Biomedical subjects

J L Zweier

Publications and source records attributed to J L Zweier.

At least 37 records · Page 2Linked to original sources

PubMed2000

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells.

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.

Calcium↗

PubMed2000

Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems.

While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.

Dimethyl Sulfoxide↗

PubMed2000

Regulation of xanthine oxidase by nitric oxide and peroxynitrite.

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.

Electron Spin Resonance Spectroscopy↗

PubMed2000

Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue.

Tissue oxygen content is a central parameter in physiology but is difficult to measure. We report a novel procedure for spatial mapping of oxygen by electron paramagnetic resonance (EPR) utilizing a spectral-spatial imaging data set, in which an EPR spectrum is obtained from each image volume element. From this data set, spatial maps corresponding to local spin density and maximum EPR spectral line amplitude are generated. A map of local EPR spectral linewidth is then computed. Because linewidth directly correlates with oxygen concentration, the linewidth image provides a map of oxygenation. This method avoids a difficulty inherent in other oxygen content mapping techniques using EPR, that is, the unwanted influence of local spin probe density on the image. We provide simulation results and data from phantom studies demonstrating the validity of this method. We then apply the method to map oxygen content in rat tail tissue and vasculature. This method provides a new, widely applicable, approach to direct visualization of oxygen concentration in living tissue. Magn Reson Med 43:804-809, 2000.

Animals↗

PubMed2000

Measurements of free radicals in isolated, ischemic lungs and lung mitochondria.

Previous studies in isolated, ventilated lungs have demonstrated by indirect measurements that oxidant generation occurs during pulmonary ischemia before reperfusion. To identify and quantify the types of free radical species generated during ischemia, we used electron paramagnetic resonance (EPR) spectroscopy in the presence and absence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR spectra obtained from the vascular effluent of isolated ferret lungs, contained a doublet signal (g = 2.005) indicative of ascorbyl radical. This signal doubled in magnitude after 180 min of ischemia, providing evidence of oxidant formation during ischemia. When DMPO, which reacts with radicals including superoxide anions and hydroxyl radicals, was added to the perfusate, the spectra contained ascorbyl radical signals but no DMPO-adducts. To clarify the relationship between ascorbyl radical and DMPO-adduct formation, additional studies were conducted in the presence and absence of ascorbate with isolated lung mitochondria as the source of free radicals. The results showed that in the presence of ascorbate, oxygen free radicals were not detected by EPR spin trapping with DMPO because of the formation of prominent ascorbyl radical signals. These data suggest that DMPO may be useful for the detection of reactive oxygen species in isolated lungs, provided the ascorbate can be sufficiently depleted. Alternatively, as shown by our results, EPR studies that directly monitor ascorbyl radical formation may be used as a marker of oxidative stress in the lung.

Animals↗

PubMed2000

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase.

Whereas altered nitric oxide (NO.) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO. formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO. synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO. formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by the L-[(14)C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO. generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO. synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.

Animals↗

PubMed2000

Targeted antioxidant properties of N-[(tetramethyl-3-pyrroline-3-carboxamido)propyl]phthalimide and its nitroxide metabolite in preventing postischemic myocardial injury.

We investigated the cardioprotective efficacy of a new compound based on 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC-NH). Biochemical studies using electron paramagnetic resonance (EPR) spectroscopy suggest that TPC-NH is a scavenger of reactive oxygen species. In vitro cellular studies show that TPC-NH protects isolated cardiomyocytes against oxidative damage caused by superoxide radicals. Ex vivo EPR studies on the isolated rat heart indicate that the TPC-NH is metabolically oxidized to the nitroxide form. Studies were also performed in the isolated rat heart model to measure the efficacy of TPC-NH and its metabolites in preventing postischemic reperfusion injury. Serial measurements of contractile function were performed on hearts subjected to ischemia-reperfusion. Hearts were either untreated or treated with 50 microM TPC-NH or with its metabolites for 1 min before ischemia and during the first 5 min of reflow. TPC-NH showed marked protection with a more than 3-fold increased recovery of contractile function compared with control hearts, whereas its oxidative metabolites exhibited significant but lower protection. Thus, TPC-NH and, to a lesser extent, its oxidation metabolites exhibit potent membrane-targeted antioxidant action and exert marked protection against myocardial injury in the postischemic heart.

Animals↗

PubMed2000

Myocardial postischemic injury is reduced by polyADPripose polymerase-1 gene disruption.

BACKGROUND: PolyADPribose polymerase (PARP) is activated by DNA strand breaks to catalyze the addition of ADPribose groups to nuclear proteins, especially PARP-1. Excessive polyADPribosylation leads to cell death through depletion of NAD+ and ATP. MATERIALS AND METHODS: In vivo PARP activation in heart tissue slices was assayed through conversion of [33P]NAD+ into polyADPribose (PAR) following ischemia-reperfusion (I/R) and also monitored by immunohistochemical staining for PAR. Cardiac contractility, nitric oxide (NO), reactive oxygen species (ROS), NAD+ and ATP levels were examined in wild type (WT) and in PARP-1 gene-deleted (PARP-1(-/-)) isolated, perfused mouse hearts. Myocardial infarct size was assessed following coronary artery occlusion in rats treated with PARP inhibitors. RESULTS: Ischemia-reperfusion (I/R) augmented formation of nitric oxide, oxygen free radicals and PARP activity. I/R induced decreases in cardiac contractility and NAD+ levels were attenuated in PARP-1(-/-) mouse hearts. PARP inhibitors reduced myocardial infarct size in rats. Residual polyADPribosylation in PARP-1(-/-) hearts may reflect alternative forms of PARP. CONCLUSIONS: PolyADPribosylation from PARP-1 and other sources of enzymatic PAR synthesis is associated with cardiac damage following myocardial ischemia. PARP inhibitors may have therapeutic utility in myocardial disease.

Adenosine Triphosphate↗

PubMed2000

A pyrroline derivative of mexiletine offers marked protection against ischemia/reperfusion-induced myocardial contractile dysfunction.

The efficacy and mechanism of protection of a new 2,2,5, 5-tetramethylpyrroline derivative of mexiletine, MEX-NH, against ischemia/reperfusion-induced cardiac dysfunction are reported. The MEX-NH and its nitroxide metabolite are membrane-permeable antioxidants. Studies were performed in an isolated rat heart model to measure the efficacy of MEX-NH in preventing postischemic injury. Serial measurements of contractile function and coronary flow were performed on hearts subjected to 30 min of global 37 degrees C ischemia followed by 45 min of reperfusion. Hearts were either untreated or treated with 25 microM MEX-NH or MEX for 1 min before ischemia. The hearts treated with MEX-NH showed marked recovery of left ventricular developed pressure (96.3 +/- 2.7% of preischemic value) compared with untreated (13.7 +/- 1.0%) or MEX-treated (19.9 +/- 2.7%) hearts. The cardiac sarcolemmal Na(+),K(+)-ATPase activity showed that the enzyme activity was fully restored in hearts treated with MEX-NH compared with 65 +/- 5.3% inhibition in the untreated hearts. Competitive inhibition of [(3)H]ouabain binding revealed that the MEX-NH binds at the K(+)-binding site of the enzyme. The present study establishes that the compound MEX-NH provides marked protection against ischemia/reperfusion-induced contractile dysfunction in isolated hearts. A combination of reversible inhibition of Na(+)/K(+)-ATPase activity during ischemia and site-targeted antioxidative effect upon reperfusion seems to contribute to this cardioprotection.

Animals↗

PubMed1999

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide.

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.

Animals↗

PubMed1999

A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats.

Many human diseases are associated with the overproduction of oxygen free radicals that inflict cell damage. A manganese(II) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) was designed to be a functional mimic of the superoxide dismutase (SOD) enzymes that normally remove these radicals. M40403 had high catalytic SOD activity and was chemically and biologically stable in vivo. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage. Such mimics may result in better clinical therapies for diseases mediated by superoxide radicals.

Animals↗

PubMed1999

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.

Animals↗

PubMed1999

Non-enzymatic nitric oxide synthesis in biological systems.

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac 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 enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Animals↗

PubMed1999

Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging.

EPR imaging has emerged as an important tool for noninvasive three-dimensional (3D) spatial mapping of free radicals in biological tissues. Spectral-spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be mapped. We report the development of EPR imaging instrumentation enabling 3D spatial and spectral-spatial EPR imaging of small animals. This instrumentation, along with the use of a biocompatible charcoal oximetry-probe suspension, enabled 3D spatial imaging of the gastrointestinal (GI) tract, along with mapping of oxygenation in living mice. By using these techniques, the oxygen tension was mapped at different levels of the GI tract from the stomach to the rectum. The results clearly show the presence of a marked oxygen gradient from the proximal to the distal GI tract, which decreases after respiratory arrest. This technique for in vivo mapping of oxygenation is a promising method, enabling the noninvasive imaging of oxygen within the normal GI tract. This method should be useful in determining the alterations in oxygenation associated with disease.

Animals↗

PubMed1999

Bicarbonate is required for the peroxidase function of Cu, Zn-superoxide dismutase at physiological pH.

Cu,Zn-superoxide dismutase (SOD1) acts as a peroxidase in the presence of H2O2 at high pH (pH > 9). The high pH species of H2O2, HO2-, was previously implicated as the reactive species. However, recent EPR studies of the enzyme performed in the physiological pH range 7.4-7.6 with the spin trap 5,5'-dimethyl-1-pyrolline-N-oxide attributed the intense EPR signal of 5, 5'-dimethyl-1-pyrolline-N-oxide-OH obtained from SOD1 and H2O2 to the peroxidase activity of the enzyme. The present study establishes that this intense signal is obtained only in the presence of bicarbonate. To explore the critical role of HCO3-, a comprehensive EPR investigation of the radical production and redox state of the active site copper was performed. The results indicate that HCO3- competes with other anions for the anion-binding site of SOD1 (Arg141) but does not bind directly to the copper. Structurally different anions that bind to Arg141 did not stimulate, but rather blocked, peroxidase function, ruling out an effect due to mere anion binding. However, the structurally similar anions HSeO3- and HSO3- mimic HCO3- in stimulating peroxidase function. These data suggest that HCO3- bound to Arg141 anchors the neutral H2O2 molecule at the active site copper, enabling its redox cleavage. Thus, SOD1 acquires peroxidase activity at physiological pH only in the presence of HCO3- or structurally similar anions. Alterations in pH that shift the HCO3-/CO2 equilibrium as occur in disease processes such as ischemia, sepsis, or shock would modulate the peroxidase function of SOD1.

Animals↗

PubMed1999

A tunable reentrant resonator with transverse orientation of electric field for in vivo EPR spectroscopy.

There has been a need for development of microwave resonator designs optimized to provide high sensitivity and high stability for EPR spectroscopy and imaging measurements of in vivo systems. The design and construction of a novel reentrant resonator with transversely oriented electric field (TERR) and rectangular sample opening cross section for EPR spectroscopy and imaging of in vivo biological samples, such as the whole body of mice and rats, is described. This design with its transversely oriented capacitive element enables wide and simple setting of the center frequency by trimming the dimensions of the capacitive plate over the range 100-900 MHz with unloaded Q values of approximately 1100 at 750 MHz, while the mechanical adjustment mechanism allows smooth continuous frequency tuning in the range +/-50 MHz. This orientation of the capacitive element limits the electric field based loss of resonator Q observed with large lossy samples, and it facilitates the use of capacitive coupling. Both microwave performance data and EPR measurements of aqueous samples demonstrate high sensitivity and stability of the design, which make it well suited for in vivo applications.

Animals↗

PubMed1999

Oxygen free radicals in interleukin-1beta-induced glycosaminoglycan production by retro-ocular fibroblasts from normal subjects and Graves' ophthalmopathy patients.

Graves' ophthalmopathy (GO) is attributed to an autoimmune process that results in the accumulation in retro-ocular tissue of glycosaminoglycans (GAG) that are in turn responsible for the development of clinical signs and symptoms. Retro-ocular fibroblasts are thought to be the source of GAG production and deposition in GO. In the present study, we investigated interleukin (IL)-1beta-induced oxygen free radical production and the role of oxygen free radicals in IL-1beta-induced GAG production in retro-ocular fibroblasts from both normal subjects and patients with GO. Normal retro-ocular fibroblasts demonstrated no measurable oxygen free radicals whereas GO retro-ocular fibroblasts showed detectable signals by electron paramagnetic resonance (EPR) spectroscopy. IL-1beta increased the free radical production in both cells. Superoxide dismutase (SOD) activity in GO retroocular fibroblasts was higher than that in normal cells. IL-1beta dose- and time-dependently stimulated the SOD activity in both cells, with GO retro-ocular fibroblasts showing less responsiveness. IL-1beta dose-dependently increased [3H]glucosamine incorporation into GAG by both cells. An exogenous oxygen free radical-generating system failed to increase GAG. Scavenging oxygen free radicals by the use of SOD (100 U/mL) and catalase (300 U/mL) partially blocked the IL-1beta-induced GAG production in both cells. These results suggest that stress related oxygen free radicals are present in the retro-ocular tissue in GO and that oxygen free radicals are involved in GAG accumulation induced by cytokine IL-1beta.

Catalase↗

PubMed1998

Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process.

It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (O-2) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O-2. Moreover, the mechanism and control of this process are not fully understood. Therefore, we performed electron paramagnetic resonance spin-trapping experiments to directly measure and characterize the O-2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O-2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O-2 formation required Ca2+/calmodulin and was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not its non-inhibitory enantiomer D-NAME. A parallel process of Ca2+/calmodulin-dependent NADPH oxidation was observed which was also inhibited by L-NAME but not D-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O-2 generation. BH4 exerted dose-dependent inhibition of the O-2 signals generated by eNOS. Conversely, in the absence of BH4 L-arginine did not decrease this O-2 generation. Thus, eNOS can also catalyze O-2 formation, and this appears to occur primarily at the heme center of its oxygenase domain. O-2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than L-arginine.

Arginine↗