Endothelium- and nitric oxide-dependent vasorelaxing activities of gamma-butyrobetaine esters: possible link to the antiischemic activities of mildronate.

Mildronate [3-(2,2,2-trimethylhydrazine) propionate (THP)] is an antiischemic drug acting mainly via inhibition of fatty acid beta-oxidation. Some effects of the drug cannot be explained by the latter mechanism. We tested the eventual nitric oxide (NO) dependence of the mildronate action. Mildronate, gamma-butyrobetaine (GBB) and GBB methyl ester induced transient increases in nitric oxide (NO) concentrations in rat blood and myocardium. In vitro, these compounds neither modified the activities of purified neuronal and endothelial recombinant nitric oxide synthases (NOSs) nor were able to interact with their active site. GBB induced vasodilatation at high concentrations only (EC50 = 5 x 10(…

Glycyrrhetinic Acid Reverses the Lipopolysaccharide-Induced Hypocontractility to Noradrenaline in Rat Aorta: Implications to Septic Shock

Abstract.: Septic shock and associated vascular hyporeactivity to vasoconstrictor agonists remain a major problem of critical care medicine. Here we report that glycyrrhetinic acid (GA), the active component of licorice, effectively restores vascular contractility in the model of lipopolysaccharide (LPS)-treated rat aorta. GA was as effective as the NO synthase inhibitor NG-nitroarginine methylester. GA did not affect the vascular NO levels (measured by EPR spin trapping) and relaxations to l-arginine in LPS-treated rings as well as relaxation to S-nitroso-Nacetylpenicillamine in control rings. Thus, GA may represent an interesting alternative to NO synthase inhibitors in sepsis-associated …

Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats.

Nebivolol is a β 1 -receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II–induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, …

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2−/− mice

Background and purpose: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. Experimental approach: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) an…

Dinitrosyl-iron triggers apoptosis in Jurkat cells despite overexpression of Bcl-2

Cells expressing the cytokine-inducible NO synthase are known to trigger apoptosis in neighboring cells. Paramagnetic dinitrosyl nonheme iron complexes (DNIC) were found in tumor tissue about 40 years ago; however, the role of these NO(+)-bearing species is not completely understood. In the human Jurkat leukemia cell line, the application of the model complex DNIC-thiosulfate (50-200 microM) induced apoptosis (defined by phosphatidylserine externalization) in a concentration- and time-dependent manner. In Jurkat cells, the pan-caspase inhibitor, zVADfmk (50 microM), and/or stable transfection of antiapoptotic protein, Bcl-2, was unable to afford protection against DNIC-induced apoptosis. Th…

O44. Inhibition of CD98-associated amino acid transporters by dinitrosyl iron complexes

Normalization of endothelial dysfunction and vascular oxidative stress by chronic atorvastatin treatment in a rat model of streptozotocin-induced diabetes mellitus (type I)

Heparin–polynitroxides: Synthesis and preliminary evaluation as cardiovascular EPR/MR imaging probes and extracellular space-targeted antioxidants

We report here the synthesis of heparin-polynitroxide derivatives (HPNs) in which nitroxide moieties are linked either to uronic acid or glycosamine residues of the heparin macromolecule. HPNs have low anticoagulant activity, possess superoxide scavenging properties, bind to the vascular endothelium/extra-cellular matrix and can be detected by EPR and MRI techniques. As the vascular wall-targeted redox-active paramagnetic compounds, HPNs may have both diagnostic (molecular MRI) and therapeutic (ecSOD mimics) applications.

Cyclooxygenase 2-selective and nonselective nonsteroidal anti-inflammatory drugs induce oxidative stress by up-regulating vascular NADPH oxidases.

Cyclooxygenase 2-selective inhibitors (coxibs) and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with an increase in cardiovascular events. The current study was designed to test the effect of coxibs and nonselective NSAIDs on vascular superoxide and nitric oxide (NO) production. mRNA expression of endothelial NO synthase (eNOS) and of the vascular NADPH oxidases was studied in spontaneously hypertensive rats (SHR) and in human endothelial cells. The expression of Nox1, Nox2, Nox4, and p22phox was increased markedly by the nonselective NSAIDs diclofenac or naproxen and moderately by rofecoxib or celecoxib in the aorta and heart of SHR. The up-regulation of NADPH …

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4.

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expressio…

Heparin–polynitroxide derivatives: a platform for new diagnostic and therapeutic agents in cardiovascular disease?

Vascular wall extracellular oxidative stress Cardiovascular disease (CVD; mainly atherosclerosis, hypertension and diabetes mellitus) remains a major cause of death in western society [1]. Despite substantial progress achieved, the diagnosis of CVD often comes too late, when the disease has already advanced to therapeutically incurable stages. The development of efficient diagnostic probes allowing early non-invasive diagnostics, as well as drugs which can prevent or reverse CVD and/or its complications (e.g., myocardium infarctus and stroke) are highly desired tasks of the modern cardiovascular medicinal chemistry.

Gp91phox-containing NAD(P)H oxidase increases superoxide formation by doxorubicin and NADPH

Doxorubicin is a highly effective antineoplastic drug associated with a dose-dependent cardiotoxicity that may result in irreversible cardiomyopathy and heart failure. Gene variants of the superoxide-generating enzyme NAD(P)H oxidase have recently been associated with this phenotype. We investigated the mechanism of this association using lucigenin-enhanced chemiluminescence, spectrophotometry, electrochemical sensor, and electron paramagnetic resonance spectroscopy. Superoxide production was measured in female wild-type and NAD(P)H oxidase-deficient (gp91phox knockout) mice. The magnitude of the increase in superoxide production on the addition of doxorubicin was much higher in hearts of w…

Electron paramagnetic resonance in a biomedical laboratory

Sirolimus-Induced Vascular Dysfunction

Objectives This study sought to analyze mechanisms that mediate vascular dysfunction induced by sirolimus. Background Despite excellent antirestenotic capacity, sirolimus-eluting stents have been found to trigger coronary endothelial dysfunction and impaired re-endothelialization. Methods To mimic the continuous sirolimus exposure of a stented vessel, Wistar rats underwent drug infusion with an osmotic pump for 7 days. Results Sirolimus treatment caused a marked degree of endothelial dysfunction as well as a desensitization of the vasculature to the endothelium-independent vasodilator nitroglycerin. Also, sirolimus stimulated intense transmural superoxide formation as detected by dihydroeth…

Development of a Polarizer and Biocompatible Polarizing Agents for Use in Dynamic Nuclear Polarization DNP-Enhanced NMR and MRI

The application of 13C (or other low γ nuclei) NMR spectroscopy and imaging for clinical diagnosis has been constrained by the extremely long imaging and spectroscopy acquisition times that are required to obtain high SNR under physiological conditions (low natural abundance of 13C, low concentration of 13C-compounds, physiological temperature etc.). However, this obstacle could be overcome by in vitro hyperpolarization of a 13C-containing molecule with long spin lattice relaxation time via dynamic nuclear polarization (DNP) and subsequent injection into the animal or patient of investigation [1, 2]. DNP is achieved by resonant excitation of electron spins of radicals (electron paramagnetic…

Mechanisms of Increased Vascular Superoxide Production in an Experimental Model of Idiopathic Dilated Cardiomyopathy

Objective— In the present study, we sought to identify mechanisms underlying increased oxidative stress in vascular tissue in an experimental animal model of chronic congestive heart failure (CHF). Methods and Results— Superoxide and nitric oxide (NO) was measured in vessels from cardiomyopathic hamsters (CHF hamsters) and golden Syrian hamsters. We also determined expression of endothelial nitric oxide synthase (NOSIII), the soluble guanylyl cyclase, the cGMP-dependent kinase, and the NADPH oxidase. To analyze the contribution of the renin-angiotensin system to oxidative stress, CHF hamsters were treated with the angiotensin-converting enzyme inhibitor captopril for 200 days (120 mg · kg …

NADPH Oxidase Accounts for Enhanced Superoxide Production and Impaired Endothelium-Dependent Smooth Muscle Relaxation in BKβ1 −/− Mice

Objective— Nitric oxide (NO)-induced vasorelaxation involves activation of large conductance Ca 2+ -activated K + channels (BK). A regulatory BKβ1 subunit confers Ca 2+ , voltage, and NO/cGMP sensitivity to the BK channel. We investigated whether endothelial function and NO/cGMP signaling is affected by a deletion of the β1-subunit. Methods and Results— Vascular superoxide in BKβ1 −/− was measured using the fluorescent dye hydroethidine and lucigenin-enhanced chemiluminescence. Vascular NO formation was analyzed using electron paramagnetic resonance (EPR), expression of NADPH oxidase subunits, the endothelial NO synthase (eNOS), the soluble guanylyl cyclase (sGC), as well as the activity a…

Deficiency of glutathione peroxidase-1 accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice.

Background— We have recently demonstrated that activity of red blood cell glutathione peroxidase-1 is inversely associated with the risk of cardiovascular events in patients with coronary artery disease. The present study analyzed the effect of glutathione peroxidase-1 deficiency on atherogenesis in the apolipoprotein E-deficient mouse. Methods and Results— Female apolipoprotein E-deficient mice with and without glutathione peroxidase-1 deficiency were placed on a Western-type diet for another 6, 12, or 24 weeks. After 24 weeks on Western-type diet, double-knockout mice (GPx-1 −/− ApoE −/− ) developed significantly more atherosclerosis than control apolipoprotein E-deficient mice. Moreover…

Spin‐Labeled Heparins as Polarizing Agents for Dynamic Nuclear Polarization

A potentially biocompatible class of spin-labeled macromolecules, spin-labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser-type dynamic nuclear polarization (DNP). All presented SL-heparins show high 1 H DNP enhancement factors up to E=-110, which validates that effectively more than one hyperfine line can be saturated even for spin-labeled polarizing agents. The parameters for the Overhauser-type DNP are determined and discussed. A striking result is that for spin-labeled heparins, the off-resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non-negligible part…

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

Nitric oxide (NO) is a free radical species with multiple physiological functions. Because of low concentrations and short half-life of NO, its direct measurement in living tissues remains a difficult task. Electron paramagnetic resonance (EPR) spin trapping is probably one of the best suitable platforms for development of new methods for quantification of biological NO. The most reliable EPR-based approaches developed so far are based on the reaction of NO with various iron complexes, both intrinsic and exogenously applied. This review is focused on the current state and perspectives of EPR spin trapping for experimental and clinical NO biology.