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J L Zweier

Publications and source records attributed to J L Zweier.

At least 55 records · Page 3Linked to original sources
PubMed1998

Hydrogen peroxide decreases pHi in human aortic endothelial cells by inhibiting Na+/H+ exchange.

Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pHi, the effect of hydrogen peroxide on endothelial pHi was examined. Hydrogen peroxide (100 micromol/L for 10 minutes) decreased pHi from 7.24+/-0.01 to 7.02+/-0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 micromol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pHi. A 2-minute exposure to 100 micromol/L H2O2 decreased intracellular ATP levels by approximately 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pHi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pHi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.

Adenosine Triphosphate↗
PubMed1998

The natural polyamine spermine functions directly as a free radical scavenger.

The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.

Azides↗
PubMed1998

Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems.

Recently it was shown that the formation of nitric oxide (NO) is increased in biological tissues during ischemia, and that there is an enzyme-independent pathway of NO generation due to reduction of tissue nitrite under the acidic conditions which occur. To investigate the quantitative importance of this mechanism of NO generation in biological systems where pH and nitrite concentrations vary, electron paramagnetic resonance and chemiluminescence studies were performed to characterize the kinetics and magnitude of the nitrite disproportionation process. The reaction process and the corresponding rate law of NO formation from nitrite were derived. The generation and accumulation of NO from typical nitrite concentrations found in biological tissues increased 100-fold when pH decreased from normal values of 7.4 to the acidic values found in ischemic tissues, such as the heart, where pH falls to 5.5. It was also observed that ischemic heart tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions the magnitude of this enzyme-independent NO generation may exceed that which can be generated by tissue concentrations of nitric oxide synthase. Thus, in ischemic tissues nitrite can be primarily a source rather than a product of NO.

Animals↗
PubMed1998

Inducible nitric-oxide synthase generates superoxide from the reductase domain.

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.

Animals↗
PubMed1998

An improved assay for measurement of nitric oxide synthase activity in biological tissues.

While the L-arginine conversion assay has been utilized to measure nitric oxide synthase (NOS) activity in isolated enzyme and pure cell preparations, this method often fails to provide accurate measurements in whole tissues. Biological tissues contain variable amounts of unlabeled substrate and enzymes are present which can compete for substrate or independently form the product L-citrulline. NOS-independent conversion of radiolabeled L-arginine to L-citrulline occurs due to arginase- and ornithine transcarbamylase-mediated reactions and this limits the accuracy of this assay for measurement of NOS activity. In heart tissue, NOS-independent L-citrulline formation was observed which could not be blocked by the NOS inhibitor L-NAME but was blocked by the arginase inhibitor L-ornithine. To eliminate the effect of arginase-mediated L-citrulline formation, KCl-washed membrane particulate fractions were obtained by high-speed centrifugation. While arginase-mediated nonspecific activity was 85% concentrated in the cytosol, 93% of NOS activity was localized within the particulate fraction of the heart. The remaining arginase activity found in the crude pellet was mostly removed by a one-step KCl wash purification and when incubation periods of 8 min were utilized specific and accurate measurements of NOS activity were obtained. NOS enzymatic properties were defined for rat heart preparations with a Km of 2.9 microM for L-arginine. All NOS activity detected was calcium-dependent suggesting it originated from the constitutive endothelial isoform. Thus, NOS-independent activity can be largely eliminated from the heart tissue by assaying KCl-washed membrane particulate fractions and this enables accurate quantitation of NOS activity.

Animals↗
PubMed1998

Measurement and characterization of superoxide generation in microglial cells: evidence for an NADPH oxidase-dependent pathway.

While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.

Animals↗
PubMed1998

Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.

While nitrosothiol compounds have been hypothesized to be important in the transport and function of nitric oxide (NO) in biological systems many important questions regarding their mechanism of formation and functional importance remain. In view of these fundamental questions there has been a great need for simple, sensitive, and specific methods for quantitation of nitrosothiols in biological samples. We report the development of two methods, for the measurement of nitrosothiol compounds using a chemiluminescence nitric oxide analyzer with a standard purging vessel. The first method is based on treatment with acidified solutions of potassium iodide in the presence or absence of dissolved free iodine. Quantitative release of NO occurs either from both nitrite and nitrosothiols or from nitrite alone, respectively. Subtraction of the amount of NO released without iodine from NO released in the presence of iodine allows estimation of the nitrosothiol concentration. To selectively measure nitrosothiols, we developed a redox quinone-hydroquinone alkaline reactant that selectively releases NO from nitrosothiols. This reactant quantitatively converts nitrosothiols to NO at elevated temperature, > 60 degrees C. Both methods were shown to detect nitrosothiols in biological buffers or blood plasma down to 10 nM concentration with high accuracy and reproducibility, variability less than 5%. These assays should be a useful addition to techniques used to characterize the biochemistry of NO.

Hydrogen-Ion Concentration↗
PubMed1998

Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postischemic injury.

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper, zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Animals↗
PubMed1998

In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model.

Nitroxides are redox-sensitive probes, which are useful in noninvasively delineating tissue heterogeneity especially with respect to metabolic activity and tissue oxygenation. Recent studies have shown that nitroxides are in vitro and in vivo radioprotectors and selectively protect normal tissue compared to tumor tissue. It has been postulated that the basis for selective radioprotection of normal tissues is greater bioreduction of nitroxides in tumor tissue compared to normal tissue. The aim of the present study was to investigate the distribution and lifetime of nitroxides in tumor and normal tissues. Mice were implanted with tumor cells (RIF-1) in the thigh, and the tumor was allowed to grow to about 10-15 mm in diameter. After i.v. infusion of nitroxides, in vivo electron paramagnetic resonance spectroscopy and imaging of the tumor were performed using a specially built bridged-loop surface resonator. The pharmacokinetic and spatial distribution of the nitroxides in tumor tissue were followed and compared with those in normal tissue. Three-dimensional spatial images showed significant heterogeneity in the nitroxide distribution as well as reduction rates. The nitroxide reduction rates were significantly higher in tumors than in the normal tissue. Measurements using spin label oximetry showed a substantial difference in the level of oxygenation between normal tissue (muscle) and tumor tissue. Average pO2 levels in tumor tissue were found to be 3-fold lower than in a corresponding volume of normal tissue. The lower pO2 levels in tumor compared to normal tissue may explain the more rapid reduction of nitroxides in these tissues. This study demonstrates that electron paramagnetic resonance imaging can perform noninvasive anatomical as well as functional imaging and provide in vivo physiological information regarding cellular metabolism in tumor and normal tissues.

Animals↗
PubMed1998

Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy.

In view of the important role of superoxide in cellular injury, there has been a great need for methods suitable for quantitation of superoxide production from cells. Previous methods have had limited sensitivity or specificity as well as problems with side reactions in cellular systems. Recently, we have shown that the new spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide has ideal properties for quantitative superoxide measurement in chemical/biochemical systems; however, its suitability and potential for measurements in cellular systems has not been determined. Therefore, we evaluated the use of DEPMPO for quantitative measurement of superoxide formed by polymorphonuclear leukocytes. After activation of these cells with the phorbol ester (PMA, 200 ng/ml) or opsonized zymosan (1 mg/ml) at 24 degrees C a strong signal of the superoxide adduct, DEPMPO-OOH, was observed. This technique was highly sensitive and enabled measurement of superoxide generation from as few as 2 x 10(3) cells. The kinetics of adduct formation and decay were measured which enabled quantitation of superoxide formation. Spin label electron paramagnetic resonance (EPR) oximetry was used to measure the oxygen consumption from these cells. With PMA activation rapid onset of superoxide generation occurred with a rate of 0.78 nmol/min/10(6) cells while with zymosan a slower gradual onset of activation was seen to a peak rate of 0.061 nmol/min/10(6) cells. With both stimulators the ratios of superoxide production to oxygen consumption were similar with values of approximately 50% obtained. Thus, EPR spin trapping with DEPMPO together with EPR oximetry methods can be used to provide sensitive and specific quantitation of cellular superoxide generation and oxygen consumption. These methods provide a promising new approach for the measurement of oxygen reduction and superoxide generation in cellular systems.

Cyclic N-Oxides↗
PubMed1998

Hydrogen peroxide induces intracellular calcium oscillations in human aortic endothelial cells.

BACKGROUND: Because the vascular endothelium is exposed to oxidant stress resulting from ischemia/reperfusion and from the products of polymorphonuclear leukocytes or monocytes, studies were performed to examine the effect of hydrogen peroxide (1 micromol/L to 10 mmol/L) on endothelial Ca2+ signaling. METHODS AND RESULTS: At low concentrations (1 to 10 micromol/L), hydrogen peroxide did not affect intracellular Ca2+ concentration in subconfluent, indo 1-loaded human aortic endothelial monolayers. At a concentration of 100 micromol/L hydrogen peroxide, intracellular free Ca2+ gradually increased from 125.3+/-6.8 to 286.3+/-19.9 nmol/L over 4.2+/-0.9 minutes before repetitive Ca2+ oscillations were observed, consisting of an initial large, transient spike of approximately 1 micromol/L followed by several spikes of decreasing amplitudes at a frequency of 0.7+/-0.1 min-1 over 12.0+/-1.1 minutes. After these oscillations, intracellular Ca2+ reached a plateau of 543.4+/-64.0 nmol/L, which was maintained above baseline levels for >5 minutes and then partially reversible on washout of hydrogen peroxide in most monolayers. Intracellular Ca2+ oscillations were typically observed when monolayers were exposed to 100 to 500 micromol/L hydrogen peroxide. Higher concentrations of hydrogen peroxide (1 and 10 mmol/L) increased intracellular Ca2+ but only rarely (2 of 6 monolayers at 1 mmol/L) or never (at 10 mmol/L) stimulated intracellular Ca2+ oscillations. Removal of Ca2+ from the buffer either before hydrogen peroxide stimulation or during an established response did not block intracellular Ca2+ oscillations in response to 100 micromol/L hydrogen peroxide, but prior depletion of an intracellular Ca2+ store with either caffeine, histamine, or thapsigargin abolished Ca2+ oscillations. CONCLUSIONS: Hydrogen peroxide induces concentration-dependent intracellular Ca2+ oscillations in human endothelial cells, which results from release of an endoplasmic reticulum Ca2+ store. Because oxidant production appears to occur in the micromolar range in the postischemic/anoxic endothelium and is associated with impaired endothelium-dependent relaxation, the effects of micromolar concentrations of hydrogen peroxide on endothelial Ca2+ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

Aorta↗
PubMed1998

Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.

Acridines↗
PubMed1998

In vivo topical EPR spectroscopy and imaging of nitroxide free radicals and polynitroxyl-albumin.

Piperidine nitroxides have considerable clinical potential, both as antioxidant therapeutic compounds and contrast agents in magnetic resonance imaging. However, their development has thus far been limited by their rapid bioreduction in vivo. Recently, it was reported that polynitroxyl albumin (PNA) can reverse the bioreduction of the reduced 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) in the rat heart, enabling the performance of high resolution EPR imaging for prolonged time (Kuppusamy et al., Biochemistry 35, 7051-7057 (1996)). In this report, the efficacy of PNA in maintaining Tempol concentrations in vivo in mice was demonstrated, using L-band (1.25 GHz) EPR spectroscopy and imaging. The EPR signal of intravenous Tempol had a half-life of 1.0+/-0.2 min and became undetectable within 6 min. Subcutaneous Tempol, however, decayed at a slower rate (half-life, 5.0+/-0.5 min) suggesting that Tempol had been bioreduced to the corresponding hydroxylamine form, Tempol-H. Subcutaneously injected PNA restored 20% of the Tempol signal in the vicinity of the PNA deposit. In vivo topical EPR imaging demonstrated that the Tempol signal was restored at the site of PNA injection, but not at locations remote from the PNA injection site. The ability of PNA to maintain Tempol in its paramagnetic state in vivo should enable a wide range of therapeutic and diagnostic applications of piperidinyl nitroxides.

Albumins↗
PubMed1998

Electron paramagnetic resonance imaging of the rat heart.

It has been hypothesized that free radical metabolism, oxygenation and nitric oxide generation in biological organs such as the heart may vary over the spatially defined tissue structure. To address fundamental questions regarding the role of spatially localized alterations in radical metabolism, oxygenation and nitric oxide in the pathophysiology of cellular injury during ischaemia, we have developed instrumentation optimized for 3D spatial and 3D or 4D spectral-spatial imaging of free radicals in the isolated perfused rat heart at 1.2 GHz. Using this instrumentation, high-quality 3D spectral-spatial imaging of nitroxide metabolism was performed as well as spatially localized measurements of oxygen concentrations, based on the oxygen-dependent linewidth broadening observed. In these spectral-spatial images, submillimetre resolution was observed enabling visualization of the left ventricular and right ventricular myocardium. With 3D spatial imaging using single-line labels, resolutions down to 100 to 200 microm were obtained enabling visualization of the ventricles, aortic root and proximal coronary arteries. Using metal complexes which trap nitric oxide, measurement and imaging of nitric oxide generation during ischaemia was performed. With the use of 15N isotope labelling it was possible to map the metabolic pathway of this nitric oxide generation. Thus, EPR imaging is a powerful tool which can provide unique information regarding the spatial localization of free radicals, oxygen and nitric oxide in biological organs and tissues.

Animals↗
PubMed1998

In vivo measurement of arterial and venous oxygenation in the rat using 3D spectral-spatial electron paramagnetic resonance imaging.

Electron paramagnetic resonance imaging (EPRI) instrumentation, enabling the performance of three-dimensional spectral-spatial images of free radicals, has been developed to study spatially defined differences in tissue metabolism and oxygenation. Using this instrumentation 3D images of nitroxides in rat tail were obtained. The images visualize the arterial and venous vasculature in the tail segment. Based on the exchange broadening influence of oxygen on the EPR linewidth of nitroxides, we performed localized oxygen measurements in the in vivo rat. An oxygen concentration of 300+/-30 microM was measured in the arteries and 50+/-20 microM in the veins. These results demonstrate the feasibility of performing in vivo, non-invasive measurements and mapping of localized oxygenation in small animals using spectral-spatial EPR imaging techniques.

Animals↗
PubMed1998

Development of functional electron paramagnetic resonance imaging.

The potential use of electron paramagnetic resonance imaging (EPRI) to obtain physiological information noninvasively is reviewed. EPR, a spectroscopic technique similar to nuclear magnetic resonance (NMR), is useful in detecting and characterizing free radical species. The ability to obtain information about tissue redox and oxygen status using nontoxic free radical spin probes is presented. The capability to encode this information spatially using magnetic field gradients, similar to magnetic resonance imaging (MRI), gives this technique the ability to overlay functional information of tissue with anatomic information. The noninvasive and quantitative nature of EPRI makes it a potentially useful technique for obtaining physiological information from tumors. The requirements for the magnetic field strengths are approximately 600 times lower than that for proton MRI at an identical frequency, making this a low-cost diagnostic tool.

Journal Article↗