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• W2018474491 abstract "The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411–18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO2 ≤ 2 mm Hg) is preceded by increased inducible NO synthase and NO synthase activity. This effect is accompanied by oxidation of intracellular glutathione and formation of S-nitrosothiols. Incubation of cells with a selective inhibitor of inducible NO synthase (S-(2-aminoethyl)-isothiourea) or a NO scavenger ([2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide]) significantly attenuated the increase in heme oxygenase activity caused by reduced oxygen availability. A series of antioxidant agents did not prevent the elevation in heme oxygenase activity by hypoxia; however, the precursor of glutathione synthesis and thiol donor,N-acetylcysteine, completely abolished HO-1 induction. We also found that the hypoxia-mediated increase in endothelial heme oxygenase activity was potentiated by the presence ofS-nitrosoglutathione. These results indicate that intracellular interaction of thiols with NO is an important determinant in the mechanism leading to HO-1 induction by reduced oxygen levels. We suggest that in addition to oxidative stress, HO-1 gene expression can be regulated by redox reactions involving NO andS-nitrosothiols (nitrosative stress), emphasizing a versatile role for the heme oxygenase pathway in the cellular adaptation to a variety of stressful conditions. The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411–18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO2 ≤ 2 mm Hg) is preceded by increased inducible NO synthase and NO synthase activity. This effect is accompanied by oxidation of intracellular glutathione and formation of S-nitrosothiols. Incubation of cells with a selective inhibitor of inducible NO synthase (S-(2-aminoethyl)-isothiourea) or a NO scavenger ([2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide]) significantly attenuated the increase in heme oxygenase activity caused by reduced oxygen availability. A series of antioxidant agents did not prevent the elevation in heme oxygenase activity by hypoxia; however, the precursor of glutathione synthesis and thiol donor,N-acetylcysteine, completely abolished HO-1 induction. We also found that the hypoxia-mediated increase in endothelial heme oxygenase activity was potentiated by the presence ofS-nitrosoglutathione. These results indicate that intracellular interaction of thiols with NO is an important determinant in the mechanism leading to HO-1 induction by reduced oxygen levels. We suggest that in addition to oxidative stress, HO-1 gene expression can be regulated by redox reactions involving NO andS-nitrosothiols (nitrosative stress), emphasizing a versatile role for the heme oxygenase pathway in the cellular adaptation to a variety of stressful conditions. hypoxia inducible factor-1 nitric oxide nitric-oxide synthase inducible nitric-oxide synthase endothelial constitutive nitric-oxide synthase carbon monoxide heme oxygenase-1 ([2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide]) manganese (III) tetrakis(4-benzoic acid) porphyrin S-nitrosoglutathione [1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one] tin protoporphyrin IX N G-nitro-l-arginine methyl ester S-(2-aminoethyl)isothiourea [l-N6-(1-iminoethyl)-lysine dihydrochloride] S-nitrosothiols phosphate-buffered saline 5,5′-dithiobis-(2-nitrobenzoic acid) Tissue hypoxia is a condition of reduced oxygen levels which characterizes several pathophysiological states including ischemia, atherosclerosis, and cancer. Mammalian organisms have evolved a series of stratagems to counteract the negative effect of intracellular oxygen deficiency and favoring the adaptation of tissues to low oxygen tension. For example, there exist sensitive genes that, in the absence of oxygen, are readily stimulated to encode for modulators of erythropoiesis (erythropoietin), promoters of angiogenesis (vascular endothelial growth factor), and proteins involved in alternative metabolic pathways for ATP generation (glycolytic enzymes) (1.Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1043) Google Scholar). Thus, the ultimate purpose of these inducible systems is to increase the oxygen-carrying capacity of the blood and improve oxygen delivery to hypoxic tissues. The expression of hypoxic-sensitive genes appears to require redox modification and phosphorylation of specific transcription factors such as hypoxia inducible factor-1 (HIF-1)1 (1.Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1043) Google Scholar). In addition to the above mentioned systems, low oxygen or anoxic conditions affect the protein expression of nitric-oxide synthase (NOS) and heme oxygenase, two enzymatic pathways responsible for the synthesis of signaling molecules that regulate important biological activities (2.Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2198) Google Scholar). Nitric oxide (NO), generated from the oxidation ofl-arginine by NOS enzymes, is a multifunctional interactive molecule involved in the modulation of vascular tone, inhibition of platelet aggregation, and oxygen transport to tissues (3.Palmer R.M.J. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9289) Google Scholar, 4.Ignarro L.J. Buga G.M. Wood K.S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9265-9269Crossref PubMed Scopus (4313) Google Scholar, 5.Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1460) Google Scholar). Under most physiological conditions, NO derives mainly from constitutively expressed endothelial NOS (ecNOS); however, the inducible isoform (iNOS) can generate substantial amounts of NO once appropriately stimulated by cytokines or other inflammation-mediated stimuli. Hypoxia has also been shown to affect differentially the expression and activity of the diverse NOS isoforms. Exposure of human umbilical vein endothelial cells to hypoxia results in decreased transcription of the ecNOS protein as well as reduced mRNA stability (6.Mcquillan L.P. Leung G.K. Marsden P.A. Kostyk S.K. Kourembanas S. Am. J. Physiol. 1995; 267: H1921-H1927Google Scholar, 7.Phelan M.W. Faller D.V. J. Cell. Physiol. 1996; 167: 469-476Crossref PubMed Scopus (109) Google Scholar). Similarly, bovine pulmonary artery endothelial cells exposed to low oxygen levels show a marked repression in the ecNOS transcript and decreased NOS activity (8.Liao J.K. Zulueta J.J., Yu, F.S. Peng H.B. Cote C.G. Hassoun P.M. J. Clin. Invest. 1995; 96: 2661-2666Crossref PubMed Scopus (198) Google Scholar). In contrast, the iNOS gene appears to be up-regulated by conditions of low oxygen availability. Indeed, a functional hypoxia-responsive element has been detected in the promoter region of iNOS in murine macrophages (9.Melillo G. Musso T. Sica A. Taylor L.S. Cox G.W. Varesio L. J. Exp. Med. 1995; 182: 1683-1693Crossref PubMed Scopus (540) Google Scholar), and induction of the iNOS transcript in vascular tissue was found using a chronic hypoxia model of pulmonary hypertension (10.Palmer L.A. Semenza G.L. Stoler M.H. Johns R.A. Am. J. Physiol. 1998; 274: L212-L219PubMed Google Scholar). Heme oxygenase is the rate-limiting step in heme degradation; it catalyzes the oxidation of the α-meso carbon of the protoporphyrin ring leading to the formation of carbon monoxide (CO), free iron, and biliverdin (11.Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar). The inducible isoform of heme oxygenase, HO-1, is a ubiquitous heat shock protein (HSP32) that is highly induced by diverse stress-related conditions (2.Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Crossref PubMed Scopus (2198) Google Scholar, 12.Abraham N.G. Drummond G.S. Lutton J.D. Kappas A. Cell. Physiol. Biochem. 1996; 6: 129-168Crossref Scopus (231) Google Scholar). The end products of heme oxygenase may have crucial biological functions in the cardiovascular system. Bilirubin, which is formed from biliverdin by biliverdin reductase, is a potent antioxidant (13.Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Science. 1987; 235: 1043-1046Crossref PubMed Scopus (2900) Google Scholar) and has been shown recently to ameliorate postischemic myocardial dysfunction in a model of isolated heart (14.Clark J.E. Foresti R. Sarathchandra P. Kaur H. Green C.J. Motterlini R. Am. J. Physiol. 2000; 278: H643-H651Crossref PubMed Google Scholar). Moreover, enhanced CO production following HO-1 induction in vascular tissue effectively contributes to the suppression of both aortic vasoconstriction in vitro (15.Sammut I.A. Foresti R. Clark J.E. Exon D.J. Vesely M.J.J. Sarathchandra P. Green C.J. Motterlini R. Br. J. Pharmacol. 1998; 125: 1437-1444Crossref PubMed Scopus (212) Google Scholar) and acute hypertensive responses in vivo (16.Motterlini R. Gonzales A. Foresti R. Clark J.E. Green C.J. Winslow R.M. Circ. Res. 1998; 83: 568-577Crossref PubMed Scopus (245) Google Scholar). Reports have also demonstrated that the HO-1/CO pathway is markedly up-regulated by hypoxia in vascular smooth muscle cells, cardiomyocytes, and heart tissue (17.Morita T. Perrella M.A. Lee M.E. Kourembanas S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1475-1479Crossref PubMed Scopus (665) Google Scholar, 18.Borger D.R. Essig D.A. Am. J. Physiol. 1998; 274: H965-H973PubMed Google Scholar, 19.Lee P.J. Jiang B.H. Chin B.Y. Iyer N.V. Alam J. Semenza G.L. Choi A.M.K. J. Biol. Chem. 1997; 272: 5375-5381Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). It has been suggested that aortic vasoconstriction following chronic hypoxia in rats involves the induction of endothelial HO-1 and the enhanced production of CO (20.Caudill T.K. Resta T.C. Kanagy N.L. Walker B.R. Am. J. Physiol. 1998; 275: R1025-R1030PubMed Google Scholar). In view of the vasoregulatory effects of both NO and CO, it appears that the heme oxygenase and NOS pathways could actively participate in the maintenance of local tissue oxygenation; however, how these two systems interact mutually in response to limited availability of oxygen has not been examined previously. In previous studies, we demonstrated that various NO-releasing compounds induce endothelial HO-1 protein expression and heme oxygenase activity and that thiols are important regulators of this effect (21.Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,22.Foresti R. Motterlini R. Free Rad. Res. 1999; 31: 459-475Crossref PubMed Scopus (240) Google Scholar). In the present study we examined the temporal pattern of ecNOS/iNOS and HO-1 expression in aortic endothelial cells exposed to hypoxia. Having established that iNOS expression precedes the up-regulation of HO-1 protein, we investigated a possible involvement of endogenously generated NO, glutathione, andS-nitrosothiols in modulating hypoxia-mediated HO-1 induction. Inhibitors of ecNOS and iNOS, carboxy-PTIO (CPTIO), manganese (III) tetrakis(4-benzoic acid) porphyrin (MnTBAP),S-nitrosoglutathione (GSNO), and [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ) were obtained from Alexis Corporation (Bingham, Nottingham, U. K.). Tin protoporphyrin IX (SnPPIX) was from Porphyrin Products Inc. (Logan, UT). N-Acetylcysteine, uric acid, 1,3-dimethyl-2-thiourea, and all other chemicals were obtained from Sigma unless otherwise specified. Bovine aortic endothelial cells were purchased from the European Collection of Animal Cell Culture (ECACC, Salisbury, U. K.), cultured in 75-cm2 flasks, and grown in Iscove's modified Dulbecco's medium supplemented with 8% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Endothelial cells at confluence were transferred to an air-tight chamber (Billups-Rothenberg Inc., Del Mar, CA) and flushed with a mixture of 95% N2and 5% CO2. The gas was infused continuously into the air-tight chamber at a flow rate of 5 liters/min for the first 2 h and at 1 liter/min for the following hours of incubation. In preliminary experiments conducted under these conditions, it was found that the pO2 measured in the media by an oxygen electrode was 2 mm Hg after a 2-h exposure to hypoxia and did not fluctuate from this value throughout the remaining incubation period. Within the hypoxia chamber, cells were maintained in a humidified atmosphere at 37 °C. A time course (0–24 h) of heme oxygenase activity, HO-1, cNOS, and iNOS protein expression was determined in endothelial cells exposed to hypoxia. At specific time points, NOS activity was also measured. To examine the involvement of endogenously produced NO on heme oxygenase activation, cells were exposed to hypoxia (18 h) in the presence of ecNOS/iNOS inhibitors.N G-nitro-l-arginine methyl ester (l-NAME, 1 mm) was used as the inhibitor of ecNOS, whereas S-(2-aminoethyl)isothiourea (ITU) and [l-N 6-(1-iminoethyl)-lysine dihydrochloride] (l-NIL) at concentrations of 20 and 40 μm were used as selective inhibitors of iNOS. In an additional set of experiments, heme oxygenase activity was measured in endothelial cells 18 h after hypoxic treatment in the presence of 100 μm CPTIO, a NO scavenger. To assess a possible role of intracellular redox changes in the stimulation of the heme oxygenase pathway by hypoxia, total glutathione (GSH + GSSG) and glutathione disulfide (GSSG) were measured at the indicated time points. In addition, heme oxygenase activity was determined in endothelial cells exposed to low oxygen tension in the presence of the following compounds: N-acetylcysteine (1 mm), a precursor of glutathione synthesis and permeable reducing agent; uric acid (1 mm), a scavenger of peroxynitrite; 1,3-dimethyl-2-thiourea (1 mm), a specific scavenger of hydroxyl radical; MnTBAP, a cell-permeable superoxide dismutase mimetic and peroxynitrite decomposition catalyst; and the antioxidant enzymes superoxide dismutase and catalase. S-Nitrosothiols (RSNO) were also measured during hypoxia at specific time points. To examine the possible role of S-nitrosation in the modulation of heme oxygenase activity, cells were exposed to hypoxia for 6 h in the presence of 0.5 mm GSNO and compared with cells treated with the compound in normoxic conditions. In additional experiments, cells were exposed to hypoxia in the presence of SnPPIX (50 μm), an inhibitor of heme oxygenase activity, or ODQ (100 nm–10 μm), a potent inhibitor of guanylate cyclase activity. Heme oxygenase activity assay was performed as described previously (23.Motterlini R. Foresti R. Intaglietta M. Vandegriff K. Winslow R.M. Am. J. Physiol. 1995; 269: H648-H655PubMed Google Scholar). Briefly, microsomes from harvested cells were added to a reaction mixture containing NADPH, rat liver cytosol as a source of biliverdin reductase, and the substrate hemin. The reaction was conducted at 37 °C in the dark for 1 h, terminated by the addition of 1 ml of chloroform, and the extracted bilirubin was calculated by the difference in absorbance between 464 and 530 nm (ε = 40 mm−1 cm−1). Samples of endothelial cells treated for the heme oxygenase activity assay were also analyzed by Western immunoblot technique as described previously (21.Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Briefly, an equal amount of proteins (30 μg) for each sample was separated by SDS-polyacrylamide gel electrophoresis, transferred overnight to nitrocellulose membranes, and the nonspecific binding of antibodies was blocked with 3% non-fat dried milk in PBS. Membranes were then probed with a polyclonal rabbit anti-HO-1 antibody (Stressgen, Victoria, Canada) (1:1,000 dilution in Tris-buffered saline, pH 7.4) for 2 h at room temperature. When probed for NOS proteins, membranes were incubated for 2 h with anti-ecNOS and anti-iNOS antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:20,000 dilution in Tris-buffered saline (pH 7.4). After three washes with PBS containing 0.05% (v/v) Tween 20, blots were visualized using an amplified alkaline phosphatase kit from Sigma (Extra-3A) and the relative density of bands analyzed by an imaging densitometer (model GS-700, Bio-Rad). Endothelial cells were seeded onto Lab-Tek® chamber slides (Nunc Inc., Naperville, IL) at a density of 106 cells/ml. They were grown to confluence and exposed to hypoxia for 18 h in the presence of various agents. The medium was then discarded, and the upper part of the slide well frame was removed. Slides were washed twice very gently with cold (4 °C) PBS for 5 min. Cells were then fixed in cold 95% v/v ethanol for 10 min and gently washed again in cold PBS for 5 min. Slides were immersed in cold 3% v/v H2O2 in PBS for 5 min followed by 5% v/v normal goat serum (Vector Laboratories Ltd., Peterborough, Cambridgeshire, U. K.) in PBS for 20 min at room temperature. Next, slides were covered with rabbit anti-HO-1 antibody (1:1,000 dilution in PBS) and left for 18 h at 4 °C. Slides were then covered with a 1:100 dilution in PBS of biotinylated goat anti-rabbit IgG antibody (Vector Laboratories Ltd.) and left for 60 min at room temperature on an orbital shaker. After incubation with the secondary antibody, slides were washed again with PBS at room temperature, and the tertiary avidin-biotin-antibody complex was applied (Vectastain® ABC Elite Kit, Vector Laboratories Ltd.) for 60 min at room temperature on an orbital shaker. Slides were washed in PBS for 5 min and finally incubated with 3,3′-diaminobenzidine (DAB Substrate Kit, Vector Laboratories Ltd.) until suitable staining developed (about 10 min) and analyzed by an imaging densitometer. Total RNA was isolated by phenol-chloroform using the method described by Chomczynski and Sacchi (24.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). Total RNA was run on a 1.3% denaturing agarose gel containing 2.2 m formaldehyde and transferred onto a nylon membrane. The membrane was hybridized using [α-32P]dCTP-labeled cDNA probes to rat HO-1 gene as described previously (25.Vesely M.J.J. Exon D.J. Clark J.E. Foresti R. Green C.J. Motterlini R. Am. J. Physiol. 1998; 275: C1087-C1094Crossref PubMed Google Scholar, 26.Vesely M.J.J. Sanders R. Green C.J. Motterlini R. FEBS Lett. 1999; 458: 257-260Crossref PubMed Scopus (21) Google Scholar), and staining of the 18 S rRNA band was used to confirm integrity and equal loading of RNA. The hybridized membrane was exposed to radiographic film and bands analyzed using an imaging densitometer. Total glutathione (GSH + GSSG) was measured in endothelial cells at various times of hypoxia using a modification of a method described previously (27.Adams J.D. Lauterburg B.H. Mitchell J.R. J. Pharmacol. Exp. Ther. 1983; 227: 749-754PubMed Google Scholar). Cells were harvested in ice-cold PBS, centrifuged at 10,000 × g for 5 min, and the pellet was resuspended in 100 mm potassium phosphate buffer (pH 7.5), containing 5 mm EDTA (buffer 1). The cell suspension was freeze-thawed three times, and an aliquot (60 μl) was added to an equal volume of 100 mm potassium phosphate buffer (pH 7.5), containing 12 mm EDTA and 10 mm5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The sample was mixed by tilting and centrifuged at 12,000 × g for 2 min. The supernatant was added to a cuvette containing 0.5 unit of glutathione reductase in buffer 1, equilibrated for 1 min, and the reaction initiated by adding NADPH (220 nmol). The change in absorbance at 412 nm was recorded over a period of 5 min using a reference cuvette containing equal concentrations of NADPH, DTNB, and enzyme. To assay GSSG, an aliquot of the cell suspension (400 μl) was added to an equal volume of 100 mm potassium phosphate buffer (pH 6.8), containing 17.5 mm EDTA and 10 mm N-ethylmaleimide. The sample was mixed, centrifuged and the supernatant passed through a C18 Sep-Pak cartridge (Waters, Watford, U. K.) to remove the excess N-ethylmaleimide and washed with buffer 1. The sample was added to a cuvette containing DTNB and glutathione reductase and the assay performed as for the measurement of total GSH. The intracellular glutathione content, expressed as nmol/mg of protein, was determined by comparison with a standard curve obtained with GSH and GSSG solutions. NOS activity was determined using the hemoglobin assay as described previously (28.Hevel J.M. Marletta M.A. Methods Enzymol. 1994; 233: 250-258Crossref PubMed Scopus (424) Google Scholar). The assay is based on the oxidation of oxyhemoglobin with NO and is performed spectrophotometrically by measuring the formation of methemoglobin under initial rate conditions. Briefly, cells were washed with PBS, gently scraped using a rubber policeman, and the cellular pellet separated by centrifugation. The pellet was resuspended in a solution containing 0.32 msucrose, 10 mm Tris (pH 7.4), 0.5 mmphenylmethylsulfonyl fluoride, and homogenized. The supernatant obtained after centrifugation at 12,000 × g for 30 min was used for NOS activity measurements. The reaction mixture contained (in a final volume of 1 ml): 1 mm l-arginine, 1 mm CaCl2, 0.1 mm NADPH, 12 μm tetrahydro-l-biopterin, 5 μmoxyhemoglobin, 4 μm FAD, 100 mm Hepes (pH 7.5), and 0.5 mg of protein sample. The formation of methemoglobin was monitored by the change in absorbance which occurred over time between 411 and 401 nm using a double beam spectrophotometer (Perkin-Elmer 559). Detection of S-nitrosothiols in cell extracts was performed after cleavage of the S-nitroso bond by measuring the NO released using the chemiluminescence reaction between NO and the luminol-hydrogen peroxide system (29.Clancy R.M. Levartovsky D. Leszczynskapiziak J. Yegudin J. Abramson S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3680-3684Crossref PubMed Scopus (305) Google Scholar, 30.Kikuchi K. Nagano T. Hayakawa H. Hirata Y. Hirobe M. Anal. Chem. 1993; 65: 1794-1799Crossref PubMed Scopus (192) Google Scholar). Differences in the data among the groups were analyzed by using one-way analysis of variance combined with the Bonferroni test. Values were expressed as a mean ± S.E. and differences between groups were considered to be significant atp < 0.05. Exposure of aortic endothelial cells to severe hypoxia (pO2≤ 2 mm Hg) resulted in a time-dependent increase of heme oxygenase activity and HO-1 protein expression (Figs.1 A and2 B). The increase was evident at 12 h, and both HO-1 protein and heme oxygenase activity continued to rise in the following hours of incubation. Northern blot analysis (Fig. 1 B) indicated that up-regulation of the HO-1 gene by hypoxia occurs at the transcriptional level and precedes protein expression, as HO-1 mRNA was markedly visible at 6 h and continued to increase for the entire period of the hypoxic treatment.Figure 2Time course of endothelial iNOS , ecNOS , and HO-1 protein expression during hypoxia. Western blot analysis of cells exposed to hypoxia for different times using specific antibodies for iNOS and ecNOS (panel A) and HO-1 (panel B) is shown. Samples were processed as described under “Experimental Procedures.” Each blot is representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A time-dependent increase in the expression of endothelial iNOS protein was also observed during hypoxia (Fig. 2 A); this was accompanied by enhanced NOS activity (see Fig. 7). However, iNOS protein was significantly up-regulated at 6 and 12 h hypoxia and started to decrease at 18 and 24 h. It is important to emphasize that at 6 h hypoxia, iNOS expression was already markedly elevated, whereas no changes in heme oxygenase activity and HO-1 protein expression were yet to be detected (Figs. 1,2 A, and 2 B). In contrast, endothelial ecNOS protein, which is highly expressed under control conditions (normoxia), was down-regulated by hypoxia in a time-dependent manner (Fig. 2 A). These data reveal that stimulation of iNOS protein by hypoxia occurred very rapidly and preceded both the increased expression of HO-1 protein and heme oxygenase activity. Our group has demonstrated the ability of various NO-releasing agents to increase heme oxygenase activity and HO-1 protein expression in bovine aortic endothelial cells, aortic tissue, and other cell types (15.Sammut I.A. Foresti R. Clark J.E. Exon D.J. Vesely M.J.J. Sarathchandra P. Green C.J. Motterlini R. Br. J. Pharmacol. 1998; 125: 1437-1444Crossref PubMed Scopus (212) Google Scholar, 21.Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 25.Vesely M.J.J. Exon D.J. Clark J.E. Foresti R. Green C.J. Motterlini R. Am. J. Physiol. 1998; 275: C1087-C1094Crossref PubMed Google Scholar, 31.Motterlini R. Foresti R. Intaglietta M. Winslow R.M. Am. J. Physiol. 1996; 270: H107-H114Crossref PubMed Google Scholar,32.Clark J.E. Green C.J. Motterlini R. Biochem. Biophys. Res. Commun. 1997; 241: 215-220Crossref PubMed Scopus (62) Google Scholar). Because a transient increase in iNOS protein and NOS activity preceded the induction of heme oxygenase during hypoxia, we wanted to verify whether iNOS-derived NO is required for triggering the expression of HO-1. As shown in Fig. 3, exposure of endothelial cells to hypoxia in the presence of a selective inhibitor of iNOS (ITU) resulted in a concentration-dependent attenuation of heme oxygenase activity (p < 0.05). We could not use higher concentrations of ITU because in preliminary experiments we found that concentrations above 40 μm can directly affect heme oxygenase activity in an in vitro assay. l-NIL, another inhibitor of iNOS activity, had a less pronounced effect; however, the presence of the NO scavenger CPTIO (100 μm) during hypoxia decreased endothelial heme oxygenase activity from 2,231 ± 140 to 969 ± 60 pmol of bilirubin/mg of protein/h (p < 0.05, Fig. 3). In contrast, l-NAME (1 mm), an inhibitor of cNOS, had no effect on heme oxygenase activation by hypoxia (Fig. 3). These data suggest that augmented NO production following stimulation of iNOS contributes to increased HO-1 expression and heme oxygenase activity under conditions of reduced oxygen tension. This effect appears to be cGMP-independent because ODQ, a selective inhibitor of guanylate cyclase activity, did not prevent hypoxia-mediated heme oxygenase activation (data not shown). Several lines of evidence suggest that glutathione is a crucial intracellular modulator of HO-1 gene expression both in vitro and in vivo (21.Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 33.Lautier D. Luscher P. Tyrrell R.M. Carcinogenesis. 1992; 13: 227-232Crossref PubMed Scopus (180) Google Scholar, 34.Ewing J.F. Maines M.D. J. Neurochem. 1993; 60: 1512-1519Crossref PubMed Scopus (169) Google Scholar, 35.Rizzardini M. Carelli M. Cabello Porras M.R. Cantoni L. Biochem. J. 1994; 304: 477-483Crossref PubMed Scopus (92) Google Scholar, 36.Oguro T. Kaneko E. Numazawa S. Imaoka S. Funae Y. Yoshida T. J. Pharmacol. Exp. Ther. 1997; 280: 1455-1462PubMed Google Scholar). Therefore, we examined glutathione levels after exposure of endothelial cells to low oxygen tension. Fig.4 shows the changes in total glutathione (GSH + GSSG), oxidized glutathione (GSSG), and the ratio between reduced and oxidized glutathione (GSH:GSSG) in endothelial cells at various time points of hypoxia. Hypoxia caused a substantial change in GSSG content (Fig. 4, center) which was markedly increased at 6 h hypoxia (14-fold, p < 0.05versus control), transiently diminished at 12 h, and significantly elevated after 24 h hypoxia (24.5-fold,p < 0.05). It can be observed that this effect was associated with a gradual and significant increase in total glutathione levels (Fig. 4, top) that were maximal at 12 h hypoxia (95%, p < 0.05 versus control), suggesting that reduced oxygen tension stimulates the enzymatic activities responsible for glutathione synthesis. However, at all time points examined, hypoxia caused a substantial change in the GSH:GSSG ratio, an index of the redox status of the cell. As shown in Fig. 4(bottom), the GSH:GSSG ratio decreased significantly from 115 ± 10 (control) to 11.3 ± 0.9 after 6 h hypoxia (p < 0.05); although this index transiently increased at 12 h hypoxia, a further decrease of this parameter was observed at 24 h hypoxia (5.8 ± 0.8, p < 0.05). The fact that the transient recovery of the GSH:GSSG ratio at 12 h hypoxia correlated with a rise in total glutathione is indicative of the inherent ability of cells to produce new glutathione in response to augmented GSSG. Taken together, these data reveal that exposure of cells to severe and prolonged hypoxic conditions results in the rapid formation of GSSG, leading to a marked alteration in the redox status of the intracellular milieu. Because thiol residues are important targets for NO-mediated biological activities, and we observed radical changes in GSH:GSSG ratio during hy" @default.
• W2018474491 created "2016-06-24" @default.
• W2018474491 creator A5006907344 @default.
• W2018474491 creator A5034436289 @default.
• W2018474491 creator A5041692767 @default.
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• W2018474491 date "2000-05-01" @default.
• W2018474491 modified "2023-10-15" @default.
• W2018474491 title "Endothelial Heme Oxygenase-1 Induction by Hypoxia" @default.
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