate tolerance with the formation of ROS induced by pentaerithrityltetranitrate (PETN) or nitroglycerin (GTN) in vitro in porcine smooth muscle cells, endothelial cells, washed ex vivo platelets and whole blood. By examining cGMP formation, it was found that 24-hr treatment with GTN but not PETN induced significant nitrate tolerance, which was prevented by parallel treatment with Vit C. Incubation of vascular cells acutely with 0.5 mM GTN doubled the rate of ROS generation, whereas PETN had no such effect. The rate of ROS (peroxynitrite and O2) formation detected by specific spin traps in tolerant smooth muscle cells, treated for 24 hr with 0.01 mM GTN, was substantially higher (30.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also increases the formation of ROS both in vascular cells and in whole blood. ROS formation is minimally stimulated by PETN comparable to data obtained in Vit C-suppressed GTN tolerance. ROS formation induced by organic nitrates seems to be a key factor in the development of nitrate tolerance.

Dietary supplement with vitamin C prevents nitrate tolerance.

Enhanced formation of superoxide radicals has been proposed to play a major role in the development of nitrate tolerance in humans. We tested the effects of vitamin C (Vit-C) supplementation on glyceroltrinitrate (GTN)-induced hemodynamic effects during 3-d nonintermittent transdermal administration of GTN (0.4 mg/h) in nine healthy subjects. Tolerance development was monitored by changes in arterial pressure, dicrotic digital pulse pressure, and heart rate. Studies with GTN, Vit-C, or GTN/Vit-C were successively carried out at random in three different series in the same subjects. GTN treatment caused an immediate rise in arterial conductivity (a/b ratio of dicrotic pulse), but within 2 d of initiating GTN, the a/b ratio progressively decreased and reached basal levels. In addition, there was a progressive loss of the orthostatic decrease in blood pressure. However, coadministration of Vit-C and GTN fully maintained the GTN-induced changes in the orthostatic blood pressure, and the rise of a/b ratio was augmented by 310% for the duration of the test period. Changes in vascular tolerance in GTN-treated subjects were paralleled by upregulation of the activity of isolated platelets, which was also reversed by Vit-C administration. These findings demonstrate that dietary supplementation with Vit-C eliminates vascular tolerance and concomitant upregulation of ex vivo-washed platelet activity during long-term nonintermittent administration of GTN in humans.

Detection of superoxide radicals and peroxynitrite by 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine: quantification of extracellular superoxide radicals formation.

The reactions of the new sterically hindered hydroxylamine 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) with superoxide radical and peroxynitrite have been studied. These reactions produce the nitroxide 4-phosphonooxy-2,2,6, 6-tetramethyl-piperidinyloxy. The rate constant for reaction of superoxide with PP-H is determined as (8.4+/-0.6).10(2) M-1s-1. It was found that PP-H provides almost the same spin trapping efficacy as 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background oxidation of PP-H in blood is much less than for CP-H. The extremely slow PP-H penetration into the cells makes possible the study of extracellular formation of superoxide radical. The acute treatment of blood with nitroglycerin is shown to induce an extracellular superoxide radical formation. PP-H is more sensitive for detection of reactive oxygen species as compared with CP-H. PP-H is an effective scavenger of superoxide radical and of peroxynitrite, and can be used to quantify the extracellular formation of these reactive oxygen species.

Intrinsic neural regulation of the heart in the chronic, conscious dog.

The present experiments were performed to examine the capability of the intrinsic cardiac nerves (ICN) to modify cardiac performance in the resting chronic, conscious dog. Control and cardiac-denervated dogs were instrumented for recording of left atrial (LA) and ventricular (LV) contractility, heart rate, and atrioventricular (AV) conduction time. Acetylcholine (ACh) and nicotine (Nic) were administered via an indwelling coronary artery catheter. Limited distribution from the injection site only allowed access to the LA, LV, and AV node. Both beta-blockade with timolol and cardiac denervation were used to separate direct effects of ICN stimulation from indirect (e.g., reflex) effects. ACh produced the expected negative inotropic and dromotropic changes. ICN stimulation with Nic caused large decreases in LA on but only trivial effects on the LV. We concluded that the ICN has limited effects on cardiac performance in the resting animal under minimal sympathetic drive. It is likely, however, that the ICN is capable of significantly depressing cardiac function under conditions of elevated sympathetic tone as would be encountered in exercise.