A prospective study of contrast preservation using ultra-low contrast delivery technique versus standard automated contrast injector system in coronary procedures.

BACKGROUND: We aimed to assess the decrease in contrast media volume (CMV) with ultra-low contrast delivery technique (ULCD) developed at our institution versus the usual automated contrast injector system (ACIS) contrast delivery in coronary procedures. METHODS: We analyzed the amount of contrast given in the consecutive 204 patients of the operators who use ULCD technique versus consecutive 200 patients of the other operators who use ACIS without ULCD technique for coronary angiograms and/or percutaneous coronary interventions (PCIs) from May 2017 to July 2018 at our center. We calculated the mean CMV between these groups. RESULTS: We observed a significant reduction in mean CMV with ULCD technique versus standard ACIS, respectively: angiogram 24.8 ± 15.8 mL (n = 194) vs 42.3 ± 25.1 mL (n = 200) (p < 0.0001); PCI 23.5 ± 19.7 mL (n = 52) vs 48.2 ± 30.8 mL (n = 16) (p < 0.0070); angiogram with ad hoc PCI 53.4 ± 32.1 mL (n = 23) vs 89.7 ± 35.6 mL (n = 16) (p < 0.0024); and overall angiogram and PCI 27.4 ± 20.5 mL (n = 204) vs 44.9 ± 28.0 mL (n = 181) (p < 0.0001). CONCLUSION: Our study showed a highly significant reduction in CMV using ULCD technique compared to standard ACIS contrast delivery in coronary invasive procedures. Even in the standard ACIS arm, CMV was significantly lower than values reported in literature, possibly due to operators' bias toward contrast preservation.

The favorable kinetics and balance of nebivolol-stimulated nitric oxide and peroxynitrite release in human endothelial cells.

BACKGROUND: Nebivolol is a third-generation beta-blocker used to treat hypertension. The vasodilation properties of nebivolol have been attributed to nitric oxide (NO) release. However, the kinetics and mechanism of nebivolol-stimulated bioavailable NO are not fully understood. METHODS: Using amperometric NO and peroxynitrite (ONOO⁻) nanosensors, ß3-receptor (agonist: L-755,507; antagonists: SR59230A and L-748,337), ATP efflux (the mechanosensitive ATP channel blocker, gadolinium) and P2Y-receptor (agonists: ATP and 2-MeSATP; antagonist: suramin) modulators, superoxide dismutase and a NADPH oxidase inhibitor (VAS2870), we evaluated the kinetics and balance of NO and ONOO⁻ stimulated by nebivolol in human umbilical vein endothelial cells (HUVECs). NO and ONOO⁻ were measured with nanosensors (diameter ~ 300 nm) placed 5 ± 2 µm from the cell membrane and ATP levels were determined with a bioluminescent method. The kinetics and balance of nebivolol-stimulated NO and ONOO⁻ were compared with those of ATP, 2-MeSATP, and L-755,507. RESULTS: Nebivolol stimulates endothelial NO release through ß3-receptor and ATP-dependent, P2Y-receptor activation with relatively slow kinetics (75 ± 5 nM/s) as compared to the kinetics of ATP (194 ± 10 nM/s), L-755,507 (108 ± 6 nM/s), and 2-MeSATP (105 ± 5 nM/s). The balance between cytoprotective NO and cytotoxic ONOO- was expressed as the ratio of [NO]/[ONOO⁻] concentrations. This ratio for nebivolol was 1.80 ± 0.10 and significantly higher than that for ATP (0.80 ± 0.08), L-755,507 (1.08 ± 0.08), and 2-MeSATP (1.09 ± 0.09). Nebivolol induced ATP release in a concentration-dependent manner. CONCLUSION: The two major pathways (ATP efflux/P2Y receptors and ß3 receptors) and several steps of nebivolol-induced NO and ONOO⁻ stimulation are mainly responsible for the slow kinetics of NO release and low ONOO⁻. The net effect of this slow kinetics of NO is reflected by a favorable high ratio of [NO]/[ONOO⁻] which may explain the beneficial effects of nebivolol in the treatment of endothelial dysfunction, hypertension, heart failure, and angiogenesis.

Deletion of the activated protein-1 transcription factor JunD induces oxidative stress and accelerates age-related endothelial dysfunction.

BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.

Amorphous silica nanoparticles aggregate human platelets: potential implications for vascular homeostasis.

BACKGROUND: Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO(-)]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO(-)] balance and platelet aggregation. METHODS: Nanoparticle-platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO(-) released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy. RESULTS: Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO(-) leading to an unfavorably low [NO]/[ONOO(-)] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size. CONCLUSIONS: The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO(-)] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets.

Amorphous silica nanoparticles trigger nitric oxide/peroxynitrite imbalance in human endothelial cells: inflammatory and cytotoxic effects.

BACKGROUND: The purpose of this study was to investigate the mechanism of noxious effects of amorphous silica nanoparticles on human endothelial cells. METHODS: Nanoparticle uptake was examined by transmission electron microscopy. Electrochemical nanosensors were used to measure the nitric oxide (NO) and peroxynitrite (ONOO(-)) released by a single cell upon nanoparticle stimulation. The downstream inflammatory effects were measured by an enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and flow cytometry, and cytotoxicity was measured by lactate dehydrogenase assay. RESULTS: We found that the silica nanoparticles penetrated the plasma membrane and rapidly stimulated release of cytoprotective NO and, to a greater extent, production of cytotoxic ONOO(-). The low [NO]/[ONOO(-)] ratio indicated increased nitroxidative/oxidative stress and correlated closely with endothelial inflammation and necrosis. This imbalance was associated with nuclear factor κB activation, upregulation of key inflammatory factors, and cell death. These effects were observed in a nanoparticle size-dependent and concentration-dependent manner. CONCLUSION: The [NO]/[ONOO(-)] imbalance induced by amorphous silica nanoparticles indicates a potentially deleterious effect of silica nanoparticles on vascular endothelium.

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction.

BACKGROUND: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. METHODS: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. RESULTS: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p<0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, l-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p<0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p<0.05 vs. PQ treated PARP-1(+/+). CONCLUSION: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.

Effect of enhanced glycemic control with saxagliptin on endothelial nitric oxide release and CD40 levels in obese rats.

AIM: Endothelial cell (EC) dysfunction contributes to insulin resistance in diabetes and is characterized by reduced nitric oxide (NO) release, increased nitroxidative stress and enhanced inflammation. The purpose of this study was to test the effect of improved postprandial glucose control on EC function in insulin-resistant rats as compared to fasting glucose (FG) changes. METHODS: Obese Zucker rats were treated with 10 mg/kg/day saxagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, for 4 or 8 weeks and compared to lean rats. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was measured ex vivo using amperometric approaches and correlated with FG, postprandial glucose, insulin, soluble CD40 (sCD40) and L-citrulline levels. RESULTS: Saxagliptin treatment improved NO production and reduced ONOO(-) release prior to any observed changes in FG levels. In untreated obese animals, NO release from aortic and glomerular ECs decreased by 22% and 31%, respectively, while ONOO(-) release increased by 26% and 40%. Saxagliptin increased aortic and glomerular NO release by 18% and 31%, respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio, an indicator of NO synthase coupling, increased by >40%. Improved glycemic control was further associated with a reduction in sCD40 levels by more than ten-fold (from 300 ± 206 to 22 ± 22 pg/mL, p < 0.001). CONCLUSION: These findings indicate that enhanced glycemic control with DPP4 inhibition improved NO release and reduced inflammation in a manner not predicted by FG changes alone.

Synergistic effect of amlodipine and atorvastatin in reversing LDL-induced endothelial dysfunction.

PURPOSE: Statins and certain calcium channel blockers may improve nitric oxide (NO) release and endothelial function through various mechanisms, but their combined effects are not well understood. METHODS: The separate versus combined effects of amlodipine (AML) and atorvastatin (AT) on NO and peroxynitrite (ONOO-) were measured in human umbilical vein endothelial cells (HUVEC) in the presence and absence of low-density lipoprotein (LDL) using electrochemical nanosensors. RESULTS: The combination of AML (5 micromol/l) and AT (3-6 micromol/l) directly stimulated NO release that was about twofold greater than the sum of their separate effects (p < 0.05). This synergistic activity is attributed to enhanced endothelial NO synthase (eNOS) function and decreased cytotoxic ONOO-. LDL (100 mg/dl) caused a dysfunction of HUVEC manifested by a 60% reduction in NO and an almost twofold increase in ONOO-. Treatment with AML/AT partially reversed the effects of LDL on endothelial function, including a 90% increase in NO and 50% reduction in ONOO-. Small-angle X-ray diffraction analysis indicates that AML and AT are lipophilic and share an overlapping molecular location in the cell membrane that could facilitate electron transfer for antioxidant mechanisms. CONCLUSION: These findings indicate a synergistic effect of AML and AT on an increase in NO concentration, reduction of nitroxidative stress. Also, AML/AT partially restored the NO level of LDL-induced dysfunctional endothelium. Their combined effects may be enhanced by antioxidant properties related to their intermolecular actions in the cell membrane and an increase in the expression and coupling of endothelial nitric oxide synthase.

Electropolymerization on microelectrodes: functionalization technique for selective protein and DNA conjugation.

A critical shortcoming of current surface functionalization schemes is their inability to selectively coat patterned substrates at micrometer and nanometer scales. This limitation prevents localized deposition of macromolecules at high densities, thereby restricting the versatility of the surface. A new approach for functionalizing lithographically patterned substrates that eliminates the need for alignment and, thus, is scalable to any dimension is reported. We show, for the first time, that electropolymerization of derivatized phenols can functionalize patterned surfaces with amine, aldehyde, and carboxylic acid groups and demonstrate that these derivatized groups can covalently bind molecular targets, including proteins and DNA. With this approach, electrically conducting and semiconducting materials in any lithographically realizable geometry can be selectively functionalized, allowing for the sequential deposition of a myriad of chemical or biochemical species of interest at high density to a surface with minimal cross-contamination.

S-nitroso human serum albumin attenuates ischemia/reperfusion injury after cardioplegic arrest in isolated rabbit hearts.

BACKGROUND: Depletion of nitric oxide (NO) is associated with ischemia/reperfusion injury. The novel NO donor, S-nitroso human serum albumin (S-NO-HSA), could bridge NO depletion during reperfusion in cardiac transplantation and minimize ischemia/reperfusion injury. METHODS: In an isolated erythrocyte-perfused working heart model, rabbit hearts were randomly assigned after assessment of hemodynamic baseline values to receive S-NO-HSA (0.2 micromol/100 ml, n = 8), L-arginine (10 mmol/100 ml, n = 8) or albumin (control) (0.2 micromol/100 ml, n = 8). After 20 minutes of infusion, the hearts were arrested and stored in Celsior (4 degrees C) enriched with respective drugs for 6 hours, followed by 75 minutes of reperfusion. Hemodynamic values were assessed and biopsy specimens were taken to determine calcium-ionophore stimulated release of NO and superoxide. RESULTS: During early reperfusion, recovery of cardiac output (75% +/- 6% vs 49% +/- 5%, p < 0.05) and coronary flow (99% +/- 8% vs 70% +/- 5%, p < 0.05) were higher, and myocardial oxygen consumption was reduced in the S-NO-HSA Group compared with Control (4.08 +/- 0.46 ml/min/0.1 kg vs 6.78 +/- 0.38 ml/min/0.1 kg, p < 0.01). At the end of the experiment cardiac output (53% +/- 5% vs 27% +/- 5%, p < 0.01) was higher and left atrial pressure (115% +/- 9% vs 150% +/- 8%, p < 0.05) was lower in the S-NO-HSA Group compared with Control. NO release was increased (1,040 +/- 50 nmol/liter and 1,070 +/- 60 nmol/liter vs 860 +/- 10 nmol/liter, p < 0.01) and superoxide release diminished (31 +/- 5 nmol/liter and 38 +/- 5 nmol/liter vs 64 +/- 5 nmol/liter, p < .01) in the S-NO-HSA and L-arginine Groups compared with Control. CONCLUSION: S-NO-HSA improved hemodynamic functions after prolonged hypothermic cardiac arrest by supplementing NO and thereby decreasing ischemia/reperfusion injury.

Restoration of endothelial function via enhanced nitric oxide synthesis after long-term treatment of raloxifene in adult hypertensive rats.

Raloxifene (CAS 84449-90-1, RAL), a selective estrogen receptor modulator (SERM) effective for the prevention of post-menopausal osteoporosis, also has been shown to acutely stimulate nitric oxide (NO) synthesis associated with improved endothelium-dependent relaxation. The effect of a 3-month RAL treatment (10 mg/kg/d) on basal blood pressure, measured via the carotid artery, and its challenge with increasing doses of intravenous bradykinin (1, 3 and 10 nmol/kg) was investigated. Furthermore, aortic NO bioavailability and relaxation in 9-month-old male and female ovarectomized (OVX) spontaneously hypertensive rats (SHR) was tested. Calcium ionophore stimulated NO release from aortic endothelial cells and aortic superoxide (O2-) production was directly assessed by using electrochemical nanosensors. Relaxation studies were performed with acetylcholine (10(-8) to 10(-5) mol/L) following precontraction with phenylephrine (10(-7) mmol/L). Whereas basal blood pressure (BP) was not significantly decreased in RAL treated SHR, the dose-dependent challenge with bradykinin induced an enhanced BP reduction in either gender. In contrast to female animals, aortic segments from RAL treated male animals showed significantly improved relaxaSHR) to 360 +/- 15 nmol/L. Vice versa, O2- was decreased from 110 +/- 15 to 22 +/- 1 nmol/L. In female SHR, ovarectomy led to an increase/decrease of NO/O2- from 130 +/- 5 to 180 +/- 10 nmol/L and 82 +/- 7 to 68 +/- 3 nmol/L, respectively. These effects were significantly amplified by RAL treatment (NO: 370 +/- 10 and O2-: 25 +/- 2 nmol/L). The results show that long-term treatment with RAL has beneficial affects on the cardiovascular system in old male and female OVX SHR via an increased NO bioavailability.

Endothelial NADH/NADPH-dependent enzymatic sources of superoxide production: relationship to endothelial dysfunction.

There is growing evidence that endothelial dysfunction, which is often defined as the decreased endothelial-derived nitric oxide (NO) bioavailability, is a crucial factor leading to vascular disease states such as hypertension, diabetes, atherosclerosis, heart failure and cigarette smoking. This is due to the fact that the lack of NO in endothelium-dependent vascular disorders contributes to impaired vascular relaxation, platelet aggregation, increased vascular smooth muscle proliferation, and enhanced leukocyte adhesion to the endothelium. During the last several years, it has become clear that reduction of NO bioavailability in the endothelium-impaired function disorders is associated with an increase in endothelial production of superoxide (O(2)(*-)). Because O(2)(*-) rapidly scavenges NO within the endothelium, a reduction of bioactive NO might occur despite an increased NO generation. Among many enzymatic systems that are capable of producing O(2)(*-), NAD(P)H oxidase and uncoupled endothelial NO synthase (eNOS) apparently are the main sources of O(2)(*-) in the endothelial cells. It seems that O(2)(*-) generated by NAD(P)H oxidase may trigger eNOS uncoupling and contribute to the endothelial balance between NO and O(2)(*-). That is maintained at diverse levels.

Combined L-arginine and antioxidative vitamin treatment mollifies ischemia-reperfusion injury of skeletal muscle.

Enhanced production of superoxide in L-arginine-depleted environments and concomitant reduction of nitric oxide (NO) concentration are involved in ischemia-reperfusion (I/R) injury. Treatment with L-arginine or antioxidative vitamins alone and in combination was used to mollify I/R injury in skeletal muscle. Untreated rabbits were compared with those treated with L-arginine/antioxidative vitamin cocktail Omnibionta only, or a combination of L-arginine/ antioxidative vitamins during hind limb I/R (2.5 hours/2 hours). NO was continuously measured in vivo. Plasma malondialdehyde (MDA) served as the measure of oxygen free radical formation. Interstitial edema formation, microvessel diameter alterations, microvessel plugging, and blood flow changes were used as indicators of I/R injury. The MDA level in untreated animals 2 hours after reperfusion was significantly higher than in control animals (0.81 micromol/L +/- 0.14 micromol/L vs 0.57 micromol/L +/- 0.11 micromol/L; P<.05), indicating enhanced production of oxygen free radicals. This sequela paralleled the decreasing concentration of NO, which dropped below the detection limit (1 nmol/L) after reperfusion. Microvascular changes during I/R injury were expressed as a 40% decrease in microvessel diameter and adhesion of neutrophils in 20% of microvessels, which led to a consequent 60% reduction in blood flow, demonstrating "no reflow" (reperfusion failure after restoration of blood flow). The increase in the fraction of muscle interfiber area by 85% indicated prominent edema formation. Treatment with antioxidative vitamins alone had a minimally positive effect on edema formation and microvascular plugging, possibly by suppression of oxygen free radical production, as expressed by the reduction in plasma MDA levels. However, this therapy failed to preserve basal NO production and to protect from microvascular constriction and no reflow. Treatment with L-arginine alone had a stronger protective effect, maintaining basal NO production, further reduction of neutrophil plugging, abolition of microvascular constriction, and no reflow. The combination of antioxidative vitamins and L-arginine was the best treatment against I/R injury, expressed not only by the protection of microvessel constriction, but also by abolition of microvascular plugging, increase in NO production (68 nmol/L +/- 5 nmol/L) over the basal level (52 nmol/L +/- 7 nmol/L), and higher blood flow, as compared with treatment with L-arginine or antioxidative vitamins alone.

Effect of chronic treatment with the vasopeptidase inhibitor AVE 7688 and ramipril on endothelial function in atherogenic diet rabbits.

Cardiovascular disease is the major cause of death in Western nations, although improved possibilities regarding diagnosis and therapy now exist. Endothelial dysfunction is triggered by cardiovascular risk factors such as hypercholesterolaemia, hypertension, adiposity and smoking, contributing to the common endpoint of atherosclerosis. This study examined the pharmacological effects of angiotensin-converting enzyme (ACE) and combined ACE-neutral endopeptidase (NEP) (vasopeptidase) inhibitors on endothelial dysfunction in the model of hyperlipidaemic rabbits. The focus of the study was to assess endothelial function after treatment with the ACE-NEP inhibitor AVE 7688 (30 mg/kg/day) in comparison to the ACE inhibitor (ACE-I) ramipril (1 mg/kg/day). Different parameters, such as endothelial function, blood pressure (BP), expansion of plaques, endothelial nitric oxide (NO) and superoxide (O2-) release and plasma levels of various lipidaemic parameters were analysed. Control groups consisted of one group fed only with normal diet, one group fed only with atherogenic diet and the direct control group fed with varied diets (six weeks atherogenic diet followed by 12 weeks normal diet). Since for the treatment of atherosclerosis, a change in feeding is absolutely necessary, in the present study, at the start of the treatments with AVE 7688 and ramipril, the rabbits food was changed to a normal diet. At the end of the study, mean arterial blood pressure (MAP) was measured in the anaesthetised animals. The values in standard, atherogenic and varied diet-fed rabbits were around 73 2 mmHg. Angiotensin I (Ang I) given intravenous (i.v.) induced a strong increase in MAP of about 20%. In both the treated groups Ang I-induced BP increase was inhibited. In contrast, i.v. bradykinin led to a strong reduction in MAP in both the treated groups of around 50%. Six weeks feeding with an atherogenic diet in the rabbits induced an enduring endothelial dysfunction despite the food subsequently being changed to a normal chow. All measured parameters indicated a significant favourable effect on endothelial dysfunction as a result of the two treatment regimens. Endothelial function measured in the organ chamber showed somewhat greater improvement in the ACE-NEP treated group than in the ACE-I treated group. The treatment with ramipril, as well as with AVE 7688, restored endothelial function by increasing the ratio of NO to O2- concentration and bioavailability of NO. In this study, a similar protective effect on endothelial function was shown by ACE-NEP inhibition as already seen with ACE inhibitors in an animal model of atherosclerosis.

Effect of aspirin on constitutive nitric oxide synthase and the biovailability of NO.

INTRODUCTION: Aspirin decreases the activity of iNOS and the formation of prostanoids. Constitutive nitric oxide synthase (cNOS) is present in endothelial cells, platelets, leukocytes and neurons, yet no data are available on the effect of aspirin on cNOS and the bioavailability of NO produced by this enzyme. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs), rat adrenal gland pheochromocytoma cells (PC-12) and human platelets were incubated with different aspirin concentrations. The kinetics of NO, O2- and ONOO- release were measured simultaneously in single cells or platelet suspensions using tandem electrochemical nanosensors. The NO, O2- and ONOO- release from cells and platelets was stimulated with calcium ionophore and collagen, respectively. cNOS expression was estimated by Western blot analysis. RESULTS: Incubation of HUVECs and PC-12 with 10(-5) mol/l of aspirin increased cNOS expression by 70 +/- 7% and 50 +/- 5, respectively. However, the NO concentration increased only by 33% in HUVECs incubated with the same aspirin concentration. Incubation of HUVECs with aspirin also increased the O2- and ONOO- production. Therefore the bioavailability of NO increased only slightly in endothelium and did not reflect the increase in eNOS. This was in contrast to platelets, where maximal NO bioavailability almost doubled after incubation with aspirin. CONCLUSIONS: Aspirin did not have a significant effect on the NO bioavailability in endothelial cells. However, aspirin highly improved the NO production in platelets. The high NO production in platelets may counteract the effect of thromboxane, inhibit platelet aggregation, and compensate for the reduction of prostacycline concentration by aspirin.

Third-generation beta-blockers stimulate nitric oxide release from endothelial cells through ATP efflux: a novel mechanism for antihypertensive action.

BACKGROUND: Nebivolol and carvedilol are third-generation beta-adrenoreceptor antagonists, which unlike classic beta-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. METHODS AND RESULTS: Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [3H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both beta-blocker-induced glomerular vasorelaxations and beta-blocker-stimulated NO release from GECs. Both beta-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the beta-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188+/-14 and 226+/-17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd3+, inhibited the beta-blocker-dependent release of ATP and NO from GECs. CONCLUSIONS: These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO release from GECs.

Ischemia/reperfusion injury of skeletal muscle: plasma taurine as a measure of tissue damage.

BACKGROUND: Cell membrane rupture by oxygen-derived free radicals is a systematic feature of ischemia/reperfusion (I/R) injury. High taurine concentration gradients in skeletal muscle prompted us to evaluate whether plasma taurine levels (pTau) are a useful marker of I/R injury after different periods of ischemia. METHODS: Rabbits were randomly assigned to either 1 or 2.5 hours of hind-limb ischemia followed by 2 hours of reperfusion (groups IR1 [n = 12] and IR2.5 [n = 13], respectively). Corresponding sham groups (SHAM1 [n = 8] and SHAM2.5 [n = 9]) were used as controls. Analyzed parameters included histomorphometry and electron microscopy of skeletal muscle biopsies, pTau, and plasma level of malondialdehyde. Skeletal muscle function was assessed 3 weeks after I/R injury. RESULTS: No significant morphologic changes were detectable at the end of ischemia. After reperfusion, mild interstitial edema with intact muscle cell membranes developed in IR1 group; pTau was not increased. IR2.5 group, by contrast, showed severe interstitial edema formation (interfiber area increased by 112%, P <.005), microvascular constriction (microvessel area decreased by 33%, P <.0005), and damage to the muscle cell membranes that was confirmed by the increased plasma malondialdehyde. pTau was higher than in the SHAM2.5 group (P <.0005). Pronounced cell damage in IR2.5 group resulted in impaired muscle function (maximal tetanic tension was reduced 2 times, P <.005) but not in IR1 group. CONCLUSION: Skeletal muscle tolerates 1 h/2 h but not 2.5 h/2 h of I/R, the latter resulting in interstitial edema formation, microvascular constriction, and a late muscle dysfunction. Cell membrane rupture through stimulated lipid peroxidation promotes leakage of intracellular taurine, leading to increased pTau after reperfusion and may be considered as prognostically unfavorable in terms of organ function reversibility. In the rabbit model, pTau seems to be a sensitive marker of I/R injury to skeletal muscle.

S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release.

BACKGROUND: Peroxynitrite generated from nitric oxide (NO) and superoxide (O2-) contributes to ischemia/reperfusion (I/R) injury. Feedback inhibition of endothelial NO synthase by NO may inhibit O2- production generated also by endothelial NO synthase at diminished local L-arginine concentrations accompanying I/R. METHODS AND RESULTS: During hindlimb I/R (2.5 hours/2 hours), in vivo NO was monitored continuously (porphyrinic sensor), and high-energy phosphates, reduced and oxidized glutathione (chromatography), and I/R injury were measured intermittently. Rabbits receiving human serum albumin (HSA) (controls) were compared with those receiving S-nitroso human serum albumin (S-NO-HSA) beginning 30 minutes before reperfusion for 1 hour or 30 minutes before ischemia for 3.5 hours (0.1 micromol x kg(-1) x h(- 1)). The onset of ischemia led to a rapid increase of NO from its basal level (50+/-12 nmol/L) to 120+/-20 and 220+/-15 nmol/L in the control and S-NO-HSA-treated groups, respectively. In control animals, NO dropped below basal levels at the end of ischemia and to undetectable levels (<1 nmol/L) during reperfusion. In S-NO-HSA-treated animals, maximal NO levels never decreased below basal concentration and on reperfusion were 100+/-15 nmol/L (S-NO-HSA preischemia group, 175+/-15 nmol/L). NO supplementation by S-NO-HSA led to partial and in the preischemia group to total preservation of high-energy phosphates and glutathione status in reperfused muscle (eg, preischemia groups: ATP, 30.23+/-5.02 micromol/g versus control, 15.75+/-4.33 micromol/g, P<0.0005; % oxidized glutathione, 4.49+/- 1.87% versus control, 22.84+/-6.39%, P<0.0001). S-NO-HSA treatment in all groups led to protection from vasoconstriction and reduced edema formation after reperfusion (eg, preischemia groups: interfiber area, 12.94+/-1.36% versus control, 27.83+/-1.95%, P< 0.00001). CONCLUSIONS: Long-lasting release of NO by S-NO-HSA provides significant protection of skeletal muscle from I/R injury.