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Biomedical subjects

N V Gorbunov

Publications and source records attributed to N V Gorbunov.

At least 19 recordsLinked to original sources
PubMed2000

Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

Animals↗
PubMed1999

Mechanisms of nitric oxide protection against tert-butyl hydroperoxide-induced cytotoxicity in iNOS-transduced human erythroleukemia cells.

We studied nitric oxide-mediated protection against tert-butyl hydroperoxide (t-BuOOH)-induced cytotoxicity in a subline of human erythroleukemia K562 cells (K/VP.5) and in K/VP.5 cells transduced with a retroviral vector containing the human iNOS gene (K/VP. 5-iNOS). K/VP.5-iNOS cells were 2-fold less sensitive to the cytotoxic effects of t-BuOOH compared to K/VP.5 cells. A nitric oxide-donor, NOC-15 ((Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1- ium-1, 2-diolate), protected K/VP.5 cells against t-BuOOH-induced cytotoxicity and provided an additional increment of protection in K/VP.5-iNOS cells. Under conditions of excess t-BuOOH and deficiency of iron catalytic sites (hemoglobin, Hb) in K/VP.5-iNOS cells, the increase of intracellular Hb concentration is the main contributor to enhanced sensitivity of the cells to t-BuOOH-induced cytotoxicity (despite the effects of small amounts of endogenously produced nitric oxide). Protection against t-BuOOH-induced cytotoxicity in K/VP.5-iNOS cells was diminished by treatment with an iNOS inhibitor, L-N(G)-monomethylarginine (L-NMA), but was restored upon addition of NOC-15 to L-NMA-treated cells. Incubation of K/VP.5 cells with NOC-15 resulted in the production of dinitrosyl complexes of non-heme iron and hexacoordinated heme iron nitrosyl complexes based on low-temperature EPR spectra. In K/VP.5-iNOS cells, only a weak EPR signal of dinitrosyl complexes of non-heme iron was observed in the absence of NOC-15. In addition, no heme iron nitrosyl complexes were discernible in the EPR spectra from K/VP.5-iNOS cells. Upon addition of NOC-15 to K/VP.5-iNOS cells, the EPR signal of dinitrosyl complexes of non-heme iron was enhanced, and the EPR signal of nitrosylated heme iron became discernible. It was determined that levels of non-heme and heme (hemoglobin) iron were dramatically decreased in K/VP.5-iNOS cells compared to K/VP.5 cells, thus explaining the decreased intensities of EPR signals of nitrosylated species. In addition, t-BuOOH-induced oxoferryl-Hb-associated protein-centered free radical species as well as t-BuO(*) alkoxyl radicals were observed in these two cell lines. These t-BuOOH-induced radical species were greatly reduced in K/VP.5-iNOS cells compared to K/VP.5 cells, consistent with a reduction in heme iron levels in the iNOS-expressing cells. Most importantly, the combined action of NOC-15 and t-BuOOH resulted in complete elimination of both oxoferryl-associated radical EPR signals as well as those from dinitrosyl complexes of non-heme iron and nitrosylated heme iron in both K/VP.5-iNOS cells and K/VP.5 cells. We conclude that two independent pathways operate in erythroleukemia cells for nitric oxide-mediated protection against t-BuOOH-induced cytotoxicity. First, prolonged endogenous production of nitric oxide results in a decreased content of catalytic non-heme iron and heme iron sites through posttranscriptional regulation of iron homeostasis. Second, nitric oxide can chemically reduce t-BuOOH-induced oxoferryl and t-BuO(*) alkoxyl radicals.

Alcohols↗
PubMed1999

Nitric oxide prevents myoglobin/tert-butyl hydroperoxide-induced inhibition of Ca2+ transport in skeletal and cardiac sarcoplasmic reticulum.

Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) vesicles, we demonstrated by EPR the formation of alkoxyl radicals and protein-centered peroxyl radicals in the presence of myoglobin (Mb) and tert-butyl hydroperoxide (t-BuOOH). The low temperature EPR signal of the radicals was characterized by major feature at g = 2.016 and a shoulder at g = 2.036. In the presence of SR vesicles, the magnitude of the protein-centered peroxyl radical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxidative damage, as evidenced by accumulation of 2-thiobarbituric acid-reactive substances (TBARS) and the inhibition of Ca2+ transport. We have shown that nitric oxide (NO), reacting with redox-active heme iron, can prevent peroxyl radical formation activated by Mb/t-BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non-thiol compound that releases NO) at 200-500 microM completely prevented the t-BuOOH-dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibition of Ca2+ transport.

Animals↗
PubMed1999

tert-butyl hydroperoxide/hemoglobin-induced oxidative stress and damage to vascular smooth muscle cells: different effects of nitric oxide and nitrosothiols.

The goal of the present work was to determine whether nitric oxide (NO) released from different donors (NONOates and nitrosothiols) can act as a protective antioxidant against oxidative stress and cytotoxicity induced by extracellular hemoglobin/tert-butyl hydroperoxide (Hb/tert-BuOOH) in vascular smooth muscle cells (VSMCs). No changes in phospholipid composition were found in VSMCs incubated with oxyhemoglobin (oxyHb)/tert-BuOOH. Using our newly developed HPLC-fluorescence technique for measurement of site-specific oxidative stress in membrane phospholipids, we produced VSMCs in which endogenous phospholipids were metabolically labeled with an oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. In these cells, we were able to reliably quantitate oxidative stress in major phospholipid classes-phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol-induced by tert-BuOOH in the presence of oxyHb or methemoglobin (metHb). The oxidative stress was accompanied by cytotoxic effects of oxyHb/tert-BuOOH and metHb/tert-BuOOH on VSMCs. We further found that an NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen 1-ium-1,2-diolate (PAPANONO), but not nitrosothiols, protected VSMCs against oxidative stress and cytotoxicity induced by Hb/tert-BuOOH. The protective effect of PAPANONO was most likely due to its ability to form NO-heme Hb (detectable by low temperature EPR spectroscopy and visible spectrophotometry). These findings are important for further understanding the physiological antioxidant role of NO against oxidative stress induced by hemoproteins as well as for pathological hypertensive events induced by extracellular Hb via NO depletion.

Animals↗
PubMed1998

Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatidylserine.

We studied protective effects of nitric oxide against tert-butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species--alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals--were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis-parinaric acid. An NO-donor, (Z)-1-[N-(3-ammonio-propyl)-N-(n-propyl) amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert-butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.

Alcohols↗
PubMed1997

A proposed biochemical mechanism involving hemoglobin for blast overpressure-induced injury.

Blast overpressure (BOP) is the abrupt, rapid, rise in atmospheric pressure resulting from explosive detonation, firing of large-caliber weapons, and accidental occupational explosions. Exposure to incident BOP waves causes internal injuries, mostly to the hollow organs, particularly the ears, lungs and gastrointestinal tract. BOP-induced injury used to be considered of military concern because it occurred mostly in military environments during military actions or training, and to a lesser extent during civilian occupational accidents. However, in recent years with the proliferation of indiscriminate terrorist bombings worldwide involving civilians, blast injury has become a societal concern, and the need to understand the biochemical and molecular mechanism(s) of injury, and to find new and effective methods for treatment gained importance. In general, past BOP research has focused on the physiological and pathological manifestations of incapacitation, thresholds of safety, and on predictive modeling. However, we have been studying the molecular mechanism of BOP-induced injury, and recently began to have an insight into that mechanism, and recognize the role of hemoglobin released during hemorrhage in catalyzing free radical reactions leading to oxidative stress. In this report we discuss the biochemical changes observed after BOP exposure in rat blood and lung tissue, and propose a biochemical mechanism for free radical-induced oxidative stress that can potentially complicate the injury. Moreover, we observed that some antioxidants can interact with Hb oxidation products (oxy-, met- and oxoferrylHb) and act as prooxidants that can increase the damage rather than decrease it.

Air Pressure↗
PubMed1997

Nitric oxide prevents oxidative damage produced by tert-butyl hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron. Electron paramagnetic resonance evidence.

We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-iu m-1, 2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2, 2'-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.

Cell Survival↗
PubMed1997

Air blast-induced pulmonary oxidative stress: interplay among hemoglobin, antioxidants, and lipid peroxidation.

Blast overpressure (BOP) is a phenomenon that describes the instantaneous rise in atmospheric pressure above ambient, resulting from the firing of large caliber weapons or from military or civilian explosions. Exposure to BOP results in injury to the gas-filled organs, such as the lungs, which exhibit a contusion-type injury. We examined the effects of BOP in rats at 5 and 60 min after exposure to a low-level BOP (62 +/- 3 kPa). The exposure was found to cause oxidative stress in the lung that was characterized by 1) a 3.5-fold decrease in total antioxidant reserves, 2) a depletion of the major water-soluble antioxidants ascorbate and glutathione (GSH) by 50 and 75%, respectively, 3) a depletion of lipid-soluble antioxidant vitamin E by 30%, 4) a 2.5-fold increase of fluorescent end products of lipid peroxidation, and 5) an increased methemoglobin (metHb) content at 60 min after exposure. To elucidate the role of released hemoglobin (Hb) in blast-induced oxidative stress, we studied the interactions of oxyhemoglobin (oxyHb), metHb, and the oxoferryl from of Hb free radical species with two physiologically important reductants, ascorbate and GSH. We found that both ascorbate and GSH were able to convert oxyHb to metHb in a reaction that yielded the one-electron oxidation intermediates semidehydroascorbyl radical and glutathionyl radical, respectively. This reaction did not occur under anaerobic conditions, suggesting that oxyHb-bound O2 acted as the electron acceptor. OxyHb induced peroxidation of cis-parinaric acid in the presence but not absence of ascorbate or GSH. Thus the prooxidant action of water-soluble antioxidants via redox cycling of oxyHb and metHb may promote oxidative stress rather than prevent it.

Animals↗
PubMed1996

Nitric oxide/carbon monoxide. A molecular switch for myocardial preservation during ischemia.

BACKGROUND: In heart, NO is produced from L-arginine catalyzed by NO synthase, and CO is formed during the conversion of bilirubin from heme by the action of heme oxygenase. NO, which exerts its biological actions through cGMP and heme, has recently been implicated in myocardial protection during ischemia and reperfusion. We hypothesized that the intracellular signaling by NO may be modulated by heme oxygenase. METHODS AND RESULTS: To test this hypothesis, isolated rat hearts were perfused for 10 minutes with one of the following: (1) buffer alone; (2) 3 mmol/L L-arginine, a precursor for NO; (3) 650 mumol/L zinc protoporphyrin, a heme oxygenase inhibitor; (4) 3 mmol/L L-arginine plus 650 mumol/L zinc protoporphyrin; (5) 15 mumol/L methylene blue, a cGMP inhibitor; or (6) 3 mmol/L L-arginine plus 15 mumol/L methylene blue. Hearts were then made ischemic for 30 minutes, followed by 30 minutes of reperfusion. L-Arginine afforded significant myocardial protection, as evidenced by increased developed pressure (DP) (53.3 +/- 4.3 versus 35.4 +/- 1.8 for control), dP/dtmax (2405 +/- 125 versus 1758 +/- 117 for control), aortic flow (23 +/- 1.5 versus 9.4 +/- 1.6 for control), and coronary flow (CF) (23.0 +/- 0.8 versus 19.0 +/- 1.6 for control) at the end of reperfusion. Protoporphyrin tended to reduce these values compared with L-arginine alone (DP, 27.5 +/- 1.4; dP/dtmax, 1400 +/- 78; CF, 17 +/- 0.5), suggesting a contribution of heme oxygenase in addition to NO for myocardial preservation. Increased mRNAs for the heme oxygenase were noticed in the ischemic reperfused myocardium. Contents of cGMP, the second messenger for NO signaling, increased in the L-arginine group (1.6 +/- 0.1 versus 1.1 +/- 0.1 for control) and were reduced by protoporphyrin. cGMP was completely inhibited by methylene blue, which also retarded postischemic myocardial functional recovery. Malonaldehyde formation, a presumptive marker for free radical generation, was decreased in the L-arginine group (0.053 +/- 0.003) compared with control (0.089 +/- 0.005) but was increased in the protoporphyrin group (0.09 +/- 0.003) compared with the L-arginine group. In vitro studies demonstrated that NO was able to reduce the reactive oxygen species produced by myoglobin, especially oxoferrylmyoglobin, which either are present in heart or are formed in high concentrations during the reperfusion of ischemic myocardium. CONCLUSIONS: The results suggest that NO contributes to myocardial preservation by both cGMP-dependent and cGMP-independent mechanisms, the former being modulated by CO signaling and the latter by virtue of its antioxidant action.

Animals↗
PubMed1996

NO-redox paradox: direct oxidation of alpha-tocopherol and alpha-tocopherol-mediated oxidation of ascorbate.

Nitric-oxide (NO) can act as both a pro- or an antioxidant, yielding either cytotoxic or protective effects, respectively. The previously unrecognized redox interactions of NO with antioxidants, and not solely its well-known reactions with oxygen radicals, peroxyl radicals and transition metal centers, may be essential for its dual mechanisms in cells. Since the alpha-tocopherol/ascorbate redox cycle is central to antioxidant protection, we studied the direct effects of NO on alpha-tocopherol, ascorbate and combinations thereof in aqueous, micellar environments using ESR spectral and HPLC quantitative techniques. We found that NO does not directly oxidize ascorbate under anaerobic conditions. alpha-Tocopherol, however, in the presence of NO and under anaerobic conditions, was oxidized to the alpha-tocopheroxyl radical. Under conditions where NO oxidized alpha-tocopherol, the subsequent production of the alpha-tocopheroxyl radical depleted ascorbate, yielding the semidehydroascorbyl radical and regenerating alpha-tocopherol. Thus, NO interacts with the redox cycle involving alpha-tocopherol and ascorbate in a pro-oxidant manner.

Antioxidants↗
PubMed1995

Reduction of ferrylmyoglobin and ferrylhemoglobin by nitric oxide: a protective mechanism against ferryl hemoprotein-induced oxidations.

The reactions of metmyoglobin (metMb) and methemoglobin (metHb), oxidized to their respective oxoferryl free radical species (.Mb-FeIV = O/.Hb-4FeIV = O) by tert-butyl hydroperoxide (t-BuOOH), with nitric oxide (NO.) were studied by a combination of optical, electron spin resonance (ESR), ionspray mass (MS), fluorescence, and chemiluminescence spectrometries to gain insight into the mechanism by which NO. protects against oxidative injury produced by .Mb-FeIV = O/.Hb-4FeIV = O. Oxidation of metMb/metHb by t-BuOOH in a nitrogen atmosphere proceeded via the formation of two protein electrophilic centers, which were heme oxoferryl and the apoprotein radical centered at tyrosine (for the .Mb-FeIV = O form, the g value was calculated to be 2.0057), and was accompanied by the formation of t-BuOOH-derived tert-butyl(per)oxyl radicals. We hypothesized that NO. may reduce both oxoferryl and apoprotein free radical electrophilic centers of .Mb-FeIV = O/.Hb-4FeIV = O and eliminate tert-butyl(per)oxyl radicals, thus protecting against oxidative damage. We found that NO. reduced .Mb-FeIV = O/.Hb-4FeIV = O to their respective ferric (met) forms and prevented the following: (i) oxidation of cis-parinaric acid (PnA) in liposomes, (ii) oxidation of luminol, and (iii) formation of the tert-butyl(per)oxyl adduct with the spin trap DMPO. NO. eliminated the signals of tyrosyl radical detected by ESR and oxoferryl detected by MS in the reaction of t-BuOOH with metMb. As evidenced by MS of apomyoglobin, this effect was due to the two-electron reduction of .Mb-FeIV = O by NO. at the oxoferryl center rather than to nitrosylation of the tyrosine residues. Results of our in vitro experiments suggest that NO. exhibits a potent, targetable antioxidant effect against oxidative damage produced by oxoferryl Mb/Hb.

Animals↗
PubMed1993

[The effect of a structural modification in the membrane proteins on the lipid-protein interaction in human erythrocyte membranes].

The effect of resonance energy transfer from excited tryptophan residues of proteins towards the pyrene molecules which took place in a close association, has been used to study lipid-protein interaction in human erythrocyte membranes. Diamide, N-ethylmaleimide, and t-butyl hydroperoxide were utilized to modify membrane proteins. The disturbance of membrane protein architecture induced by N-ethylmaleimide, and t-butyl hydroperoxide resulted in the increase of the mobility of pyrene molecules being present in the microenvironment of membrane proteins. In the bulk lipid matrix no detectable changes were observed. The influence of the reagents on the erythrocyte membranes was controlled using merocyanine 540 and spin-probe techniques.

Blood Proteins↗
PubMed1992

[Effects of nitroxyl radicals on pathogenic properties of herpesvirus (the spin-label technique in the study of virus-macrophage interactions)].

The effect of various concentrations of both methyl ether of 5-doxyl-stearic acid (M5DS) and 4-maleimido-TEMPO (4MT) on the pathogenicity of herpes simplex virus (HSV-1) was studied. It is known that the reagents modify the lipid matrix and the proteins of virion envelope. The decrease of the HSV-1 pathogenicity was shown when using the concentration of reagents more 5 x 10(-5) M. HSV-1 having high pathogenicity and cytotoxicity was obtained when the concentrations of the reagents were less 5 x 10(-5) M.

Animals↗
PubMed1992

[Participation of tyrosine residues of myoglobin in the disproportionateness reaction of heme iron (II) and (IV)].

By means of the comparison of the constant oxidation reactions of both the myoglobin modified by N-acetylimidazole and the intact myoglobin in the presence of H2O2 or ferrylmyoglobin we characterized the role of the tyrosine residues (Tyr) of myoglobin in the synproportionation reaction between heme iron (II) of one molecule and heme iron (IV)--of another. It was demonstrated that Tyr derivatization resulted in the decrease of the velocity of redox interaction between deoxymyoglobin (II) and ferrylmyoglobin (IV) and led to the decrease of the efficiency of oxymyoglobin deoxygenation. The effects were shown to be independent on Tyr quantity in myoglobin molecule and to have the same character for both the sperm-whale myoglobin and the horse myoglobin.

Animals↗