Biomedical subjects

Eva Nozik-Grayck

Publications and source records attributed to Eva Nozik-Grayck.

12 recordsLinked to original sources

PubMed2006

Duodenal cytochrome b: a novel ferrireductase in airway epithelial cells.

Catalytically active iron in the lung causes oxidative stress and promotes microbial growth that can be limited by intracellular sequestration of iron within ferritin. Because cellular iron uptake requires membrane ferrireductase activity that in the gut can be provided by duodenal cytochrome b (Dcytb), we sought Dcytb in the lung to test the hypothesis that it contributes to epithelial iron regulation by reducing Fe(3+) for cellular iron transport. Dcytb expression was found in respiratory epithelium in vitro and in vivo and was responsive to iron concentration. Iron transport was measured in human bronchial epithelial (HBE) cells using inductively coupled plasma atomic emission spectroscopy and was demonstrated to be partially inhibited in the presence of Dcytb-blocking antibody, suggesting that Dcytb reduces Fe(3+) for cellular iron transport. A definite source of reducing equivalents for Dcytb was sought but not identified. We found no evidence that ascorbate was involved but did demonstrate that O(2)(-). production decreased when Dcytb function was blocked. The presence of Dcytb in airway epithelial cells and its regulation by iron therefore may contribute to pulmonary cytoprotection.

Animals↗

PubMed2006

Electrocardiographic response to enzyme replacement therapy for Pompe disease.

PURPOSE: Electrocardiogram (ECG) abnormalities are universal in infantile Pompe disease or glycogen storage disease type II, a fatal genetic muscle disorder caused by deficiency of acid alpha-glucosidase (GAA). Hallmarks of this disease include a shortened PR interval, an increased QT dispersion (QTd), and large left ventricular (LV) voltages. We evaluated the effect of recombinant human GAA (rhGAA) enzyme replacement therapy (ERT) on these ECG parameters in patients with infantile-onset Pompe disease. METHODS: A total of 134 ECGs were evaluated from 19 patients (5 females and 14 males) with a median age of 5.5 months at the time of enrollment in open-label clinical trials exploring the safety and efficacy of ERT at a single center from 1999 to 2004. rhGAA was purified from genetically engineered Chinese hamster ovary cells overproducing GAA and infused intravenously at doses ranging from 10 mg/kg per week to 20 to 40 mg/kg every 2 weeks in patients with infantile-onset Pompe disease. The PR interval, QTd (longest to shortest QT), and LV voltage (SV1 + RV6) were blindly determined by two independent observers. RESULTS: The median follow-up period was 6 months (range 2-30 months). The PR interval lengthened from 83 (42-110) ms to 107 (95-130) ms (P < .001), and the QTd decreased from 83 (40-125) ms to 53 (20-80) ms (P = .003). There were significant decreases in LV voltage (67 [17-83] mV vs. 48 [18-77] mV, P = .03), which correlated with decrease in LV mass on two-dimensional echocardiogram. There was no evident change in the QTc interval (429 [390-480] ms vs. 413 [370-450] ms, P = not significant). CONCLUSION: rhGAA ERT for infantile Pompe disease results in an increase in PR interval and a decrease in both the QTd and the LV voltage. These results suggest that these ECG parameters may be useful markers of the severity of cardiac disease and the response to ERT treatment in patients with infantile Pompe disease.

Cohort Studies↗

PubMed2005

S-nitrosoglutathione inhibits alpha1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery.

Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to G(q), we hypothesized that S-nitrosothiols modify the alpha1-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10(-8)-10(-6) M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or LY-83583} or G kinase inhibitor (KT-5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the alpha1-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of alpha1-adrenergic receptor subtype expression and receptor density by saturation binding with 125I-HEAT showed that GSNO decreased alpha1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible alpha1-adrenergic receptor inhibition by S-nitrosothiols.

Adrenergic alpha-Agonists↗

PubMed2005

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia.

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.

Aging↗

PubMed2005

Extracellular superoxide dismutase.

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.

Animals↗

PubMed2004

Discordant extracellular superoxide dismutase expression and activity in neonatal hyperoxic lung.

Antioxidant defenses in the neonatal lung are required to adapt to the oxygen (O(2))-rich postnatal environment, and oxidant/antioxidant imbalance is a predisposition to lung injury when high concentrations of inspired O(2) are used in neonatal lung diseases. The lung's main extracellular enzymatic defense against superoxide, extracellular superoxide dismutase (EC-SOD), is closely regulated during development. In testing the hypothesis that developmental change in EC-SOD expression and activity in the immature lung would be disrupted by hyperoxia, we found a doubling of lung EC-SOD protein in newborn rats exposed to 95% O(2) for 1 week. Furthermore, EC-SOD protein secretion increased, but EC-SOD enzyme activity did not change with O(2) exposure. EC-SOD mRNA did not change at multiple points between 6 hours and 8 days. Lung EC-SOD recovered by immunoprecipitation after 1 week of O(2) showed strong increases in protein nitrotyrosine and variable, nonsignificant differences in protein carbonyl content. These data provide the first direct evidence that EC-SOD is itself a target of nitration in hyperoxia, and offer a plausible explanation for low EC-SOD activity despite its increased secretion by O(2)-exposed neonatal lung.

Animals↗

PubMed2003

Bicarbonate-dependent superoxide release and pulmonary artery tone.

Pulmonary vasoconstriction is influenced by inactivation of nitric oxide (NO) with extracellular superoxide (O2-*). Because the short-lived O2-* anion cannot diffuse across plasma membranes, its release from vascular cells requires specialized mechanisms that have not been well delineated in the pulmonary circulation. We have shown that the bicarbonate (HCO3-)-chloride anion exchange protein (AE2) expressed in the lung also exchanges O2-* for HCO3-. Thus we determined whether O2-* release involved in pulmonary vascular tone depends on extracellular HCO3-. We assessed endothelium-dependent vascular reactivity and O2-* release in the presence or absence of HCO3- in pulmonary artery (PA) rings isolated from normal rats and those exposed to hypoxia for 3 days. Lack of extracellular HCO3- in normal PA rings significantly attenuated endothelial O2-* release, opposed hypoxic vasoconstriction, and enhanced acetylcholine-mediated vasodilation. Release of O2-* was also inhibited by an AE2 inhibitor (SITS) and abolished in normoxia by an NO synthase inhibitor (NG-nitro-L-arginine methyl ester). In contrast, hypoxia increased PA AE2 protein expression and O2-* release; the latter was not affected by NG-nitro-l-arginine methyl ester or other inhibitors of enzymatic O2-* generation. Enhanced O2-* release by uncoupling NO synthase with geldanamycin was attenuated by hypoxia or by HCO3- elimination. These results indicate that O2-* produced by endothelial NOS in normoxia and unidentified sources in hypoxia regulate pulmonary vascular tone via AE2.

Animals↗

PubMed2003

Superoxide-dependent iron uptake: a new role for anion exchange protein 2.

Lung cells import iron across the plasma membrane as ferrous (Fe2+) ion by incompletely understood mechanisms. We tested the hypothesis that human bronchial epithelial (HBE) cells import non-transferrin-bound iron (NTBI) using superoxide-dependent ferri-reductase activity involving anion exchange protein 2 (AE2) and extracellular bicarbonate (HCO3-). HBE cells that constitutively express AE2 mRNA by reverse transcriptase-polymerase chain reaction and AE2 protein by Western analysis avidly transported NTBI after exposure to either Fe2+ or Fe3+, but reduction of Fe3+ to Fe2+ was first required. The ability of HBE cells to reduce Fe3+ and transport Fe2+ was inhibited by active extracellular superoxide dismutase (SOD). Similarly, HBE cells that overexpress Cu,Zn SOD after adenoviral infection with AdSOD1 showed diminished iron uptake. The role of AE2 in iron uptake was indicated by three lines of evidence: (i) lack of both iron reduction and iron transport in bicarbonate-free buffer at controlled pH, (ii) failure of HBE cells treated with stilbene AE inhibitors to reduce Fe3+ or transport iron, and (iii) inhibition of iron uptake in HBE cells by inhibition of AE2 protein expression with antisense oligonucleotides. We thus disclose a novel ferri-reductase mechanism of NTBI uptake by human lung cells that employs superoxide exchange for HCO3- by AE2 protein in the plasma membrane.

Adenoviridae↗

PubMed2002

Extracellular superoxide dismutase protects lung development in hyperoxia-exposed newborn mice.

We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.

Animals↗

PubMed2002

Prenatal hypoxia decreases lung extracellular superoxide dismutase expression and activity.

Extracellular superoxide dismutase (EC-SOD), which scavenges extracellular superoxide (O.), is highly regulated in the developing lung. In the prenatal rabbit, EC-SOD is predominantly intracellular and inactive, and postnatally, active EC-SOD is secreted. We hypothesized that prenatal hypoxia would delay the normal postnatal secretion of active EC-SOD in the lung. Pregnant New Zealand White rabbits were exposed to hypobaric hypoxia (15,000 ft x 36 h) to alter fetal O(2) tension or were maintained in room air. Lungs were harvested from preterm (28 days), term (30 +/- 1 day), and 1-wk-old kits. After prenatal hypobaric hypoxia, EC-SOD mRNA expression was significantly decreased in lungs of full-term kits, whereas EC-SOD protein decreased at all ages. Immunohistochemical staining for EC-SOD showed that hypoxia delayed secretion of the isoenzyme in the airways and pulmonary vasculature. Furthermore, pulmonary EC-SOD enzyme activity was significantly decreased in the 1-wk-old kits exposed to prenatal hypoxia. We conclude that prenatal hypoxia downregulates EC-SOD expression at both the transcriptional and posttranslational levels. Furthermore, prenatal hypoxia delays secretion of active EC-SOD enzyme. These findings have important implications for the effects of prenatal asphyxia on postnatal response to oxidant stress.

Animals↗

PubMed2002

Pulmonary vasoconstriction by serotonin is inhibited by S-nitrosoglutathione.

Nitric oxide (NO) functions as an endothelium-derived relaxing factor by activating guanylate cyclase to increase cGMP levels. However, NO and related species may also regulate vascular tone by cGMP-independent mechanisms. We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to serotonin (5-HT) in the intact lung through chemical interactions with 5-HT(2) receptors. In isolated rabbit lung preparations and isolated pulmonary artery (PA) rings, 50-250 microM S-nitrosoglutathione (GSNO) inhibited the response to 0.01-10 microM 5-HT. The vasoconstrictor response to 5-HT was mediated by 5-HT(2) receptors in the lung, since it could be blocked completely by the selective inhibitor ketanserin (10 microM). GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in PA rings. In PA rings, inhibition by GSNO could be reversed by treatment with the thiol reductant dithiothreitol (10 mM). 3-Morpholinosydnonimine (100-500 microM), which releases NO and O simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT(2) G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.

Animals↗

PubMed2002

Oxidative stress activates anion exchange protein 2 and AP-1 in airway epithelial cells.

Anion exchange protein 2 (AE2) is a membrane-bound protein that mediates chloride-bicarbonate exchange. In addition to regulating intracellular pH and cell volume, AE2 exports superoxide (O.) to the extracellular matrix in an HCO-dependent process. Given this ability to export O., we hypothesized that expression of AE2 in the lung is regulated by oxidative stress. AE2 mRNA and protein expression was measured by RT-PCR and Western blot analysis, respectively, in differentiated human bronchial epithelial cells exposed to H(2)O(2) (100 microM). Alterations in in vivo AE2 protein expression were evaluated in lung tissue of rats exposed to 70% O(2). The role of transcription factor activator protein (AP)-1 in oxidant regulation of AE2 was evaluated by EMSA and by immunoblotting of nuclear phospho-c-jun. Results show increased AE2 mRNA and protein expression after oxidant exposure. This was preceded by transient increases in DNA binding of AE2-specific AP-1 and phosphorylation of c-jun. This study demonstrates that AE2 expression is regulated by oxidative stress in airway epithelial cells and that this regulation correlates with activation of AP-1.

Anion Transport Proteins↗